LIBRARY Michigan State University *- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE MAY 0 4 2006 JUN 0 8 2006 ‘J‘DUS -I” 6/01 cJCIRC/DateDuepGS-p. 15 ROLE OF OVEREXPRESSION OF THE Sp1 AND Sp3 TRANSCRIPTION FACTORS IN THE MALIGNANT TRANSFORMATION OF HUMAN FIBROBLASTS By Zhenjun Lou A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry and Molecular Biology 2004 ABSTRACT ROLE OF OVEREXPRESSION OF THE Sp1 ANO Sp3 TRANSCRIPTION FACTORS IN THE MALIGNANT TRANSFORMATION OF HUMAN FIBROBLASTS By Zhenjun Lou Two kinds of genes are involved in the malignant transformation of a normal cell: oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function. Alteration in functions Of these genes breaks the tight control over the proliferation and death of normal cells, and finally leads to the progressive conversion to cancer cells. Sp1 is a transcription factor for many genes, including genes that play an important role in tumorigenesis. Recently, overexpression or higher binding activity Of Sp1 has been found in several types of human cancers, including pancreatic adenocarcinoma, breast carcinoma, gastric carcinoma, and thyroid carcinoma. To investigate the role of overexpression of Sp1 in the malignant transformation of human fibroblasts, l transfected an Sp1 UtsnRNA/Ribozyme into two human fibrosarcoma cell lines, malignantly transformed in culture by a carcinogen or overexpression of an oncogene, and into a patient-derived fibrosarcoma cell line. Several cell strains expressing the Sp1 U1snRNA/ Ribozyme showed significant decrease in Sp1 level. When injected into athymic mice, these cell strains with near normal levels of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. They showed spindle-shaped morphology and exhibited increased apoptosis and decreased expression of several genes linked to cancer, viz., epithelial growth factor receptor, urokinase plasminogen activator (uPA), uPA receptor, and vascular endothelial growth factor. These results strongly suggest that overexpression of Sp1 plays a causal role in malignant transformation of human fibroblasts and that for cancers in which it is overexpressed, Sp1 constitutes a target for therapy. TO determine how expression of Sp3 is regulated and the basis for the coordinated expression Of Sp1 and Sp3 in human fibrosarcoma cell lines, I isolated 2.1 kb of the 5’-flanking region of the human Sp3 gene and inserted it upstream of the firefly luciferase reporter gene. Deletion analysis showed that the fragment (minimal promoter) spanning nt -—339 to —39 (relative to the ATG translation start codon) conferred the same activity as that of the 2.1 kb promoter. Within the minimal promoter there are two putative Sp1/Sp3 binding sites. Mutation studies demonstrated that each Of the two putative Sp1/Sp3 binding sites is required for promoter activity. Gel shift assays showed that both Sp1 and Sp3 bound to these two sites. ln human embryonic kidney (HEK 293) cells and SL2 insect cells, Sp1 caused strong activation Of the Sp3 minimal promoter. In contrast, in insect cells, Sp3 was a weak activator for the Sp3 minimal promoter, and in HEK 293 cells, it repressed activation. This suggests that Sp3 requires secondary modification(s) in order to function as a repressor. DEDICATION TO my mother, Shulan Wu, who gives me life, love, and everything she has TO my wife, Huiying Zhang, for her love and support TO my beautiful daughter, Jia Lou, and son, Matthew J. Lou, they are the joy Of my life ACHNOWLEDGEMENTS .I thank my major professor, Dr. J. Justin McCormick, for his guidance throughout the course Of my Ph.D study in Michigan State University. His dedication to science inspires me to strive to become the kind of scientist he is. His enthusiasm, energy and dedication to helping people Show me what kind of person I should be. I also want to express my sincerest gratitude to Dr. Veronica M. Maher for all her advice and encouragement. Because of her wonderful presentation for graduate students during the BMB graduate student orientation in 1999, I decided to come to the Carcinogenesis Laboratory. Furthermore, I give my special thanks to other members Of my graduate committee, Dr. Kathleen A. Gallo, Dr. Min-Kao Kuo and Dr. Katheryn Meek, for their help and invaluable time during my Ph.D study. I also thank Dr. Meek for sharing ideas and equipments, especially for her advice for my future career. The members of the Carcinogenesis Laboratory give me tremendous help from the beginning Of my research here. I thank Dr. Sandra O’Relly for her scientific training and instructions and insightful advice for my research. I thank Dr. Hongyan Liang, Dr. Jeannine Scott, Dr. Igor Zlatkin, Jessica Apostol, Dan Appledom, Kathy Bergdolt, Clarissa Dallas, Bethany Heinlen, Suzanne Kohler, PirO Lito, Terry McManus, Kristin McNally, Lijuan Wang, Yun Wang and Jie Zhang for your generous assistance. I appreciate the help from Angela Taylor and Bryan D. Mets. I am very grateful that my mother, one Of the greatest women in my life, brought me to the world because I am the tenth kid in my family. Her greatest love will be with me in all my life. I thank my wife, Huiying Zhang, for her love and endless support. I give my very special thanks to my daughter, Jia Lou, and my son, Matthew J. Lou. They are the joy of my life. vi TABLE OF CONTENT LIST OF TABLES .................................................................................. xi LIST OF FIGURES ............................................................................... xii LIST OF ABBREVIATIONS ..................................................................... xiv INTRODUCTION .................................................................................. 1 REFERENCES .................................................................................... 8 CHAPTER 1: LITERATURE REVIEW ................................................... 12 I. Molecular Biology of Cancer ............................................................. 12 A. Genetic Alterations That Play a Role in Cancer ................................. 12 1. Chronic Myelogenous Leukemia and the BCFI/ABL Gene ....................... 12 2. Retinoblastoma and the Rb Gene ..................................................... 14 3. Malignant Transformation in Culture .................................................... 15 4. Transgenic Mice .............................................................................. 16 5. Summary ..................................................................................... 16 B. DNA Damage Leads to Gene Mutations .......................................... 17 1. DNA Damage ................................................................................ 17 2. Cell Cycle and Mutations .................................................................. 18 3. DNA Repair Mechanisms .................................................................. 18 4. Summary ..................................................................................... 19 C. Oncogenes and Tumor Suppressor Genes ........................................ 20 1. Typical Oncogenes ......................................................................... 20 1.1. Fias ........................................................................................ 20 1.2. c-Myc ........................................................................................ 22 2. Typical Tumor Suppressor Genes ..................................................... 23 2.1. p53 .......................................................................................... 23 2.2. INK4a and ARF .......................................................................... 25 D. Telomeres and Telomerase ........................................................... 26 1. Telomeres ..................................................................................... 27 2. Telomerase .................................................................................. 28 3. Telomeres, Telomerase and Cancer ................................................... 28 E. The Multistep Process of Carcinogenesis ....................................... 29 1. Mouse Skin Model Of Experimental Carcinogenesis ............................... 3O 2. Human Colon Cancer ....................................................................... 31 2.1. FAP and Sporadic Colon Cancer ................................................... 32 a. The APC Tumor Suppressor Gene ................................................ 32 b. Methylation Status ....................................................................... 32 c. The K-ras Oncogene .................................................................. 33 d. The p53 Tumor Suppressor Gene ................................................ 33 2.2. HNPCC .................................................................................. 34 3. Malignant Transformation of Human Fibroblasts in Culture ..................... 34 vii II. Sp1 and Sp/KLF Transcription Factors .............................................. 38 A. The Finding of Sp1 (Specificity_ Protein1 1) ........................................ 38 B. Characterization Of the Functional Domains of the Sp1 Protein ............. 39 1. DNA Binding Domain ...................................................................... 39 2. Transcriptional Domains ................................................................. 39 3. Other Functional Domains ................................................................ 40 C. Sp1 Transcriptional Activity ............................................................... 41 1. Interactions with the Components of Basal Transcription Machinery .........41 2. Interactions with Site-specific Transcription Factors ............................... 41 3. Interactions with Chromatin Modifier Complex ...................................... 42 4. Summary .................................................................................... 43 D. Post-Translational Modifications ..................................................... 43 1. Glycosylation ............................................................................... 44 2. Phosphorylation ............................................................................. 44 3. Acetylation .................................................................................... 46 E. Physiological Function of Sp1 protein .............................................. 47 F. Sp3 ......................................................................................... 47 1. Transcriptional Activity ................................................................... 49 2. Physiological Function Of Sp3 ......................................................... 50 3. Sp1/Sp3-mediated GeneExpression .................................................. 50 G. Other Sp/KLF Members .............................................................. 51 1. DNA Binding Domains ................................................................... 52 2. Transcriptional Regulatory Domains .................................................. 53 3. Expression patterns ........................................................................ 54 H. Sp/KLF proteins and Cancer ......................................................... 54 1. Sp/KLF Proteins and Cell Growth ..................................................... 56 2. Sp/KLF Proteins and Cell Apoptosis .................................................. 58 3. Sp/KLF Proteins and Cell lnvasiveness and Metastasis .......................... 60 4. Sp/KLF Proteins and Angiogenesis ..................................................... 62 5. Summary ....................................................................................... 63 REFERECES ..................................................................................... 65 CHAPTER 2: DOWN-REGULATION OF OVEREXPRESSED Sp1 PROTEIN IN HUMAN FIBROSARCOMA CELL LINES INHIBITS TUMOR FORMATION ..107 ABSTRACT ...................................................................................... 109 INTRODUCTION .......................................................................... 110 MATERIALS AND METHODS .......................................................... 114 Cells and cell culture ......................................................................... 114 Preparation of Sp1 ribozyme antisense construct .................................... 114 Transfection ..................................................................................... 1 15 Western blot analysis ......................................................................... 1 15 Preparation of conditioned medium ........................................................ 116 ELISA ............................................................................................. 1 16 RT-PCR analysis of Sp1 mRNA .......................................................... 116 Luciferase assay .............................................................................. 1 17 viii Assay for anchorage-independence ........................................................ 1 17 Assay for tumorigenicity ........................................................................ 1 18 Cell morphology ............................................................................... 1 18 Cell death assay ............................................................................... 118 Apoptosis assay ................................................................................. 1 19 RESULTS ................................................................................... 120 Overexpression of Sp1 in human fibrosarcoma cell lines ........................... 120 Construction of the Sp1 U1snRNA/ribozyme vector ................................... 120 Down-regulation of Sp1 level and transactivating activity ........................... 125 Down-regulation Of Sp1 expression reduces expression of Sp3 ................... 127 The Sp1 U1snRNA/ribozyme acts as a ribozyme and as antisense ............... 127 H-Ras expression in PH2MT cell line and its derivatives ........................... 128 Cell strains with reduced Sp1 levels no longer forrn large colonies in agarose .......................................................................................... 132 Down-regulation of Sp1 inhibits the tumorigenicity of cell lines PH2MT and y2- 3A/SB1 .......................................................................................... 135 Cell morphology changes following the down-regulation of Sp1 level ............ 137 Down-regulation Of Sp1 expression induces apoptosis ............................. 140 The expression of HGF/MET, uPA/uPAR, EGFR and VEGF in the cell strains with reduced Sp1 levels ..................................................................... 144 Down-regulation of Sp1 expression in a patient-derived fibrosarcoma cell line inhibits its tumorigenicity ..................................................................... 147 DISCUSSION ................................................................................ 152 ACKNOWLEDGEMENTS ................................................................ 158 REFERENCES ............................................................................... 159 CHAPTER 3: IDENTIFICATION OF THE PROMOTER OF HUMAN TRANSCRIPTION FACTOR Sp3 AND CHARACTERIZATION OF THE ROLE OF THE Sp1 AND Sp3 IN THE EXPRESSION OF Sp3 PROTEIN ............... 166 ABSTRACT ................................................................................... 167 INTRODUCTION .......................................................................... 168 EXPERIMENTAL PROCEDURES ................................................... 171 Cells and Cell Culture .......................................................................... 171 Isolation of the Human Sp3 and Sp1 Promoters and Construction of Plasmids ....................................................................................... 171 Constructs for Identifying the Minimal Sp3 Promoter .................................. 172 Mutagenesis of the Sp1/Sp3 Binding Sites .............................................. 172 5’ RLM-RACE ................................................................................... 173 Transient Transfection and Lluciferase Assay of Promoter Activity ............... 173 Preparation Of Nuclear Extracts and EMSA .............................................. 174 Preparation of an Sp3 Ribozyme Antisense Construct ................................ 175 Stable Transfection and Western Blot Analysis ......................................... 175 Database Mining ................................................................................ 176 RESULTS ................................................................................... 177 Cloning of the 5’-Flanking Region of the Human Transcription Factor Sp3 Gene ......................................................................................... 177 ix Putative Transcription Factors Binding Elements in the 2.1 kb Fragment ~ 5’ to the Human Sp3 Gene .................................................................. 177 Multiple Transcription Iinitiation Sites ..................................................... 180 Functional Mapping of the Human Sp3 Promoter .................................... 183 The Binding of Sp1 and Sp3 to the Proximal Region of Sp3 Promoter ........... 186 Role of Sp1 and Sp3 in the Regulation of Sp3 Promoter Activity .................. 189 Coordinated Expression of Sp1 and Sp3 in Human Fibrosarooma Cell Llines ......................................................................................... 193 Construction Of the Sp3 U1snRNA/Ribozyme Vector .................................. 193 Evidence That Sp1 and Sp3 Proteins Play a Role In the Coordinate Expression of Sp3 and Sp1 .................................................................. 193 Multiple Sp1/Sp3 Binding Sites In 5’- -F|anking Regions of the Genes of the Sp/KLF Family Members ............................................................... 198 DISCUSSION ................................................................................ 201 FOOTNOTES ................................................................ ‘ ................ 2 05 ACKNOWLEDGEMENTS ................................................................. 206 REFERENCES .............................................................................. 207 LIST OF TABLES CHAPTER 2 Table 1: Inhibition of tumor formation by down-regulation Of Sp1 expression in malignant cell lines ............................................... 136 Table 2: Inhibition Of tumor formation by downregulation of Sp1 expression in patient-derived fibrosarcoma cell line SHAC ............................... 151 xi LIST OF FIGURES CHAPTER 2 Fig. 1: Evidence that Sp1 is overexpressed in the malignant human fibroblast cell lines and diagram Of the Sp1 U1snRNA/Ribozyme and its predicted structure ........................................................... 122 Fig. 2: Evidence that stable transfection of malignant cell lines with the Sp1 U1snRNA/Ribozyme reduces expression of Sp1 protein and its transactivating activity, and expression Sp3 protein, but does not affect that Of a H-ras oncogene ......................................... 129 Fig. 3: Evidence that down-regulation Of overexpressed Sp1 protein inhibits anchorage-independent growth ................................................... 133 Fig. 4: Evidence that down-regulation of Sp1 protein affects cell morphology, but not through B-actin ............................................. 138 Fig. 5: Evidence that down-regulation of Sp1 expression induces apoptosis ................................................................................. 141 Fig. 6: Effect of down-regulation of Sp1 protein on the expression of cancer-related genes .................................................................. 145 Fig. 7: Evidence that the Sp1 U1snRNA/Ribozyme reduces expression , Of Sp1 in a human patient-derived fibrosarcoma SHAC and inhibits its ability to grow in agarose ......................................................... 149 CHAPTER 3 Fig. 1: Nucleotide sequence Of the human Sp3 promoter ............................ 178 Fig. 2: Identification of the transcription start sites by 5’-RLM-RACE assay ....................................................................................... 181 Fig. 3: Deletion analysis of human Sp3 promoter activity in SHAC cells .......... 184 Fig. 4: The binding Of Sp1 and Sp3 to the Sp3 proximal promoter ................ 187 Fig. 5: Role of Sp1 and Sp3 in the regulation Of human Sp3 promoter activity ......................................................................... 191 Fig. 6: Expression of Sp1 and Sp3 in human fibrosarcoma cell lines and reduced expression by the use of an Sp3-specific ribozyme .......... 195 xii Fig. 7: Alignment of the sequences of the 5’-flanking regions or promoters of the genes of 21 Sp/KLF family members .................................... 199 xiii LIST OF ABBREVIATIONS ALT, alternative lengthening Of lelomeres APC, adenomatous golyposis goli ARF, alternative leading lrame ATM, ataxia lelangiectasia _nlutated ATR, ataxia lelangiectasia [elated BCL-2, B-gell lymphoma -2 BER, _base excision lepair BTEBS, Qasic transcription alement (BTE)-Qinding protein 5 CAM, gall-cell adhesion molecules CBP, _QREP-llinding grotein CDK, gyclin-gependent _lginase CIAP, _calf intestine alkaline ghosphatase CML, ahronic _m_yelogenous leukemia CRC, gologectal gancer CRSP, gofactor lequired for S91 DMBA, 7,12-gimethyll2enz[alanthracene DRS, geath [eceptor 5 ECM, axtragellular matrix EGF, apidennal growth factor EGFR, _e_pidermal growth lactor [eceptor ERK, axtracellular signal-[egulated I_<_inase xiv FAP, lamilial adenomatous golyposis FCC, familial golon gancer GAP, (_iTPase-activating groteins GEF, guanine nucleotide axchange [actors HAT, histone acetyllransferase HDAC1, histone ge_a_c_etylase 1 Hdm2, human gouble minute 2 HG F, hypatocyte growth factor HNPCC, hereditary honpolygosis golon gancer HSV-1, herpes aimplex yinIs-1 IGF II, lnsulin-like growth lactor ll RB, [etinohlastoma XP, heroderma pigmentosum LDL, low gensity lipoprotein MAPK, mitogen-activated grotein hinase MAX, myc associated factor X MDM2, mouse gouble minute 2 MEK, MAP/ERK hinase MGMT, 05-methylguanine-DNA methyllransferase MMR, mismatch [epair MMP, matrix metallogroteases MNNG, N-methyl-N’-hitro-N-hitrosoguaniding MSV, murine aarcoma yirus NER, hucleotide axcision I_'epair NDF, heu _clifferentiation lactor NF-Y, huclear lactor-_Y_ PDGF, glatelet-gerived growth lactor PKA, grotein _Iginase _A_ PKCC, grotein hinase Q g PI3K, ghosphatodyllnsitol 3-hinase Sp/KLF, Sg/lgrupel-like lactor SREB-1a, ateroI-[esponsive alement-hinding protein-1a TAF, IBP-associate l‘actor TBP, IATA box-hinding grotein TERC, telomerase RNA component TERT, laiomerase _r_everse lranscriptase TF, lranscription [actor TG F, transforming growth factor TNF R, lumor hecrosis lactor [eceptor TPA, 12- O-letradecanoylghorbOl-1 3-acetate uPA, grokinase-type glasinnogen activator uPAR, urokinase-type plasinnogen activator receptor VEGF, yascular andothelial growth lactor VSMC, yascular _s_mooth muscle gell YY1, Xin Xang 1 INTRODUCTION Cancer is a disease involving genetic changes that confer upon cells with new Characteristics, such as loss of control Of cell growth and cell death, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (1). The genes that are usually altered in cancer cells are divided into two categories: oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function (2). Both classes of genes have been identified through their alteration in human and animal cancer cells and by their elicitation of cancer phenotypes in experimental models (3). In general, these genes involved in cancer formation are those that regulate cell cycle progression, apoptosis, DNA damage repair and telomere function (2). Alteration in function Of these genes breaks the tight control over the proliferation and death of the normal cells, and finally leads to the progressive conversion to cancer cells (1, 4). Tumorigenesis is a multistep process (5). With the exception Of an eye tumor, retinoblastoma, that only requires each allele Of the Rb tumor suppressor gene in a retina cell to be inactivated (mutated) in order to form a tumor (6), all tumor cells have undergone progressive selections and clonal expansions such that they have acquired all the genetic Changes to confer the malignant phenotype (7). It is estimated that four to seven genetic changes are needed for a normal human cell to become malignant (8, 9). Evidence that tumorigenesis is a multistep process comes from the observation that most human cancers occur with an age-dependent incidence, as seen in the studies of colon cancer (9), and the development of the MSU1 lineage of cells in tissue culture (7). Sp1 was the first transcription factor to be purified, cloned, and * characterized in mammalian cells (10). Ubiquitously expressed, it binds the 60- box (GGCGGG) and GT-box (CACCC) DNA sequences via its CyszHisz zinc- finger DNA binding domain and activates transcription of the target genes (11). Sp1 belongs to the Sp/KriippeI-like factor (Sp/KLF) family, consisting of 21 members that share high homology in their DNA binding domains, ranging from 67% to 95% in similarity (12). These proteins are present in species ranging from C. elegans to humans (13—16). In humans, 21 Sp/KLF genes have been found by a variety Of cloning approaches (12), whereas the Drosophila melanogaster only has three Sp1/KLF proteins (15, 17). Sp3, a member Of the Sp/KLF family, was cloned in 1992 (18). Sp3 and Sp1 are closely related: 1) both genes are found to be linked to the homeobox gene cluster on respective chromosome (Sp1 to HOX C and Sp3 to HOX D) (19), indicating that these two genes arise from a single ancestor; studies that showed that the exon-intron structures of the Sp1 and Sp3 genes are conserved also supports this hypothesis, 2) the Sp1 and Sp3 proteins are ubiquitously expressed (20), 3) the Sp1 and Sp3 proteins share a similar modular structure (21) and share 95% similarity in the zinc-finger DNA binding domains; consistent with this, they both bind to GC-box and GT-box with the same affinity. Sp1 is a transcription activator (20), whereas Sp3 has been reported not only to positively regulate transcription (22-25), but also to act as a repressor (26). Recent studies using mammalian cells indicate that the Sp3 protein is modified by phosphorylation (27, 28), acetylation (29, 30), and sumoylation (31, 32) and that these modifications determine the role that Sp3 plays in transcription. The nucleotides adjacent to the GC-box and the GT-box influence the role Of Sp3 in transcription (20). Consistent with the differences in transcriptional activity, the physiological roles of Sp1 and Sp3 are different. Sp1" mouse embryos are severely retarded in growth and die after 9.5 days Of development (33). Sp34‘ mouse embryos survive, but they die immediately after birth as the result of respiratory failure. Defects in tooth and bone development are found in such Sp3" mouse embryos (34, 35) Indirect evidence that Sp1 and other members of the Sp/KLF family play a role in the malignant transformation of normal cells comes from studies showing that Sp1 and other members of the Sp/KLF family regulate the expression Of more than 1,000 genes involved in a broad spectrum of cell functions, including cell cycle control, cell growth and proliferation, cell apoptosis, angiogenesis, and invasiveness and metastasis (36). Recently, overexpression or higher binding activity of Sp1 was found in human pancreatic cancer cell lines and cancer tissue (37), breast cancer cell lines and cancer tissue (38), gastric carcinoma (39), and thyroid carcinoma (40). Several of these studies also showed that overexpression of Sp1 or up-regulation of Sp1 transcriptional activity is closely correlated with up-regulation of vascular endothelial growth factor (VEGF) (37), urokinase plasminogen activator (uPA) and uPA receptor (uPAR) (38), and epithelial growth factor receptor (EGFR) (39). In addition, upregulation Of KLF4 was detected in dysplastic epithelium and squamous cell carcinoma of oral cavity, and in breast carcinoma, suggesting that upregulation Of KLF4 contributes ‘_I"—' " ‘-""'“'_“ contributes to the malignant phenotype Of squamous cell carcinomas (41) and '- breast tumors (41, 42). KLF6 acts as a tumor suppressor gene in prostate carcinoma, and the ability Of KLF6 to inhibit cell growth is reduced by mutations within its transcriptional regulatory domain (43). Taken together, these studies suggest that changes in the expression of Sp1 or other members of the Sp/KLF family contribute to tumor formation. However, until my studies, direct evidence that overexpression of Sp1 contributes to the malignant transformation of normal human cells had not been determined. Liang et al. (44) in the MSU Carcinogenesis Laboratory recently showed that four of six malignantly transformed cell lines of the MSU1 lineage examined overexpressed Sp1 (The MSU1 lineage of cell lines used for investigation Of the genetic changes involved in malignant transformation are discussed in detail in CHAPTER 1, Literature Review). In addition, three of five patient-derived fibrosarcoma cell lines examined showed a high level of Sp1 compared to normal human fibroblasts. These results suggest that overexpression of Sp1 plays a role in the malignant transformation of human fibroblasts, not only in culture, but also in the human body. The first part Of my dissertation research focused on the role of overexpression of Sp1 in the malignant transformation of human fibroblasts. For these studies, I chose two human fibrosarcoma cell lines, designated PH2MT and v2-3A/SB1, which belong to the MSU1 lineage. PH2MT cells were derived from tumors formed in athymic mice by injection of MSU-1.1 cells malignantly transformed by overexpression of an H-Fias oncogene (45). v2-3A/SB1 cells were similarly derived from a tumor formed by MSU-1.1 cells malignantly transfonned by y-irradiation (46). These cells exhibited an Sp1 level 3- to 6-fold (PH2MT) or 7- to 10-fold (y2-3A/SBI) higher than that Of the normal parental MSU-1.1 cells. To investigate whether the up-regulation of Sp1 expression plays a causal role in the malignant transformation of human fibroblasts, I stably transfected the two cell lines with an Sp1 U1snRNA/Ribozyme construct. I also transfected the same construct into a patient-derived fibrosarcoma cell line, SHAC, that expresses a high level of Sp1. Transfected cell clones with significantly reduced Sp1 protein levels were isolated and their ability to grow in agarose and to form tumors in athymic mice was examined. The PH2MT- and v2-3A/SB1-derived cell strains with significantly reduced Sp1 protein level (<20% compared with that Of the parental cell lines) from cell lines could nO longer grow in agarose. They formed only small colonies, similar to those formed by MSU-1.1 cells, while their malignant parental and vector control cells formed large-sized colonies. When injected into the flank regions Of athymic mice, the cells with significantly reduced Sp1 protein levels did not form tumors within six months after injection, whereas, the parental and vector control cells gave rise to tumors within 4-6 weeks. These results indicate that overexpression of Sp1 in human fibrosarcoma cell lines plays a causal role in malignant transformation. Evidence that the inhibition of tumorigenicity of the human fibrosarcoma cell lines is Sp1-leveI-dependent comes from my finding that the PH2MT- and SHAC-derived cell strains with intermediate levels of Sp1 could form tumors in athymic mice, but at a greatly reduced frequency and with a much longer latency. The cell strains in which the overexpressed Sp1 has been " significantly reduced regained the spindle-shaped morphology of normal fibroblasts and exhibited increased apoptosis and decreased expression of several genes linked to cancer, viz., EGFR, uPA, uPAR, and VEGF. Taken together, my results indicate that overexpression Of Sp1 plays a causal role in the malignant transformation Of normal human fibroblasts. Its contribution is through the regulation of Sp1-targetted genes involved in the regulation Of cell morphology, cell growth and death, angiogenesis, invasiveness and metastasis. The second part Of my dissertation research focused on the regulation of transcription Of the human Sp3 gene. In my studies of Sp1, I found that the PH2MT- and y2-3A/SB1-derived cell strains with significantly reduced Sp1 level also showed reduced expression of Sp3. I also showed that Sp1 and Sp3 are coordinately expressed in human fibrosarcoma cell lines. These results suggest that Sp1 is involved in the regulation of transcription Of the human Sp3 gene. To determine how transcription of Sp3 is regulated and the basis for the coordinated expression of Sp3 and Sp1, l isolated a 2.1 kb DNA fragment containing the 5’-flanking region of the human Sp3 gene, and by deletion analysis, I found that region spanning nt —339 to —39 (relative to the ATG translation start codon) conferred the same activity as that Of the 2.1 kb promoter. This result indicated that the DNA responsive elements within this region (minimal promoter) play an important role in the regulation Of transcription Of the human Sp3 gene. There are two putative Sp1/Sp3 binding sites within this region; mutation studies and gel shift assays showed that both Sp1 and Sp3 bind to these two sites and contribute to the minimal promoter activity. I also found that Sp1 caused strong activation of the Sp3 minimal promoter in SL2 insect cells and in human embryonic kidney (HEK) 293 cells. In contrast, Sp3 was a weak activator in SL2 insect cells, and in HEK 293 cells, it repressed activation. This suggests that Sp3 requires secondary modification(s) in order to function as a repressor. This dissertation consists of three parts. Chapter 1 is a literature review of the molecular biology of cancer, Sp1, Sp3, and other members of the Sp/KLF family, and their roles in tumorigenesis. Chapter 2 is a copy Of a manuscript submitted to Cancer Research. The title of the paper is “Down-regulation of overexpressed Sp1 protein in human fibrosarcoma cell lines inhibits tumor formation”. Chapter 3 is a copy of a manuscript submitted to Journal of Biological Chemistly. The title of the paper is “Identification of the promoter Of human transcription factor Sp3 and Characterization of the role of Sp1 and Sp3 in the expression of Sp3 protein”. 10. 11. 12. 13. 14. REFERENCES Hanahan, D. and Weinberg, R. A. The hallmarks of cancer. Cell, 100: 57- 70, 2000. Kopnin, B. P. Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biochemistry (MOSC), 65: 2-27, 2000. Bishop, J. M. and Weinberg, R. A. Molecular Oncology. New York: cientific American, Inc, 1996. Bertram, J. S. The molecular biology of cancer. MOI Aspects Med, 21: 167-223, 2000. Foulds, L. The natural history of cancer. J Chronic Dis, 8: 2-37, 1958. Brantley, M. A., Jr. and Harbour, J. W. The molecular biology of retinoblastoma. 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Blood, 98: 1792-1801, 2001 . Ossipova, 0., Stick, R., and Pieler, T. XSPR-1 and XSPR-2, novel Sp1 related zinc finger containing genes, are dynamically expressed during Xenopus embryogenesis. Mech Dev, 115: 117-122, 2002. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Schock, F., Pumell, B. A., Wimmer, E. A., and Jackle, H. Common and diverged functions of the Drosophila gene pair D-Sp1 and buttonhead. Mech. Dev., 89: 125-132, 1999. Brown, D. D., Wang, Z., Furlow, J. D., Kanamori, A., Schwartzman, R. A., Remo, B. F., and Pinder, A. The thyroid hormone-induced tail resorption programduring Xenopus laevis metamorphosis. Proc Natl Acad Sci U S A, 93: 1924-1929, 1996. Wimmer, E. A., Jackle, H., Pfeifle, C., and Cohen, S. M. A Drosophila homologue Of human Sp1 is a head-specific segmentation gene. Nature, 366: 690-694, 1993. Hagen, G., Muller, S., Beato, M., and Suske, G. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res., 20: 5519-5525, 1992. O'Brien, S. J., Womack, J. E., Lyons, L. A., Moore, K. J., Jenkins, N. A., and Copeland, N. G. Anchored reference loci for comparative genome mapping in mammals. Nat Genet, 3: 103-1 12, 1993. Suske, G. The Sp-family of transcription factors. Gene, 238: 291-300, 1999. Bouwman, P. and Philipsen, S. Regulation of the activity of Sp1-related transcription factors. Mol. Cell. Endocrinol., 195: 27-38, 2002. Kwon, H. S., Kim, M. S., Edenberg, H. J., and Hur, M. W. Sp3 and Sp4 can repress transcription by competing with Sp1 for the core cis-elements on the human ADH5/FDH minimal promoter. J. Biol. Chem., 274: 20-28, 1999. Kumar, A. P. and Butler, A. P. Enhanced Sp1 DNA-binding activity in murine keratinocyte cell lines and epidermal tumors. Cancer Lett., 137: 159-165, 1999. Pagliuca, A., Gallo, P., and Lania, L. Differential role for Sp1/Sp3 transcription factors in the regulation Of the promoter activity Of multiple cyclin-dependent kinase inhibitor genes. J. Cell. Biochem., 76: 360-367, 2000. Yajima, S., Lee, S. H., Minowa, T., and Mouradian, M. M. Sp family transcription factors regulate expression Of rat D2 dopamine receptor gene. DNA Cell. Biol., 17: 471-479, 1998. Ammanamanchi, S. and Brattain, M. G. Sp3 is a transcriptional repressor of transforming growth factor-beta receptors. J. Biol. Chem., 276: 3348- 3352, 2001. -.‘A ~ n- 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Ge, Y., Jensen, T. L., Matherly, L. H., and Taub, J. W. Synergistic '- regulation Of human cystathionine-beta-synthase-1b promoter by transcription factors NF-YA isoforrns and Sp1. Biochim. Biophys. Acta, 1579: 73-80, 2002. Bakovic, M., Waite, K., and Vance, D. E. Oncogenic Ha-Ras transformation modulates the transcription of the CTPzphosphOCholine cytidylyltransferase alpha gene via p42/44MAPK and transcription factor Sp3. J. Biol. Chem., 278: 14753-14761, 2003. Braun, H., Koop, R., Ertmer, A., Nacht, S., and Suske, G. Transcription factor Sp3 is regulated by acetylation. Nucleic Acids Res., 29: 4994-5000, 2001. Ammanamanchi, S., Freeman, J. W., and Brattain, M. G. Acetylated Sp3 is a transcriptional activator. J. Biol. Chem, 2003. Sapetschnig, A., Rischitor, G., Braun, H., Doll, A., Schergaut, M., Melchior, F., and Suske, G. Transcription factor Sp3 is silenced through SUMO modification by PIAS1. EMBO J., 21: 5206-5215, 2002. Ross, 8., Best, J. L., Zon, L. l., and Gill, G. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. MOI. Cell., 10: 831-842, 2002. Marin, M., Karis, A., Visser, P., Grosveld, F., and Philipsen, S. Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell, 89: 619-628, 1997. Bouwman, P., Gollner, H., Elsasser, H. P., Eckhoff, G., Karis, A., Grosveld, F., Philipsen, S., and Suske, G. Transcription factor Sp3 is essential for post-natal survival and late tooth development. EMBO J., 19: 655-661, 2000. Gollner, H., Dani, C., Phillips, 8., Philipsen, S., and Suske, G. Impaired ossification in mice lacking the transcription factor Sp3. Mech. Dev., 106: 77-83, 2001. Black, A. R., Black, J. D., and Azizkhan-Clifford, J. Sp1 and kruppel-Iike factor family of transcription factors in cell growth regulation and cancer. J. Cell. Physiol., 188: 143-160, 2001. Shi, 0., Le, X., Abbruzzese, J. L., Peng, Z., Qian, C. N., Tang, H., Xiong, Q., Wang, 8., Li, X. C., and Xie, K. Constitutive Sp1 activity is essential for differential constitutive expression Of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res, 61: 4143-4154, 2001 . 10 F71: 38. 39. 40. 41. 42. 43. 44. 45. 46. Zannetti, A., Del Vecchio, S., Carriero, M. V., Fonti, R., Franco, P., Botti, G., D'Aiuto, G., Stoppelli, M. P., and Salvatore, M. Coordinate up- regulation Of Sp1 DNA-binding activity and urokinase receptor expression in breast carcinoma. Cancer Res., 60: 1546-1551, 2000. Kitadai, Y., Yasui, W., Yokozaki, H., Kuniyasu, H., Haruma, K., Kajiyama, G., and Tahara, E. The level of a transcription factor Sp1 is correlated with the expression of EGF receptor in human gastric carcinomas. Biochem Biophys Res Commun, 189:1342-1348, 1992. Chiefari, E., Brunetti, A., Arturi, F., Bidart, J. M., Russo, D., Schlumberger, M., and Filetti, S. Increased expression of AP2 and Sp1 transcription factors in human thyroid tumors: a role in NIS expression regulation? BMC Cancer, 2: 35, 2002. Foster, K. W., Ren, S., Louro, I. D., LObO-Ruppert, S. M., McKie-Bell, P., Grizzle, W., Hayes, M. R., Broker, T. R., Chow, L. T., and Ruppert, J. M. Oncogene expression Cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation Of a host with epithelial features by c-MYC and the zinc finger protein GKLF. Cell Growth Differ, 10: 423-434, 1999. Foster, K. W., Frost, A. R., MCKie-Bell, P., Lin, C. Y., Engler, J. A., Grizzle, W. E., and Ruppert, J. M. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res, 60: 6488- 6495,2000. Narla, G., Heath, K. E., Reeves, H. L., Li, D., Giono, L. E., Kimmelman, A. C., Glucksman, M. J., Narla, J., Eng, F. J., Chan, A. M., Ferrari, A. C., Martignetti, J. A., and Friedman, S. L. KLF6, a candidate tumor suppressor gene mutated in prostate cancer. Science, 294: 2563-2566, 2001. Liang, H., O'Reilly, 8., Liu, Y., Abounader, R., Laterra, J., Maher, V. M., and McConTIick, J. J. Sp1 regulates expression of MET, and ribozyme- induced down-regulation of MET in fibrosarcoma-derived human cells reduces or eliminates their tumorigenicity. lntemational J. Oncol., 24: 1057-1068, 2004. ‘ Hurlin, P. J., Maher, V. M., and McCormick, J. J. Malignant transformation of human fibroblasts caused by expression Of a transfected T24 HRAS oncogene. Proc. Natl. Acad. Sci. USA, 86: 187-191, 1989. O'Reilly, 8., Walicka, M., Kohler, S. K., Dunstan, R., Maher, V. M., and McCormick, J. J. Dose-dependent transformation of cells Of human fibroblast cell strain MSU-1.1 by cobalt-60 gamma radiation and characterization of the transformed cells. Radiat Res, 150: 577-584, 1998. 11 CHAPTER 1 LITERATURE REVIEW I. Molecular Biology Of Cancer A. Genetic Alterations That Play a Role in Cancer Evidence accumulated in the past few decade reveals that all cancers harbor genetic changes which may be inherited or have resulted from somatic mutations. Alteration in gene function in normal cells can disrupt control over proliferation and death, and eventually lead to conversion of normal cells to cancer cells (1, 2). The theory that cancer results from genetic alterations is supported by the results Of a large number Of studies. Examples include: 1) investigation Of the cause Of chronic myelogenous leukemia (CML) and the hereditary predisposition to certain kinds Of cancer, such as retinoblastoma, and 2) the discovery of oncogenes and tumor suppressor genes, and their roles in the development Of cancer. 1. Chronic Myelogenous Leukemia and the BCR/ABL Gene CML is a Clonal hematopoietic stem cell disorder. It was first recognized as a distinct entity Of leukemia in 1845 when two patients were found tO have massive splenomegaly—associated leukocytosis (3). The principal cause Of CML was linked tO genetic change by studies showing that 95% Of the patients are Philadelphia chromosome (Ph) positive, which is the result Of the reciprocal translocation between the long arms of Chromosomes 9 and 22, t (9:22) (q34zq11) (4, 5). The molecular consequence is generation Of the BCR/ABL fusion gene formed by juxtaposition of ABL on chromosome 9 at exon 2 with 12 ‘M'T «..z BCR on chromosome 22 at exon 13 or 14 (6, 7). The product of the proto- oncogene c-ABL, c-ABL, is a non-receptor tyrosine kinase and is involved in the processes of cell differentiation, cell division, cell adhesion, cell death, and stress response (8, 9). At the N-tenninus of c-ABL, 80 amino acid residues that are required to achieve and maintain the inhibition Of kinase activity are lost in BCR/ABL fusion proteins. This loss leads to the constitutive tyrosine kinase activity Of c-ABL (10) and turns it into an oncogenic protein (11-14). It is generally accepted that generation of the BCR/ABL fusion gene is sufficient tO initiate CML. But because the aggressiveness of the chronic phase disease varies from patient to patient, other events may also Contribute (15). This conclusion is strongly supported by studies showing that transgenic mice that express the BCR/ABL fusion gene in hematopoietic cells, bone marrow, and myeloid progenitors can produce a CML-like disease (16-18) . Clinically, CML progresses through three distinct phases, referred to as chronic (stable), accelerated, and blast crisis (acute myoblastic leukemia). The progression from the chronic to the blast state is caused by the accumulation of molecular abnormalities. This accumulation leads to a progressive loss of capacity for terminal differentiation in the leukemic Clone (19, 20). Because of the causal role Of the BCR/ABL fusion proteins in the formation Of CML, a chemical agent called Gleevec, which specifically inhibits the tyrosine kinase activity Of the fusion proteins, has been developed and successfully applied to treat patients with CML in clinics (21, 22). 13 2. Retinoblastoma and the Rb Gene Retinoblastoma, a rare pediatric eye tumor, has served as a very useful model tO study the origin and nature Of human cancers (23). Through careful statistical analysis and insightful mathematical modeling Of the age distribution of unilateral and bilateral cases, Knudson hypothesized that “two hits” (two mutations) are required for the formation Of retinoblastoma (24). This “two hits” hypothesis was verified in 1986 by the identification Of the retinoblastoma (Rb) gene, the first tumor suppressor gene to be identified (25-28). Investigation on the DNA, RNA, and protein levels also supported the idea that the inactivation of the Rb gene was associated with retinoblastoma (29-31). The Rb protein is part of a family that includes two other members, p107Rb and p130”, which collectively co-repress the transcription of genes that regulate programs governing cell cycle progression, apoptosis, and differentiation. Active, hypophosphorylated Rb family proteins physically interact with transcription factors, e.g., E2Fs and sequester them from activating the downstream genes that promote GI tO S transition in the cell cycle (32, 33). Recent studies Show that Rb family proteins also help to repress gene expression by recruiting histone deacetylases and chromatin-remodeling factors to these loci (34). In contrast, phosphorylation of Rb family proteins by mitogen-activated, cyclin-dependent kinases (CDKS) releases the bound transcription factors, allowing expression Of Rb-mediated genes. The Rb pathway has been found to be functionally inactivated in almost all types of cancers (23), and Rb mutations occur at high frequency in a selected subset Of human cancers, e.g., small cell and non-small 14 cell lung cancers, pancreatic cancers, and breast cancers (35, 36). Evidence that loss of Rb gene function contributes to tumor progression also comes from studies showing that re-introducing Rb into Rb-deficient cells impairs growth Characteristics Of the malignant phenotype (37). Retinoblastoma may be hereditary (familial) or nonhereditary (sporadic). In the familial cases, a gerrnline mutation in one Rb allele is inherited from a parent. The loss Of the second allele function occurs spontaneously and is the rate-limiting step (38). The patients with inherited mutation of an Rb allele develop bilateral disease within an average span Of 12 months (23). In the nonhereditary (sporadic) cases, two de novo somatic mutations in both alleles are required, and patients usually develop unilateral disease within 18 months after birth (23). As a childhood cancer, retinoblastoma accounts for approximately 11% of cancers that occur in the first year Of life (39). If they survive the tumor in the eye, nonhereditary patients have a near-normal life expectancy, whereas hereditary patients harboring the Rb mutation in cells throughout the body succumb to second primary malignancies, such as osteosarcoma and melanoma (40). 3. Malignant Transformation in Culture Evidence that alterations in genes that control cell proliferation and cell death play a causal role in the formation Of cancer also comes from studies of oncogenic proteins in cells in culture. For example, Ras, the first oncogene ever identified in human tumor cells (4143), (discussed below in detail in Section C), was shown to be involved in multiple intracellular signal transduction pathways, 15 and aberrant Ras function has been detected in 30% Of all human cancers (44, .. 45). Also, Ras has been shown to malignantly transform rodent fibroblasts (46, 47) and human fibroblasts in culture (48). In the latter case, overexpression Of the oncogenic H-ras protein in MSU-1.1 cells, an infinite life-span human fibroblast cell strain, transformed the cells into malignantly tumorigenic cells. These cells formed large-sized colonies in soft agarose at a high frequency and gave rise to progressively growing, invasive fibrosarcomas in athymic mice (48). 4. Transgenic Mice Transgenic and gene knockout mice are two powerful tools for studying the molecular mechanisms Of tumorigenesis. One excellent example is a 1999 study by Felsher and Bishop who generated mice that conditionally expressed a c-Myc oncogene in their hematopoietic cells. Expression of the transgene led to malignant lymphoid and myeloid tumors. Subsequent inactivation Of the transgene caused regression of the established tumors. These data indicate that overexpression of the c-Myc oncogene in the transgenic mice plays a causal role in formation Of the malignant tumors (49). 5. Summary Cancer cells contain many genetic changes. These alterations in gene functions confer upon the tumor cells a growth advantage, the ability to resist apoptosis, sustained angiogenesis, and the ability to invade and metastasize (1). The gene defects in cancer cells may be inherited in the gennline, induced by chemical or physical mutagens, or the result Of spontaneous mutations. 16 B. DNA Damage Leads to Gene Mutations 1. DNA Damage Alterations in gene functions which modify the birth rate or the death rate Of individual cells have been firmly implicated as causative of carcinogenesis. The vast majority Of mutations are not inherited, but arise as the consequences Of DNA damage (2). Damage to DNA can occur in two ways, spontaneously, or as a result Of mutagens. Spontaneous DNA damage results from the inherent instability Of DNA molecules, particularly at 37°C, e.g., depurination as a result Of breaking of the N-glycosidic bond connecting purine to deoxyribose, and deamination Of cytosine or methylcytosine to uracil or thymine, respectively (50, 51). The consequence Of depurination is random base insertions across from the apurinic sites, and the consequence Of deamination is G-A or C-T mutations during DNA replication (51, 52). Mutagenic agents are generally divided into two categories: chemical carcinogens and physical carcinogens (2). Chemical carcinogens include a wide range Of chemicals which directly form chemical-DNA adducts, or which can be metabolized to create compounds which form such adducts (53). Physical carcinogens, such as ionizing radiation and ultraviolet irradiation, cause DNA damage in different ways. Ionizing radiation causes the formation of free radicals in the water inside of cells as well as the water outside Of cells. These free radicals can cause single and double-stranded DNA breaks as well as indirect DNA damage (54, 55). Ultraviolet (UV) irradiation is absorbed by the DNA 17 approximately 10‘6/cell division (2, 52). High fidelity DNA replication is achieved by cells through efficient mechanisms: 1) high fidelity of DNA polymerases, 2) proteins, such as p53, that survey the genome, 3) and proteins that repair DNA damage. DNA polymerase 6, the major eukaryotic DNA replication polymerase, is capable Of 3'-5’ exonuclease activity, which allows it to excise misincorporated nucleotides (62). In addition, DNA-repair genes have evolved whose products’ purpose is to survey the genome and/or repair DNA damage induced by DNA replication, endogenous or exogenous Chemical agents, and physical carcinogens (57). One group of genes whose products sense DNA damage and regulate DNA repair include p53, ataxia telangiectasia mutated (ATM) (63, 64), ataxia telangiectasia related (ATR) (65, 66). The other group of DNA-repair genes includes those whose products are associated with distinct DNA repair mechanisms, e.g., 06-methylguanine-DNA methyltransferase (MGMT) in reversion repair (67, 68), DNA glycosylases in base excision repair (BER) (69, 70), MutSa (MSH2 and MSH6), MutLa (MLH1 and PMSZ) in mismatch repair (MMR) (71-73), and K070, Ku80, XRCC4 and DNA-PKCS in DNA double-strand break repair (74, 75). Mutations in genes involved in DNA repair are responsible for the development Of several types of tumors and various hereditary diseases (63, 73, 76-87). 4. Summary DNA damage caused by endogenous reagents and exogenous carcinogens is responsible for gene mutations. The products of DNA-repair 19 bases, this provides sufficient energy to induce chemical reactions, such as the .. formation Of pyrimidine dimers between adjacent pyridines in DNA (56). The DNA damage can be reversed or excised (57). Failing to do so may give rise to mutations, which contribute to the malignant transformation Of cells. For example, a rare inherited disease, Xeroderrna pigmentosum (XP), is a result Of defects in the DNA excision repair system. Patients with this disease are very sensitive to ultraviolet light and other DNA-disturbing damage and, therefore, prone to cancers (58, 59). 2. Cell Cycle and Mutations DNA replication is a necessary process for the formation and passing on Of mutations. In responding tO extracellular signals, eukaryotic cells undergo G1, S, 62, and M phases during cell division. DNA is replicated and chromosomes are duplicated during the S phase; sister Chromatics are separated during the M phase (60, 61). Ionizing radiation can cause mutations directly by inducing single or double-stranded DNA breaks if the breaks are not rejoined without losing a nucleotide (55). DNA damage caused by chemicals or ultraviolet irradiation, however, is not a mutagenic event until the cell undergoes DNA synthesis that converts the DNA damage to an inheritable mutation (2). 3. DNA Repair Mechanisms DNA-repair is required for normal cells to maintain genome integrity (57). Considering the large number Of cells (~1015 cells) and cell divisions (107 cell divisions occur per second) in a human adult, DNA damage is expected to occur at a high frequency. In fact, mutation Of a gene in a normal cell is very rare, 18 genes are essential for maintaining the integrity Of a genome. Defects in these .. genes along with exposure to specific carcinogens result in specific types Of cancers. C. Oncogenes and Tumor Suppressor Genes It is now widely understood that the activation Of human proto-oncogenes and lost function Of tumor suppressor genes lead to the development Of cancer (88). Evidence shows that the majority Of proto-oncogenes and tumor suppressor genes code for components Of a few common signal transduction pathways that control the cell cycle progression, cell growth, apoptosis, genome integrity, and cell differentiation. Changes in these signal pathways cause the formation Of cancer (1, 88, 89). 1. Typical Oncogenes Proto-oncogenes code for normal regulatory proteins such as growth factors, growth factor receptors, transcription factors, proteins involved in signal transduction and in the regulation Of apoptosis, cyclins and cyclin-dependent kinases (CDKS) etc. Such proteins act to promote cell growth. If the activity of such proteins is increased as the result of a mutation in the gene, gene amplification, gene rearrangement, or by overexpression, a proto-oncogene becomes an oncogene (90, 91), Le, they act dominantly. 1.1 . Ras The best-studied oncogenes are the Ras oncogenes. In 1964, Harvey Observed that a murine leukemia virus induced sarcomas in new-bom mice (92). In 1967, Kristen and Mayer found that a murine erythroblastosis virus induced 20 sarcomas in mice (93). In 1975, Scolnick and his associates found that the Harvey (Ha-MSV) and Kirsten (Ki-MSV) strains were recombinant viruses that carry sequences derived from the rat genome (94). The human homologs Of murine H-Ras (41-43) and K-Ras (95) were cloned in 1982, and a third human Ras gene, N-Ras, in 1983 (96, 97). These three Ras oncogenes, H-Ras, N-Ras and K-Ras, are closely related, with 84% identity in their protein products (98). The proto-oncogene Ras, eg. the normal Ras gene, encodes a small GTPase, whose activity is regulated by the binding of GTP (active form) and GDP (inactive form). Normally, the activity Of Ras is tightly regulated by guanine nucleotide exchange factors (GEFs), which cause the exchange Of GDP for GTP to produce the active, GTP-bound state, and by GTPase-activating proteins (GAPS), which catalyze hydrolysis Of bound GTP to GDP, thereby turning Off Ras activity (99). Activated Ras recruits an elaborate array Of effector proteins, including Raf/MEK/ERK, PI3K, Rho family, RaIGEFs, and NORE/MST1, and either activates them directly or positions them to induce specific signal pathways involved in cell proliferation, transformation, differentiation, senescence, or apoptosis (100, 101). For example, GTP-bound Ras activates the serine/threonine kinase Raf, which, in turn, activates the dual-specificity tyrosine/threonine kinase MEK. Active MEK then phosphorylates ERKIMAPK, which translocates to the nucleus and regulates the transcription Of a variety of genes, including up-regulation Of cyclin D1 and p21, which promote cell-cycle progression through phosphorylation of retinoblastoma (Rb) proteins (98). 21 Mutations in Ras, particularly at positions 12, 13 or 61, cause Ras to .. become insensitive to negative regulation by GAP and remain constitutively GTP bound and active. Gain-of-function of Ras might also become functional through the deregulation Of signaling from upstream activators Ordownstream negative regulators; which lead to excessive levels of GTP-Ras (101). As a result, the constitutively active Ras causes uncontrolled cell proliferation and differentiation which contribute to malignant transformation. Oncogenic Ras can be found in ~20-30% Of all human cancers and in >90% of some, such as pancreatic cancer (45, 102). 1.2. c-Myc Another well-studied proto-oncogene is c-Myc. The c-Myc proto- oncogene encodes a basic-helix-IOOp-helix-zipper transcription factor, and was originally identified as the cellular homolog of the viral oncogene (v-Myc) of the avian myelocytomatosis retrovirus (103). Although gain-of-function mutations in C-MYC have been found in some types Of tumors (104), the typical oncogenic form results from the constitutive or deregulated expression Of c-MYC. Overexpression of c-MYC has been associated with many human tumors, including breast cancer (105, 106), prostate cancer (106), gastrointestinal cancer (107), and melanoma (108). The C-MYC protein and its partner, MAX (myc associated factor X), form a MYC-MAX heterodimer which is capable Of binding Specific DNA sequence, such as the E-box (CACCTG) (109), and activating a variety Of known target genes involved in cell growth (110, 111), cell proliferation (111-113), inhibition of cell differentiation (114, 115), and apoptosis (116-119). 22 Both transactivational activity and DNA binding activity are required for biological function Of c-MYC. Mutations in these functional domains abolish the effects Of c-MYC on cell proliferation, cell growth, cell differentiation, and apoptosis (120- 123) 2. Typical Tumor Suppressor Genes Tumor suppressor genes regulate diverse cellular activities, including cell cycle checkpoint responses, protein ubiquitination and degradation, mitogenic signaling, cell specification, differentiation and migration, and tumor angiogenesis (124). To be classified as a tumor suppressor gene, a gene must meet the following criteria: 1) acts recessively and undergoes biallellic inactivation in tumors, 2) accelerates tumor susceptibility when a single mutant allele that reduces protein function or causes loss Of its activity is inherited, and 3) frequently exhibits inactivation in sporadic cancers Of the same type (124). Twenty one tumor suppressor genes, including p53, INK4a and ARF, have been identified (125). 2.1. p53 The p53 gene is the most extensively studied tumor suppressor gene. In human colorectal cancers one allele of p53 was found to be deleted, and analysis Of the second allele in tumor cells showed that it had sustained mutations, suggesting that loss of function Of both alleles is required for p53 to contribute to the formation of human colorectal cancer (126). Patients with familial Li-Fraumeni Syndrome inherit a mutant allelic copy Of p53. Consequent- ly, they have a much higher predisposition of acquiring various types Of cancers, 23 indicating that reduction in p53 activity accelerates tumor susceptibility (127-129). -. Furthermore, mutations Of p53 are found in 50-55% Of all human tumors, and mutation-induced changes in the p53 pathway have been Observed in over 80% of human tumors (129, 130). These studies give strong evidence that p53 is a very effective tumor suppressor gene. The p53 protein forms a homotetramer and acts as a transcription factor to regulate the expression of genes involved in cell cycle arrest (131, 132), apoptosis (133-135), and DNA repair (136). In normal cells under the physiological conditions, p53 is expressed at a very low level, is highly unstable, and has a low DNA binding capacity. The level of p53 accumulates in response to DNA damage induced by cellular stresses (137-142). Cellular stress-induced accumulation Of p53 occurs as the result Of inactivation Of human double minute 2 (Hdm2), a protein with E-3 ubiquitin ligase activity which promotes the proteasome-mediated degradation Of p53 (142). The accumulation of stable and transcripttionally active p53 leads to the up-regulation of the expression Of p21WAF 1, a protein that inhibits cyclin-dependent kinase 2 (CDK 2) and cell division cycle 2 (CDC 2) (143-146). The result Of such inhibition is G1 arrest, which allows DNA repair to take place. Alternatively, the cells undergo apoptosis by activating the expression Of death receptors Fas/APO1 (138, 147-149) and DR5/KILLER, and pro-apoptotic proteins Bax, NOXA, and PUMA of the Bcl-2 family proteins (138, 140). However, the mechanisms by which cells under stress halt cell division and allow DNA repair, or those which cause the cell to commit suicide through apoptosis are not yet understood. 24 2.2. INK4a and ARF INK4a and ARF are located on human Chromosome 9 at position p21 (150, 151). The coding sequences of these two genes are partially overlapping, with alternative first exons (10 for INK4a and 18 for ARF). INK4a and ARF code for two unrelated proteins, p16'NK‘a and p14ARF (p19ARF in mouse), because the start codons within 10 and 18 exons are in different reading frames (151-154). The p16'NK4a and p14""RF proteins are involved in tumor suppression through different pathways. The p16'NK‘a protein directly inhibits the ability Of cyclin D-dependent kinases, CDK4 and CDK6, to phosphorylate Rb, a Change that maintains Rb in its active, hypophosphorylated state in which the Rb protein can bind to the transcription factor E2F (155). The sequestration of E2F represses its transcriptional activity and results in cell cycle arrest (156). The role Of INK4a as a tumor suppressor gene is further supported by the finding that mice lacking both alleles Of INK4a have an ~3-fOId higher predis-position to cancer than do p16’"*4°*" mice (157, 158). The ARF gene product, p14ARF, suppresses tumorigenicity through the p53 pathway (124). Hdm2, the negative regulator of p53, can bind to p53. The binding Of Hdm2 tO p53 inhibits its transcriptional activity, induces its ubiquitination, and accelerates its exportation from the nucleus to the cytoplasm, where it is degraded (159). The pi4ARF protein directly associates with Hdm2. The binding Of p14ARF to Hdm2 inhibits its E3 unibiquitin protein ligase activity and blocks its ability to interact with p53 (160). This association results in the 25 accumulation of p53, which leads to the p53-dependent cell cycle arrest and -. apoptosis (161, 162). Functional disruption Of p16'NK4a-CDK4/6-Rb and p14ARF-Hdm2-p53 pathways is a very common feature in cancer. The chromosome 9p21 locus is the target of deletion in many types of human cancers (150). Gerrnline point mutations Of INK4a and ARF have been associated with familial melanoma (163- 169). Somatic loss Of INK4a and ARF (through deletion, point mutation, or promoter methylation) is one of the most common events in human cancers (170). D. Telomeres and Telomerase In eukaryotic cells, genomic DNA forms highly condensed structures called chromosomes. Each eukaryotic chromosome contains a single linear DNA molecule. The building block of a chromosome is the nucleosome, consisting a histone octamer (2xH2A, 2xH2B, H3, and H4) around which is wrapped 146 base pairs of DNA. Nucleosomes, with linker DNA between them and histone H1, form a more condensed structure called chromatin, which is further organized into large units, hundreds to thousands of kilobases in length, called Chromosomes. The ends of Chromosomes, called telomeres, have unique properties which distinguish them from the damage-induced DNA ends (171, 172). In mammalian cells, telomeres consist Of tandem DNA repeats (TTAGGG) with a single-stranded 3’ overhang, and binding proteins which function to maintain and regulate its unique structure (173-177). Extensive studies indicate that telomeres protect chromosome ends from fusing with each other or with new 26 DNA ends created by double-strand breaks, prevent nucleases from degrading chromosomes from their ends, preserve chromosome stability, and prevent chromosome ends from triggering checkpoint-induced cell cycle arrest or apoptosis (178). 1. Telomeres Telomeres shorten with every replication cycle, because the extreme end Of the chromosome is not replicated, a phenomenon called “end-replication problem” which results from the lack of DNA beyond the ends to serve as a template for an additional Okazaki fragment that may function to fill the gap between the last Okazaki fragment and the end Of the chromosome (179). Human telomeres range from 6-12kb in length, and 50-100bp are lost with each cell cycle (180, 181). After a finite number of cell divisions, normal cells enter a process called cellular senescence caused by telomere shortening (180, 182, 183). The shortened telomere activates a DNA damage response and induces the accumulation Of p53, which, in turn, causes cell cycle arrest. Inactivation Of p53 (by SV4O T antigen or E6/E7 papillomavirus protein) leads to further cell divisions, until the cells reach a stage referred to as “crisis”. At this point, many telomeres have been critically shortened and can no longer protect the chromosomes from fusing. Such cells eventually undergo apoptosis (184). Normal human cells are estimated to have the potential to undergo an average of 60-70 divisions before undergoing apoptosis (180). This counting mechanism limits the number Of normal cell divisions in vivo and in vitro (183). Cells with a 27 finite life-span can not accumulate all the necessary genetic alterations required a for malignant transformation (1 , 183, 185). 2. Telomerase Telomerase is an RNA-dependent DNA polymerase. It consists Of two essential components: one is the functional RNA component (in humans called hTR or hTERC) (186), which serves as a template for telomeric DNA synthesis; the other is a catalytic protein (hTERT) with reverse transcriptase activity (187- 189). In addition, other telomerase-associated proteins are required for full telomerase activity, the assembly of an active telomerase complex, and access Of telomerase to its substrate (190). The hTERT protein catalyzes the addition of hexameric repeats, TTAGGG, to chromosome ends using an hTR RNA as a template (191). Expression Of hTERT prevents shortening of telomeres, and such cells acquire the phenotype of cellular immortalization (180, 192). hTR is highly expressed in all tissue regardless Of telomerase activity (193). In cancer cells, the hTR expression level is five-fold higher than that Of normal cells (194). In contrast, the expression (mRNA) Of the hTERT in cancer cells is estimated at less than 1 to 5 copies per cell, and is closely associated with telomerase activity (194). 3. Telomeres, Telomerase and Cancer Acquisition of an unlimited replication potential is an earty step in the multistep process of carcinogenesis in vivo and in vitro (1, 195, 196). Cells need to maintain telomeres to overcome the senescence caused by the shortening that occurs with each division. In fact, telomere maintenance has been found in 28 virtually all types Of malignant cells (197). The majority Of them (85-90%) maintain their telomeres by upregulation Of telomerase expression (197-199), and the others dO so through recombination-based interchromosomal exchanges of sequence information, a mechanism termed alternative lengthening of telomeres (ALT) (200). The evidence of the role Of telomerase in cell immortalization also comes from in vitro studies. Ectopic expression of hTERT in human fibroblasts, renal epithelial cells, or T lymphocytes led to a greatly extended life-span, apparently limitless, while control cells transfected with an empty vector underwent senescence as expected (201-203). Other proteins or pathways may also be involved in the telomerase-induced immortalization. Cells derived from tumors formed in p16’NK4‘4' mice also showed elevated telomerase activity, and the incidence was reduced when carcinogens were applied to the mice lacking both p16INK4a and telomerase (204). Telomerase activity was also detected in MSU-1.0 cells, an infinite life-span human fibroblast cell line, which expresses an oncogene v-Myc. E. The Multistep Process Of Carcinogenesis The mutant genes involved in cancer may be inherited from one parent, or may result from somatic mutations. Also, epigenic changes in DNA may cause phenotypic Changes in cells, which contribute to the carcinogenesis process (1, 205). The theory Of carcinogenesis involving a multistep process is supported by the Observations indicating that most human cancers occur with an age- dependent incidence, and that multiple genetic changes are required for tumor formation (206-208). Evidence that tumor formation is a multistep process 29 directly comes from the studies of mouse skin model of experimental - carcinogenesis (209), the studies of human colon cancer (210), and the development of the MSU1 lineage Of human fibroblasts in culture (196). 1. Mouse Skin Model of Experimental Carcinogenesis Reviewed by Hennings in 1993, the first experimental evidence that carcinogenesis is a multistep process comes from studies of the induction of mouse skin carcinomas through repeated treatment with chemical agents (211, 212). The carcinogenesis of mouse skin can be divided into three broad stages: initiation, promotion, and progression (malignant conversion) (213). During the initiation stage, a single dose of a carcinogen was topically applied to the mouse Skin. The carcinogens used in this stage became known as initiators, e.g., 7,12- dimethylbenz[a]anthracene (DMBA) , benzo(a)pyrene (BaP), and N-methyl-N’- nitrO-N-nitrosoguaniding (MNNG) (209). It was later found that the initiators acted as mutagens to induce mutations in a critical gene or genes. The mutation spectrum was mutagen-dependent. More than 90% Of the papillomas (benign tumors) initiated by application of DMBA contain an active H—ras (214), whereas less than 50% Of the papillomas initiated by MMNG contain H-ras mutations (215). After a single carcinogen treatment to mouse skin at a dose which causes no tumors, the treated area of the mouse skin is typically treated repeatedly with an agent such as phorbol ester 12-O-tetradecanoylphorbOl-13-acetate (T PA). This series of treatments resulted in the development Of many squamous papillomas and a few malignant squamous cell carcinomas (216). This Stage is now referred as promotion, and the agents, as promoters (217). Promoters are 30 not mutagens. They promote a clonal expansion Of the initiated cells. Alterations Of gene expression in such tumors have been found, but there appears to be no single critical gene Change (209). The conversion of benign tumors to malignant tumors occurs spontaneously in 50% Of such tumors over a period Of a year (216). Treatment Of such mice topically and systemically with carcinogens (e.g., Cisplatin, MNNG, urethane) accelerates the rate Of carcinoma formation (209). The conversion Of a benign tumor to a malignant tumor is associated with the new genetic changes, including the chromosomal aberrations (218), and loss Of heterozygosity such as H-Ras (219), p53 (220), and transforming growth factorB (TGF- 8) (221 ). These studies demonstrate that the formation of the mouse skin squamous carcinoma by treatment Of carcinogens is a multistep process involving progressive accumulation of genetic defects. 2. Human Colon Cancer Another well-studied model system is human colorectal cancer (CRC). CRC provides an excellent system in which one can Observe normal colon epithelium, dysplastic epithelium (hyperplasia), small, medium and large adenomas (benign tumors), and invasive carcinomas (222). Clinical and histopathological studies Show that most, perhaps all, malignant colorectal tumors (carcinomas) arise from pre-existing adenomas (223). A particular advantage Of this system is that tissue of all stages, including metastatic carcinomas, can be Obtained for studies (210, 224). A further complexity is that in addition to spontaneous colon cancers, there are two different inherited gene defects which give rise to colon cancers e.g., familial adenomatous polyposis 31 Tnfl 5.“ . (FAP) and hereditary nonpolyposis colon cancer (HNPCC) (225). Using large a L numbers Of samples from FAP patients and Sporadic tumors, Vogelstein and colleagues identified the critical genetic events driving colorectal carcinogenesis (Fig. 1) (210, 226). “tit-fl w It.» 9:: - 2.1. FAP and Sporadic Colon Cancer .th c.9- a. The APC Tumor Suppressor Gene tremor 7v FAP patients develop hundreds to thousands of colorectal adenomas in their teen and twenties and consequent early-onset colon cancers (222, 227). The locus linked to FAP has been mapped to chromosome 5q21, and the gene is called adenomatous polyposis coli (APC) (228-230). Gerrnline APC mutations ' . f t. uji‘r, It} 7 :7 £719“??? 0154:4386“ . . with result in FAP (231), also mutations in the APC has been found in 60-80% Of I I I.- ' WI: Sporadic colorectal cancers (232-234). The APC tumor suppressor gene product a is a 312 kD protein (235) with multiple functions including signal transduction, cytoskeletal organization, chromosomal segregation, and cell adhesion (231). Mutations of APC including tmncations (236, 237) result in the deregulation Of APC functions (235, 238), which in turn affect the downstream effectors, e.g. 8- catenin, EB1, microtubules (231 ). b. Methylation Status Evidence shows that general Changes in patterns Of DNA methylation follow the APC mutations (210, 239). The mechanisms of this demethylation and its role are unknown, however, changes in methylation can impact tumor development in 3 numbers of ways, including activation Of oncogenes, silencing of tumor suppressor genes, and causing genome instability (240). 32 c. The K-Ras Oncogene The Ras family of proteins is involved in signal transduction and is a part of the signaling pathways Of a large number Of functionally diverse molecules (discussed in Section C). Up to 50% Of sporadic colorectal tumors are found to contain mutations Of the K-Ras gene (226, 241, 242). The majority Of K-Ras mutations occur tO codons 12 and 13 (102). Dysplasia of colon tissue is seen when K-Ras mutations occur in association with APC mutations, implying that the K-Ras mutations give cells with APC mutations a selective growth advantage resulting in clonal expansion. d. The p53 Tumor Suppressor Gene The tumor suppressor gene, p53, can monitor cell stresses and, then, lead to appropriate cell responses, e.g., cell cycle arrest, apoptosis (discussed in Section C). In human colon cancers, mutations Of p53 are associated with the progression from adenoma (benign) to carcinoma (malignant) (222, 227). In sporadic colorectal cancers, 70% exhibit p53 mutations (227). Vogelstein suggests that mutations of p53 give colorectal tumor cells a selective growth advantage (222). Patients with p53 mutations in their tumors have a worse outcome and shorter survival than persons whose tumors do not have a p53 mutation (243). Fearon and Vogelstein conclude that at least four to five Specific genetic Changes are required to form a malignant human colorectal cancer (210). Fewer changes suffice for benign tumorigenesis. Genetic alterations Often occur in a 33 preferred sequence (e.g., APC-K-Ras-p53), however, it is the accumulation of ., these specific genetic mutations, rather than the sequence, within a single cell which is ultimately important to the formation Of a malignant tumor (Fig. 1) (210). 2.2. HNPCC Hereditary nonpolyposis colorectal cancer (HNPCC) arises as a result Of germline mutation in one Of several DNA mismatch repair genes, e.g., hMSH2, hMLH1, hPMS1, hPMSZ, hMSH3, and hMSH6 (GTBP) (73, 244-248). A somatic mutation or promoter methylation of the wild type allele results in a complete loss of the mismatch repair function, and, in turn, leads to microsatellite instability and an increased mutation rate, which ultimately leads to the formation Of cancer (248-250). This is supported by finding that the genes for type II TGF-,8 receptor (T GF-B R2), IGF II receptor (IFGIIR), Bax protein (Bax) and E2F4 cell cycle regulator (E2F4) are mutated in colon cancers from patients with HNPCC (251- 254). In such patients’ tumors, mutations in APC and K-Ras appear to be a later event (245). Once the cells Obtain a growth advantage, they progress rapidly from adenomas to carcinomas in 3-5 years for HNPCC, compared with 20-30 years for FAP (222). 3. Malignant Transformation of Human Fibroblasts in Culture TO investigate the number and kinds of genetic Changes required for the malignant transformation Of human cells, McCormick and Maher in the Carcinogenesis Laboratory at Michigan State University attempted to transform normal human skin fibroblasts into malignant cells by carcinogen treatment, following the methods that Takeo Kakunaga Of the National Cancer Institute had 34 reported (255). They did not succeed, but in the process, they discovered that no one had ever done so (256), not even Kakunaga (257). It became very Clear that for a normal human cell to be changed into a malignant cell it required such a cell to acquire at least five or more independent genetic changes, but evidence Showed that a normal human cell in culture could only undergo two or at most three sequential clonal selections and expansions before senescence occurred (196,210) In an effort to Obtain a human fibroblast cells strain with an infinite life Span in culture, McCormick and Maher and their colleagues transfected a foreskin-derived cell line from a normal newborn, designated LG1, with a v-Myc oncogene and a gene coding for resistance to G418, selected cells for drug- resistance, and grew five Of the latter clones, along with their parent, LG1, and a vector-transfected control tO the end Of their life span. The vast majority of the cells from all five v-Myc-transfected clones senesced at the same time as LG1 cells and the vector control cells. But in one cell of the v-Myc-transfected populations, they detected “young cells”. This cell had spontaneously turned on its telomerase gene, giving rise to an infinite life span diploid cell strain, designated MSU-1.0. Escape from senescence cannot have been conferred directly by upregulated expression of the v-MYC protein because the vast majority of progeny of the G418 grug-resistant clone from which the MSU-1.0 cell arose senesced at the same time as the control populations (258). MSU-1.0 cells have maintained a diploid karyotype over more than 155 population doublings since crisis. They express telomerase, have normal growth control, 35 ”fin-1W and cannot be transformed into malignant cells by carcinogen treatments or by «- oncogene transfection. The immortalized MSU-1.0 cells have the normal morphology Of human fibroblasts, and do not display the characteristics that are typical Of transformed cells, such as growth without exposure to exogenous growth factors or anchorage indepen-dent colony formation. Karyotype analysis showed that MSU-1.0 cells are diploid. They do not contain any gross chromosomal aberrations. Subcutaneous injection Of MSU-1.0 cells into athymic mice has never produced tumors. However, from MSU-1.0 cells, eventually, a faster-growing, spontaneous variant cell strain overgrew the culture. This strain, designated MSU-1.1, like its predecessor MSU-1.0 cells, expresses v-MYC, and has an unlimited life span in culture. It displays partial growth factor independence, but does not form large- sized colonies in soft agarose or tumors in athymic mice (258). Southern blot analysis showed that the sites Of integration of the v-Myc and G418 genes are identical in MSU-1.0 and MSU-1.1 cells, indicating that the MSU-1.0 and MSU- 1.1 cells are derived from the same transfectant (258). Karyotype analysis shows that MSU-1.1 cells contain 45 chromosomes, including two marker chromosomes, M1, (t (1;11) (1qter —1p13:11p15-11qter)) and M2, t(12;15) (12qter-12q11.2:15p11.2-15qter) (258). This indicates that at least two or more genetic events occurred during the progression from MSU-1.0 cells to MSU-1.1 cells. Although it is possible that these genetic changes are responsible for the important difference between MSU-1.1 cells and its predecessor, MSU-1.0 cells, as yet there is no direct evidence that this is the case (258). 36 In contrast to MSU-1.0 cells, MSU-1.1 cells can be successfully transformed into malignant cells by an exposure tO a carcinogen (259-262), or by overexpression Of an oncogene (48, 263, 264). The transformed MSU-1.1 cells proliferate rapidly in growth factor-free medium, form large-sized colonies in soft agarose, and form malignant tumors in athymic mice within 3-5 weeks (265). The oncogene-transformed MSU-1.1 cells and their tumor-derived cells are chromosomally stable and maintain a wild type p53 gene. MSU-1.1 cells malignantly transformed by carcinogens and their tumor-derived cells no longer contain a wild type p53 gene and are chromosomally unstable (265), probably as a result of the homozygous loss of the wild type p53 genes (260). Beginning with the human normal fibroblasts LG1 and ending with tumor- derived cell lines, the MSU1 lineage consists Of the two intermediate cell strains, MSU-1.0 and MSU-1.1. Additionally, there are MSU-1.1 cell-derived cell strains which express low levels Of H-Ras or K-Ras and are not malignantly transformed unless an Src-family oncogene is expressed (264). This makes the MSU1 lineage Of cells an excellent model for studying the nature of multistep carcinogenesis of human cells. Because cell stains in this lineage were sequentially derived one-from-another, comparisons between LG1 parental cells and the tumorigenic cells provide direct information Of the genetic changes that are involved in the malignant transformation of human fibroblasts. Studies carried out in the MSU Carcinogenesis Laboratory indicate that multiple genes are involved in this malignant transformation, including activation Of telomerase, upregulation of transcription factors Sp1 and Sp3, upregulation of MET, 37 tin—T’— inactivation Of p53, and loss Of expression of fibulin 1. By careful calculation, McCormick and Maher suggest that at least six genetic changes are required for the malignant transformation Of human fibroblasts (196, 265). Furthermore, the two cell strains, MSU-1.0 and MSU-1.1, which are intermediates in the process Of malignant transformation allow interesting questions to be addressed. Elucidation Of the critical genetic events involved in the transition Of non- transfonnable MSU-1.0 cells to transforrnable MSU-1.1 cells may provide valuable insight into the multistep process Of carcinogenesis, and the identification Of the genes that are involved in this process. II. Sp1 and SleLF Transcription Factors A. The Finding Of Sp1 (Specificity Erotein 1) In 1983, Dynan and Tjian reported that a component of uninfected HeLa whole cell lysates is required for specific transcription Of the SV40 early and late promoters, but not for the human B-globin promoter and the avian sarcoma virus LTR promoter (266). In a second paper, they showed that Sp1 recognized and specifically bound to a 21 bp GC-rich repeat located 70-110 bp upstream Of the SV40 eany promoter (267). In 1985, Tjian and his colleagues analyzed the DNA binding activity and in vitro transcription property Of Sp1 using wild type and mutant SV40 DNA templates. These experiments demonstrated a functional link between the Sp1 DNA binding and transcriptional activation (268). Since the discovery of the Sp1 transcription factor, several other cellular proteins which bind selectively to promoter elements and potentiate transcription have been identified through similar biochemical and genetic analyses (269-272). 38 In contrast to the general transcription factors, e.g., TBP and TAFS, Sp1 is promoter-specific, and only activates transcription Of a selective set Of genes. Detailed studies of Sp1 have provided, and continue to provide, insight into the mechanisms Of gene-specific transcriptional regulation in higher eukaryotes (273- 275) B. Characterization Of the Functional Domains of the Sp1 Protein Although the crystal structure of Sp1 is not yet available, the functional domains have been studied in some details. In 1986, Briggs et al. (276) purified two peptides with Sp1 DNA binding activity (105-kD and 95-kD) from HeLa cell nuclear extract, and in 1990 it was found that the 105-kD band represents the phosphorylated form of Sp1 (277). Sp1, a single peptide composed of 785 amino acids, contains a DNA binding domain, transactivation domains, and regulatory domains (access number, NP 6124822) (278). 1. DNA Binding Domain The Sp1 DNA binding domain is localized to the C-terrninal 168 amino acid residues. Analysis of Sp1 CDNA reveals that this DNA binding domain has three contiguous Zn(ll) finger motifs (279) which contact the DNA binding elements (GC-Box,GGCGGG and GT-Box CACCC) through direct interaction between the amino acid residues and the bases of the nucleotides (280, 281 ). 2. Transcriptional Domains Sp1 also contains several other domains that are required for Sp1 transactivation and posttranslational modifications. Deletion of domain A, B, C, or D dramatically decreases Sp1-responsive promoter activity, indicating that 39 these domains are essential for Sp1 transactivational activity (282, 283). .. Domains A and B are glutamine-rich sequences, which are also found in other transcription factors, such as the products of the Drosophila zeste gene and homeobox-containing genes Antp and Cut (282-285). Domain C is located in the N-terrninal part of the molecule adjacent to the zinc finger DNA binding domain, and is highly charged (282). Within domain C is the so-called Buttonhead box, an 11 amino acid residue stretch first identified in the Drosophila Sp1 homologue Buttonhead (Btd) (286). Evidence shows that domain C, and more specifically the Btd element, is involved in synergistic activation by the Sp1 with other proteins (287-289). Domain D is located at the C-terrninus Of the protein, and is involved in the synergistic activation of Sp1 (290) 3. Other Functional Domains The amino terminus Of Sp1 is commonly thought to be involved in the indirect regulation Of Sp1 transcriptional activity. Within this region, an Sp box containing an endoproteolytic cleavage site was identified (289) close to a region at the N-terrninus which targets proteasome-dependent degradation in vitro (291 ), suggesting that it is involved in the regulation Of Sp1 protein stability (292). In addition, an inhibitory domain (ID) found in the N-terrninal region may be involved in the recruitment Of repressors that inhibit Sp1 transcriptional activity (293). 40 C. Sp1 Transcriptional Activity The Sp1 DNA responsive element is one Of the most common elements within the promoters Of mammalian genes (290). The Sp1 transcription factor binds to its DNA responsive element through the zinc-finger DNA binding domain and functions to activate transcription through several mechanisms including direct interaction with the basal transcription machinery, coordination with other transcription factors and regulatory proteins, and recruitment of the chromatin modifier complexes. 1. Interactions with the Components of Basal Transcription Machinery Sp1 transcription activation requires interactions with the components of basal transcription machinery. For example, Hoey et al. (294) found that transcriptional activation by Sp1 requires TFIID. Other studies showed that Sp1 directly interacts with the TAP" (T ATA box-binding protein (T BP)—associate factor) subunits Of TFIID through the glutamine-rich domains Of both proteins (294, 295). The glutamine-rich domains of Sp1 have also been reported to bind specifically and directly to the C-terrninal evolutionarily conserved domain Of the human TBP (296). In addition to interactions with the components of the basal machinery, Sp1 recruits a multi-subunit complex, CRSP, cofactor required for Sp1, which contains nine subunits and interacts with the basal transcription apparatus to promote efficient transcription activation (297, 298). 2. Interactions with Site-specific Transcription Factors Evidence shows that sequence-specific transcription factors and other regulatory proteins are also involved in the Sp1 transcription activation. A study 41 by Sorensen et al. (299) showed that the hamster thymidine kinase gene -. promoter contains three Sp1 binding sites and one NF-Y binding site. Mutation Of the NF-Y site, or the Sp1 site which is near it, significantly reduces promoter activity (299). Sequence-specific transcription factors, such as Ets (300), E2F1 (301-305), YY1 (306, 307), NF-KB (308, 309), p53 (310-312), and Oct-1 (313), interact with Sp1 and together activate their target genes. Sp1 and the other transcription factor binding sites are usually separated by less than 20 nucleotides within the promoters the target genes. Together with Sp1, these transcription factors synergistically activate the transcription Of the target genes. In addition, several regulatory proteins are reported to interact with Sp1. Chang et al. (314) showed that Sp1 forms a complex with histone deacetylase 1 (HDAC1) and non-phosphorylated Rb in serum-starved CHOC400 cells. Upon serum stimulation, the Sp1-Rb interaction is lost due to the phosphorylation of Rb, whereas the Sp1-HDAC1 association persists through S phase (314, 315). In contrast, overexpression of Rb in NIH3T3 cells with amplified MDM2 genes restores Sp1 activity by binding MDM2 which releases Sp1 from the MDM2-Sp1 complex (316). Taken together, these results show that Rb is involved in the regulation Of Sp1 transcriptional activity. Another Rb-related protein, p107Rb, has been found to bind Sp1 through a domain instead Of through the pocket region, and to inhibit Sp1 activity (317). 3. Interactions with Chromatin Modifier Complex The third group of proteins which are involved in the regulation of Sp1 transcriptional activity is the chromatin modifier complex. Both Sp1 and sterOI- 42 responsive element-binding protein-1a (SREB-1a) are required for synergistic activation of the low density lipoprotein (LDL) receptor promoter in vivo and in vitro (318, 319). In addition, a multiprotein coactivator complex that includes CREP-binding protein (CBP) was found to be required for Sp1 and SREBP—1a induced synergistic activation of the LDL receptor promoter. Even if the coactivator SREBP-1a harbors histone acetyltransferase (HAT) activity, it is not Clear whether histone acetylation has any functional relevance for Sp1/SREBP- 1a synergy (318). Sp1 can also be a target for HDAC1. The binding OfHDAC-1 to Sp1 inhibits the Sp1 transcriptional activity (304). These results indicate that Sp1 can recruit protein complexes that regulate chromatin accessability for the RNA polymerase complex and other transcription factors. 4. Summary Sp1 is a transcription activator that can interact with the components of the basal transcription machinery and other sequence-specific transcription factors to activate the target gene transcription. In addition, Sp1 can recmit the proteins that are involved in the modifications Of chromatin. D. Post-Translational Modifications Sp1 is subject to post-translational modifications. Kadonaga et al. were the first to Observe that E.coIi-synthesized human Sp1 was less effective as a transactivator in vitro than protein purified from HeLa cells (320). In their 2002 review, Bouwman and Philipsen (292) described modifications to Sp1 by glycosylation, phosphorylation, and acetylation. These modifications regulate Sp1 nuclear localization, protein stability, and transcription activity. 43 '1 "31': ‘7; Simfoiltf -‘.‘_§:r‘~t;~lt"r-'r~ltiwtli”. “ 1. Glycosylation Sp1 is modified by multiple O-linked N-acetylglucosamine (GlcNAc) monosaccharide residues. Wheat germ agglutinin, a lectin, can specifically bind such residues, and this inhibits Sp1 transcriptional activity, indicating that glycosylation is involved in the regulation Of Sp1 transcriptional ability (321). In contrast, glycosylation of the B domain Of Sp1 inhibits its interactions with dTAF(Il)110, and reduces Sp1 transcriptional potential in vitro (315, 322) and in vivo (315). The mechanism involved in the regulation Of Sp1 function by glycosylation is not clear. An early study by Jackson and Tjian suggests that the glycosylation Of Sp1 regulates the interactions between Sp1 and the basal transcription machinery, and the recruitment Of other coactivators (321 ). Glycosylation Of Sp1 is also involved in the regulation Of Sp1 protein stability. Glucose deprivation in combination with adenylate cyclase stimulation results in reduced glycosylation Of Sp1, and this is associated with an increased susceptibility to proteasome-dependent degradation (323). Sug-1, a subunit Of 268 proteasome (which is also found to bind to Sp1), is involved in this process (291, 324). It has been suggested that glycosylation blocks protein interactions and prevents Sp1 from entering into protein complexes that are readily degradaded by proteasomes (322). 2. Phosphorylation The subjection of Sp1 to phosphorylation was first indicated by the Observation Of the purified Sp1 protein migrates on SDS-PAGE as two polypeptide species (279). Phosphatase treatment Of the purified protein led to 44 the disappearance of the upper band (277). Sp1 can be phosphorylated through different pathways, and by different types Of kinases. Upon SV40 infection, the proportion Of phosphorylated Sp1 increased significantly in monkey CV1-L cells, and the phosphorylation Of Sp1 required binding to GC-Box-containing DNA, suggesting that the Sp1 kinase is a DNA-dependent protein kinase. Both phosphorylated Sp1 and unphosphorylated Sp1 show equal ability to bind to the GC-Box and tO activate the transcription of SV40 early promoter, indicating that the phosphorylation by the DNA-dependent protein kinase has no effect on Sp1 function (277). In contrast, the infection of Vero cells with HSV-1 does not affect the abundance of Sp1, but the proportion Of phosphorylated Sp1 increases with an increase in the amount of the vimses. Sp1 purified from the infected cells is less active in transcription than that from the uninfected cells, indicating that the phosphorylation Of Sp1 upon infection with HSV-1 inhibits its transcriptional activity (325). The phosphorylation of Sp1 is also regulated through cell cycle progression. Upon serum stimulation, the phosphorylation of the C-terminus of Sp1 increases in mid-G1 in Balb/C 3T3 cells, and the phosphorylation Of Sp1 correlates with the induction Of dihydrofolate reductase (326). The phosphorylation Of Sp1 can be stimulated by the neu differentiation factor (NDF) (327) and EGF (328). Several specific kinases have been reported to phosphorylate Sp1 protein, including PKA (329, 330), PKCg’ (330, 331), cyclin D1 kinase (332), cyclin A—CDK (333), and ERK2 (334). In these cases, the phosphorylation of Sp1 correlates with the up-regulation of its transcriptional activity. In other reports, the 45 I -‘1I."0" ‘4 . 'I...IE."R" ' t - a | t. t I ‘ , ' , ‘ _ I ll. ‘ A Dina ‘ 8"." '1 r; . a '1' |_'i'r.“_ -I-,. ,t I_ .L. 135:. "Ink-11:01.1};- 11.1. e a ".17 '..'Z ' dephosphorylation Of Sp1 is linked to increased Sp1 DNA binding activity. For .. example, phosphatase treatment of nuclear extracts from rat livers resulted in a 10-fold increase in the DNA binding affinity Of Sp1 for its cognate site (335). Other reports Show that, in vivo, the dephosphorylation Of Sp1 with the up- regulation Of phosphatase 2A (336) and phosphotase type 1 (337) induces transcription Of the Sp1 target gene. Taken together, these results indicate that phosphorylation of the Sp1 protein is involved in the modulation Of Sp1 transcriptional activity. The fact that Sp1 is phosphorylated by various kinases suggests that the phosphorylation of different domains (activation domains, zinc finger DNA binding domain, or inhibition domain) and of different amino acids (Ser, Thr, and Tyr) by different kinases may account for the different effects on Sp1 transcription activity. Recently, Milanini-Mongiat et al. (338) showed that p42/p44 MAPK directly phosphorylated Sp1 on threonines 453 and 739 both in vitro and in vivo. Mutation of these sites to alanines decreased the MAPK- dependent transcriptional activity Of Sp1 by half. This study indicates that p42/p44MAPK phosphorylates Sp1 and, thus, positively regulates Sp1 transcrip- tional activity. Obviously, many such studies regarding the phosphorylation sites are needed for further elucidating this issue. 3. Acetylation Studies showing that Sp1-dependent transcription is activated by an acetyl transferase and repressed by histone deacetylase 1 indicate that acetylation Of Sp1 may be involved in the regulation of Sp1 transcription activity (304). Direct evidence that Sp1 is subjected to acetylation comes from the studies of Ryu and 46 Ferrante (339, 340), which showed that the acetylation Of Sp1 is enhanced under oxidative stress. The enhanced acetylation Of Sp1 may result from a decrease in HDAC activity or an increase in acetyl transferase activity, or both (340). Hyperacetylated Sp1 can bind DNA responsive elements more avidly, and lead to activation Of its target genes. E. Physiological Function Of Sp1 Protein Sp1 is essential for normal mouse embryogenesis. Sp1"' mouse embryos can survive until day 9.5-10 after embryogenesis (E9.5-10); by E10.5-11, all Of the Sp1-deficient embryos have died (341). The Sp1‘" mouse embryos show a remarkable heterogenicity and are always much smaller than their littemtates (341). The broad range Of abnormalities suggests that no particular cell lineage or developmental process is affected by Sp1-deficiency; rather, it causes a general cellular defect that precludes normal development and survival (341). Sp1‘" mouse embryo stem cells are viable, and when injected into blastocytes, these cells can contribute efficiently to the development Of Chimeric embryos at the eany stages, but after day E11 they rapidly decline with no contribution to newborn mice (341). Taken together, these studies suggest that Sp1 is a transcription factor whose function is essential for differentiated cells after day 10 of development. F. Sp3 Sp1 is the prototypical member Of the 21-member Sp/KLF family of zinc finger proteins which function as transcription factors in mammalian cells (342). The gene of. another member of the Sp/KLF family, Sp3, was Cloned in 1992 by 47 recognition site screening using a GT-Box motif Of the uterglobin promoter as a - probe (343). Sp1 and Sp3 are evolutionally Closely related. Sp1 is found on the paralogous Chromosomal region of human chromosome 12q13 (344), and Sp3, on the paralogous Chromosomal region Of human Chromosome 2q31 (345). Both genes are linked to the homeobox gene cluster on the corresponding chromosome (Sp1/HOX C and Sp3/HOX D) (346). The fact that the exon-intron structures of Sp1 and Sp3 are conserved also supports the hypothesis that the two genes arose from a single ancestor gene. Additionally, Sp1 and Sp3 are ubiquitously expressed (290). Also, both Sp1 and Sp3 Share a similar modular stnIcture; the major difference is the inhibitory domain, located at the N-terrninal of the first zinc finger domain of Sp3, and at the N-terrninal region Of Sp1 (292). Finally, Sp1 and Sp3 share 95% similarity in the zinc-finger DNA binding domain. Consistent with this, Sp1 and Sp3 bind to GC-Box and GT-Box with the same affinity. Three Sp3 isoforms exist; a full-length protein with a molecular weight Of 110-115kDa and two other proteins with molecular weights Of 60-70kDa (347). Evidence shows that the two smaller Sp3 species arise from the internal ATG translation start codons (348). All the three isoforms can bind to the GC-Box and the GT-Box (290, 348), but the two intemally-initiated Sp3 proteins have little or no capacity to stimulate transcription of Sp-regulated genes in vivo (348), suggesting that these proteins function as potent inhibitors of Sp1/Sp3- mediated transcription. Other than this, little is known about the two smaller Sp3 isoforms. 48 For this reason, the following discussion regarding the Sp3 protein will focus on the full-length Sp3 protein. 1. Transcriptional Activity Sp3 and Sp1 are highly homologous, with the same binding affinity to the same DNA responsive elements, but the transcriptional activity Of Sp1 and Sp3 is strikingly different. Sp1 is an activator. Sp3 has been reported to positively regulate transcription (349-352). Upon CO-transfection with Sp1, additive (353, 354) and synergistic (355, 356) effects were Observed. However, Sp3 can also act as a repressor, or at best, a weak activator (357-360). The mechanisms that are involved in the regulation Of Sp3 transcriptional activity are not yet clear. Studies show that whether the Sp3 acts as an activator or as a repressor depends on the stmcture Of the promoter Of the target gene, the arrangement Of the recognition sites (358, 359), and other undefined factors (361). Recent reports indicate that Sp3 in mammalian cells is modified by acetylation (362, 363), sumoylation (364, 365) and phosphorylation (366, 367). Acetylated Sp3 acts as a transcriptional activator for TGF-B receptor type II gene in MCF-7L cells (363), whereas unmodified Sp3 acts as a transcriptional repressor in the same cells (357). When SUMO-1 was removed from the Sp3 protein by mutation Of the SUMO acceptor Iysines or expression of the SUMO-1 protease SuPr—1, Sp3 was converted to a strong activator (365). Sp3 is a downstream effector of a Raslp42/44(MAPK) signaling pathway, and the phosphorylation of Sp3 correlates with an increase in Ctalpha (CTP:phosphochOline cytidylyltransferase ) gene 49 transcription (367). These studies indicate that the posttranslational modifica- -. tions are important in regulation of Sp3 transcriptional activity. The mechanism(s) by which Sp3 functions as a transcription factor is (are) not clear. Sp3 was shown to interact with dTAF(Il)110, suggesting that Sp3 regulates the transcription Of target genes through physical interaction with components Of the basal transcription machinery (358). Fingerless Sp3 can still repress Sp1-mediated transcription, indicating that Sp3 can directly interact with Sp1 protein (368). Furthermore, studies by Rotheneder et al show that E2F 1 can bind directly to the Sp3 DNA binding domain (304). Taken together, these results show that Sp3 may function as a transcription factor through protein- protein interactions. 2. Physiological Function of Sp3 Sp3-deficient mouse embryos are growth retarded and invariably die after birth due to respiratory failure. These mice also show a pronounced defect in late tooth formation and late bone development (369, 370). A comparison Of mice with Sp1-knockout phenotype to mice with and Sp3-knockout phenotype showed that Sp1 and Sp3 have distinct functions in vivo, but also suggests a degree of functional redundancy (369). 3. Sp1/Sp3-mediated Gene Expression Extensive evidence shows that Sp1 and Sp3 may act together to regulate gene expression (290) As noted above, both Sp1 and Sp3 are ubiquitously expressed and have the same binding affinity to the same DNA responsive element. However, because Sp3 acts as an activator or a repressor, whereas 50 Sp1 is only an activator, changes in Sp1 and Sp3 abundance, as well as alterations in the ratio Of Sp1 and Sp3 levels can be expected to affect Sp1/Sp3- mediated gene expression. A study by Discher et al. (371) showed that the muscle-specific pyruvate kinase-M and B—enolase are upregulated in 02012 myocytes under hypoxic conditions. They also found that the Sp1 protein level remained the same whereas the Sp3 protein was depleted by hypoxia (371). A study by Ye et al. (372) showed that the expression Of aZ-integrin was down- regulated in mammary epithelial cells by expression Of Erb-B2 and v-H-ras. This down-regulation Of the aZ-integn’n gene expression is mediated by two adjacent Sp1/Sp3 binding sites within aZ-integn'n gene promoter. Upon expression of Erb- 82 and v-H-Ras, the Sp3 DNA binding activity remained unaltered, whereas the Sp1 DNA-binding activity was reduced (372). Taken together, these studies indicate that at least three factors must be considered to predict the effect of Sp1 and Sp3 proteins on the expression of a target gene: 1) the changes of Sp1 and Sp3 levels, 2) the change Of the ratio of Sp1/Sp3, and 3) the transcriptional activity of Sp3. G. Other SpIKLF Members Sp1 was the first transcription factor to be purified and cloned from, and characterized in, mammalian cells (266, 279). It is the prototypical member Of the Sp/KLF transcription factor family, which consists Of at least 21 members which are present in species ranging from the nematode Caenorhabditis elegans to humans (342). These proteins are grouped into a family because all of them have zinc-finger motifs very similar to those of Sp1 and can bind various GC-rich 51 DNA responsive elements (342). In humans, 21 Sp/KLF genes have been .. isolated by a variety Of cloning techniques (342). The Drosophila melanogaster has three Sp1-like/KLF proteins (286, 373). SO far, homologs Of 17 Of the 21 human Sp1-like/KLF proteins have been found in mice, and 11 in rats. Other species, e.g., zebra fish, have fewer members Of the Sp1-Iike/KLF family. Based on sequence and function similarities, the members Of the Sp/KLF family have been divided into three subgroups: 1) including Sp1-Sp4 and mSp5-mSp6, 2) including KLF1-8 and KLF 12, 3) including BTEBS, KLF9-11, KLF13, and KLF15- 16 (342). Four additional human genes, Sp5-Sp8, have been identified in the present version of human genome database (www.ensembl.orq) (292). Sp1 and Sp/KLF family members are reported to regulate the expression Of more than one thousand genes which are involved in a broad number of functions including cell cycle control, cell growth and proliferation, cell apoptosis, angiogenesis, and invasiveness and metastasis. 1. DNA Binding Domains The defining feature of Sp/KLF proteins is a highly conserved DNA- binding domain at the carboxyl terminus which contains three tandem Cys2His2 zinc-finger motifs (279, 374). DNA binding domains among Sp/KLF members are highly homologous (67% to 95% simalarity to Sp1) (342), and the length Of each motif is invariant: the first two zinc-finger motifs contain 23 amino acids and the third, 21 amino acids (342). Seven amino acids linkers separating the zinc fingers are highly conserved (342). As a consequence Of the conserved DNA- binding motif, these Sp/KLF members recognize the same DNA responsive 52 elements although with different affinities because Of the differences in the amino acids in the zinc fingers. For example, Sp1, Sp3 and Sp4 preferentially recognize the GC—Box, whereas, Sp2, which has a leucine residue within the first zinc- finger motif in place of the histidine found In the corresponding region Of Sp1, preferentially binds the GT-Box rather than the GC-Box (290, 347). In addition, the subgroup ll proteins preferentially recognize the GT-Box (375, 376). In fact, Sp1 and Sp3 compete for the same DNA responsive elements (290), as do Sp1 and KLF 4 (377), Sp1 and KLF13 (378), and KLF9 and KLF13 (379), KLF1 and KLF3 (380) (whether Sp3 can compete for the same DNA responsive element with KLF4 or KLF 13 was not determined by these studies). This suggests that the competition for DNA binding sites and the difference in expression levels and transcriptional activity Of Sp/KLF proteins play an important role in the regulation Of the transcription Of their target genes. 2. Transcriptional Regulatory Domains Although the DNA binding domains are highly homologous, the transcriptional activity of the Sp/KLF proteins varies significantly. Sp1 is a transcription activator (290, 292), whereas KLF 11 functions as a potent repressor (381, 382). Sp3 acts as either an activator or a repressor, depending on the cellular context, the promoters it binds to, and the post-traslational modifications of Sp3 protein (357, 363). The amino-terminal domains Of Sp/KLF proteins are highly variable and contain functional domains which can interact with components of the basal transcription machinery to activate transcription, e.g., Sp1 and Sp3 (294, 295, 358), or recruit the coactivators, e.g., Sp1 (298) or 53 corepressors, e.g., KLF10, KLF11, KLF9, KLF13 and KLF16, to mediate the .. transcription activation Orrepression (379, 381, 383). 3. Expression Patterns The expression Of some members can be detected in all the organs and tissues. For example, Sp1 is ubiquitously expressed in murine cells. In agreement with this, Sp1-knockout mouse embryos are severely retarded in growth and Shows gross global morphological defects (341). This suggests that these transcription factors play a general role in growth-regulatory or developmental processes Of a large number of tissues. Other members are specifically expressed in certain types Of cells, such as Sp4 in brain (384) and KLF1 in erythroid cells (309), suggesting a cell-type-specific function for these factors. H. SpIKLF Proteins and Cancer As noted above, Sp1 and other members of the Sp/KLF family have been reported to regulate more than 1,000 of genes involved in a multitude of cellular processes (290, 385). Gene knockout studies of several Sp/KLF family members Show that these genes are involved in growth-regulatory and developmental processes Of many tissues, suggesting that the Sp/KLF family members participate in the malignant transformation Of normal cells (385). Recent studies show that several members Of the Sp/KLF family are up-regulated or down- regulated in cancers or cancer cell lines. For example, overexpression or higher binding activity Of Sp1 was found in human pancreatic cancer cell lines and cancer tissue (386), breast cancer cell lines and cancer tissue (387), gastric carcinoma (388), and thyroid carcinoma (389). These studies also showed that overexpression of Sp1 or an increase in the transcriptional activity of Sp1 is correlated with up-regulation Of VEGF (386), uPA and uPAR (387), and EGFR (388), proteins which are known to play an important role in tumorigenesis. In addition, upregulation Of KLF4 was detected in dysplastic epithelium and squamous cell carcinoma Of the oral cavity and also in breast carcinoma, suggesting that upregulation Of KLF4 contributes to the malignant phenotype of squamous cell carcinomas (390) and breast carcinoma (390, 391 ). KLF6 acts as a tumor suppressor gene in prostate carcinoma, and the ability Of KLF6 to inhibit cell growth is reduced by mutations within its transcrip-tional regulatory domain (392). The transformation of normal human cells into malignant cancers is a multistep process. In their review, Hanahan and Weinberg (1) proposed that cancer cells acquire six characteristics, which together confer the malignant phenotype: self-sufficiency in growth Signals, insensitivity to growth-inhibitory signals, evasion Of programmed cell death, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (1). Cancer cells acquire these capabilities through mutations which produce oncogenes with dominant gain Of function, and tumor suppressor genes with recessive loss of function. Evidence shows that Sp1 and other members Of the Sp/KLF family regulate the expression Of many of the genes that are involved in the regulation Of cell growth, cell apoptosis, [tumor cell invasiveness and metastasis, and angiogenesis (385). 55 1. SpIKLF Proteins and Cell Growth As discussed above, Sp1-knockout mouse embryos died on day 11 of gestation. The broad range Of abnormalities in the embryos indicated that Sp1- deficiency caused a general cellular defect which precludes normal development and survival, suggesting that Sp1 is required for the growth and maintenance of differentiated cells (341). Gene knock-out studies Of other members Of the SP/KLF family also support the theory that these proteins play a role in the growth and development Of specific organs, e.g., bone and marrow (Sp3) (369), lung, blood vessels, and immune system (KLF2) (393-397). These results indicate that Sp1 and the other members Of the Sp/KLF family are involved in the regulation Of normal cell growth. The implication that Sp1 and other members of the Sp/KLF family contribute to the formation Of cancer comes from studies which showed that Sp1 and other members of the Sp/KLF family regulate the expression of genes, which, when overexpressed, play an important role in the malignant transformation. For example, EGFR is a transmembrane glycoprotein and is one of four members Of the ErbB family of tyrosine kinase receptors. Binding Of EGFR to its cognate ligands, e.g., EGF, leads to autophosphorylation Of receptor tyrosine kinase, and subsequent activation Of Signal transduction pathways which are involved in regulating cellular proliferation, differentiation, and survival. Although present in normal cells, EGFR is overexpressed in a variety Of tumor cell lines and has been associated with poor prognosis and decreased survival (398). The EGFR gene promoter contains a GC-rich element, and Sp1 is 56 involved in the activation of EGFR transcription (399, 400). A study by Kitadai et al. (388) showed that an increased level Of Sp1 correlated with the up-regulation of EGFR expression in human gastric carcinomas, suggesting that overexpression Of Sp1 induces the overexpression Of EGFR in human gastric carcinomas, and this contributes to the malignant transfromation. Sp1 and Sp3 regulate the expression of Hepatocyte growth factor/Scatter factor (HGF/SF) and its receptor MET (401-403). HGF/SF, through its receptor MET, induces a wide spectmm of biological events, including, invasion, proliferation, branching morphogenesis, transformation and angiogenesis (404, 405). HGF is synthesized by mesenchymal cells, and MET has been demonstrated to be overexpressed in malignant human musculoskeletal tumors, as well as several other types Of soft tissue sarcomas, suggesting that MET is an important oncogene in these kinds of cancers (406). A study by Liang et al. (407) carried out in the MSU Carcinogenesis Laboratory showed that ten Of eleven human fibrosarcoma cell lines tested expressed significantly higher levels Of MET than normal human fibroblasts. In 6 Of the ten fibrosarcoma cell lines that overexpressed MET, Sp1 was markedly overexpressed. Furthermore, inhibition Of Sp1 binding to DNA, using an Sp1 decoy, dramatically reduced MET expression. These results indicate that Sp1 can function to control the level of MET, and that overexpression of Sp1 protein may contribute to the malignant transformation Of human fibroblasts through up-regulation of the expression of MET. 57 In addition, Sp1 and Sp3 are also involved in regulation of the expression - Of PDGFB (408) and PDGFB receptor (409). These proteins together participate in the signal transduction pathways which regulate cell growth and cell proliferation (385). 2. SpIKLF Proteins and Cell Apoptosis In addition to having an increased cell proliferation rate, cancer cells increase in number by circumventing cell death (apoptosis). Several lines Of evidence indicate that Sp/KLF proteins are involved in the regulation Of apoptosis. Upregulation Of Sp1 transcription activity induces Fas-mediated apoptosis in vascular smooth muscle cells (VSMCS) (410). Increased Sp1 DNA binding activity after ionizing radiation (IR), however, inhibits lR-induced apoptosis in U1-Mel melanoma cells. Also, expression Of the human papillomavirus E7 gene decreased Sp1 binding activity, increased apoptosis, and increased radiosensitivity in U1-Mel melanoma cells (411). In another case, L- tyrosine-induced apoptosis in B16 melanoma cells was linked to the decreased Sp1 DNA binding activity (412). Additionally, overexpression Of KLF10 and KLF4 induced apoptosis in multiple types Of cells, including pancreatic epithelial cells (413, 414), and colon cancer cells (415). Also, KLF2-knockout T cells undergo spontaneous apoptosis (394-397). The diverse apoptotic effects Of Sp1 and other members Of the Sp/KLF family in different cell types may be the result Of: 1) differences in their expression patterns, 2) differences in their abilities to recognize the target genes, 3) differences in their functions in regulation of target genes' transcription. 58 The promoter activity of many pro-apoptotic and anti-apoptotic genes is regulated by Sp1 or the other Sp/KLF proteins. For example, the cell-surface protein Fas (APO-1) is a member Of the tumor necrosis factor receptor (T NFR) superfamily, and functions to induce apoptosis following binding Of Fas ligand or exposure to certain anti-Fas antibodies. Studies Show that both Fas and FasL gene promoters contain Sp1 binding sites and are regulated by Sp1 (416, 417). In support Of these Observations, upregulation Of Sp1 DNA binding activity is linked to Fas-mediated apoptosis in VMSCS (410). The Bcl-x gene, a member Of the Bcl-2 family, is highly regulated during lymphoid development. Its expression modulates apoptosis in lymphoid and other cell populations. There are multiple Sp1 binding motifs located within mouse BcI-x gene promoter (418), indicating that Sp1 and/or other Sp/KLF proteins are involved in the regulation of the transcription Of BcI-x. In addition, several other pro-apoptotic genes are reported to contain Sp- binding Sites in their promoters, including BcI-2 (419), TGF-B and its receptor (357, 420), TNF a (421), and TRAIL (422). The promoters Of some anti- apoptosis genes also contain Sp-binding sites, e.g. Bax (423, 424), BCL-3 (425), and survivin (426). Given that the transcriptional functions and the expression patterns of Sp/KLF proteins vary so widely, the overall effects on apoptosis may depend on the cell types, the functions of the Sp/KLF proteins, and the genes which are regulated. 59 3. SpIKLF Proteins and Cell lnvasiveness and Metastasis Most cancers eventually escape the primary tumor mass and form metastases in distant sites Of the body. Most cancer deaths (~90%) are caused by metastasis (427). Several classes Of proteins are involved in invasiveness and metastasis of cancer cells, including cell-cell adhesion molecules (CAMS), which mediate cell-tO-cell interactions (428, 429), integrins, which link cells to extracellular matrix (430), and extracellular proteases, which degrade extracellular matrix (ECM) (431, 432). Changes in expression Of CAMS, integrins, and extracellular proteases have been evident in invasive and metastatic cells (1 ) The urokinase-type plasinnogen activator (uPA) and its cognate receptor (uPAR) play a key role in tumor invasion and progression. Upon binding to each other at the cell surface, the two proteins lead to a very efficient plasmin- mediated degradation of extracellular matrix components such as fibrin and collagen IV (433, 434). Thus, these proteins can promote invasion and metastasis. Up-regulation Of uPA and uPAR have been repeatedly shown to be predictive of a poor clinical prognosis of breast carcinoma (435), pancreatic carcinoma (436), and gastrointestinal cancer (437). Promoters of the uPA and uPAR genes contain Sp1 binding Sites, suggesting that Sp1 and/or other members of the Sp/KLF family are involved in the upregulation Of both proteins in cancers (438-440). In fact, upregulation Of Sp1 protein level or Sp1 DNA binding activity has been shown to be correlated with inCreases in the uPA and uPAR protein levels in different cancers, including breast cancer (387), cancer Of the 60 ‘- -‘-v' " exocrine pancreas (441), and gastrointestinal cancer (442). Such data suggest that Sp1 and other members Of Sp1/KLF family are involved in the tumor cell invasiveness and metastasis through regulation Of their target genes. Metalloproteases (MMPS) are a family of over 20 enzymes which are Characterized by their ability to degrade ECM and their dependence upon Zn” binding for proteolytic activity. MMPs facilitate tumor cell metastasis by destroying the basement Of membrane and other components Of the ECM (443). For example, overexpression Of MMP2, a member Of MMPs family, has been shown to strongly correlate with glioma progression (444) and cervical cancer invasiveness (445). The MMP2 gene promoter contains GC-boxes, and Sp1 and Sp3 are required for constitutive expression Of this gene (446). Direct evidence linking Sp1 and MMP2 to cancer progression comes from studies which showed that a single nucleotide polymorphism identified in the promoter Of MMP2 (~1306C-T) disrupted an Sp1 binding site (GT-Box), and this mutation strikingly decreased MMP2 promoter activity (447). The variant MMP2 genotype (-1306C-T) was associated with substantially reduced risk Of breast cancer (448) and lung cancer (449), compared to the CC wild-type genotype. These results suggest that Sp1 and Sp3 play a critical role in the regulation of MMP2 expression and contribute to cancer progression. In addition, expression Of some 0 subunits and 8 subunits Of integrins is subjected to regulation by Sp1 and Sp3, possibly other members Of the Sp/KLF family (450-452). The integrins consist Of two transmembrane glycoprotein subunits, the a chain and the ,8 Chain. The 0 subunits consist 01-11 and av, aL, 61 aM, 0E, 00, 0X, and alib, and the 8 subunits contain 81-8. One a and one 8 a subunit form a heterodimeric receptor, and it functions in cell adhesion and signal transduction. Different integrin subtypes have distinct substrate preferences. Cancer cells frequently exhibit expression Of integrins which favor the surrounding cells in the host rather than expression Of integrins which prefer binding to the ECM present in origin; this shift in the expression Of integrins facilitates the formation Of the metastases (453, 454). Sp1, Sp3 and other members Of the Sp/KLF family may facilitate the shift Of the expression Of integrins by regulating the expression of the subunit genes. 4. SleLF Proteins and Angiogenesis Angiogenesis is the process Of developing new blood vessels from pre- existing vasculature. The angiogenic phenotype depends on the balance Of proangiogenic growth factors such as vascular endothelial growth factor (VEGF), and angiogenic inhibitors such as thrombospondin (1). Integrin signaling also contributes to this balance (1). Active angiogenesis has been shown to be an important process for new blood vessel formation, tumor growth, tumor progression, and spread of tumor (455). This is also supported by experiments showing that the anti-VEGF antibody or the dominant negative form of VEGF receptor 2 (flk-1) is able to impair neovascularization and growth of subcutaneous tumors in mice (456, 457), and by the Observation that antiangiogenic substances are able to inhibit the growth Of tumor cells inoculated in mice (458). 62 VEGF and its receptors play a key role in the regulation of angiogenesis. The promoters Of VEGF and its receptors flk-1 and VEGFR2 contain Sp1 binding sites; mutations Of the Sp1/Sp3 binding sites significantly affect their promoter activity (386, 459-462). In human pancreatic adenocarcinoma cells, Sp1, Sp3, and VEGF are found to be upregulated, and constitutive Sp1 activity is required for constitutive expression Of VEGF (386). In addition, Sp1 binding sites are required for the expression Of VEGF and its receptors, which is induced by different cytokines, growth factors, and cell stresses (462-469). These results clearly indicate that Sp1, Sp3, and other members Of the Sp/KLF family are involved in angiogenesis through regulating the expression of the key factors for angiogenesis. In support Of this conclusion, studies showed that a Sp1 decoy Oligonuleotide suppresses expression Of VEGF in mouse melanoma tumors cells, in human lung adenocarcinoma (A549) cells, and in glioblastoma multiform (U251 ) cells (470, 471 ) 5. Summary As transcription factors, Sp1, Sp3, and other Sp/KLF proteins are reported to regulate the expression Of more than 1,000 genes. Among these genes, some play an important rOle in the regulation Of cell growth and cell development, tumor cell invasiveness and metastasis, cell apoptosis, and angiogenesis. During tumor development, the tumor cells acquire these capabilities through different strategies, including overexpression and/or downregulation Of genes that contribute to Obtaining these capabilities. 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J., Kaneda, Y., Nakashima, Y., Shirasuna, K., and Sueishi, K. Sp1 decoy transfected tO carcinoma cells suppresses the expression Of vascular endothelial growth factor, transforming growth factor beta1, and tissue factor and also cell growth and invasion activities. Cancer Res, 60: 6531-6536, 2000. Novak, E. M., Metzger, M., Chammas, R., da Costa, M., Dantas, K., Manabe, C., Pires, J., de Oliveira, A. C., and Bydlowski, S. P. Downregulation Of TNF-alpha and VEGF expression by Sp1 decoy Oligodeoxynucleotides in mouse melanoma tumor. Gene Ther, 10: 1992- 1997,2003. 106 CHAPTER 2 Down-regulation of Overexpressed Sp1 Protein in Human Fibrosarooma Cell Lines Inhibits Tumor Formation Zhenjun Lou, Sandra O’Reilly, Hongyan Liang, Veronica M. Maher, Stuart D. Sleight, and J. Justin McCormick. Carcinogenesis Laboratory Department of Microbiology and Molecular Genetics and Department of Biochemistry and Molecular Biology Michigan State University East Lansing, Michigan 48824-1302 Running Title: Reducing Overexpressed Sp1 Inhibits Tumorigenicity Grant support: This work was supported by United States Department Of Health and Human Services Grants CA82885 and CA098305 from the National Cancer Institute (J.J.M) Request for reprints: J. Justin McCormick, Carcinogenesis Laboratory, Food Safety and Toxicology Bldg., Michigan State University, East Lansing, MI 48824- 1302; Phone: (517) 353-7785; Fax: (517) 353-9004; E-mail: mccormi1@msu.edu 107 1EGF, epithelial growth factor; EGFP, enhanced green fluorescent protein; EGFR, EGF receptor; HGF, hepatocyte growth factor; Sp/KLF, Sp Krtlppel-like factor; uPA, urokinase plasminogen activator", uPAR, uPA receptor; VEGF, vascular endothelial growth factor. 108 ABSTRACT Sp1 is a transcription factor for many genes, including genes involved in tumorigenesis. We found that human fibroblast cells malignantly transformed in culture by a carcinogen or by stable transfection Of an oncogene express Sp1 at 8 to 18-fold higher levels than their parental cells. These cell lines form fibrosarcomas in athymic mice with a very short latency, and the cells from the tumors express the same high levels Of Sp1. Similar high levels Of Sp1 were found in the patient-derived fibrosarcoma cell lines tested, and in the tumors formed in mice by these cell lines. To investigate the role Of overexpression Of Sp1 in malignant transformation Of human fibroblasts, we transfected an Sp1 U1snRNA/Ribozyme into two human cell lines, malignantly transformed in culture by a carcinogen or overexpression of an oncogene, and into a patient-derived fibrosarcoma cell line. The level Of expression Of Sp1 in these transfected cell lines was reduced to near normal. The cells regained the spindle-shaped morphology and exhibited increased apoptosis and decreased expression Of several genes linked to cancer, i.e. epithelial growth factor receptor, urokinase plasminogen activator, urokinase plasminogen activator receptor, and vascular endothelial growth factor. When injected into athymic mice, these cell lines with near normal levels Of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. These data indicate that overexpression of Sp1 plays a causal role in malignant transformation Of human fibroblasts and suggest that for cancers in which it is overexpressed, Sp1 constitutes a target for therapy. 109 INTRODUCTION It is now commonly accepted that cancer results from multiple genetic changes. Many Of the genes involved have been identified, but whether all Of the primary genes involved in the malignant transformation Of any type Of human cancer have been identified remains unclear. To address such concerns, McCormick and Maher and their colleagues have utilized human fibroblasts in culture tO study the process by which these cells become malignant. By means of sequential clonal selection, they identified many genetic changes required for malignant transformation of human fibroblasts. They began these studies with a finite-lifespan human fibroblast cell line, designated LG1, derived from the foreskin Of a normal neonate. LG1 cells were transfected with a vector can'ying a v—Myc oncogene and a selectable marker. Several oncogene transfectant clones, vector controls, and an LG1 clone were expanded to the end of their lifespan. From a population derived from a clone of cells that expressed the v-MYC oncoprotein, a telomerase positive, infinite life span, chromosomally-stable, diploid cell strain arose which was designated MSU-1.0. A more rapidly-growing, chromosomally-stable variant Of MSU-1.0 cells arose spontaneously, and this strain was designated MSU-1.1 (1 ). Transfection of MSU-1.1 cells with an H-Ras (2), N-Ras (3), or v-K-Ras (4) oncogene in vectors engineered for high expression of the oncogene, the MSU-1.1 cells became malignantly transformed, i.e., able to form sarcomas when injected so. into athymic mice. A single exposure Of MSU-1.1 cells to a cobalt-60 gamma radiation (5, 6) or a Chemical carcinogen (7, 8) transformed them into cells able to form distinct foci on a 110 monolayer Of cells. When the cells from the foci are expanded and injected into .. athymic mice, they form sarcomas after a short latency. Collectively, the cell lines/strains from LG1 cells to the tumor-derived malignant cells are referred to as the MSU1 lineage. A strong advantage Of this lineage is that it allows one to identify genetic or epigenetic Changes that occur during the transformation process by comparing the malignant cell lines with the normal founder cell population, LG1, and the non-tumorigenic intermediate cell lines derived from it, MSU-1.0 and MSU-1.1. Using this approach, we have been able to correlate many Of the steps in the transformation process with specific genetic Changes. Examples include loss Of wild-type p53 (6, 8) and overexpression of MET and Sp1 (9). In 1983, Sp1 was identified as a general transcription factor (10). Sp1 was the first transcription factor to be purified, cloned, and characterized in mammalian cells (11). Ubiquitously expressed, it binds the GC-box (GGCGGG) and GT-box (CACCC) via its Cys2His2 zinc-finger DNA binding domain (12). Sp1 belongs to the Sp/KLF1 family, consisting of 21 members that share high homology in their DNA binding domains (13). These proteins are present in species ranging from C. elegans to humans (14-17). Recently, overexpression or higher binding activity Of Sp1 was found in human pancreatic cancer cell lines and cancer tissue (18), breast cancer cell lines and cancer tissue (19), gastric carcinoma (20), and thyroid carcinoma (21). Several Of these studies also showed that overexpression Of Sp1 protein or up-regulation of Sp1 transactivating ability is closely correlated with up-regulation Of VEGF (18), uPA and uPAR (19), and 111 EGFR (20), proteins that are known tO play important roles in tumorigenesis. A recent study Of MET and Sp1 expression in this laboratory by Liang et al. (9) Showed that in six Of the six malignantly transformed cell lines Of the MSU1 lineage examined, MET is overexpressed, and that four of the six also overexpressed Sp1, a transcription factor for met. In addition, three Of the five patient-derived fibrosarcoma cell lines examined showed a high level of Sp1 compared tO normal human fibroblasts, suggesting that Sp1 plays a role in the malignant transformation of human fibroblasts, not only in culture, but also in the human body. We designed and constructed an Sp1 U1snRNA/Ribozyme and stably transfected it into two human fibrosarcoma cell lines found tO express high levels Of Sp1. These cell lines had been derived from tumors formed in athymic mice by injection of MSU-1.1 cells that we had transformed by transfection Of the H-Ras oncogene (2) or by gamma irradiation (6). We also transfected the Sp1 U1snRNA/Ribozyme into a patient-derived fibrosarcoma cell line (SHAC) that expresses a high level Of Sp1 protein. From all three groups, we identified transfectants in which the expression Of Sp1 had been reduced to the level found in non-transformed parental MSU-1.1 cells. We tested them for their ability to produce large-sized colonies in agarose. None of them could do so. When injected into athymic mice, these cell lines with near normal levels Of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. We also found that the inhibition Of the tumorigenicity of these cell lines correlates with decreased expression of specific proteins known to play a 112 role in the malignant transformation Of such cells, i.e., EGFR, uPA, uPAR, and .. VEGF. 113 MATERIALS AND METHODS Cells and Cell Culture. The derivation of human fibroblast cell line MSU-1.1 has been described (1). PH2MT cells were derived from a tumor formed in athymic mice by injection Of MSU-1.1 cells malignantly transformed by an overexpressed H-Ras oncogene (2). y2-3A/SB1 cells were similarly derived from a tumor formed by MSU-1.1 cells malignantly transformed by y-irradiation (6). SHAC cells are derived from a patient’s fibrosarcoma. The cells were routinely cultured in Eagle’s minimal essential medium, supplemented with L-aspartic acid (0.2 mM), L-serine (0.2 mM) and pyruvate (1 mM), (modified Eagle’s medium) and 10% supplemented calf serum (Hyclone, Logan, UT), hydrocortisone (1 pg/ml), penicillin (100 U/ml) and streptomycin (100 lug/ml) (culture medium), at 37°C in a humidified incubator with 5% 002. For selection of transfected cell strains, blasticidin (10 pg/ml) was added to this culture medium. To be sure that the cells used in each experiment maintained the drug resistance and presumably Sp1 ribozyme expression, 10 or more vials Of each cell strain were frozen before experiments were carried out. When they were used, they were cultured in medium containing blasticidin (10 pg/ml) for at least three days before experiments were carried out. A new vial was used to provide cells for each experiment. Preparation Of Sp1 Ribozyme Antisense Construct. The Sp1 U1snRNA/ Ribozyme construct was prepared following a published procedure (22). The complementary Oligonucleotides that encode the antisense sequence of human Sp1 (GenBank number, AJ272134), including the hammerhead ribozyme, were 114 synthesized and the double-stranded DNA was inserted between the EcoR l and - Spe I sites Of the pU1 vector (gift of Dr. Laterra) containing the human U1snRNA and its endogenous promoter sequences. The U1snRNA/Sp1 antisense/ham- merhead ribozyme fragment was excised by BamHl digestion and inserted into BamHI site Of pCMV/Bsd vector (lnvitogen, Carlsbad, CA). The construct was sequenced using an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The structure of the chimeric RNA (U1snRNA/Sp1 antisense/ hammerhead ribozyme) was analyzed using MulFOld and Loop-D-Loop programs. Transfection. Transfection was performed using Lipofectamine (lnvitogen, Carlsbad, CA) following manufacturer's procedure. Transfectants were selected in medium containing 10 pg/ml blasticidin, and their Sp1 protein levels were determined by Western blot analysis. Western Blot Analysis. Whole cell lysates were prepared using single- detergent lysis buffer as described by Liang et al. (9). Conditioned-medium was prepared as described below. Protein content was quantified using the Bicinchoninic Acid Protein Assay Reagent Kit (Pierce, Rockford, IL), and 50 pg total protein or 20 pg conditioned-medium was loaded and separated by 7.5% SDS-PAGE. Protein was transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA), and Western blot analysis was performed using standard techniques. The signal was detected using SuperSignal reagent (Pierce, Rockford, IL). Antibodies against Sp1, Sp3, HGF, uPA, uPAR and EGFR were purchased from Santa Cruz (Santa Cruz, CA); CMET, from Upstate 115 (Waltham, MA); H-Raswz, from Oncogene; Ku80, from SeroteC (Raleigh, NC); and 8-actin, from Sigma (St. Louis, MO). The latter two proteins served as loading controls. Blots were quantified by densitometry, and the signal of each band was normalized to that Of its loading control. All experiments were repeated at least three times. Preparation Of Conditioned Medium. Cells were plated in culture medium at a density Of 5 X 105 cells per 100 mm-diameter culture dish. After 24 h, the medium was changed to serum-free medium. After another 48 h, the medium was collected and concentrated 15-20-fold using concentrators (VIVASPIN 20ML, 5,000 MWCO, Vivascience AG, Hannover, Germany). ELISA. The level Of secreted VEGF in the medium was determined using the DuoSet ELISA Development System (R&D, Minneapolis, MN) following manufacturer's procedures. To determine the level Of VEGF in each sample, 100 pl of concentrated conditioned-medium was used. TO create a standard curve, a series of two-fold serial dilutions Of recombinant human VEGF (2000 ng/ml to 125 ng/ml) was included in each set Of samples assayed. The concentration of VEGF in each sample was calculated by comparing the optical density Of each sample to that Of the standard curve and then normalizing that value to the protein concentration Of each sample. RT-PCR Analysis Of Sp1 mRNA. Total RNA was extracted from logarithmically-growing cells, and 1 ug of total RNA was transcribed into CDNA using Oligo dTm-Ha). Sp1 CDNA was amplified by PCR for 26 cycles with the following primers: 5’-TAATGGTGGTGGTGCCTTT-3’ and 5’-GAGATGATCTGC 116 CAGCCATT-3’, which span the proposed hammerhead cutting site. 8-actin, .. which served as an integrity and loading control, was amplified for 21 cycles (8- actin primers: 5'-AGGCCAACCGCGAGAAGATGACC-3’ and 5’-GAAGTCCAGG GCGACGTAGC-3’). The PCR products were separated by 2% agarose gel, and the gel was stained with ethidium bromide. Luciferase Assay. The Luciferase assay was carried out using the procedure described by Liang et al. (9). Cells were transiently transfected using FuGene6 (Roche, Indianapolis, IN), following manufacturer's procedure. Briefly, cells in triplet were grown to 50-60% confluence in six-well plates. pRL-TK vector (Promega, Madison WI), 0.5 pg, was added to the cells in 1.5 pl FuGene6 transfection reagent (1:3). In parallel wells, 0.5 pg Of plasmid DNA (0.01 pg pRL- CMV vector and 0.49 pg pGL2-Basic vector (Promega, Madison WI)) was added to cells to serve as transfection efficiency controls. The cells were incubated for 48 h, and cytosolic fractions were prepared with passive lysis buffer (Promega, Madison WI). The luciferase activity was analyzed using the Dual-Luciferase® Reporter Assay System (Promega, Madison WI) and a luminometer. The luciferase activity Of each sample was normalized to the protein concentration. , The luciferase activity Of each sample (pRL-TK) was normalized to the luciferase activity Of control (pRL-CMV) to adjust for transfection efficiency. Assay for Anchorage-Independence. Cells were assayed for ability to form colonies in 0.33% agarose essentially as described (23). Briefly, 5,000 cells were plated in 0.33% top agarose per 60 mm-diameter culture dish, and that layer was covered with 2 ml Of culture medium. The culture medium was replaced weekly. 117 MSU-1.1 cells were included in each assay as a negative control. After 3 wk, the cells were fixed with 2.5% glutaraldehyde, and the colonies in five randomly chosen areas of each dish were photographed using NIH Image 1.62 software. The numbers and size Of the colonies in each area were calculated by Quantity One software (Bio-Rad website). All experiments were carried out at least three times. Assay for Tumorigenicity. Cells were assayed for the ability to form tumors in athymic mice as described by Liang et al. (9), except the mice were examined weekly for tumor growth, and the tumors were removed when they reached 1 cm in diameter. If nO tumor was Observed in 6 mO following injection, the mice were sacnficed. Cell Morphology. Cells were plated in culture medium at a density Of 2 X 104 cells per well of a Chamber slide and incubated at 37°C in a humidified incubator with 5% 002. When the cells reached ~90% confluence, they were fixed with neutral-buffered formalin at 4°C for 10 min. The cell morphology was Observed under a Nikon eclipse TE300 microscope, and the images were recorded with a digital camera. Cell Death Assay. Cells were plated in culture medium at 5 X 105 cells per 100 mm-diameter culture dish (total six dishes/cell line/experiment) and incubated at 37°C as above. After 24 h, the medium in half the dishes was changed to serum- free medium; the cells in the other half received fresh culture medium. After 48 h Of incubation, the cells floating in the medium were collected and counted, and then the attached cells were dislodged with trypsin and counted. 118 Apoptosis Assay. Cells were plated at a density Of 2 X 105-5 X 105 cells per 60 .. mm—diameter culture dish and incubated as described. After 24 h at 37°C, the medium was removed in order tO remove any unattached cells and fresh medium was added tO the culture dishes. After 24 h, the cells were detached with trypsin, stained with Anexin V-EGFP following manufacturer’s procedure (Clontech, Palo Alto, CA), and assayed for evidence Of apoptosis using Flow Cytometry. Apoptotic cells are stained by EGFP. Non-stained cells and Fas antibody-treated cells served as negative and positive controls, respectively. The cells were stained with propidium iodide to determine whether or not the cell membrane was intact. All experiments were carried out three or four times. 119 RESULTS Overexpression Of Sp1 in Human Fibrosarooma Cell Lines. TO confirm the report by Liang et al. (9) that the level of Sp1 protein is higher in human fibrosarcoma cell lines PH2MT and y2-3A/SB1 than in their parental MSU-1.1 cells, we carried out Western blot analysis using lysates from these three cell lines, as well as from LG1, the finite life span parental cell line, from which the MSU-1.1 cells were derived (1). The results showed that the Sp1 level was ~2- fold higher in the MSU-1.1 cells than in the LG1 cells (Fig. 1, A and B). The PH2MT cells had an Sp1 level 3- to 6-fold higher than the Sp1 level in the MSU- 1.1 cells, and the V2-3A/SB1 cells had an Sp1 level 7- to 10-fold higher than that Of the MSU-1.1 cells. Construction Of the Sp1 U1snRNA/Ribozyme Vector. To determine whether the high expression Of Sp1 observed was causally involved in the malignant transformation of these cells, we designed and constructed an Sp1 specific ribozyme to down-regulate Sp1 expression. The Sp1 U1snRNA/Ribozyme consists Of three parts, an Sp1-specific antisense sequence with the hammerhead ribozyme in its center, and the two flanking regions Of the U1snRNA (Fig. 1C). The Sp1-specific antisense is complementary to the 165- 205 sequence of human Sp1 mRNA (Genbank accession number AJ272134). A BLAST search Showed that there is no significant similarity between this sequence and that Of other genes. Use Of the Mulfold and Loop-D-Loop programs (24-26) to analyze the Sp1 UISnRNA/Ribozyme structure revealed that the U1snRNA structure is well conserved. To make an expression vector we 120 inserted the Sp1 U1snRNA/Ribozyme with the human U1snRNA endogenous .. promoter into the BamH I site Of the pCMV/Bsd vector containing the gene for blasticidin dmg resistance. 121 Fig. 1. Evidence that Sp1 is overexpressed in the malignant human fibroblast cell lines and diagram Of the Sp1 U1snRNA/Ribozyme and its predicted structure. A, Example Of Western blot analysis Of Sp1 expression in whole cell lysates (50 pg protein/lane) from foreskin-derived normal human fibroblast cell line LG1, its non- transforrned, infinite life span derivative cell strain, MSU-1.1, and cell line PH2MT, derived from a tumor formed in an athymic mouse after so. injection Of by MSU-1.1 cells malignantly-transformed by transfection Of an H-Ras oncogene. 8-actin served as the loading control. B, Identical that shown in A, except that cell line y2-3A/SB1, derived from a tumor formed in athymic mice by MSU-1.1 cells that had been malignantly-transformed by exposure to 60Cobalt radiation, was used. C, Schematic representation and sequence of the Sp1 U1snRNA/ Ribozyme construct used to reduce expression Of Sp1. The antisense sequence is complementary to the 165-205 sequence of human Sp1 mRNA (Genbank No. AJ272134) and contains a hammerhead ribozyme sequence in its center, flanked by the U1snRNA. Cleavage is predicted to occur at nucleotide 185, 5' to the sequence GUC (arrow). 122 I £89m. n a mom I2... I a Em I 2:. I one. lac IE «on Ia: I no I 2: Em 123 3:303 3:33.? O: < O OIO oio thwon< DO< eOoiO3O95% Of the empty vector transfectants in these three experiments did not exhibit a decrease in the 125 level Of expression of Sp1 indicates that the reduction in the level Of expression Of Sp1 protein Observed. in the Sp1 U1snRNA/Ribozyme-transfected clonal populations is not the result of random variation between Clonal populations, but rather results from the expression Of the Sp1 U1snRNA/Ribozyme. Figure 2A shows the results of a Western blot for Sp1 expression for the PH2MT cell line, two empty vector-transfected cell strains, and two ribozyme- transfected cell strains. Figure 28 shows the results Of a similar Western blot for the y2-3AISB1 cell line, two empty vector-transfected cell strains, and three ribozyme-transfected cell strains. These five clonal populations Of Sp1 ribozyme- transfected cell strains, as well as the two parental cell lines, and two empty vector clonal populations from each parental strain, were used for further study. TO determine whether the Sp1 transactivating activity is reduced by down- regulation Of Sp1 protein levels, we transiently transfected the same cell strains as shown in Fig. 2, A and B with a luciferase reporter construct in which the Renilla luciferase gene is driven by an HSV-TK promoter, which responds to the level Of Sp1 protein. As shown in Fig. 2, C and D, Sp1 transactivating activity was reduced 70-90% in these Sp1 ribozyme-transfected cell strains, compared to their respective parental cell line. The vector-transfected control cell strains, designated V1 from each cell line exhibited Sp1 activity levels equal to that Of their respective parental cell strain; the strains designated V2 exhibited an intermediate level of Sp1 activity. These results demonstrate that the Sp1 U1snRNA/Ribozyme down-regulates the level Of Sp1 protein and that this reduced level correlates with reduced transactivating activity. 126 mm as. ‘51 - Down-regulation of Sp1 Expression Reduces EXpression Of Sp3. Sp3 is a ubiquitously expressed transcription factor that binds tO the same DNA responsive element as Sp1 and with the same affinity (27). Unlike Sp1, which always acts as a transcription activator, Sp3 can function as an activator or a repressor of transcription, depending on secondary modifications to the Sp3 protein (28-30). Because both Sp1 and Sp3 are ordinarily expressed in mammalian cells, the expression of genes that have the Sp1/Sp3 response elements is modulated by the cOmbined action of Sp1 and Sp3. TO determine the relative expression levels Of Sp1 and Sp3, we probed the Sp1 blots shown in Fig. 2, A and B with an antibody specific for the human Sp3. As shown in Fig. 2, A and B, the level of Sp3 expressed correlated with the level of Sp1 expressed, and the cell strains exhibiting down-regulation Of Sp1 exhibited a parallel down- regulation of Sp3. We carefully examined the antisense sequence Of the Sp1 ribozyme tO determine whether there was a homologous sequence in the Sp3 gene. None was found. The Sp1 U1snRNA/Ribozyme Acts as a Ribozyme and as Antisense. To analyze the mechanisms involved in the inhibition Of Sp1 expression by the Sp1 U1snRNA/Ribozyme, we determined the Sp1 mRNA levels by semi-quantitative RT-PCR. Total RNA extracted from the cell strains with reduced Sp1 levels and from their parental and vector control cell strains was subjected to reverse transcription, and the levels of Sp1 mRNA were determined using a pair of Sp1- Specific primers which amplify the DNA fragment spanning the proposed ribozyme cutting site (5’GUC3’). 8-actin served as the loading control. 127 Two of the five cell strains with reduced Sp1 expression, PH2MT, SpR1 and ._ y2-3A/SB1, SpR1 Showed reduced Sp1 mRNA levels compared to the parental and vector control cell strains. However, the other three cell strains showed no Change. These data suggest that in the former cell strains, the Sp1 U1snRNA/Ribozyme cut the Sp1 mRNA, resulting in degradation Of the mRNA, whereas in the latter three cell strains, the antisense sequence inhibited translation Of the Sp1 mRNA. The latter mechanism has been reported for other ribozymes (31 ). H-Ras Expression in PH2MT Cell Line and Its Derivatives. The PH2MT V12 cells overexpress the oncogene H-Ras (2). The H-Ras expression vector contains SV40 enhancers that are regulated by Sp1 (32, 33). TO determine if down-regulation of Sp1 level in PH2MT cells decreases H-RasV12 expression, we carried out Western blot analysis. As shown in Fig. 26, as expected, the MSU- 1.1 cells didn’t express H-Rasv‘z. The H-RasV12 levels in PH2MT cells and the transfectants showed no Change [the relative H-Ras levels range from 0.9-1.0 in the transfectants, compared to that in PH2MT cells (1.0)]. This result indicates that the down-regulation Of Sp1 protein level doesn’t cause loss Of transformed V12 characteristics by reducing H-Ras expression. 128 Fig. 2. Evidence that stable transfection of malignant cell lines with the Sp1 U1snRNA/Ribozyme reduces expression Of Sp1 protein and its transactivating activity, and expression Sp3 protein, but does not affect that Of a H-ras oncogene. A, Western blot analysis Of Sp1 and Sp3 protein expression in whole cell lysates (50 pg/Iane) Of tumor-derived cell line PH2MT, two derivative cell strains transfected with an empty vector as controls (V1 and V2), and two Sp1 ribozyme-transfected clonal derivative strains (SpR1 and SpR2). 8-actin was used as a loading control. B, Same type of analysis as in A, except tumor-derived cell line y2-3AISB1 was used as the parental cell line along with two of its derivative strains transfected with an empty vector as controls, and three Sp1 U1snRNNRibozyme-transfected clonal derivatives were compared. Ku80 was used as a loading control. C, Sp1/Sp3 transactivational activity found in the series of cell lines shown in above A. The cells were grown to 50% confluence and transiently transfected with an HSV-TK promoter luciferase construct and a control vector. After 48 h, whole cell lysates were prepared, and the luciferase activity was analyzed as described in Experimental Procedures. 0, Same type Of analysis as in C, but using the series of cell lines shown above in B. E, Level Of Sp1 mRNA in the cell lines shown in A above. Total RNA was extracted, and the relative level of Sp1 mRNA was assayed by RT-PCR. 8-actin served as the control for quantitation and determination Of the integrity of the RNA. Only cell strain SpR1 showed a decreased level of Sp1 mRNA. F, Same type of analysis as in E, except cell line y2-3A/SB1 and its derivatives Shown in 3 above were used. Only SpR1 Showed a decreased level Of Sp1 mRNA. G, Western blot 129 analysis Of the level of expression of H-ras oncoprotein in MSU-1.1 cells and in -. the five cell strains shown in Fig. A above (30 pg protein/lane). Ku80 was used as the loading control. 130 « K at s c .4 at 1 -l Sp1 "_" -- .2... .. ”4'38“: .— Ku80 - 105 : 110- K 9'} 45 3' 4K 4" 99$ 99 99¢. --- .. —-— "I' 0 131 Cell Strains with Reduced Sp1 Levels No Longer Form Large Colonies in Agarose. Cell lines PH2MT and y2-3A/SB1 are highly tumorigenic and form large-sized colonies in agarose. In contrast, their non-tumorigenic parental cell strain, MSU-1.1, forms only very small colonies (2, 6). Figure 3, A and B show that the two ribozyme transfectants Of PH2MT cells and the three from y2- 3A/SB1 cells formed very small colonies in agarose, identical to those formed by MSU-1.1 cells, whereas the vector control cell strains formed the large-sized colonies, similar to those Of their parental malignant cell lines, PH2MT and y2- 3A/SB1. 132 Fig. 3. Evidence that down-regulation Of overexpressed Sp1 protein inhibits anchorage-independent growth. A, The cell lines/strains shown in Fig. 2A were assayed for ability to form colonies in agarose, as described in Experimental Procedures. After 21 days, photographs were taken of the colonies in five randomly-Chosen areas Of each of the five dishes used per cell strain, and the average number of colonies with diameters of the designated sizes was calculated and plotted as percent Of the total number. 8, Same type of analysis as in A above, but using the cell lines/strains shown in Fig. 28. The error bars Show the standard error Of the mean. 133 >200|.I.m I 120400th El <4ottm m 40-120pm 48 w _ _ b a 9 2 .......... a wowowowowowowowcwowow. ' ‘ ‘ ‘ . ‘ ‘ ‘ ‘ . .wcucuowowowchHOw. .— o 1 8295:. 33:94.3 £3 3.5.00 Luna Co c9359.?"— 134 Down-regulation Of Sp1 Inhibits the Tumorigenicity of Cell Lines PH2MT .. and y2-3AISB1. To determine whether high expression level of Sp1 plays a role in tumor formation, we injected athymic mice with the five Sp1 U1snRNA] Ribozyme transfectants showing the largest reduction in Sp1 levels (Fig. 2, A and B) as well as three ribozyme-transfected derivatives Of PH2MT cells that exhibited intermediate levels of Sp1 (Table 1, Western blot data not shown), the two parental cell strains (Fig. 2, A and B) and four vector control cell lines (Fig. 2, A and 8). Six months after injection, these five cell strains with markedly reduced Sp1 expression had not produced any tumors (Table 1). In contrast, the parental and vector control cell strains formed large-sized tumors within 4-6 wks and three ribozyme-transfected PH2MT strains with intermediate levels Of Sp1, i.e., Sp1 R3, R4, and R5, produced tumors in approximately half Of the sites after a longer latency. 135 Table 1 Inhibition Of tumor formation by down-regulation Of Sp1 expression in malignant cell lines Cells (106) were injected 3.0. into the right and left rear flank regions Of athymic mice (two sites/mouse). Mice were examined weekly for tumors. When tumors reached ~1 cm in diameter, they were removed. Relative Sp1 Frequency Cell Lines Latency” (Wk) Level“ (Tumors/Sites) PH2MT 1.0 6/6 4 V1 0.9 4/4 4-6 V2 1.2 6/6 4-6 SpR4 0.7 4/6 5-8 SpR3 0.6 4/4 8-10 SpR5 0.4 6/16 1231 SpR 1 0.2 0/6‘ NA” SpR 2 0.2 0/6 NA fl-AISBI 1.0 6/6 5—6 V1 1.0 5/6 4-5 V2 1.0 6/6 4-5 SpR1 0.1 0/6 NA SpR2 0.1 0/6 NA SpR3 0.1 0/6 NA ° Relative Sp1 level determined by Western blotting. " Time required for tumors to reach ~1 cm. ‘ NO tumors formed within the 26-30-wk Observation period. ” Not applicable. V, vector control. SpR, transfectants expressing Sp1 U1snRNA/Ribozyme. 136 Cell Morphology Changes following the Down-regulation Of Sp1 Level. As .. shown in Fig. 4, A-J, four cell strains with reduced Sp1 levels (D, E, I, and J) (Of. Western blotting, Fig. 2, A and 8) showed dramatic Changes in morphology compared tO their malignant parental strains (A, F) and the vector transfectants (B and C; G and H). The cell lines shown in D, E, and I had acquired a spindle- shaped morphology, similar tO that of the MSU-1.1 cells from which their respective parental cell strains had been derived. The cells shown in J were flatter than those Shown in D, E, and l, and had a round shape, intermediate between normal fibroblasts and fibrosarcoma cells. All four transfectants with reduced Sp1 level were larger than their parental cell lines and the vector control cell lines (Fig. 4, A-C and F-H). Figure 4, K and L Show that these changes in cell morphology in the four cell strains with reduced levels of Sp1 were not the result of Changes in their levels of 8-actin. Such levels were constant among the parental, vector control cell strains, and ribozyme transfectants with reduced Sp1 levels. 137 Fig. 4. Evidence that down-regulation Of Sp1 protein affects cell morphology, but not through 8-actin. A, Morphology Of the malignant PH2MT cell line, B and C, PH2MT cells transfected with an empty vector, 0 and E. PH2MT cells transfected with the Sp1 U1snRNA/ribozyme, F, morphology of the y2-3AlSB1 cells, G and H, y2-3A/SB1 cells transfected with an empty vector, l and J, y2-3AISB1 cells transfected with the Sp1 U1snRNA/ribozyme. Cells were grown to ~90% confluence, fixed with neutral buffered formalin at 4°C for 10 min., and photographs were taken at a magnification of 200. The bar indicates 40 pm., K, Level Of 8-actin protein in whole cell lysates (50 pgllane) from the cells shown in A-E, with Ku80 as the loading control, L, Same as in K except using the cells shown in F-J. 138 .3" n“- nn"a 139 Down-regulation Of Sp1 Expression Induces Apoptosis. As Shown in Fig. 5, A and B, with or without serum in the growth medium, 8-22% of the Sp1 U1snRNA/Ribozyme transfectants of PH2MT and V2-3AISB1 cells detached from the dish. In the parental and vector control cell strains <8% of the cells detached from the dish. TO determine whether the floating cells were dead, we collected the floating cells by centrifugation and plated them in new dishes. None Of the cells attached (data not shown). TO determine if cell death was caused by apoptosis, the cells were grown in medium with 10% supplemented calf serum for 48 h, collected and labeled with Anexin V-EGFP and analyzed by Flow Cytometry. The ribozyme transfectants of PH2MT and IQ-3AISB1 cells displayed a 20°/o-40°/o increase in EGFP positive cells, indicating they died by apoptosis (Fig. 5, C and D). The parental and vector control cells had <5°/o EGFP positive cells (Fig. 5, C and D). These results demonstrate that down-regulation of Sp1 levels correlates with increased apoptosis. 140 Fig. 5. Evidence that down-regulation of Sp1 expression induces apoptosis. A and B, The cell strains shown in Fig. 2, A and B were grown in medium with or without 10% serum. The cells floating in the medium and the attached cells were collected and counted separately. The number Of floating cells is shown as percentage Of the total. C and D, The same series of cells were grown in medium with 10% serum for 48 h, and then collected and stained with Anexin V-EGFP and analyzed by Flow Cytometry for evidence Of apoptosis. The marker position was based on the fluorescence of unlabeled cells and cells treated with Fas antibody (100 ng/ml). The data shown are representative Of 3-4 independent experiments. 141 I gift» a. 5M“:- 8 ..\° ‘3: ,e ' ’4 ,,\° 0 El ’9‘, ’8- . . J". a a 2 ° 8r suoo wheels I0 9531000191! 0) j- ‘it» I 4- g ”a L. 8 e g 4 $2 I ’4 °\° ~( 0 [3 ’ie . . 4'9' 8 R 2 ° BIIOO Gannon Io 953W°°J°d 142 3.2.3:. 353802“. v war 62. ”.5 m mg m w r q 5.3. . m . .38 . N 5— &3 E8 .mmzni n— bficsc. 8:33.62“. . n8 «2 .2 o2 «2 n2 «2 .2 ..8 fl: no. we .2 :2 r z a . I . . . N . q . . am .\.~ 3 3e 8 , 4 .9 an S 5%.. U 143 The Expression of HGF/MET, uPA/uPAR, EGFR and VEGF in the Cell .. Strains with Reduced Sp1 Levels. Sp1 is a transcription factor and regulates more than a thousand of genes, some of which play an important role in tumorigenesis (34). To determine whether the expression level of proteins coded by Sp1-regulated genes that are thought to play a role in cancer formation is reduced in Sp1 U1snRNA/Ribozyme transfectants that exhibited down-regulated Sp1 levels, whole cell lysates were prepared and analyzed by Western blotting. Conditioned-medium from these cell strains was also prepared, concentrated, and analyzed for secreted proteins by Western blotting or ELISA. The level of the cMET protein, which has been shown to be higher in human fibrosarcoma cell lines (9), did not change. The level of HGF protein, the ligand for cMET, also showed no change (Fig. 6, A-D). However, the level of uPA, which was found to be high in 11 out of 11 human fibrosarcoma cell lines (35), was reduced in the Sp1 U1snRNA/Ribozyme transfectants (Fig. 6, C and D). The level of EGFR, VEGF and uPAR was strikingly decreased in the transfectants of y2-3A/SB1 cell line but not in those of the PH2MT cell line (Fig. 6, A and B; E and F). 144 Fig. 6. Effect of down-regulation of Sp1 protein on the expression of cancer- related genes. A, Whole cell lysates were prepared from the cells used in Fig. 2A, and analyzed (50 ,ug/lane) by Western blotting for expression of MET, EGFR and uPAR, with Ku80 as the loading control, B, Same as A, above except that cell lines used were those shown in Fig. 28, C and 0, Expression of HGF and uPA in the cell lines/strains shown in A and B, above. Cells were plated at 3 X 105 to 5 X 105 cells per 100 mm diameter dish in culture medium. After 24 h, the medium was changed to serum-free medium. The conditioned-medium (CM) was collected 48 h later, and concentrated at 4°C. The samples (20 ,ug) were analyzed by Western blotting with anti-HGF and anti-uPA antibodies. E and F, Expression of VEGF in the same series of cell lines/strains shown in A and B. CM was obtained as described above and the VEGF levels were analyzed by ELISA. The data shown are the average values of three independent experiments. 145 so A" 4“ 9° 9° A ,a‘ Q MET —|.....'...._...) EGFR—‘~~ o o...- n um- m t! a Ku80- -—-——-1 c $§ HGF-[t .—. , NJ 1 uPA-L..~«w ' .- «be 4" 4'” 9° 99 E VEGF nglmg CM B 9 s '3? 4" 4" (99$ (’33. MET - [- - - - VEGF nglmg CM 146 Down-regulation of Sp1 Expression in a Patient-derived Fibrosarcoma Cell Line Inhibits Its Tumorigenicity. Because the Sp1 ribozyme successfully blocked tumor formation by human fibrosarcoma cells malignantly transformed in culture, we transfected the Sp1 UlsnRNA/Ribozyme construct into a patient- derived fibrosarcoma cell line (SHAC), which expresses a high level of Sp1 protein, compared with the normal human fibroblast cell line, LG1 (data not shown), and tested them for tumorigenicity. As shown in Fig. 7A, the parental and two vector controls expressed high levels of Sp1 protein, and formed large- sized colonies (>50 pm in diameter, 30%-40%, Fig. 7B). The two clonal populations with the largest reduction in Sp1 protein levels (Fig. 7A, SpR1 and SpR2) couldnot form large colonies (>50 pm in diameter, 10%-15%, Fig. 78), as did MSU-1.1 cells (>50 pm in diameter, 10%, Fig. YB) . The transfectant with intermediate level of Sp1 protein (Fig. 7A, SpR3) formed large-sized colonies (>50 pm in diameter, 35%, Fig. 7B). These results demonstrate that down- regulation of Sp1 level in human patient-derived fibrosarcoma cell line inhibited their ability to grow in agarose. To examine their ability to form tumors in vivo, we injected these cells into the flank regions of athymic mice. The two clonal populations with the largest reduction in Sp1 protein levels (Fig. 7A, SpR1 and SpR2) formed tumors with a greatly increased latency and a decreased frequency, compared to the parental SHAC cells and the two vector control cell lines (Table 2). The transfectant with an intermediate level of Sp1 (Fig. 7A, SpR3) formed tumors with an increased latency and a decreased frequency compared to the controls (Table 2). These results demonstrate that decreased 147 expression of Sp1, caused by the Sp1 U1snRNA/Ribozyme, is effective in .. blocking tumor formation by a patient-derived human fibrosarcoma cell line that overexpressed Sp1. 148 Fig. 7. Evidence that the Sp1 U1snRNA/Ribozyme reduces expression of Sp1 in a human patient-derived fibrosarcoma SHAC and inhibits its ability to grow in agarose. A, The Sp1 levels in patient-derived fibrosarcoma cell line SHAC and derivatives were analyzed by Western blotting (whole cell lysates, 50pg/lane) with anti-Sp1 antibody. Ku80 served as loading control. V, empty vector control; SpR, transfectants expressing Sp1 ribozyme. B, SHAC and derivatives were assayed for ability to form colonies in agarose, as described in Experimental Procedures and Fig. 3. V, empty vector control; SpR, transfectants expressing Sp1 ribozyme. 149 0 K '5 Y' ‘3‘ 4" 4" 93.994093. 3P1 ‘ggz_‘-a~n bu fl K080 — g... “—— cull-bun.“ B C] <50pm m 50-100pm llOO-150pm I>150~200pm 100 :r I E 80 r .3 F 5 so 2'9 40 ._ ‘6 .\° 20 I o N N :v 4 $9 150 Table 2 Inhibition of tumor formation by down-regulation of Sp1 expression in patient-derived fibrosarcoma cell line SHAC Cells (106) were injected so into the right and left rear flank regions of athymic mice (two sites/mouse). Mice were examined for tumors, weekly. When tumors reached ~1 cm in diameter, they were removed. The data in parentheses are from a second experiment with the same cell lines. Frequency SHAC Relative Sp1 Levela Latency” (Wks) (Tumors/Sites) Parental 1 .0 6/6 9 V1 0.8 3/4 6-8 V2 1 .0 3/4 9-1 1 SpR3 0.6 5/12 11-17 SpR1 0.4 3/10 18-30 SpR2 0.4 1/12 17 ' Relative Sp1 level determined by Western blotting. " Time required for tumors to reach ~1 cm. V, vector control, SpR, transfectants expressing Sp1 U1 snRNA/Ribozyme. 151 DISCUSSION In the present study, we successfully down-regulated Sp1 levels by >80% without significantly affecting the doubling time of the cells in culture. We found that the cell strains in which the level of Sp1 was near normal, no longer formed tumors in athymic mice and lost the ability to form colonies in agarose. In transient assays, lshibashi et al. (36) found that an Sp1 decoy suppressed the invasive activity of human lung adenocarcinoma cell line A549 and human glioblastoma cell line U251. However, the Sp1 decoy Oligonucleotides would be expected to inhibit all the members of the Sp/KLF family that are able to bind the ‘Sp1 site’ (GC-box or GT-box). Therefore, this study argues for a role for ‘Sp1 site'-dependent transcription rather than directly implicating the Sp1 itself. A study by Abdelrahim et al. (37) showed that transient transfection of small interfering RNA duplexes for Sp1 mRNA decreased Sp1 protein in nuclear extracts of MCF-7 cells to 30—50% of that of the parental cells. This was accompanied by a decrease in the percentage of cells in the S phase and an increase in the percentage in cells in Go/G1 (37). In our studies using the Sp1 U1snRNA/Ribozyme which is specific for the Sp1 gene, we provide direct evidence that up-regulation of Sp1 expression is involved in the malignant transformation of the fibroblasts cell lines we examined. These data are consistent with the fact that when the non-tumor-forming MSU-1.1 cells are malignantly transformed, Sp1 expression is at least increased in 65% of the cases. It is likely that the malignantly-transformed cells that did not show a higher level of Sp1 became malignant by an alternative route. Further evidence of the 152 importance of overexpression of Sp1 in malignant transformation comes our finding the Sp1 U1snRNA/Ribozyme transfectants of SHAC cells with reduced Sp1 levels formed tumors in athymic mice with a greatly increased latency and decreased frequency, indicating up—regulation of Sp1 also plays a causal role in the formation of fibrosarcoma in vivo. We found that when we reduced the level of Sp1 by >80%, the human fibrosarcoma cells reverted to a spindle cell morphology characteristic of the non- tumorigenic MSU-1.1 cells from which they were derived (2, 6). In one case, the Sp1-U1snRNA/Ribozyme-expressing cell strains exhibited a morphology intermediate between normal fibroblasts and fibrosarcoma. Malignant transformation of cells commonly results in a morphological change (38). The data in Figure 4 clearly indicate that the change in cell morphology in the cells with reduced Sp1 protein level is not the result of alterations of B—actin expression. It would be interesting to know if the down-regulation of Sp1 level affects the expression of other genes that are related to the regulation of cell morphology and if the down-regulation of Sp1 level causes rearrangement of cell skeleton (38). The promoters of many pro-apoptotic and anti-apoptotic genes contain ‘Sp1 site’ (39-47), which suggests that Sp1 and other members of Sp/KLF family are involved in the regulation of apoptosis. Here we found that down-regulation of Sp1 expression resulted in 20-40% of the cells undergoing apoptosis. However, in another type of mesenchymal cell (vascular smooth muscle), up-regulation of Sp1 activity is linked to Fas-mediated apoptosis (48). Whether the difference 153 reflects a difference in cell type, or in the nature of the malignant change is not ._ clear. The HGF/SF receptor, cMET, has been demonstrated to be overexpressed in malignant human musculoskeletal tumors as well as several other types of soft tissue sarcomas (49). Studies carried out in our laboratory (9) as well as other laboratories (50, 51) showed that the expression of MET and HGF is regulated by Sp1. Surprisingly, the expression of both the HGF/SF and MET showed no change in the transfectants with reduced Sp1 protein levels. These results suggest that other transcription factors are mainly responsible for the HGF/MET promoter activity, or that a minimal level of Sp1 protein is sufficient for transcription of both genes. The uPA protein is a key player in the regulation of cancer cell invasion and metastasis (52). Elevated levels of uPA protein and/or mRNA have been reported in colorectal cancer (53), gastric cancer (54), breast cancer (55-57), prostate cancer (58), head and neck adenoid cystic carcinoma (59), and non small-cell lung carcinoma (60). Inhibition of uPA activity by uPA inhibitors or down-regulation of uPA expression has been shown to suppress tumor growth in vivo and cell invasiveness in vitro (61-63). An earlier study in our laboratory (35) showed that 11 out of 11 fibrosarcoma cell lines derived from the MSU1 lineage, as well as cell lines from patients’ tumors, exhibited significantly higher levels of active (receptor bound) uPA than the cell strains from which they were derived or the other nonmalignant cell strains. In the present studies, we found that uPA expression was significantly decreased with the down-regulation of Sp1 protein 154 level in both cell lines. Taken together, these data suggest that higher uPA expression is very important for the malignant transformation of human fibroblasts. We also found that uPAR, EGFR, and VEGF, which contribute to tumor growth and angiogenesis (64), display dramatic decreases in the transfectants of y2- 3AISB1 cell line with reduced Sp1, but show no change or only a slight decrease in the transfectants of the PH2MT cells with reduced levels of Sp1. These results suggest that the uPAR, EGFR and VEGF play different roles in the inhibition of the tumorigenicity of human fibrosarcoma cell lines caused by down-regulation of Sp1 expression. The y2-3A/SB1 cells express wild type H-Ras and the PH2MT cells express wild type p53. Because both types of transformed cell lines are derived from MSU-1.1 cells, they both express the v—Myc oncogene, telomerase, and perhaps other as yet unidentified genetic changes. The Sp1 belongs to human SpIKLF family consisting at least 21 members (13). Among these members, the Sp3 shares the same expression patterns and the same binding affinity to the same DNA responsive elements as Sp1, but has different transcriptional activity (27). We observed that Sp3 protein levels were high in the human fibrosarcoma cell lines with elevated levels of Sp1 protein; and low in the cells with low levels of Sp1 protein (data not shown). The Sp3 protein levels decreased when the expression level of Sp1 was down-regulated by the Sp1 ribozyme antisense. The inhibition of Sp3 expression cannot be caused directly by the Sp1 U1snRNA/Ribozyme because there is no similarity between the sequences of the Sp1 U1snRNA/Ribozyme sequence and the Sp3 cDNA. 155 We hypothesize that the Sp1 acting as a transcription factor regulates the .. transcription of the Sp3 gene, and that the down-regulation of the Sp1 protein level reduces the level of Sp3 gene transcription. Additional studies on this problem are underway. The finding that a transcription factor Sp1 acts as an oncoprotein when it is over expressed is not surprising. The c-Myc oncogene encodes a transcription factor, which activates a diverse group of genes involved in the regulation of cell proliferation, differentiation and apoptosis and acts as an oncoprotein when up- regulated (65). Other transcription factors known to act as oncoproteins when up- regulated include c-JUN, and STATS (66). What is surprising is that the Sp1 protein, when functioning as an oncoprotein, can exhibit specificity in up- regulation of other genes (e.g. oncogenes) although it controls more than a thousand genes (27). Sp1 is ubiquitously expressed and regulates the expression of genes that have a single Sp1 site in their promoters as well as genes that have multiple Sp1 sites in their promoters. We propose that Sp1 functions more like a ‘switch’ to turn on and off the transcription of genes with a single ’Sp1 site’ in their promoter, whereas in genes with multiple ‘Sp1 sites’ in their promoters, function in a synergistic manner to regulate expression. This would explain why the known oncogenes (e.g. VEGF, EGFR, uPAR and uPA) that we found to be modulated by Sp1 U1snRNA/Ribozyme expression all have multiple Sp1 sites in their promoters. Overexpression of Sp1 or up-regulation of Sp1 binding activity has been reported in multiple cancer types or cancer cell lines, including human pancreatic 156 cancer cell lines and pancreatic cancer tissue specimens (18), breast cancer cell lines and breast cancer tissue specimens (19), gastric cancer (20), thyroid cancer (21). The studies presented here provide direct evidence that up- regulation of Sp1 expression plays a causal role in the malignant transformation of human fibroblasts. 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Oncogene, 22: 5967-5975, a 2003. lbanez-Tallon, l., Ferrai, C., Longobardi, E., Facetti, l., Blasi, F., and Crippa, M. P. Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells. Blood, 100: 3325—3332, 2002. Aguirre-Ghiso, J. A., Estrada, Y., Liu, D., and Ossowski, L. ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK). Cancer Res, 63: 1684-1695, 2003. Pelengaris, S., Khan, M., and Evan, G. c-MYC: more than just a matter of life and death. Nat Rev Cancer, 2: 764-776, 2002. Darnell, J. E., Jr. Transcription factors as targets for cancer therapy. Nat Rev Cancer, 2: 740-749, 2002. 165 CHAPTER 3 Identification of the Promoter of Human Transcription Factor Sp3 and Characterization of the Role of Sp1 and Sp3 in the Expression of Sp3 Protein * Zhenjun Lou, Veronica M. Maher, and J. Justin McCormick 1] Carcinogenesis Laboratory Department of Microbiology and Molecular Genetics and Department of Biochemistry and Molecular Biology Michigan State University Food Safety and Toxicology Building East Lansing, Michigan 48824-1302 Running Title: Sp1 and Sp3 Regulate Sp3 Promoter Activity *This work was supported by US. DHHS Grants CA82885 and CA098305 from the National Cancer Institute (J.J.M.) 1|To whom correspondence should be addressed: Carcinogenesis Laboratory, Food Safety and Toxicology Bldg., Michigan State University, East Lansing, MI 48824-1302; Tel.: 517-353-7785; Fax: 51 7-353-9004; E-mail: mccormi1@msu.edu 166 ABSTRACT We recently observed that in two human fibrosarcoma cell lines that express Sp1 and Sp3 at greatly increased levels, specific down-regulation of transcription factor Sp1 also reduces the level of transcription factor Sp3, and that when the level of these two transcription factors is reduced to near normal, the cells no longer form tumors. The present study was undertaken to determine how expression of Sp3 is regulated and the basis for the coordinated expression of Sp3 and Sp1. We isolated 2.1 kb of the 5’-flanking region of the Sp3 gene, determined its minimal promoter, and using mutation studies, demonstrated that each of the two putative Sp1/Sp3 binding sites in that minimal promoter is required for promoter activity. Gel shift assays showed that both Sp3 and Sp1 bind to these two sites. Transcription activation studies were carried out by transiently transfecting into insect cells that do not express Sp1 or Sp3 or into human cells a reporter gene linked to the Sp3 minimal promoter with along with Sp1 or Sp3 expression vectors. In insect cells, Sp1 expression stimulated Sp3 promoter activity 4 -fold, and Sp3 expression stimulated Sp3 promoter activity 1.2-fold. In human cells, Sp1 expression stimulated Sp3 promoter activity, but Sp3 expression inhibited Sp3 promoter activity. These results suggest that secondary modification(s) of the Sp3 protein is required for it to function as a repressor. By alignment of the 5’-flanking regions and/or promoters of the 21 human Sp/Kruppel-like factor genes we found that 18 out of them have Sp1/Sp3 binding sites, suggesting that Sp1 and/or Sp3 also regulate expression of these transcription factors. 167 INTRODUCTION Sp1 is the prototypical member of the 21-member Sp/KLF1 family of zinc finger proteins that function as transcription factors in mammalian cells (1). Identified in 1983 (2), Sp1 protein was shown to recognize and specifically bind to a GC-rich region within the Simian virus 40 promoter. In 1992, the Sp3 transcription factor gene was cloned (3). The Sp1 and Sp3 genes share 95% similarity in their zinc-finger DNA binding domain. The fact that they bind with identical affinity to the GC-box (GGGGCGGGG) and GT-box (GGTGTGGGG) and are ubiquitously expressed suggests that they act together to regulate gene expression (4). However, the physiological roles of Sp1 and Sp3 appear to differ because Sp1"' mouse embryos are severely retarded in growth and die after 9.5 days of development (5), whereas Sp3"' mouse embryos survive to full term, although they die immediately after birth as the result of respiratory failure. The latter animals also show defects in tooth and bone development (6,7). Sp1 is a transcription activator (4). Sp3 has been shown to positively regulate transcription (8-11), but it can also act as a repressor (12). Recent reports of studies using mammalian cells indicate that Sp3 protein is modified by phosphorylation (13,14), acetylation (15,16), sumoylation (17,18) and that these modifications influence the role that Sp3 plays in transcription. Evidence also suggests that the nucleotides adjacent to the GC-box and the GT-box influence the role of Sp3 in transcription (4). In studies of the expression and function of Sp1 in human fibroblasts, including human fibroblastic tumor cell lines, Liang et al. (19) in this laboratory 168 recently found that 60-70% of the malignant cell lines tested express Sp1 protein a at a level 8-18-fold higher than that found in normal human skin fibroblasts. Following up on that study, we and our colleagues2 transfected an Sp1 ribozyme into human fibrosarcoma cell lines that overexpress Sp1 and found that when Sp1 expression was reduced to a near-normal level, the cells’ ability to form tumors in athymic mice was either completely eliminated, or very significantly reduced. In the latter case, the latency period for the tumors to form was lengthened. That study also demonstrated that when the level of Sp1 protein was reduced by means of the Sp1 ribozyme, the level of Sp3 protein was also decreased. These results strongly suggest that differences in the level of expression of Sp1 and Sp3 can play a very important role in cancer, but little or nothing is known about the mechanisms that regulate the transcription of Sp3. The present study was undertaken to identify the Sp3 promoter, characterize it, and determine how it is regulated. A partial human Sp3 cDNA sequence, lacking the 5’ end, was published in 1992 (3). In 2002, Oleksiak and Crawford (20), using a combination of genome-walking and database mining, identified intron and exon sequences of the 5’ end of the human Sp3 gene and located the ATG start codon. Building on this information, we have isolated the 5’-flanking region of the Sp3 gene from human genomic DNA, defined its minimal promoter, determined that it contains two putative Sp1/Sp3 binding sites, and shown that both Sp1 and Sp3 are involved in the regulation of Sp3 promoter activity. Sp1 acts as an activator and Sp3 as a repressor. Using cells that lack endogenous Sp1 and Sp3, i.e., SL2 insect cells, we show that when Sp1 induced 169 Sp3 promoter activity in these cells. Overexpression of human Sp3 protein cannot repress such activity. This suggests that to exert its repressive activity, Sp3 requires secondary modifications that are not supplied by SL2 insect cells. 170 EXPERIMENTAL PROCEDURES Cells and Cell Culture—Human fibrosarcoma cell lines SHAC and HT1080 are derived from patients. PH2MT cells are derived from a fibrosarcoma formed in athymic mice by injection of MSU-1.1 cells that had been malignantly transformed by overexpression an H-RAS oncogene (21). The derivation of the MSU-1.1 cells has been described by Morgan et al. (22). All the human fibroblastic cell lines were routinely cultured in Eagle’s minimal essential medium supplemented with L-aspartic acid (0.2 mM), L-serine (0.2 mM) and pyruvate (1 mM), containing 10% supplemented calf serum (HyClone, UT), hydrocortisone (1 pg/ml), penicillin (100 U/ml), and streptomycin (100 pg/ml). Human embryonic kidney-derived cell line HEK 293 Phoenix Ambo cells was grown in Dulbecco Modified Eagle’s Medium (high glucose) with 10% supplemented calf serum, and the two antibiotics. Cells were incubated at 37 °C in a humidified incubator with 5% 002. SL2 insect cell were cultured in Schneiders Drosophila medium (Invitrogen), supplemented with 10% fetal calf serum, with penicillin (100 U/ml) and streptomycin (100 pg/ml), and incubated in closed flasks at 22-25 °C. Isolation of the Human Sp3 and Sp1 Promoters and Construction of Plasmids—Genomic DNA was isolated from human placenta tissue using standard procedures. The Sp3 gene promoter was amplified by PCR using primers designed according to the sequences of the contig ACO16737: (forward primer, 5’ TCGCCTCGAGTGCTCTCAAGGTGGCTGGAC 3’ with a Xhol site) and the human Sp3 gene (AF494280, reverse primer, 5’ AGTGAAGCTTACAC ATGGTGAGGAGCGAAG 3’, with a Hindlll site). The ~2.1 kb DNA fragment of 171 the PCR products was inserted between the Xhol and Hindlll sites of the pGL3- Basic vector (Promega) (luciferase assay plasmid). To verify that no mutations occurred during PCR, the inserted fragments from three independent clones were assayed by DNA nucleotide sequencing. Constructs for Identifying the Minimal Sp3 Promoter—Progressive deletion constructs of the Sp3 promoter were prepared and the DNA fragments from the Sp3 promoter were cloned between the Xhol and Hindlll sites of pGL3-Basic vector (Promega). The DNA fragments which have a common 3’ end and a different 5’ end were obtained by PCR, using five different forward primers and a common reverse primer. Each fonrvard primer contains an Xhol site, and the reverse primer a Hindlll site. The numbers indicate the length of each DNA fragment: Fonrvard 1 (Sp3-P1) 5’ tcgc CTCGAG TGCTCTCAAGGTGGCTGGAC 3’ (2097 bp), Forward 2 (Sp3-P2) 5’ tcgc CTCGAG TCACAGTAATGGGAAAACTG 3’ (1421 bp), Forward 3 (Sp3-P3) 5’ tcgc CTCGAG TTCAGGGTACGTGAAAGTG 3’ (837 bp), Fonrvard 4 (Sp3-P4) 5’ tcgc CTCGAG TCTCGAAAACCTACGCTG 3’ (341 bp), Forward 5 (Sp3-P5) 5' tcgc CTCGAGAAAAAAT CCCCGGACCGCTC 3’ (90 bp), Reverse primer: 5’ agtg AAGCTTACACATGGTGAGGAGCGAAG 3’. Mutagenesis of the Sp 1/Sp3 Binding Sites—Introducing mutations into the Sp1/Sp3 sites in the Sp3 promoter (Sp3-P4) was performed using the QuickChange Il XL site-directed Mutagenesis kit (Stratagene), following the manufacturer’s procedure. Two pairs of primers: Sp1-1: 5’ GCCAAGAAGAGG ‘L/. was. - p . MGAG‘ITCCGGGCGGGC 3’ and 5’ GCCCGCCCGGAACTCILGAACCT .. CTTCTTGGC 3’; and Sp1-2: 5’ GGGGCGGGAGTTCCGA_AQ_T‘_I'GCTGTCACCC TC'I'TCC 3’ and 5’ GGAAGAGGGTGACAGCAAQECGGAACTCCCGCCCC 3’ were used to create mutations (underlined) in Sp1/Sp3 sites. 5’ RLM-RACE—Total RNA from human fibrosarcoma cell lines PH2MT, SHAC, and HT1080 was isolated with RNA-Bee (T el-Test) reagent, followed by extraction with acidic phenol. The transcription start sites were determined using the FirstChoice RLM-RACE kit, following the manufacture’s procedure (Ambion). The Sp3 gene-specific primer (5’ TCGGGAGCGGTCATAGTGTGT‘ITA 3’), complementary to the DNA sequence encompassing the translation start site was used. To carry out nested-PCR, an internal primer (5’ ACACATGGTGA GGAGCGAAG 3’), located just upstream of the gene-specific primer, was used. The DNA fragments were excised and purified using a gel extraction kit (Qiagen), and their structures were verified by DNA sequencing. Transient Transfection and Luciferase Assay of Promoter Activity— Transient transfection was performed using FuGene6 reagent, following the manufacturer’s procedure (Roche). Human fibrosarcoma cell lines SHAC and PH2MT were grown to ~60% confluence in six-well plates (three sets of plates for each cell line). Promoter reporter constructs (1 pg) and pRL-CMV (Promega) (0.01 pg), serving as transfection efficiency control, were added to the cells with FuGene6 transfection reagent (ratio: DNAzFuGene6,1:3). In experiments involving forced expression of Sp1 or Sp3 protein, 0.8 pg of promoter reporter construct (i.e. Sp3-P4, above) and 0.2 pg of pRL-TK (Promega) was co-transfected into 173 human HEK 293 cells, along with plasmid CMV-Sp1 (kindly provided by Dr. Robert Tjian, UC Berkeley, USA), or CMV-Sp3 (kindly provided by Dr. Guntram Suske, Philipps-Universitat Marburg, Germany). Cells were harvested 24 h post- transfection, and reporter gene activity was determined using a Dual-Luciferase® Reporter Assay System (Promega) and read with a luminometer. SL2 insect cells were plated at 2 X 105 cells per well in 24-well plates, and transfection was carried out 24 h later, using 0.4 pg of Sp3-P4 and 0.1 pg of pRL-TK (Promega) reporter constructs and various amounts of pPacSp1 and/or pPacUSp3 vectors (kindly provided by Dr. G. Suske). The cells were harvested 24 h post-transfection, and Luciferase reporter gene activity was determined. The protein concentration was determined with Coomassie reagent (Pierce). Preparation of Nuclear Extracts and EMSA— Nuclear extracts were prepared from human fibrosarcoma cell lines PH2MT, SHAC, and HT1080, using the Cellytic NuCLEAR Extraction kit (Sigma) following the manufacturer’s procedure. The concentrations of the nuclear extracts were determined using the Bicinchoninic Acid method (Pierce), and the extracts were stored at -80 °C until used. EMSA was performed as described (19). The two probes, Sp1-1 and Sp1- 2, were end-labeled with y-32P-ATP, using T4 polynucleotide kinase, and 1000- 2000 cpm of probe was mixed with 5 pg of nuclear extract. The mixture was incubated at room temperature for 20 min. For competition assays, unlabeled probe or Sp1 consensus Oligonucleotides (Promega) were added 20 min prior to addition of the 32P-Iabeled probe, at a concentration 50 X that of the labeled 174 probe. The complexes were separated in 5% native polyacrylamide gel at 4 °C, a and the gel was dried and exposed to film at —80 °C. Preparation of an Sp3 Ribozyme Antisense Construct—The Sp3 U1 sn- RNA/Ribo zyme construct was prepared as described (23). The complementary Oligonucleotides that encode the antisense sequence of human Sp33, including the hammerhead ribozyme, were synthesized, and the double-stranded DNA was inserted between the EcoRI and Spel sites of the pU1 vector containing the human U1snRNA (gift of Dr. John Laterra, Johns Hopkins University School of Medicine). The U1snRNA/Sp3 antisense/hammerhead ribozyme fragment was amplified and inserted into pcDNA3.1/Hygro+ (Invitrogen). The construct was sequenced using an ABI PRISM® 3100 Genetic Analyzer. The structure of the chimeric RNA (U1snRNA/Sp1 antisense/hammerhead ribozyme) was analyzed using MulFoId and Loop-D-Loop programs. Stable Transfection and Western Blot Analysis—Transfection of the PH2MT cells was performed using Lipofectamine (lnvitrogen) following the manufacturer’s procedure. The transfectants were selected by adding 100 pg/ml Hygromycin to the medium. The Sp1 and Sp3 protein levels were analyzed by Western blot analysis. Whole cell lysates were prepared using RIPA buffer as described (19). Protein content was quantified by the Bicinchoninic Acid assay (Pierce), and 50 pg total protein was loaded and separated by 7.5% Sodium dodecyl sulfate-PAGE. Protein was transferred to PVDF membrane (Millipore). Western blot analysis was performed by standard techniques with SuperSignal 175 reagent (Pierce). Antibodies against Sp1 and Sp3 were purchased from Santa Cruz, and B—actin from Sigma. Database Mining—The promoters or 5’-flanking regions of the members of Sp/KLF family were obtained by blast-searching the human genomic database using the 5’ cDNA sequences of the members as “bait”. Approximately 1,000 bp sequences upstream of the translation start site of the members were aligned, and the perfect Sp1/Sp3 binding site (GGCGGG) was located. 176 RESULTS Cloning of the 5 ’-Flanking Region of the Human Transcription Factor Sp3 Gene—To identify the 5’-flanking region of human Sp3 gene, we searched the human genome database (http://www.ncbi.nlm.nih.gov[qenome/guiclTe/human/) using the translation start site and the adjacent sequence as a virtual probe. We found that the contig ACO16737 contained the sequence of the 5’ human Sp3 gene. Primers designed according to the sequence of contig ACO16737 (forward primer) and the human Sp3 gene (AF494280, reverse primer) were used to amplify a DNA fragment containing 2.1 kb, which corresponds to the 5’-flanking region of the human Sp3 gene (based on the translation start site) (Fig. 1). The DNA fragment was cloned into pGL3-Basic vector, sequenced, and submitted to GenBank (accession number, AY628424). Putative Transcription Factors Binding Elements in the 2.1 kb Fragment 5’ to the Human Sp3 Gene—The 2.1 kb sequence was subject to computational analysis for transcription binding sites using the Matlnspector software (24). Putative DNA binding elements found in this sequence include the binding sites for Sp1/Sp3, AP1, AP2, CREB, E2F. HIF, NF1, and NF-Y (Fig. 1). 177 ill. 56 IE” Fig. 1. Nucleotide sequence of the human Sp3 promoter. Analysis of the sequence of the 5’-flanking region (according to ATG) is shown. The potential regulatory elements are underlined. 178 -2095 -2035 -l975 —1915 -1855 -l795 -1735 —1675 -l615 -1555 -1495 -1435 -1375 -l315 —1255 -1195 -1135 -1075 -1015 -955 -895 -835 -775 -715 -655 -595 -535 -475 TGCTCTCAAG ATCCCCACTC GGGAAAGAGC STATS CCCTTCCTCC TTTTTCCATT TTTGTTGTAA AATATGAGAC AGTCTCGTTT CAATTAAGAG CTTTGGAACT CACAAAATCT ACACAAAATA ACAGGTATTG TTGAAGGTAA TTTTCCCCCA GTGGCTGGAC CTGAAAAAAA TACCTTCCAC CTCCCAGTCC TTCCCCACCC AATAGTACAT TACAAGGACT CCGCATGTTT CAAAGTAACC GGGATGTAAA GGTTAAAGTG TAATAATCAC TGGACAGTGT CAGGGAGAGA GGATGACAGC TAACACTTTG CAAAAAGGGT AAACAAAACA AAACTGAAAA CCAAGAGATC CGGGTAGGAA ACTTATTTTCACCTG TCCCG MOYD CAAATCAGGT TATTTTGTGA AGGTTATACG ACTCAACTTT ATTAGAAGAG ATGAACCCTA ATGGCGATAG CGTAGAACAT TGCCTGAAAT ATCCTTTCTA CAGAGTGATAAT AGTGACTT GATA3 AP1 CTCTTCAAGG CACCCATCTC GKLF CTTTTATTTCCTT AAAAGTT STAT GAAAGTGACT TTTCCACTTA TTCTTGGACT CAGAATTTTG GTGGTTTTTAGGTTA ATTCCCTG GAAATTTGATCTTT GTA CREE NF-KE/STATG ATACAATGTT CTCAGTATCT TCTGATCCTG GATA3 TCAGCATAGG CGGTTAAGTG CACAAATG GATTACCTATTAAGTTA GGACA CTTGGACTTC AGTAATGGGA TTCCACCTCT TGTCTCTTAG ACAACCTCAT AAACTGAATC TGAATTTAAC TTGGCAAAAC GGATGCCAAA AATCCAGTAA GAGAAACAAGATTT TAAAAT AAGATACCAA GATAZ ACAACAAAAA GATA3 GGTAGTTCAC RAR GAGATATCTG CGGTTCACGA ACTTGGTACC AAATGGTTATGTGA CCGATT CREE TACCACAAGT AAAATGAAAT AAAACTCTCC TTCCGTATGT TTCAGGGTAC GTGAAAGTGG CTTTAAAAAA CGTTGGCAGT AAGAGGATAAA GAGGCCGGG GATA TTTTAAAACA CAGAATTCTG CREE CREE CCACCAGCAC ATCACATATC TTGAGCCAGATTTCTA AAAA C/EEP TTCTTTCTTGGCAA CTATAC NF1 ACACACTTAA AACCTGAGCA GAAGGAAGGA GTGTTAACCT CREE TGTTCCAATGG GGGAAAAAA NF-Y GGTTGATGGGCT TATTTCAG NF-Y AATAATACGT TTGTGCAGCA CCCCACTGAA TACATACACA ATACTTTTAA GTGCCCGTTTTGGT GCCACT GGCCAAAATT 82F GAGGTGACTT GAGATTTCTT GAGACCGGTT GGAAAACTGG TTTTGCTTTA AATGCCTGCC GGCTAAGGGG GCTGGGAGCG AGATGGCTGTGACTG AATCAGG AAGCTGGA TGTGAGAGAGAAAAA TCCTC CTCCAGCCTC AP1 AP1 GGAGGGGGTG TCTCCCCTAA TTGCTCAGTA GCGGCAGCGG GGCAGCGCAG GGAAGCGGCG TGCTGCCGGG ATGGAGGAAG ETSl CCAGCTCAGC TCCCCTACAG E2? TACCAGCTCCCTCCCCCC cc Sp1/ZBP-89 CCGCAGCAGC AGCCCCGGGG MYOD AP2 GCTCCAAGAG CGTCCAGTGG AP1 CTTGGCTAGG CCTGCTCCGA CGCGAGGCCCTGGGGAG ATC AGCAGGAGTCACC GGGGACG GCTCGTGGGGCGGGTCTGGT Sp1/SMAD3 CTGAGCAGCG GTGGCCTCCT CGGCCAAGAC NF1/SMAD4 AATTTCCTCC AP2 GCGCCTGCAA GGTGGAGGAG VDR/RXR -415 -355 -295 -235 -l75 ~155 -95 -35 GCCTGCGCCT GGCGAGCCGT TCCACCCGGG CGGGCCTGGC AGCCGCTCTC GGCAAACAGG AGCGGCAGCC GAAAACCTAC AAGCGCGCCG GGCGAGGGAGGCGGG CACAG Sp1 GCTGCCACGG CCGCTCATTG CCTGGCAGAC CGACGGACAG CGGGGTAACC TCTCTCCCCT VDR/RXR GCGCCTGGAC AP2 CAATGAGCAC NF-Y TTCCGGGCGGG CTGTCACCC Sp1 + 82 AGCCGACAAA + 32 +31/P1/Hl HIP GAGCACGGCG TCTTCCCCCC TTTTGGGCTG ZEP-89 +83/P3/H3 GCGAATGAGAGCCAAG AAGA GGGGCGGGAG NPl +P2 Sp1 GAGGCTCCAC CTTTTGTGTT 32? TCCCGCACAGTCAATCA AAA TAGGAAAAAA AAATCCCCGG ACCGCTCCGG CCGTGTCCGC NF-Y CGCCGCTTCC CGCATCCTCT CCCGCCGCCG CCGCCTTCGC TCCTCACCAT GTGTAAGGCG GCGGGGAGCC CCGCCTGAGG TGCCCTAAAC ACACTATG ‘l79 Multiple Transcription Initiation Sites—To identify the transcription start site of the human Sp3 gene, we employed the 5’-RLM-RACE assay (see experimental procedures). Total RNA prepared from human fibrosarcoma cell lines SHAC, HT1080, and PH2MT was used. A primer specific to the human Sp3 gene and a primer specific to the 5’ adaptor sequence were used to amplify the 5’ end of the Sp3 transcript. Nested PCR was carried out using an internal primer specific to the Sp3 gene and the primer to the 5’ adaptor. In addition to the two common bands in all the three samples (Fig. 2A, top bands, designated S1/P1/H1, and bottom bands, designated SS/P3/H3), a third band was detected (designated S2, P2, and H2, middle bands). The location of transcription initiation sites are shown in Figure 2B. 180 Fig. 2. Identification of the transcription start sites by 5’-RLM-RACE assay. A, RNA was isolated from human fibrosarcoma cell lines SHAC, PH2MT, and HT1080 using RNA-Bee, and subjected 5’-RLM-RACE assay. Nested-PCR was carried out using an internal 3’ primer and a 5’ adaptor primer. PCR products were separated by 2% agarose gel. B, Locations of the transcription start sites (underlined). S, SHAC cells; P, PH2MT cells; H, HT1080 cells. 181 08¢ 80¢U¢ 0¢¢<800008 00¢0800000 000(000000 0000¢<8080 8¢00<08008 0008800000 DJJDJJDJJJ ._. J ._. JJ ~¢JDJ JJ ._. ._. JDJJDJ 88.8.58 008.88% 080895 mofi. «(0.59509 998889 mm 3:? Q? mm+ 880808888wmwfl008000¢0 0800008888 0000008808 00 8m80 00000000088 Hm Hm Hm+ 0¢00000000 <0¢< 0¢<000<0<08<¢000 ml 1 H mm- 3% «a mm: 52%; mNHr mer mer mam- mew- m H d S 1 H. H H .6 V w w w o m o .. m m < 182 Functional Mapping of the Human Sp3 Promoter—To further characterize .. the promoter, we constructed a series of five reporter plasmids containing various lengths of the human Sp3 5’-flanking region (from nt —2095 to —39, Fig. 3A) upstream of the firefly luciferase (Luc) reporter gene. These constructs were transiently transfected into human fibrosarcoma-derived SHAC cells, along with, a second plasmid, i.e., the Renilla luciferase vector, pRL-CMV, which was used to control for transfection efficiency. (The firefly luciferase activity was normalized to the Renilla luciferase activity). The Sp3-P4 promoter fragment, spanning nt — 339 to —39 conferred the same the activity as that of the longest promoter, Sp3- P1. Deletion down to nt —128 reduced the activity to 35% of the activity of the longest promoter (Fig. 3B). These results indicate that the elements in the region from nt —339 to —128 play an important role in the regulation of Sp3 gene transcription. 183 Fig. 3. Deletion analysis of human Sp3 promoter activity in SHAC cells. A, The full length (Sp3-P1, 2.1 kb) of the 5’-flanking region of the human Sp3 gene was used as a template to cany out a series of deletions from the 5’ end to obtain the other four fragments Sp3-P2 (1.4 kb), Sp3-P3 (0.8 kb), Sp3-P4 (0.4 kb), and Sp3-P5 (0.1 kb). These fragments were inserted in pGL3-Basic vector (Promega) between Xho I and Hind lll sites. B, Relative luciferase activity of human Sp3 promoter constructs in fibrosarcoma cell line SHAC. The DNA construct (1 pg) was transiently transfected into SHAC cells along with pGL3- CMV (0.01 pg) serving as transfection efficiency control. The firefly luciferase activity was determined using Dual Luciferase System, and normalized to the Renilla luciferase activity. Promoter activity is shown as percentage of the activity of the longest construct (Sp3-P1). The data shown are the average of three independent experiments. 184 QNHmn «.3 H 2:. Qhflwm 9H00w {a 03.. . a m . an? man 0 03 3.8% E3» as... 034 I Q DOO- OFVFI NI M m 03 5-3m an. mmou. 185 The Binding of Sp1 and Sp3 to the Proximal Region of Sp3 Promoter— The Sp3-P4 promoter fragment contains several DNA responsive elements, including two Sp1/Sp3 putative binding sites, one at position nt —181 and the other at nt —168 (Fig. 1). To examine the role of Sp1/Sp3 binding sites in the regulation of human Sp3 gene promoter activity, we mutated one or other or both of the two sites (Fig. 4A). Mutation of either of the sites reduced the activity of the promoter in SHAC cells to ~45% of the wild type promoter. Mutation of both sites decreased the promoter activity to 16% (Fig. 4B). These results indicate that both DNA responsive elements are critical for regulation of Sp3 gene promoter activity. To verify that the transcription factors bind to the two putative Sp1/Sp3 binding sites, we performed EMSA, using nuclear extracts from the three human fibrosarcoma cell lines. Two probes, Sp3-1 and Sp3-2, which contain an Sp1/Sp3 binding site at position —181 or -168 and the surrounding nucleotides, respectively, were used. Both probes produced the typical Sp1 and Sp3 binding patterns (Fig. 4C). The Sp1 consensus Oligonucleotides carrying Sp1/Sp3 DNA binding elements completely inhibited the formation of the complexes (Fig. 4D). These results demonstrate that both Sp1 and Sp3 protein bind to the putative Sp1/Sp3 binding sites identified and strongly suggest that they are both involved in the regulation of Sp3 promoter activity. 186 Fig. 4. The binding of Sp1 and Sp3 to the Sp3 proximal promoter. A, The Sp3 proximal promoter (—339 to —39 bp) luciferase constructs. Mutations in the putative Sp1/Sp3 binding sites are shown with an X. B, Each construct shown in A was transiently transfected into SHAC cells along with the pRL-CMV vector and assayed for luciferase activity. The Firefly luciferase activity, normalized to the Renilla luciferase activity, is shown as the percentage of the activity of the wild-type construct (Sp3-P4). The results are the mean of three independent assays. C, Results of EMSA involving two probes, Sp3-1 and Sp3-2, each containing an Sp1/Sp3 putative binding site and surrounding nucleotides. Nuclear extracts from SHAC, PH2MT and HT1080 cells were incubated with 32P- Iabeled probes. Each probe gave the typical Sp1/Sp3 binding pattern. D, Results of competition assays with the designated 32P-Iabeled probes in nuclear extracts from SHAC cells. Competition assays involved 50-fold excess of unlabeled probes or the Sp1 consensus Oligonucleotides. 187 38% I++| n. + + + 1.0 «0.: + + 0N9 ad H 56' Wk «fun 88' l+ll 32.3.50 Em 8.8.38 32.. aozxéew 32.. 8.8:-8m 32.. 853% 32a o<=mlwz .\. + + 4- 080 LLH LWZHd _ 03 _r H H 852. a 373'. E 0.7;? 080 MM 4» t «.an one... + 3% one... .. mz lWZHd + DVHS + 0 ”1.48.383 «43.3.88 7393.58 E83 < 188 Role of Sp1 and Sp3 in the Regulation of Sp3 Promoter Activity—Sp1 and ,, Sp3 are ubiquitously expressed and bind to the same DNA responsive elements with the same binding affinity (4). However, the Sp1 protein is reported to act as a transcriptional activator, and the Sp3 protein as an activator or a repressor (4). To determine the role of human Sp1 and Sp3 in the regulation of Sp3 gene promoter activity, we utilized SL2 insect cells because they lack endogenous Sp1 and Sp3 proteins. An Sp3-P4 (minimal promoter) firefly luciferase construct, alone or with the Sp1 or the Sp3 expression vector, was transiently transfected into SL2 insect cells, and the Sp1/Sp3-regulated HSV TK promoter-Renilla luciferase construct was also included to serve as a positive control to detect the expression of functional Sp1 or Sp3 protein. The promoter activity was determined using the Dual Luciferase assay system. In all of these experiments, HSV TK promoter activity was detected, indicating that functional Sp1 and Sp3 protein were being expressed (Fig. 5). As shown in Figure 5A and SB, expression of Sp1 stimulates Sp3 promoter activity ~4-fold, but expression of Sp3 has little effect on the Sp3 promoter activity (~1.2-fold). These results indicate that Sp1 acts as an activator in the regulation of Sp3 promoter activity and that Sp3 acts as a very weak activator in SL2 insect cells, which lack endogenous Sp1 and Sp3. As shown in Figure SC, when both Sp1 and Sp3 expression vectors were co-transfected into. the SL2 insect cells, along with the minimal Sp3 promoter reporter construct (Sp3-P4), expression of Sp3 did not cause the expected inhibition of Sp3 promoter activity, strongly suggesting that such inhibition 189 requires some form of secondary modification that is lacking in insect cells. To test this hypothesis, we co-transfected the Sp3 promoter reporter construct (Sp3- P4) and an Sp1 or Sp3 expression vector into human HEK 293 cells, which do express endogenous Sp1 and Sp3 proteins. When the Sp3 gene promoter reporter constnrct was co-transfected into these cells, along with an Sp1 expression vector, the Sp3 promoter activity increased (Fig. 5D). In contrast, when the Sp3 gene promoter reporter construct was co-transfected into these cells, along with an Sp3 expression vector, the Sp3 gene promoter activity was inhibited (Fig. SD). These results strongly suggest that the inhibition of Sp3 promoter activity by Sp3 requires secondary modification of Sp3, as recently reported (13-18). 190 Fig. 5. Role of Sp1 and Sp3 in the regulation of human Sp3 promoter activity. Promoter activity was determined using the Dual Luciferase System, normalized to the protein concentration of each sample, and expressed as fold of the values obtained from transfection of the reporter construct only. The HSV TK promoter-Renilla luciferase construct was also included in each transfection to confirm the expression of functional Sp1 protein and/or Sp3 protein. A, SL2 insect cells were co-transfected with Sp3 promoter-firefly luciferase construct (Sp3-P4) (1 ug) with or without Sp1 expression vector pPacSp1. B, The same experiment as in A, except that the Sp3 expression vector pPacUSp3 was used. C, SL2 insect cells were transfected with Sp3 promoter-firefly luciferase construct (Sp3-P4) (1 pg) and the indicated amounts of pPacSp1 and pPacUSp3. D, Human 293 Phoenix Ambo cells were transfected with the mammalian expression vector (CMV-Sp1) or the Sp3 expression vector (CMV—Sp3) in the indicated amounts. 191 LouoEoE x... . r e. .. y. r. . 88:35 I @8383 U 95 as D 84229 Em->s_o 33895 x... 1.3m 9.28. I 95D ”amnesia LouoEoLn. x... ocoowbcom 9.29.8 95 sec 888%: Femenea V'IH 0.. ..ouoEoE x... vainnm I. 958 I 95D Ewomea IHI 2. V'IH mp e~< 192 Coordinated Expression of Sp1 and Sp3 in Human Fibrosarcoma Cell .. Lines—We compared the expression levels of the Sp1 and Sp3 proteins in five human patient-derived fibrosarcoma cell lines by Western blot analysis. The three cell lines with higher Sp1 protein levels also showed higher levels of the Sp3 protein; whereas the two cell lines with lower levels of Sp1 protein showed lower levels of the Sp3 protein (Fig. 6A). Construction of the Sp3 U1snRNA/Ribozyme Vector—To examine the effect of a decrease in expression of Sp3 protein on that of Sp1 and Sp3 protein, we designed and constructed an Sp3-specific ribozyme to down-regulate Sp3 expression. The Sp3 U1snRNA/Ribozyme consists of three parts, an Sp3- specific antisense sequence with the hammerhead ribozyme in its center, and the two flanking regions of the U1 snRNA (Fig. 68). The Sp3-specific antisense is complementary to the nt 1681-1721 sequence of human Sp3 mRNAZ. A BLAST search showed that there is no significant similarity between this sequence and that of other genes. Use of the Mulfold and Loop-D-Loop programs (25-27) to analyze the Sp3 U1snRNA/Ribozyme stmcture revealed that the U1snRNA structure is well conserved (Fig. 6B, insert). To construct an expression vector, we inserted the Sp3 U1snRNA/Ribozyme, driven by the human U1snRNA endogenous promoter, into the pcDNA6/v5-hisB vector containing the gene for blasticidin drug resistance. Evidence that Sp1 and Sp3 Proteins Play a Role in the Coordinate Expression of Sp3 and Sp1—We previously observed that the level of Sp3 protein decreased when the level of Sp1 protein was down-regulated by means 193 of an Sp1-specific ribozyme construct in human fibrosarcoma-derived PH2MT cellsz. To determine whether down-regulation of Sp3 protein reduces the expression of both Sp3 and Sp1 protein, PH2MT cells were stably transfected with the Sp3 ribozyme construct (Fig. 6B). Stable transfectants were selected for drug resistance. Western blot analysis using antibodies specific for Sp3 or Sp1 showed that the Sp3 ribozyme significantly decreased the levels of both Sp3 and Sp1 protein (Fig. 6C). These data indicate that the expression of Sp1 and Sp3 is co-regulated in human fibrosarcoma cell lines. 194 Fig. 6. Expression of Sp1 and Sp3 in human fibrosarcoma cell lines and reduced expression by the use of an Sp3-specific ribozyme. Whole cell lysates (50 pg), prepared using RIPA buffer, were loaded and separated on 10% Sodium dodecyl sulfate-PAGE gel, and electroblotted onto PVDF membranes. Sp1 and Sp3 protein were detected with anti-Sp1 and anti-Sp3 antibodies and SuperSignal substrate. B—actin served as loading control. A, Expression of Sp1 and Sp3 protein in five patient-derived fibrosarcoma cell lines. B, Schematic representation of the Sp3 ribozyme antisense construct. The inset figure shows the predicted RNA structure of the constnrct as determined using MulFold and Loop-D-Loop programs. C, PH2MT human fibrosarcoma cells transfected with an Sp3 ribozyme antisense construct showing reduced expression of Sp1 and Sp3 protein. 195 v". A ESUUNIQ ll .' fie. an --.1! .. . Oh ”am I. I... to: I + I Louoo> + I I oE>Nont new 0 .. 583 Ice h.l II I IS mam .. I... tire: ' ‘iiiumowv Fn—m M m a a .. w .. 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