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I I' ' . . . 1111114113 :3 :2: ‘ . z: . ‘ 1 3‘1“. 1, 1 ”-4 -vfi—o ..v. M “..vo— o W. —- maul—W W ——> THth-S' Ill'i'liltimfil W111 litfi'flliflflitt {iiiliflifl 3 1293 00885 2745 It This is to certify that the dissertation entitled Charaeievwzei‘tdvb a? ?V°+3;M.¢ lyroymb an‘bf‘kah'éQ-LS 'Jvu HILMM Breut' Ewruttcpt ans NWPWWW’W Tramsfiwmé. by flu. ”ea, 01»wa :‘ru gaunt») Hole as (L Tumor Smreasov. presented by 9,91,“, zka.‘ has been accepted towards fulfillment of the requirements for APRD - ?\'\or\~\acctoq {Watwloqy degreem I MM w. «Mot WM gag/W Major professor é Luz/93 Date MSU is an Affirmatiw Action /Equal Opportunity Institution 0-12771 CHARACTERIZATION OF PROTEIN TYROSINE PHOSPHATASES IN HUMAN BREAST EPITHELIAL CELLS NEOPLASTICALLY TRANSFORMED BY THE NEU ONCOGENE: THE POTENTIAL ROLE AS A TUMOR SUPPRESSOR. By Yifan Zhai A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1993 ABSTRACT CHARACTERIZATION OF PROTEIN TYROSINE PHOSPHATASES IN HUMAN BREAST EPITHELIAL CELLS NEOPLASTICALLY TRANSFORMED BY THE NEU ONCOGENE: THE POTENTIAL ROLE AS A TUMOR SUPPRESSOR By Yifan Zhai Reversible phosphorylation/dephosphorylation of protein tyrosyl residues appears to be a key regulatory mechanism in the control of signal transduction, cell proliferation, differentiation and transformation; this metabolic process is modulated by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (P'I'Pases). While the role of P'I‘Ks has been examined extensively in human breast tumorigenesis, virtually nothing is known as to the role of Pl'Pases in this tumorigenic process. To address this issue, two different approaches have been used: one approach was aimed at studying whether or not the introduction of an activated neu oncogene (a potent PTK) into an immortalized human breast epithelial cell line, could result in alterations of any P'I'Pase expression. This approach led to the finding of a significant elevation in the expression of LAR and FTP 13 in three independent neu transformed human breast carcinoma cell lines in response to neu introduction. The level of neu expression , as well as the differential expression between P185neu and LAR and FTP 18, directly correlated with .tumorigenicity. Furthermore, elevated LAR-PTPase expression was Observed in neu induced rat mammary carcinomas compared to carcinogen (7,12- dimethylbenzanthracene) induced rat mammary carcinomas. The second approach was to directly test the anti-oncogenic potential of LAR P'I'Pase. To accomplish this, the full length of human LAR cDN A, was constructed into an inducible expression vector. The level of LAR expression was modulated through transcription from a metallothionein (MT) promoter. When this LAR containing plasmid was introduced into a neu transformed human breast carcinoma cell line, a substantial increase in LAR expression in these cells was observed. This resulted in a change in the morphological appearence of these cells in vitro, and more importantly, a significant suppression of tumorigenicity of these cells inoculated into athymic nude mice. Thus, the relationship between the activities of P185neu-P1X and LAR-PTPase, may be extremely important in human breast carcinogenesis. THIS WORK IS DEDICATED TO: My husband: Dajun Yang My daughter and son: Alina Yang and Anthony Yang My Parents: Gaosheng Zhai and Yanqun Xu My country: China iv ACKNOWLEDGEMENTS I would like to express my appreciation to my co-advisors Dr. Clifford W. Welsch and Dr. Walter J. Esselman for their encouragement, financial support, friendship, guidance in science and scientific writing and many other ways of sharing through the years. I really enjoyed my mentor-mentee relationship! I also owe thanks to Dr. Richard Schwartz for his suggestions and advice as well as the other members of my graduate committee, Drs. Jay Goodman and John Thornburg for their advice and invaluable time. A special debt of gratitude is extended to Dr. Michael Streuli and Dr. Justin McCormick for providing the anti-LAR monoclonal antibody and PTB-hyg vector, respectively. Without their generous assistance, much of my work would have been impossible. I also thank Drs. Kevin Walton and Jack Dixon for their assistance on the Southern blot analysis. I would like to acknowledge all the past and the present members in Dr. Welsch’s and Dr. Esselman’s laboratories for their assistance, criticism and friendship. In particular, I would like to thank Dr. Hsun-Lang Chang and Mr. Howard Beittenmillier for their collaboration and technical advice. I am grateful to all of those who helped in one way or another during the course of this work. Finally, I wish to thank my parents for their support and encouragement. I owe an unrepayable debt of appreciation to my husband, Dr. Dajun Yang, for his love, understanding, enduring support and sharing with me scientific thoughts as well as the we of our daughter Alina and our son Anthony. Without their support, the completion of this degree would not have been possible. vi TABLE OF CONTENTS Page LIST OF TABLES ....................................... x LIST OF FIGURES ....................................... xi CHAPTER I. INTRODUCTION ............................... 1 A. HUMAN BREAST CANCER AND neu ONCOPROTEH‘I TYROSINE KINASE ..................................... l B. PROTEIN TYROSINE PHOSPHATASES (PTPases): DIVERSE FAMILY OF INTRACELLULAR AND TRANSMEMBRANE ENZYMES . . 8 1. Characterization of PTPases ....................... 8 2. Structural and functional relationship of R-linked PTPases and the interactions between PTKS and PTPases ................ 13 3. Genes of P'I'Pase - A Tumor Suppressor Gene? ............ 20 4. The potential roles of PTPases in cell differentiation and programmed cell death ............... 23 C. CELL CYCLE REGULATION ......................... 25 D. SIGNIFICANCE OF THIS STUDY ...................... 27 LIST OF REFERENCES ............................... 30 CHAPTER II. INCREASED EXPRESSION OF SPECIFIC PROTEIN TYROSINE PHOSPHATASES IN HUMAN BREAST EPITHELIAL CELLS NEOPLASTICALLY TRANSFORMED BY THE NEU ONCOGENE . . . 50 FOOTNOTES ...................................... 51 ABSTRACT ....................................... 52 INTRODUCTION ................................... 54 MATERIALS AND METHODS .......................... 57 Cell lines ..................................... 57 Animals ..................................... 57 vii Retrovirus Infection .............................. 57 Induction of rat mammary carcinomas ................... 58 Oligonucleotide Primers ............................ 58 RT-PCR analysis ................................ 59 Immunofluorescence analysis ......................... 60 Southern and Northern blot analysis .................... 60 Flow cytometric DNA analysis ....................... 61 Tumorigenicity assay ............................. 61 RESULTS ........................................ 62 Effect of neu introduction of 184B5 cell proliferation in vitro ..... 62 Effect of neu introduction on tumorigenicity of 18435 cells in athymic nude mice ............................. 62 Expression of neu and PTPases in 18485 cells and in neu transformed 184B5 cells .......................... 64 Expression of neu and PTPases in new and DMBA-induced rat mammary carcinomas ............................ 70 Expression of P185neu, LAR and PTPlB protein in 18435 cells and in neu transformed 184B5 cells ................... 70 Southern blot analysis ............................. 74 DISCUSSION ...................................... 76 LIST OF REFERENCES ............................... 82 CHAPTER III. INFLUENCE OF INCREASED EXPRESSION OF LEUKOCYTE COMMON ANTIGEN RELATED (LAR)-PROTEIN TYROSINE PHOSPHATASE ON HUMAN BREAST CARCINOMA TRANSFORMED BY THE NEU ONCOGENE ............................. 86 FOOTNOTES ...................................... 87 ABSTRACT ....................................... 88 INTRODUCTION ................................... 89 MATERIALS AND METHODS .......................... 93 Cell Culture ................................... 93 Construction of The Vector Expressing Human LAR CDNA ..... 93 DNA Transfection and Selection of Clones ................ 97 Effects of LAR Overexpression on Cell Morphology and Growth .................................... 97 Immunoprecipitation and Western Blot Analysis ............. 97 Tumorigenicity Assay ............................. 98 RESULTS ....................................... 100 Vector Construction and Verification ................... 100 Transfection and Selection of 18-Hn1 Colonies With Expression 0f MT LARED ............................... 100 Effects of Overexpression of LAR on Morphology and viii Growth In Wtro of 18-Hn1-LAR Cells ................ 104 Effect of LAR Overexpression on Tyrosine Phosphorylation in 18-Hn1 and 18-Hn1-LAR Cells ................... 109 Effect of LAR Introduction on Tumorigenicity of 18-Hn1 Cells in Athymic Nude Mice ...................... 115 DISCUSSION ..................................... l 17 LIST OF REFERENCES .............................. 124 ix LIST OF TABLES CHAPTERII 1. Expression of neu, LAR, PTPlB and TC-PTP in neu transformed 18435 human breast epithelial cells and in SK-BR-3 and MCF-7 human breast carcinoma cell lines determined by RT-PCR analysis .............. 67 2. Expression of protein P185°°“, LAR and PTPlB in mm transformed 18435 human breast epithelial cells and in SK-BR—3 and MCF-7 human breast carcinoma cell lines determined by immunofluorescence analysis ............................. 73 Chapter III 1. Multiple restriction digestion of plasmid DNA ................. 103 2. Tumorigenicity of 18-Hn1 cells and l8-Hn1-LAR cells in athymic nude mice ......................................... 1 16 LIST OF FIGURES Elam Ease CHAPTERII 1. Tumorigenicity of neu transformed 18435 human breast epithelial cells . . . 63 2. Kinetics of RT-PCR amplification of GAPDH, neu and LAR in neu transformed 18435 human breast epithelial cells (18-Rn1, 18-Hn1) and parental 18435 cells .................................. 65 3. Expression of GAPDH, neu and LAR specific RT—PCR products from neu transformed 18435 human breast epithelial cells (18-Rn1, 18-Hnl, 18-Rn2) and parental 18435 cells .......................... 66 4. Expression of GAPDH, neu, LAR PTPlB and TC-PTP specific RT-PCR products from neu transformed 18435 human breast epithelial cells (18-Rn1, 18-Hn1) and 18-Rn1 and 18-Hn1 human breast tumors ............. 69 5. Expression of GAPDH, neu, LAR and PTPlB specific RT—PCR products from neu-induced and DMBA-induced rat mammary carcinomas .......... 72 6. Southern blot analysis of LAR in neu transformed 18435 human breast epithelial cells ...................................... 76 CHAPTER III 1. Strategy for a construction of a full length LAR CDNA expression vector utilizing a MT promoter and a hygromycin selection marker ......... 95 2. Restriction enzyme digestion of the intermediate and full length LAR expression vectors .................................. 102 xi Comparison of expression of LAR and p185“‘1 in 18-Hn1 cells and PTB-LARED transfected cells (18-Hn1-LAR) .................. 106 Morphological appearance of 18-Hn1 cells and 18-Hn1-LAR maintained in vitro ......................................... 108 Effect of ZnSO4 supplementation on the growth rate of 18-I-In1-LAR cells in vitro ...................................... 110 Western blot analysis of cell density dependent LAR expression in 18-Hn1 cells and in 18-Hnl-LAR5 cells ..................... 112 Western blot analysis of phosphotyrosine-containing proteins of 18-Hnl, control plasmid containing 18-Hnl cells and 18-Hn1-LAR cells with or without ZnSO4 treatment ............. 114 xii INTRODUCTION It is well known that protein tyrosine phosphorylation is a reversible reaction catalyzed by both protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTPase). Recent findings suggest that phosphoryltyrosine levels found within cells are the result of balance between the opposing activities of PTKs and PTPases (Fisher et al. , 1991; Saito and Streuli, 1991). Although the phosphorylation on tyrosine residues within the cell occurs much less frequently than that on serine and tlrreonine residues, tyrosine phosphorylation is a strictly controlled system involved in the regulation of key cellular activities including gene expression, signal transduction, proliferation and transformation. A. HUMAN BREAST CANCER AND neu ONCOPROTEIN TYROSINE KINASE The neu oncogene was first identified as a transforming oncogene in DNA from chemically (ethylnitrosourea) induced neuroblastomas in the rat (Schechter et a1. , 1985). The human neu proto-oncogene which encodes a 185 kDa transmembrane glycoprotein, P185"“‘, shows extensive structural similarity to epidermal growth factor receptor (EGFR), a 170 kD transmembrane protein. Like EGFR, the neu protein possesses a hydrophobic transmembrane spanning sequence, a cytoplasmic portion with intrinsic protein tyrosine kinase (PTK) activity and the extracellular portion containing two 2 cysteine-rich clusters (repeat sequences) (U llrich and Schlessinger, 1990). Several neu specific ligands with a variety of sizes have been identified. The identified ligands include 30 Kd (Lupu et al. , 1990) and 45 Kd (Holmes et al. , 1992) glycoproteins from conditioned medium of a human breast carcinoma cell line MDA-MB 231, a 44 Kd neu differentiation factor from the medium of a ras transformed fibroblast cell line (Peles et al., 1992; Yarden et al., 1991), and others isolated from transformed human T lymphocytes (Dobashi et al., 1991; Davis et al., 1991), macrophages (Tarakhovsky et al., 1991), and bovine kidney cells (Huang et al., 1990). It appears that ligand-induced activation of the kinase domain in the P185"“‘ oncoprotein and its signaling potential are mediated by receptor dimerization (Hudziak et al. , 1987). Ligand binding and subsequent conformational alteration of the extracellular domain including receptor dimerization, stabilizes interactions between adjacent cytoplasmic domains and leads to activation of the kinase function (Ullrich and Schlessinger, 1990). This dimer-mediated receptor activation allows heterodimer formation between structurally very similar receptors such as P185"‘“ and EGFR, resulting in the appearance of a very high affinity EGFR leading to an increase of P185"“‘ kinase activity for autophosphorylation and various cellular substrate phosphorylation (Wada et al. , 1990; Qian et al., 1992). More recently, however, it has been demonstrated that EGF-induced heterodimerization of EGFR and P185"‘“ can promote either stimulatory or inhibitory influences on kinase activity, which lead to either activation or inhibition of the interaction of growth factor controlled cellular signaling (Spivak-Kroizman et al. , 1992). 3 The normal function of the transmembrane domain is to anchor the receptor in the plane of the plasma membrane, thereby connecting the extracellular environment with internal compartments of the cell. The point mutations containing a substitution of Glu for Val at position 664 in transmembrane region of P185"“‘ result in constitutive receptor dimerization, thus leading to an activation of PTK, which is essential for the transforming potential of neu (Weiner et al., 1989). Recently, Cao et al. (1992) observed that the lateral position and rotational orientation of Glu664 did not correlate with transformation, but the primary structure in the vicinity of this Glu664 played a significant role in this activation. This suggests that the transmembrane domain, in particular the glycine-containing motif (anrino acid 661-665) of the P185"“‘, plays an active rather than a passive role in signal transduction. The human neu prom-oncogene, also known as c-erb3-2 or HER-2, has been mapped to chromosome 17, region 21 (Coussens et al., 1985), and has been found to be amplified and/or overexpressed in approximately 30% of human primary breast cancer patients (Kraus et al., 1987; Slamon et al., 1987; Van de Vijver et al., 1987; Clark et al. , 1991). The incidence and level of neu amplification has been found to remain consistent in matched primary and metastatic tumors from the same patient, suggesting that amplification is an early and possibly initiating event in human breast cancer (Lacroix et al., 1989). The importance of the activated neu oncogene in experimental mammary carcinogenesis has previously been investigated using the transgenic mouse model (Muller et al., 1988; Bouchard et al., 1989). Little is known about its direct effects on the neoplastic transformation of human breast epithelial cells. Clark et al. (1988) 4 cotransfected v-Ha-ras and SV-40 large T antigen into an immortalized human mammary epithelial cell line, resulting in a strongly tumorigenic transformation. They claimed that complimentation between the two oncogenes (i.e. , H-ras and large T antigen ) was required to produce the fully carcinomatous phenotype. Recently, Pierce and her coworkers have demonstrated that the activated neu oncogene alone was sufficient for neoplastic transformation of immortalized human breast epithelial cells; such transfection resulted in progressively growing carcinomas in athymic nude mice. The data indicated that the overexpression of neu may directly contribute to the transformation of human breast epithelium (Pierce et al., 1991). The activated form of P185"“‘ exhibits a higher propensity to dimerize and an elevated tyrosine kinase activity (Bargmann and Weinberg 1988; Weiner et al. , 1989). By using monoclonal antibodies (Scott et al. , 1991,; Yarden et al. , 1990) or chimeric neu proteins (Lehvaslaiho et al., 1989; Peles et al., 1991; Fagioli et al., 1991), it was possible to demonstrate that P185"‘“-PTK could be stimulated and transmit growth regulatory biochemical signals. Cross-linking of P185”"“ with monoclonal antibody 4D5 on the P185"“‘ overexpressing human breast carcinoma cell line, SK-BR-3, resulted in receptor phosphorylation and internalization, as well as increased intracellular levels of the second messengers: inositol triphosphates (1P3) and diacylglycerol (DAG), and elevated expression of c-fos mRN A that directly regulated cell-cycle progression (Sarup et al., 1991; Scott et al., 1991). More interestingly, 4D5 stimulated rapid phosphorylation of P185"“‘ and an associated 56-Kda phosphotyrosyl protein (P-Tyr56), with parallel elevation of PI4-kinase activity and production of the lipid substrate for 5 phospholipase-Cy (PLCy) (Scott et al., 1991). These data suggested that P-Tyr56 and PI4-kinase could represent specific substrates of P185"“‘-PTK in its signaling cascade. The transforming potential of neu oncogene is probably manifested via multiple genetic mechanisms as follows: i) specific point mutation, a substitution of Glu for Val at position 664 in the transmembrane region (Bargmann et al., 1986; 1988; Weiner et al., 1989; Cao et al. , 1992); ii) extensive deletions of the noncatalytic portions in either the carboxyl or amino-termini (Di Fiore et al., 1987; Bargmann et al., 1988; Akiyama et al., 1991; Pierce et al., 1991). The proteolytic release of the extracellular domain is part of the mechanism by which the tyrosine kinase can become activated: a) The extracellular domain of P185” (105 Kd) was found to be shed in the human breast carcinoma cell line SK-BR-3 (Zabrecky et al., 1991); b) Expression of N- terminal truncated neu in NIH-3T3 cells resulted in a 10 fold greater transforming activity compared to the full length gene (Di Fiore et al., 1987). These data suggest that the remaining cell-associated cleavage product, probably represented an active and oncogenic form of the Pl85”“‘. iii) overexpression of the apparently normal proto-oncogene (human or rat) leading to transformation of NIH-3T3 fibroblasts (Di Fiore et al., 1987 ; Hudziak et al., 1987). iv) ligand stimulation in the context of a chimeric neu-EGFR protein (Lehvaslaiho et al. , 1989). 6 All these modes of oncogenic activation result in constitutive activation of neu intrinsic PTKs activity. Like other receptor linked PTKs, all autophosphorylation sites of P185"‘“ are located in its carboxyl terminal tail (Margolis et a1. , 1989). Autophosphorylation of P185"“‘ on tyrosine residues is absolutely required for intrinsic PTK activity (Sorkin et al., 1992) to trigger an efficient nritogenic response and high affinity PLCy substrate coupling (Segatto etal., 1992). The EGFR/erbB—2 mutant bearing multiple Tyr to Phe substitutions at P185”“‘ autophosphorylation sites was unable to deliver a sizeable rnitogenic signal when activated by EGF treatment (Segatto et al., 1992). PLC'y is one of the known substrates for P185"“‘-PTK and its catalytic activity is increased by tyrosine phosphorylation. Activation of PLCy leads to rapid hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIPZ) to generate two second messengers, i.e. diacylglycerol (DAG) and inositol triphosphate (13;), which in turn activate protein kinase C (PKC) and mobilize intracellular calcium. The combined effect provides an internal stimulus for unregulated cell proliferation and neoplastic transformation (Ullrich and Schlessinger, 1990; Peles et al., 1991; Rhee et al., 1992). Replacement of the consensus lysine residue (Lys753 to Met) of the ATP binding site within the PTK domain completely abolished neu transforming ability (Akiyama et al. , 1991). These results suggest that the mechanism of cellular neoplastic transformation by the neu oncoprotein involves tyrosine phosphorylation and activation of the PLCy mediated phosphatidylinositol signalling pathway (Peles et al., 1991). A number of studies have provided evidence that PLC'y activation is one of the critical elements in the rnitogenic signaling pathway in human breast tumorigenesis. First, higher levels of tyrosine phosphorylated PLCy proteins were detectable in the 7 majority of primary human breast carcinoma samples tested (18/21), compared to normal breast tissues (Arteaga et al., 1991). Second, tyrosine phosphorylation of PLC'y was greatly increased in human breast carcinoma cell lines as the result of EGF stimulation (Soderquist et al., 1992; Jallal et al., 1992). Third, it has been found that P185"‘“ formed a complex with PLCy (Jallal et al., 1992). In fact, Peles et al. have demonstrated that the oncogenic forms of neu tyrosine kinase are permanently coupled to PLCy, in which the tyrosine residues are constitutively phosphorylated. As tyrosine phosphorylated PLC'y mediates accelerated turnover of PI, protein kinase C and Ca+ + fluxes are constitutively activated. This may in turn result in accelerated cellular proliferation, which is characteristic of the transforming phenotype (Peles 1991). Fourth, SH2 domains of PLC'y were able to enhance substrate phosphorylation by EGFR (Rotin et al. , 1992a) and prevented tyrosine dephosphorylation of EGFR by PTPases (Rotin et al., 1992b). This suggests that SH2 domains of PLC'y could play a crucial role in P185"“‘ activation, because of its extensive structural similarity with EGFR. In view of the role of neu oncogene in human breast carcinomas, it is important to note that neu gene amplification and overexpression have been found to be highly correlated with poor prognostic factors by using multivariate statistical analysis (Pierce et al., 1991; Van de Vijver et al., 1991). These factors include large tumor size, lymph node positivity, a higher number of involved nodes, advanced stage, steroid receptor negativity, aberrant DNA content and a higher rate of cell proliferation (Slamon et al. , 1987; 1989; Berger et al., 1988, Wright et al., 1989, Thor et al., 1989; R0 et al., 1989; Tandon et al., 1989; King et al., 1989; Borg et al., 1991; Marx et al., 1990). Overexpression of neu is also strongly associated with early recurrence and death among 8 axillary lymph node positive patients (Borg et al. , 1991). More recently, NIH 3T3 cells transformed by the activated rat neu oncogene have been shown to exhibit metastatic properties both in vitro and in viva (Yu and Hung, 1991), indicating that neu oncogene expression is sufficient for the induction of metastasis in this cell line. Furthermore, human breast carcinomas overexpressing the erb32 gene were resistent to certain cytotoxic drugs, and amplified expression of neu induced resistance of NIH 3T3 cells to tumor necrosis factor a (TNFoz) and active macrophages (Hudziak et al. , 1988). Thus, neu may potentiate tumorigenesis by inducing tumor cell resistance to host defense mechanisms. These findings suggest that amplification and/or overexpression of the neu proto-oncogene, and the consequent continued over- phosphorylation of tyrosine residues in cellular proteins, is an important and critical factor in the initiation of the growth and progression of human breast carcinomas. B. PROTEIN TYROSINE PHOSPHATASES (PTPases): A DIVERSE FAMILY OF INTRACELLULAR AND TRANSMEMBRANE ENZYMES Wm PTPlB, the first recognized member of P'I‘Pase gene family, was isolated in homogeneous form from the soluble and particulate fractions of human placenta by Tonks and his collaborators in 1988 (Tonks et al. , 1988). PTPlB is active as a monomeric catalytic subunit of 321 amino acids, which is truncated product from the C-terminal. Intact PTPlB should contain 431 amino acids as predicted from the cDNA sequence. The gene coding for PTPlB has been mapped to chromosome 20ql3.1-13.2, a region containing other genes which encode factors implicated in tumorigenesis, including src, hck and plcl (Brown-Shimer et al. , 1990). Amino acid 9 sequence comparison demonstrated that PTPlB is structurally not similar to any other catalytic subunits of serine/threonine phosphatases (Charbonneau et al. , 1989), but was found to be homologous to the cytoplasnric domain of the leukocyte common antigen (CD45) (Charbonneau et al., 1988). CD45 is a transmembrane protein which possesses intrinsic tyrosine phosphatase activity (Cool et al. , 1989). By using low stringency screening methods, PTPases have now been identified in many different eukaryotic cell types in a broad range of sizes. The diverse family contains both transmembrane glycoproteins and cytosolic proteins (Fisher et al. , 1991; Saito and Streuli, 1991). Cool et al. (1989) isolated a cDNA clone, TCPTP, from a human T-cell library which encoded a protein with 415 amino acids and displayed 65 % sequence identity with PTPlB. Interestingly, the sequence similarity between PTPlB and TCPTP is only in the NHz-terminal 300 amino acids (i. e. , within catalytic domains), with the remaining C-terminal sequences being distinct. The full-length and C-terminal truncated forms of TCPTP are found in distinct subcellular fractions and display differential effects on cell division and actin assembly (Cool et al., 1990; 1992; Fisher et al., 1991; Frangioni etal., 1992). It is known that the last 19 residues of C-terminal segment are hydrophobic and appear to be critical for subcellular localization, substrate specificity and regulation of the enzyme (Fischer et al., 1991). PTPlC is another cytosolic soluble PTPase isolated from human breast carcinoma ZR-75-1 cDNA library (Shen et al. , 1991). Surprisingly, this enzyme is different from other cytosolic PTPases, in that it possesses a large noncatalytic region at NH2 terminus and contains two adjacent SH2 domains. The SH2 domains of PTPlC formed a high affinity complex with activated EGFR which had intrinsic PTK activity. The finding of the SH2 domain in 10 PTPlC as well as other PTPases (Adachi et al., 1992; Freeman et al., 1992; Matthews et al., 1992; Yi et al., 1992; Rotin et al., 1992b; Plutzky et al., 1992) indicates the linkage of PTPase to growth factor-receptors (PTKs) for regulation of signal transduction (Shen et al. , 1991). The sequence homology of cytoskeletal protein and cytosolic PTPases indicated its potential role in controlling cytoskeletal integrity (Yang and Tonks, 1991; Gu et al., 1991). Non-receptor, cytosolic PTPases possessing a single catalytic PTPase domain have been identified in viral, bacterial, yeast and mammalian species. An important question as to the origin of these genes was raised by the discovery of PTPases in pathogenic bacteria Yersinia (Guan etal., 1990) and vaccinia virus (Guan et al., 1991). What is the role of PTPases in developing and maintaining the pathological state? The transmembrane receptor type PTPases have been cloned from mammalian species and from Drosophila. They typically contain a single hydrophobic transmembrane region and two tandemly repeated conserved cytoplasmic domains fused to a variety of extracellular domains with great diversity in length and structure: several of which are related to neural cell adhesion molecules (N-CAM) (Streuli et al., 1988; 1989; 1990; Krueger et al. , 1990) and some are homologous to carbonic anhydrases (Krueger and Saito, 1992). CD45 or leukocyte common antigen (LCA) is found only in hematopoietic cells and is comprised of a family of heavily glycosylated transmembrane proteins with variable isoforms resulting from alternative splicing. CD45 contains two tandem PTPase-like domains with intrinsic PTPase activity (Charbonneau et al. , 1988; Tonks et al., 1990). CD45-mutant T cell lines failed to elicit phosphatidylinositol (PI) turnover, indicating that CD45 is essential in linking the TCR complex to P1 metabolism (Koretzky 11 et al., 1990). In 3 cells, CD45 may be a component of the antigen receptor complex (Justement et al. , 1991). Extensive studies have indicated that CD45 plays a critical role in both T and B lymphocyte signal transduction. Activation and proliferation in response to antigen stimulation requires the direct dephosphorylation of Tyr505 of P56ICk prom—oncogene by CD45 (Fisher et al., 1991; Saito and Streuli, 1991). More recently, Desai et al. reported that a chimeric protein in which the extracellular and transmembrane domains of CD45 were replaced with those of the EGF receptor (EGFR), was able to restore TCR signaling in a CD45 deficient cell. This data demonstrated that the cytoplasmic domain of CD45 is necessary and sufficient for TCR signal transduction (Desai et a1. , 1993). Leukocyte common antigen related protein (LAR) is a transmembrane molecule composed of a 1234 amino acid extracellular receptor-like region, a 24-amino acid transmembrane segment and a 623 amino acid cytoplasmic region containing two tandemly repeated PTPase domains 40% homologous to PTPlB. It was found that the LAR gene was expressed on endothelial and epithelial cells of a broad range of tissue and cell types including lung, breast, kidney, thymus, brain, intestine, muscles and different tumor cell lines (Fisher et al., 1991; Saito and Streuli, 1991; Streuli et al., 1988). Streuli et a1. (1992) have demonstrated that the LAR protein is proteolytically cleaved within the cell to produce a mature cell surface structure containing two subunits, termed the LAR-P-subunit (PTPase) and LAR-E—subunit (LAR extracellular subunit). The LAR- E-subunit can be released from the cell surface particularly in high density cell cultures. This suggests that LAR PTPase activity may be regulated by either unknown specific ligands or by shedding of the E-subunit. The extracellular region of LAR is composed 12 of three immunoglobulin (Ig) like domains and eight fibronectin type III (FN-III) domains which resemble neural cell adhesion molecules (N—CAM) (Streuli et a1. , 1988). N—CAM is a family of proteins derived from alternative mRNA splicing resulting in highly homologous extracellular domains (NHz-terminal) but different cytoplasmic domains (Edelman et al., 1985). N-CAM is found on various differentiated tissues but mainly in neurons and muscles. It facilitates cell adhesion by homophilic cell-cell interaction among Ig-like and FN-III domains (1'. e. , binding to the same molecules expressed on the surface of other cells) (Edelman et al., 1985; Cunningham et al., 1987; Owens et al., 1987). Hence, LAR may also be a cell adhesion molecule. The physiological function of LAR PTPase may involve in controlling cell growth and proliferation through cell—cell or cell-matrix interactions (Fisher et al. , 1991, Saito et al. , 1991), the loss of which could lead to unrestrained cell proliferation and transformation (Saito and Streuli, 1991). Supporting this hypothesis is the finding that the product of a colorectal tumor suppressor gene, DCC (deleted in colorectal cancer), which was shown to be frequently deleted in colorectal carcinomas, is structurally similar to the extracellular region of LAR (Fearon et al., 1990; Standbridge, 1990). DCC gene has been mapped to chromosome 18q, for which approximately 40% of human breast carcinomas showed allelic losses (Devilee et al. , 1991). More recently, Streuli et al. (1992) have demonstrated that the LAR gene is located on human chromosome 1p32—33, which contains several candidate tumor suppressor genes. Altogether, the unique structure of LAR PTPase raises the possibility that it is an ideal candidate for tumor suppressor gene. 13 Krueger et al. (1990) isolated six novel human placenta cDN A clones that encode receptor-like PTPases by cross-hybridization to Drosophila DPTP cDN A probe and named them I-IPTPa, B, c, 6, e, and f. HPTPa (RPTPa or LRP) and HPTPe have much smaller external domains than the rest. Variable cDN A structurns suggest that alternative splicing takes place in the first tyrosine phosphatase domain of LRP (Matthews at al., 1990). The human LRP gene has been assigned to chromosome 20p13 (Kaplan et al., 1990; Jirik et al., 1990). LRP and HPTPe are broadly distributed and mainly expressed in brain (Kaplan et al. , 1990). They probably have a general function in signal transduction. HPTPB is unique and has only one PTPase domain on its cytoplasmic region and 16 repeated FN-III domains on the extracellular region. Most interestingly, the gene coded for RPTPy has been localized to chromosome 3p21, and this PTPr allele was lost in approximately 50% of tumor samples examined (Laforgia et al., 1991). Such observations support the concept that certain RPTPases may be candidates for tumor suppressor gene products. - II t. -. .. r n Mn; ‘1: “'11 hi ovofR-link PTP“; . o ' in ._ tin W The structural diversity of PTPases indicates that the PTPase gene family may be similar in complexity to that of the PI‘K multigene family (Kaplan et a1. , 1990; Hunter, 1989). Although the size of each PTPase domain is about 300 amino acids (AA), significant amino acid sequence similarity is seen only within the core region of about 250 AA. Krueger et al. (1990) compared the AA sequences of the core regions of 12 PTPases and found that 42 AA positions within the core region were totally invariant. PTPases, like PTKs, usually possess a consensus AA sequence in the 14 catalytically active domains. The most notable cluster in the stretch of AA within the consensus sequence is GPMVVHCSAGVGRTG, which surrounds the critical cysteine residue. It has been shown that this stretch constitutes an important portion of the catalytic sites of PTPases by site-directed mutagenesis (Streuli et al. , 1990). The data indicates that the catalytic domains of all PTPases share significant amino acid homology and evolutionary conservation (Fisher et a1. , 1991; Krueger et al. , 1990). All receptor-linked PTPases characterized so far contain two PTPlB-like domains and all PTPlB-like sequences have two cysteine residues in the same relative positions (i.e., within the highly conserved cluster) (Krueger et al., 1990; Streuli et al., 1990). The functional importance of two domains remains unclear. There are three possibilities: a) both domains are catalytically active and behave in a cooperative manner; b) both domains are catalytically active but have different substrate specificities; c) only one domain has enzymatic activity, while the other is regulatory in function. Since there is more constraint against sequence divergence in domain 1 than in domain 2, it may be that only the first domain has enzymatic activity (Laforgia et al. , 1991; Pot et al. , 1991). In fact, Streuli et al. demonstrated that the first domains of both LCA and LAR have enzymatic activity and that one cysteine residue is absolutely required for activity. In contrast, the second PTPase-like domains do not have detectable catalytic activity but sequences within the second domains influence substrate specificity and activity (Streuli et al., 1990; Itoh et al., 1992). In addition, a deletion (LAR: 1275-1311) which is upstream of the core sequence of LAR, completely abolished its PTPase activity (Streuli et al. , 1990). On the other hand, using molecular cloning techniques, Wang et al. (1991) have shown that both a PTP domain 1 and domain 2 of HPTPa were enzymatically 15 active but differed in substrate specificity and responses to effectors. Inactivation of domain 1 may suppress domain 2 activity, suggesting that the interdependence between the two domains is important for PTPase function in signal transduction. A variety of compounds has been shown to modulate PTPase activity in both a positive or negative manner. Orthovanadate is one of the most potent inhibitors of PTPases (Gordon et al., 1991; Pot et al., 1991; Hecht et al., 1992; Itoh et al., 1992; Tahiri-Jouti et al. , 1992). The fact that thiol reducing agents are necessary for enzymatic activity suggests that cysteines play important roles in enzyme structure and function (Tonks et al., 1988). Three sulflrydryl agents ( N—ethylmaleimide, p- (hydroxymecuric)benzoate and iodoacetate) inhibited rat LAR PTPase activity irreversibly (Pot et al., 1991), whereas the loss of PTPase activity caused by diamide is reversed by 2-mercaptoethanol or EGF in fibroblasts (Monteiro et al. , 1991). It is important to note that both human and rat LAR PTPase activity is not inhibited by the divalent zinc, even at high concentration (1mM) in vitro, a characteristic which distinguishes LAR from other PTPases (Itoh et al., 1992; Pot et al., 1991; Wang et al., 1992). These results indicate that, despite the high degree of sequence conservation in the PTPase domains, these enzymes have different specific activities and respond differently to various modulators. Since the level of protein tyrosine phosphorylation of intracellular substrates is determined by the balance of PTK and PTPase activity, their roles in regulation of signal transduction, cell proliferation, differentiation and neoplastic transformation have become an extremely interesting area of study. Although the finding of SH2 domain in PTPlC indicated the linkage between PTPase and PTK (Shen et al., 1991), nothing is known 16 about the direct interaction between PTKs and PTPases. A dynamic and complicated interaction between PTPases and PTKs or ser/thr kinases are likely to be involved in signaling pathways. There are a number of possibilities for such interaction. a) As substrates, receptor linked P'I‘Ks are dephosphorylated by PTPases. b) As substrates of PTKs or other protein kinases, PTPases are phosphorylated. c) Both PTPases and PTKS have effects on common substrates (i.e. affect the state of activity of downstream secondary enzymes). Indeed, it has been demonstrated in vitro that a number of receptor linked PTKs, such as EGFR, IGF-I, PDGF and insulin receptor, are substrates of different PTPases. The autophosphorylated EGFR was dephosphorylated by PTPlB (Tappia et al. , 1991; Hashimoto et al., 1992a), by TCPTP (Zander et al., 1991), by LAR and by LRP (Hashimoto et al., 1992b) in vitro. The activated EGFR was able to form high affinity complexes with SH2 domains of PTPlC (Shen et al. , 1991). Dephosphorylation of IGF-I receptor on tyrosine residues by membrane associated PTPases resulted in either a decrease in its intrinsic activity (Peraldi et al. , 1992) or marked reduction in its autophosphorylation and rnitogenic responses (Mooney et al. , 1992). Hashimoto et al. demonstrated that LAR rapidly deactivated insulin receptor kinase by dephosphorylation on the receptor regulatory domains (Hashimoto et al. , 1992b). Interestingly, the pattern of dephosphorylation of these receptor PTKs by PTPases in vitro occurs in a sequential or ordered manner, indicating that features surrounding the dephosphorylation sites might contribute to substrate specificity in intact cells (Ramachandran et a1. , 1992). Transmembrane PTPases identified exhibit high affinity for substrates and high activities 17 in cells, suggesting that these enzymes are important in vivo in controlling or reversing auto-phosphorylating PTK-induced regulatory or signalling events. Several pieces of evidence support the second possibility. Since many possible sites of phosphorylation exist within the cytoplasmic domain of rLAR, the ability of both protein kinase C (PKC) and P43"‘“”’ PTK to phosphorylate rLAR in a time-dependent manner in vitro, highly suggested that LAR may be regulated in vivo by these or similar enzymes (Pot et al., 1991). The ability of rLAR to undergo auto- or trans- dephosphorylation on phosphotyrosine residues parallels the autophosphorylation ability of PTKs (Pot et al., 1991). Brautigan et al. have shown that both the stimulation of CAMP dependent kinase (PKA) and Ca”r +/phospholipid dependent PKC, and the inhibition of ser/thr phosphatases (especially type 2A), activated PTPlB activity in CV-l monkey kidney epithelial cells. These data were consistent with observations in which phosphorylation of PTP 13 was elevated in TPA-stimulated HeLa cells (Flint et al. , 1993). Phosphorylation of Ser/Thr residues in the regulatory subunit of PTPlB seemed sufficient to give full PTPase activity without dissociation or truncation of the catalytic subunit (Brautigan et al. , 1991). Altogether, these data indicated that crosstalk between ser/thr kinases and PTPases may not alter basal levels of tyrosine phosphorylation, but modulate the response to stimuli. More recently, it has been shown that a mouse SH2- containing PTPase, Syp, was able to bind to autophosphorylated EGFR and was rapidly phosphorylated on tyrosine in EGF-stimulated cells. Also, Syp was constitutively phosphorylated on tyrosine in mouse fibroblast A31 cells transformed by v-src (Feng et al. , 1993). These results support a model of regulation of PTPases by phosphorylation. 18 The cellular phosphorylation content may be regulated not only by PTK or PTPase activities, but also by SH2 domain containing proteins such as phospholipase C'y (PLC'y). As evidence to support this, Rotin et al. have demonstrated recently that the tyrosine dephosphorylation of EGFR was inhibited by the SH2 domain of PLC-y, suggesting that the PTPases and the SH2 domains compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of EGFR (Rotin et al. , 1992b). These data provide further support for the critical regulatory role of PLCy and its SH2 domains in signal transduction and neoplastic transformation. It is believed that the activity of cytoplasmic domains (PTPase domain) of receptor linked PTPases is regulated by specific unknown extracellular ligand, in a manner similar to the regulation of the growth factor receptor PTK activity (Hunter, 1989; Desai et al. , 1993). The function and regulation of transmembrane PTPases have been difficult to study because of the paucity of information regarding ligands and in vivo substrates. Utilizing antibody crosslinldng or chimeric protein models permits the study of regulation of PTPase without knowledge of the natural ligands. Indeed, Desai et al. recently reported that EGFR ligands functionally inactivated the EGFR-CD45 chimera in a manner that depended on dimerization of the chimeric protein. This indication resulted in the loss of TCR signaling. These data indicated that ligand-mediated regulation of receptor type PTPases may have mechanistic similarities with receptor PTKs. However, unlike receptor linked PTKs, which are activated by ligand binding, the family of transmembrane PTPases may, in general, be functionally inactivated by ligand binding (Tonks et al. , 1990; Desai et al., 1993). The model for the mechanism of action of LAR proposed by Saito and Streuli (1990) stipulates that when a cell expressing LAR PTPase 19 encounters another cell carrying an unknown ligand, ligand binding to the extracellular domain of RPTPase might activate its cytoplasmic PTPase activity. The activated PTPase could, in turn, block the growth promoting effects of PTKs by either striping phosphate groups from the kinase themselves, thus shutting down one of the enzymes driving cell proliferation, or dephosphorylate cellular proteins that are also substrates for kinases. In addition, the preliminary results of Streuli et al. (1992) suggest yet another pathway for regulation of LAR PTPase, the proteolytic release of the extracellular subunit ( similar to P185""‘). In general, PTPases could be thought of as enzymes that serve to counteract the action of the PTKs resulting in the activation or inhibition of their target enzymes. It is possible too, that PTPases may act synergistically with Pl‘Ks (Fischer et al. , 1991). This . model of interaction between PTPase and PTK is supported by the observed activation of P56” PTK via CD45 dephosphorylation of the Tyr505 of Lek, an autophosphorylation site (Fischer et al. , 1991; Saito and Streuli, 1991); and the activation of P34‘d62, a critical component involved in cell cycle regulation, which starts the GZ to M transition by cd025 PTPase dephosphorylation (Gautier et al. , 1991; Dunphy et al. , 1991). Thus, PTK or other kinase inhibition would be relieved by PTPase and the cellular response would be increased even though the level of substrate phosphorylation would decrease. On the other hand, a SH2 domain containing PTPase, PTP ID, was found to be activated via phosphorylation on its tyrosine residue by a PTK. PTP 1D however, did not dephosphorylate receptor PTKs in vitro (Vogel et al. , 1993). Thus, PTKs and PTPases do not simply oppose each other’s action, rather they may work in concert to maintain a fine balance of effector activation needed for the regulation of cell 20 growth and differentiation. Therefore, the PTPases within the multigene PTPase family would vary in structure, mechanism of activation, regulation of activation, and interaction with specific PTKs. Was} Perhaps the most intriguing activity for PTPases is their possible role as tumor suppressors. It has known that reversible protein tyrosine phosphorylation is a fundamental mechanism for regulating diverse cellular processes including signal transduction, cell proliferation and neoplastic transformation. The constitutive activation of certain PTKs causes unregulated cell proliferation which is a very important component of oncogenesis. In fact, about one-third of all known oncogenes are PTKs, such as neu, src, fins, abl. Therefore, it is possible that certain PTPase genes are tumor suppressor genes (Fischer et al. , 1991; Stanbridge, 1990; Sager et al. , 1989). Conceptually, hyperphosphorylation of a key signal transduction protein driving neoplastic growth can occur either by deregulation or overexpression of PTK activity or by the loss of PTPase activity. Therefore, one could predict that overexpression of a PTPase counteracts oncogenic PTKs, either by conferring resistance to neoplastic transformation or by reversing a neoplastically transformed phenotype. On the other hand, loss or inactivation of both copies of a PTPase gene could result in constitutively increased tyrosine phosphorylation of particular cellular proteins, thus leading to tumorigenicity. Among the multigenic PTPase family, LAR would appear to be an ideal candidate for a tumor suppressor gene because of its unique structure and broad tissue distribution (Fischer et al., 1991; Saito and Streuli, 1991; Streuli et al., 1988; 1990; Krueger et al., 1990). In contrast to LCA which is expressed only on hemopoietic lincage cells, LAR 21 is expressed on epithelial and endothelial cells of many different organ types, including lung, heart, kidney, thyroid, mammary gland, uterus, pancreas and nerve (Fischer et al., 1991; Streuli et al., 1990; 1992). Furthermore, the 72% homology of evolutionary conservation found between human LAR and its Drosophila homolog, DLAR, (Streuli et al. , 1989) suggests that the function of LAR is fundamental to basic cell physiology. In addition, the overall structure of LAR, i.e. , a cell adhesion molecule (N -CAM)-like structure on its extracellular subunit linked to a cytoplasmic PTPase domain, suggests that LAR may play an important role in the negative regulation of cell growth via cell-cell contact interaction. Further evidence to support the tumor suppressor gene concept is the finding that the product of a putative colorectal tumor suppressor gene, DCC (which was shown to be frequently deleted in colorectal carcinomas and perhaps in breast carcinomas) is structurally similar to the LAR E—subunit (Streuli et al. , 1990). In addition, the human LAR gene has been mapped on chromosome lp32-33 (Streuli et al. , 1992), a region that contains a candidate breast carcinoma suppressor gene (Genuardi et al., 1989; Weinberg, 1991). This would suggest that LAR may be critical or at least very relevant to the development of human breast carcinoma. The concept that PTPases may be tumor suppressor gene products is further supported by the following evidence: a) Chromosomal localization. Chromosomal mapping of some PTPase genes is highly correlated with lesions known to be associated with various neoplasms. For example, the gene coded for human PTPlB has been mapped on chromosome 20ql3.1-13.2 by in situ hybridization. This region contains several other genes such as src, hck and plcl which are involved in signal transduction (Brown-Shimer b) d) 22 et al. , 1990). The human RPTPa gene has been mapped to chromosome 20pter-20q12, a region involved in translocations and deletions in myeloid disorders and neoplasms (Kaplan et al., 1990). RPTPy gene is located on chromosome 3p21, a region which is also commonly deleted in renal and lung carcinomas (Laforgia et al. , 1991). Treatment with the PTPase inhibitor, vanadate, caused a phenotypic transformation of a fibroblast cell line (NRK) most probably due to increasing the amount of phosphotyrosine in these cells (Klarlund et al. , 1985; Hunter, 1989). When PTPlB or another cytosolic PTPase were introduced in NII-I3T3 cells, the overexpression of this enzyme suppressed neoplastic transformation by the neu oncogene in vivo (Brown-Shimer et al., 1992), by the v-src oncogene in vitro (Woodford-Thomas et al. , 1992) and by v-erb3 oncogene (Ramponi et al. , 1992). Microinjection of P'I'PlB into Xenopus oocytes can reverse the mitogenic effects of insulin-stimulated tyrosine kinase activity (Cicirelli et al. , 1990). Overexpression of the truncated TC PTP resulted in approximately 50% reduction in a growth rate in baby hamster kidney cells (Cool et al., 1990). A 37 Kd membrane associated nonreceptor type PTPase activity is significantly increased in Swiss 3T3 as well as normal fibroblast cells in response to density dependent contact inhibition (Pallen et al., 1991). This is consistent with the observation of Brautigan by using CV-l monkey kidney epithelial cells (Brautigan et al. , 1991). These findings suggest that PTPases have anti-oncogenic potential. However, one hallmark of a tumor suppressor gene is that they are deleted in tumors in which their 23 inactivation contributes to the malignant phenotype. To support the concept that certain PTPases are tumor suppressor gene products, Laforgia et al. have demonstrated that loss of one allele of the human RPTPy gene occurred in approximately one half of the tumor samples examined (Laforgia et al., 1991). While certain PTPases are proposed to act as tumor suppressors, it is possible that other PTPases are involved in tumorigenesis in some other way, i.e. , activation of PTPase actually stimulates the transforming process because some proto-oncogenes are known to be activated by tyrosine dephosphorylation, such as the src family tyrosine kinases, P56” and the cell cycle serine/threonine kinase P34‘d02. It has been reported recently that overexpression of the receptor linked PTPa in rat embryo fibroblast, resulted in persistent activation of P60”rc and cell transformation (Zheng et a1. , 1992). PTPases vary in structure, tissue distribution, mechanism of action and function even though they have highly homologous cytoplasmic PTP domains. There is no doubt that some of these PTPases (e.g. LAR, PTPlB and RPTPa, RPTPc), but not all, are excellent candidate tumor suppressor genes. The availability of cDN A clones for these PTPases and molecular cloning techniques will allow elucidation of the role of these PTPases, in particular receptor linked-LAR, in the initiation and growth of human breast carcinomas. 4. 11‘ III: !-._ rol; I_ ' 2.; in 11 Iiff ‘tt"I._II ._ I. III: .. my. 11 I‘--. -1 The potential role played by the changes in PTPase activity during differentiation is particularly interesting. Increased levels of certain PTPase activities have been 24 documented to be involved in morphological and functional differentiation in HL-60 cells (Frank DA et al., 1986; 1988; Buzzi et al., 1992), and other leukemia cell lines as well in response to growth inhibitory cytokines, such as IL-6 (Zafriri et al., 1993) or other maturational agents (PMA and DMSO) (Cohen et al., 1992; Butler et al. , 1990; Buzzi et al., 1992). CD45 is one PTPase that plays a crucial role in hematopoietic cell differentiation (Buzzi et al. , 1992). On the other hand, a rapid increased intracellular PTPase activity was observed during calcium induced keratinocyte differentiation (Zhao et al. , 1992). More recently, den Hertog et al. (1993) demonstrated that activation of RPT7 (LRP) and other PTPases play an important role during neuronal differentiation (Aparicio et al. , 1992). Taken together, these findings indicate that, like PTKs, PTPases indeed are involved in cell differentiation and development. Programmed cell death (apoptosis) is an active cellular mechanism that is dependent on active participation of cellular components and could potentially be regulated by PTPases. Aberrant cell survival resulting from an inhibition of apoptosis could lead to oncogenesis. In contrast, induction of apoptosis in tumor cells could be used therapeutically (Williams et al. , 1991). The morphological characteristics of apoptosis include nuclear condensation and DNA fragmentation. It has been demonstrated experimentally that expression of the bch oncogene product can inhibit apoptosis (William et al., 1991), whereas overexpression of wild type p53 tumor suppressor gene suppressed tumor growth (Shaw et al. , 1992). Based on such observations, we could speculate that one of the mechanisms for p53 suppression of tumor growth may be the triggering of tumor cell death through apoptosis. Since certain genes that encode PTPases are good candidate tumor suppressor genes, it should not be 25 a surprising to find that activation of a PTPase may be associated with or even participate in apoptosis. In supporting this hypothesis, Bronte et al. (1993) found that addition of orthovanadate and phenylarsinooxide (PTPase inhibitors) or genistein (PTK inhibitor) both induced dose-dependent reduction in apoptosis without affecting cell viability. These preliminary data indicate that a complex interaction between PTKs and PTPase may be a crucial event in programmed cell death. C. CELL CYCLE REGULATION Normal eukaryotic cells exist in either a proliferative or a non-proliferative quiescent state. Proliferating cells progress through a chain of complex and tightly controlled events called the cell cycle. During one round of the cell cycle, a single parent cell prepares for and divides into two daughter cells. A typical eukaryotic cell cycle is composed of four major phases; 61, S, G2 and M phase. A central control mechanism in the transition from interphase to mitosis is the activation of P34Cd‘2-cyclin 3 complex called maturation-promoting factor (MPF). Recent studies have demonstrated that cdc25 is a specific protein phosphatase (PTPase), which by dephosphorylating tyrosine residues on P34‘d‘2 directly activates 1,34ch (Gautier et al. , 1991). Tanks and his coworkers showed that microinjection of purified PTPlB into Xenopus oocytes retarded maturation and meiotic cell division induced by insulin, and abolished insulin stimulation (Tonks et al. , 1990). On the other hand, insulin increased mRN A expression of PTPlB in well-differentiated rat hepatoma cells, suggesting a potential mechanism for feedback desensitization of phosphotyrosine signalling through the insulin action pathway (Hashimoto et al. , 1992c). In addition, by 26 using sensitive pattem-matching methods, they were able to detect a significant homology between cdc25 and the PTPase family including LAR and PTPlB (Gautier et al. , 1991). Although it has been demonstrated that PTP 13 was associated with the endoplastic reticulum (Frangioni et al., 1992; Woodford-Thomas et al., 1992), increased phosphorylation of PTP 13 was seen to accompany the transition from 62 to M phase of the cell cycle (Flint et al., 1993). This observation indicated that PTP 13 could be involved in controlling the structural changes in microtubules and the endoplastic reticulum that are associated with the cell cycle. More interestingly, overexpression of carboxyl-terminal truncated TCPTP in baby hamster kidney cells, resulted in cytokinetic failure and asynchronous nuclear division (Cool et al. , 1992). These results suggested that certain PTPases (such as PTP 13, TCPTP) other than cdc25 are directly or indirectly involved in the regulation of cell cycle progression. Crissman and his coworkers compared the effects of staurosporine on the proliferation of non-transformed and neoplastically transformed cells and found that the kinase mediated regulation of G1 progression found in normal cells was lost as the result of neoplastic transformation (Crissman et al. , 1991). This indicated that kinase mediated mechanisms play important roles in cell cycle regulation. Many models for oncogenic transformation involve abnormal progression into the early stages of the cell cycle, such as observed with the oncogenes mos, met, ms and src. These oncogenes promote quiescent nondividing cells to enter the cell cycle at G1. In addition, altering the expression or activity of certain proto-oncogenes can result in inappropriate activation of MPF (M-phase promoting factor), which functions late in the cell cycle during the GZ/M-phase transition. It is well known that many proto-oncogene 27 products including src, abl, p53, SV40 large T antigen and mos are substrates for 1334ch phosphorylation of the catalytic subunit of MPF. The activation of these oncogenes results in activation of MPF, an essential molecule for initiation of mitosis in a variety of species (Freeman et al., 1991). Little is known about the direct effects of neu (erb32) proto-oncogene on cell cycle regulation. There is evidence that erb32 amplification in human breast cancer is associated with a high rate of cell proliferation (Borg et al., 1991; Marx et al., 1990). In a study of 539 invasive primary breast carcinomas, Borg and his colleagues (1991) demonstrated that erbB2 amplification was strongly correlated to most known prognostic risk factors, and also correlated with an increased rate of cell proliferation and aberrant DNA content (large 8 phase fraction). However, a major issue unresolved in these studies is the mechanism by which neu regulates cell proliferation. It will be necessary to determine, therefore, whether or not neu amplification and overexpression results in higher carcinoma cell proliferation and whether or not such expression has a direct effect on certain specific regulators of cell cycle regulation. By comparing the cell cycle kinetics of neu and LAR transfected cells and their parental cells, one should be able to test the hypothesis that both neu-PI‘K and LAR PTPase are involved in the coupling of external signals to proteins such as MPF, a phenomena that is directly linked with cell cycle control. D. SIGNIFICANCE OF THIS STUDY There are now a number of studies which link overexpression and/ or amplification of the neu proto—oncogene to neoplastic transformation and growth of human breast 28 carcinomas. It remains unclear how neu-PTK amplification and/ or overexpression disturbs the balance between phosphorylation and dephosphorylation of cellular proteins and how this intricate balance influences oncogenesis on the molecular level. In addition, while much progress has been made in the identification of PTPase isoforms in recent years (Fischer et al., 1991; Saito and Streuli, 1991; Brautigan et al., 1992), little is known about their mechanisms of action or regulation. For example, literally nothing is known about the role of dephosphorylation which is catalyzed by neu-responsive protein tyrosine phosphatases (PTPases) especially when compared to what is known regarding the role of neu-PTK in human breast tumorigenesis. Hence, an understanding of the functions and interaction of neu-PTK and neu-responsive PTPases in normal and transformed cells will help to elucidate the role of these enzyme systems in normal cellular function as well as in the pathogenesis of human breast cancer. Since LAR has an unique structure and broad tissue distribution, it is an ideal candidate for a tumor suppressor gene among the PTPase multigene family. The loss of this gene may confer a growth advantage on evolving human breast carcinomas cells. However, the critical role that LAR plays in normal and carcinomatous breast cellular processes has only recently been addressed and remains virtually unknown. Examination of the potential of LAR in reversing neoplastic transformation of neu transfected and/or over-expressing carcinoma cell lines, will allow one to evaluate and test the hypothesis. 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CHAPTERII INCREASED EXPRESSION OF SPECIFIC PROTEIN TYROSINE PHOSPHATASES IN HUMAN BREAST EPITHELIAL CELLS NEOPLASTICALLY TRANSFORMED BY THE NEU ONCOGENE 50 FOOTNOTES Abbreviations: PTPase, protein tyrosine phosphatase; PTK, protein tyrosine kinase; RT- PCR, reverse transcription-PCR; LAR, leukocyte-common antigen related; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMBA, 7,12-dimethylbenzanthracene.. 51 ABSTRACT Protein tyrosine phosphorylation/dephosphorylation is a fundamental mechanism in the regulation of cell proliferation and neoplastic transformation; this metabolic process is modulated by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). While the role of PTK’s has been examined extensively in human breast tumorigenesis, the role of PTPases in this process is virtually unknown. To address this issue, an activated neu oncogene was introduced into an immortalized non-tumorigenic human breast epithelial cell line (18435). This resulted in a substantial increase in P185"‘“ expression, which led to the formation of progressively growing carcinomas after such cells were inoculated into athymic nude mice. Importantly, a striking increase in the expression of specific PTPases, LAR and PTPlB, was observed in 3 independently neu transformed cell lines and their derived tumors. This elevation was verified at both the mRNA and protein levels. TC-PTP PTPase expression was only slightly increased in these neu transformed cells, and no expression of CD45 PTPase was observed. The level of neu expression, as well as the differential expression between P185"“‘ and LAR/PTPlB, directly correlated with tumorigenicity. Furthermore, rat mammary carcinomas with elevated neu expression (neu-induced) also had sharply elevated LAR-PTPase expression when compared to rat mammary carcinomas with little or no neu expression (DMBA-induced); the level of expression of LAR PTPase was directly correlated with the level of neu expression. 52 53 Thus, our results provide the first evidence that, in human breast carcinoma cells and in rat mammary carcinomas that have an induced increase in neu expression, a consistent and substantial increase in the expression of specific PTPases occurs. The relationship between P185"‘“-PTK expression and specific PTPase expression may play a critical role in human breast tumorigenesis. INTRODUCTION Reversible phosphorylation of protein tyrosyl residues is a fundamental mechanism for regulating diverse cellular processes including cell proliferation and neoplastic transformation. Modulation of protein phosphotyrosine is accomplished through the Opposing actions of protein tyrosine kinases (P'I‘Ks) and protein tyrosine phosphatases (PTPases) (l). The neu oncogene (c-erbB-2 or HER-2) is a member of the PTK class of oncogenes encoding a 185 kD transmembrane glycoprotein, P185"‘“ (2). The transforming potential of the neu oncogene is manifested via multiple genetic mechanisms, including a point mutation within its transmembrane domain (V a1664-IGlu664) (3 ,4), over-expression of the normal neu proto-oncogene product, and amino terminal truncation (5,6,7). All of these mechanisms result in constitutive activation of the neu intrinsic PTK activity, which is essential for transforming activity (2). The human neu proto-oncogene has been found to be amplified and/or over- expressed in approximately 30% of primary human breast carcinomas (8, 9,10) and to be highly correlated with a poor prognosis (7,9,11,12). However, until very recently, the effects of induced neu over-expression in cultured human breast epithelial cells and the effects of such over-expression on the pathogenesis of these cells have remained unknown. Pierce and her coworkers (7) first reported that over-expression of normal P185"“‘ alone was sufficient for neoplastic transformation of immortalized human breast epithelial cells. These findings strongly suggest that amplification and/ or over-expression of the neu proto-oncogene, and the consequent continued over-phosphorylation of 54 55 tyrosine residues in cellular proteins, is an important and critical factor in the initiation, growth and/or progression of human breast carcinomas. PTPases comprise a family of cytosolic nonreceptor and transmembrane receptor type proteins (1). LAR (13) and CD45 (14) represent receptor type PTPases whereas PTPlB (1,15) and TC-PTP (16) are cytosolic PTPases. PTPases have been identified in many different eukaryotic cell types and have been shown to participate in the various responses of cells to external stimuli (1). Recently, it was proposed that PTPases may have the potential to suppress the development and/or growth of tumor cells because of their ability to counteract the activity of PTKs ( 1). Evidence in support of this hypothesis includes the observation that the human PTPlB gene, when introduced into NIH 3T3 cells, suppressed neoplastic transformation by the neu (17) and the v-src oncogenes (18). In addition, Laforgia et al. have demonstrated that loss of one allele of the human hPTPy gene occurred in approximately one-half of the human cancer samples examined (19). There are now a number of studies which link over-expression and/ or amplification of the neu proto-oncogene to neoplastic transformation and development of human breast carcinomas. However, it remains unclear how over-expression of the P185"‘“ oncoprotein disturbs the balance between phosphorylation and dephosphorylation of specific cellular proteins, and how this intricate balance influences oncogenesis on the molecular level. In addition, while much progress has been made in the identification of PTPases and in determining the structure and enzymatic properties of PTPases, little is known about their mechanisms of action or regulation. Virtually nothing is known about the role of dephosphorylation catalyzed by PTPases, especially when compared to 56 what is known regarding the role of P185"“‘-PTK in human breast tumorigenesis. Hence, an understanding of the functions and interaction of P185"‘“-PTK and PTPases in normal and transformed cells will help to elucidate the role of these enzyme systems in normal cellular functions as well as in the pathogenesis of human breast cancer. 57 MATERIALS AND METHODS Cell lines. A benzopyrene induced, immortalized, but non-tumorigenic human breast epithelial cell line, 18435, was obtained from Dr. M. Stampfer (Livermore Laboratory, Berkeley, CA) (20). 18-Rn1, 18-Rn2 and 18-Hn1 cell lines (preparation discussed below) and 18435 cells were grown in MCDBl70, prepared by mixing equal amounts of minimum essential medium (MEM) (GIBCO, Grand Island, NY) and keratinocyte basal medium, 1'. e. modified MCDBlS3 (Clonetics Corp., San Diego,CA), supplemented with epithelial growth factor (EGF, 10 ng/ ml), insan (10 ug/ ml), transferrin (10 ug/ml),(Collaborative Research Inc. , Bedford, MA), hydrocortisone (0.5 rig/ml, Sigma Chemical Co., St.Louis, MO) and Gentamicin (5 pg/ml, GIBCO). MCF-7 and SK-BR-3 human breast carcinoma cell lines were obtained from American Type Culture Collection (Bethesda, MD). MCF-7 and SK-BR-3 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (GIBCO). All cells were incubated at 37°C in 5% C02. Animals. Female athymic nude mice (nu/ nu) and female Sprague—Dawley rats were obtained from Harlan Sprague-Dawley, Indianapolis, IN. All animals were maintained according to the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals. Retrovirus Infection. 18-Rn1 cells were derived by infecting 18435 cells with a mutationally activated replication-defective amphotropic retrovirus vector, JR-neu, containing a single amino acid substitution (Val664 to Glu664) in the transmembrane region. The rat neu oncogene was under the transcriptional control of the Moloney murine leukemia virus LTR. A neomycin resistance gene (neo') was used for selection. 58 The details for the construction of the JR-neu vector have been presented elsewhere (21). 18-Rn2 cells (obtained from Dr. C. Aylsworth, Michigan State University, E. Lansing MI) were derived by infecting 18435 cells with a mutationally activated rat neu oncogene (identical to that above) utilizing the retrovirus expression vector pSV2neuT (22). 18- Hnl cell line was derived by transfecting 18435 cells with a mutationally activated human neu (erbB-2) oncogene (7) and was the generous gift of Dr. J. Pierce (National Cancer Institute, Bethesda, MD). Induction of rat mammary carcinomas. The retrovirus vector described above (JR-neu) was infused once into the central duct of each mammary gland of 55 day old rats as described previously (21). To an additional group of rats, 7 , 12-dimethyl- benzanthracene (DMBA) (Upjohn Co., Kalamazoo, MI) was administered i.v. at a dose of 2 mg/ 100g body weight as described previously (23). Five weeks after neu infection or DMBA treatment, each rat had multiple mammary ductal carcinomas. The carcinomas were surgically excised, cleaned of connective tissues and subsequently prepared for RT-PCR analysis. Oligonucleotide Primers. All Oligonucleotide primers were synthesized in the Macromolecular Synthesis Facility, Michigan State University. The primers (including position in cDNA and the size of amplified fragment in bp) were as follows: 1. GAPDH (24), human CCATGGCACCGTCAAGGCTGAGAACG(228-253), CAATGCCAGC- CCCAGCGTCAAAGGT(964-939) 738bp, rat CCTGGCCAAGGTCATCCATG- ACAAC'ITI‘GG(501-530), CAATGCCAGCCCCAGCGTCAAAGGTG(928-903), 429bp; 2. LAR (13), human TCGAGCGCCTCAAAGCCAACG(4362-4382), GGCAGGCAC- CT CT GTGTGGCCG(5 191-5 170), 831bp, rat GAGGTGAACAAGCCCAAGAACCG- 59 CT AT(988- 1014) ,CGG'I'I‘CTTGAACTI‘GTI‘GCAGGGCAGGTIG( 1 876- 1 84% , 890bp; 3. P'I‘PlB (15), human CGGCCA’I’ITACCAGGATATCCGACA(130-154), TCAGC- CCCATCCGAAACTI‘CCTC(839-817) 711bp, rat TCGATAAGGCTGGGAAC- TGGGCGGCTA(155-181), CCGTCTGGATGAGCCCCATGCGGAA(916-892), 763bp; 4. TC-PTP (16), human GCTGGCAGCCGCTGTACTTGGAAAT(110-134), ACTAC- AGTGGATCACCGCAGGCCCA(687—663) 579bp; 5. CD45(25), human GCATC- CCGCGGGTGTICAGCAAGTI(2056—2080) ,TCCACTI‘TGTTCTCGGC'ITCCAAGC- (2719-2695) 665bp; 6. neu, rat TGCCCCATCAACTGCACCCACTCCTGT(1907-1933), TCCAGGTAGCTCATCCCCTI‘GGCAATC(2541-2515), 636bp; Primers for human neu were identical to those previously reported (26). GAPDH was used as an internal control to insure that amplification had occurred in each sample and to correct for differences in the amount of mRNA in each sample (27). RT—PCR analysis. Total RNA was isolated from cultured cells immediately before confluence and from freshly excised tumors derived from mice and rats (28), then RT-PCR was performed as previously described (29). Radioactive labeling was performed by addition of 1 pCi of (a-32P)dCTP (Dupont/NEN Research Products, Wilmington, DE) to each PCR reaction mixture. At the end of every five PCR cycles, one reaction was stopped and the amplified products were analyzed by 8 % native polyacrylamide gels and autoradiography. The amount of incorporated radioactivity of each gel band was determined by using a Betascope 603 blot analyzer (Betagen, Waltham, MA). Immunofluorescence analysis. Cells were seeded at 1 x 104 cells per chamber (8 chamber slides; Nunc, Inc. , Naperville, IL), incubated for 48 hours, then fixed with 60 100% methanol at -20°C. Anti-human c-erbB-2 monoclonal antibody was obtained from Oncogene Science, Inc. (U niondale, NY), anti-human LAR mAb 11:1A (30) and anti PTPlB-Mab AE4-2J (17) were generous gifts from Dr. M. Streuli (Dana-Farber Cancer Institute, Boston, MA) and Dr. D. Hill (Applied Biotechnology, Cambridge, MA), respectively. The second antibody for c-erbB-2, LAR and PTPlB was FITC conjugated goat anti-mouse IgG (Sigma). The amount of neu oncoprotein, LAR and PTPlB proteins was quantitated by measuring the fluorescence intensities of the samples on a single-cell basis using an ACAS 570 interactive laser cytometer (Meridian Instruments, Inc. , Okemos, MI). Southern and Northern blot analysis. DNA was extracted using sodium dodecylsulfate-proteinase K and was digested with HindIII or BamI-II, then run on 0.8% agarose gels and transferred to a nylon membrane (Schleicher & Schuell, Keene, NH). RNA was isolated using guanidine thiocyanate (28) and poly-A mRNA was isolated using a PolyATract mRNA Isolation System (Promega Co. , Madison WI). mRNA was separated on a 1% MOPS-formaldehyde agarose gel and transferred to a nylon membrane (Gene Screen, Dupont/NEN Research Products). GAPDH (73 8bp) and neu (622bp) probes were prepared by PCR of cDNA of 18-Hnl cells. The LAR (1306bp) and PTPlB (1273bp) probes were prepared by BamHI or HindIII digestion, respectively, of cloned cDNAs. Probes were labeled with a(32P)DCTP (Dupont/NEN Research Products) using a random primer labeling kit (United States Biochemical Co. , Cleveland OH). Flow cytometric DNA analysis. Bivariate flow cytometric measurement of BrdUrd incorporation and relative DNA content (propidium iodide staining) was 61 performed as previously described (31). Fluorescence intensity was determined with an Ortho cytofluorograph (Ortho Diagnostic Systems, Westwood, MA). Tumorigenicity assay. Tumorigenicity of cell lines was determined by the ability of cells to form tumors in mature female athymic nude mice. 5 x 106 cells of each cell line were inoculated s.c. into athymic nude mice. Mice were observed at weekly intervals and tumor diameters were Obtained by using a vemier caliper. The tumor volumes were calculated based on the formula V = 4/3rI-(A+B/4)3 in which A and B are two perpendicular diameters obtained from each tumor. 62 RESULTS Effect of neu introduction on 18435 cell proliferation in vitro. After introducing the activated rat neu oncogene cDNA into the immortalized human breast epithelial cell line, 18435, more than 1,000 G418 resistant colonies were observed on each 100 mm plate following a single exposure to 1 ml of JR-neu virus at 2 x 104 CFU/ ml. These colonies were pooled and designated cell line 18—Rn1. As a consequence of neu introduction, 18-Rnl and another neu transfected cell line, 18-Hn1, proliferated more rapidly compared to parental 18435 cells. The population doubling times for 18435, 18-Rn1 and 18-Hn1 were 28, 24 and 19 hrs, respectively. The neu transfection resulted in BrdUrd uptake of 30.3% and 38.9% in 18-Rn1 and 18-Hn1 cells, respectively, compared to 11.7% for 18435 cells. This was confirmed using propidium iodide staining, in which the percent of cells in S phase of the cell cycle was 30.2% and 46.5% in 18-Rn1 and 18-Hn1 cells, respectively, compared to 13.5% for 18435 cells. Thus, these data provide evidence that increased neu expression resulted in a substantial elevation of DNA synthesis and cell proliferation. Effect of neu introduction on tumorigenicity of 18435 cells in athymic nude mice. As a direct test of the transforming potential of the neu oncogene, the capability of neu transformed 18435 cells (18-Hn1 and l8-Rn1) to form tumors in athymic nude mice was determined. Both 18-Rn1 and l8-Hnl cells readily formed palpable tumors after s.c. inoculation (Fig. 1). 18-Rn1 cells formed tumors which initially grew progressively, followed by a period of slow growth; a number of these tumors ultimately regressed. 18—Hnl cells formed rapidly growing progressive tumors; no evidence of 63 1.6 v 18-Hn1 (n=17) A O 18-Rn1 (n=24) n — E 1.2 _ o 18485(n—12) J/ir Q) E 2 g 0.8 - k ‘6 r j/ 3 *- o.4 - 0.0 o 2 4 8 8 Time after implont (weeks) Fig. 1. Tumorigenicity of neu transformed 18435 human breast epithelial cells. Tumor growth of neu transformed 18-Hn1 (-v-) and 18-Rn1 (-O-) cells, and parental 18435 (-O-) cells was measured after s.c. inoculation of these cells into athymic nude mice. The number in parentheses is the number of inoculation sites for 18435 cells, or the number of tumors resulting from inoculation of 18-Hnl and 18-Rn1 cells. Data points are the mean tumor volume 1 SEM. 64 regression was observed in these tumors. Histological analysis revealed that all 18-Rnl tumors exhibited many characteristics of human breast carcinomas, including a high degree of nuclear polymorphism, central necrosis and cysts. Histological characteristics of 18-Hn1 tumors were Similar to l8-Rn1 tumors but lacking substantial cystic formation. No tumor development was observed from the parental 18435 cell line. Expression of neu and PTPases in 18435 cells and in neu transformed 18435 cells. Since virtually nothing is known about PTPases in either normal or neoplastic human breast epithelial cells, 4 different PTPases (LAR, PTPlB, TC-PTP and CD45) were studied by RT-PCR. Labeling with (o-32P)dCTP was used to determine the RT-PCR amplification efficiency and to semi-quantitatively compare amplified PCR products. The amount of radioactive amplified product produced for each PTPase, GAPDH, and neu was measured every 5 PCR cycles by scanning dried polyacrylamide gels using a Betascope radioanalytic system (Fig. 2). This allowed determination of the range in which amplification proceeded with similar efficiency. Since amplification generally reached the plateau range at more than 25 cycles, the data used for comparative analysis was at 25 cycles of amplification. Thus it was possible to compare the initial amounts of mRNA template by comparing the amounts of amplified PCR products. Based on this analysis, the expression of neu and 4 PTPases in 18—Rn1, 18-Hn1 and l8-Rn2 cells and the parental 18435 cells were compared using the amount of GAPDH as an internal control. A summary of these data is shown in Table 1. In order to compare cell lines, the expression levels of neu and the PTPases were standardized relative to 18435. Neu was elevated from 5 to 22 fold in the 3 independently 65 10 o GAPDH NEU LAR +: g 103 'U .0 b O. E» 102 E V 18—Hnl E O 18-Rn1 < , 0 18485 0/ 10 l a l l l a l l a l a l 10 20 so i 10 20 so 10 20 so Cycle number Fig. 2. Kinetics of RT-PCR amplification of GAPDH, neu and LAR in neu transformed 18435 human breast epithelial cells (18-Rn1, 18-Hn1) and parental 18435 cells. mRNA from 18435 (-O-), 18-Rnl (-O-) and 18-Hn1 (-v-) cells was transcribed and cDNA was amplified for the indicated number of cycles. (a-32P)dCTP radioactivity (cpm) incorporated into the amplified products is plotted against the number of PCR cycles. CPM were obtained by a Betascope 603 blot analyzer. neu . o . O LAR - i... Fig. 3. Expression of GAPDH, neu and LAR specific RT-PCR products from neu transformed 18435 human breast epithelial cells (18-Rnl, 18-Hn1, 18-Rn2) and parental 18435 cells. MCF-7 and SK-BR-3 human breast carcinoma cells served as positive and negative controls. PCR was performed in the presence of (a-32P)dCTP for 25 cycles and the gel separated products were analyzed using the Betascope blot analyzer. The sizes of the specific products are: GAPDH, 738 bp; neu, 266 bp; and LAR, 831 bp. 67 .358596 3:222: v 8 m we :88 2: 8a .565 £33 568..qu .P .3920 we commmoaxo as 8 Eva—cg» 203 33E Ba 53 .3 BR: .8685me a fio H N.o Nd H 0.0 ad H 06 Nd H _.N Frau: _.o H Nd m.o H _._ N.o H w; >4 H "MN mrmmtv—m 5.0 H 9m v; H 0.x _.n H 1.2 ad H o.n warm“ 9N H *6 UN H #6 wé H v.3 a; H N.NN :5er n._ H 9N 0.». H v.5 mé H N: Wm H aw 23-3 — M ~ ~ meow“ EGF mar—L Mai 3»: £00 ..szm H 58.5 Ba: 8 .888 Samoan mum Lo obs. can»? «OATS— ? Bum—58% 8:: :3 «Scamp—8 28.5 58:: >452 Ea Tam—gm 5 can 38 305:6 3.83 58:: mmvwfi 38.—£83 3»: 5 EOE 93 5E .53 e5 .3»: Lo commmoaxm ._ 29¢. 68 transformed 18435 cell lines (18-Rnl, 18-Hnl and 18-Rn2)(Table 1, Fig. 3). PTPase expression was increased in the 3 independently derived neu transformed 18435 cell lines, to similar levels, even though the expression of neu was much higher in 18-Hnl cells compared to 18-Rn1 and 18-Rn2 cells. Of the 4 PTPases examined, LAR expression increased the most (as 15 fold), followed by PTPlB expression (2 8 fold). TC-PTP expression was observed to increase slightly and CD45 was not expressed in these cells (data not shown). SK-BR-3 and MCF-7 are two well-known human breast carcinoma cell lines; substantial expression of PTPases was not observed in these cell lines despite the fact that one of these lines (SK-BR-3) has high neu expression. In an effort to determine whether or not a correlation exists between neu and PTPase expression and tumor growth (in athymic nude mice), we subjected total cellular RNA from 18-Rn1 and 18-Hn1 tumors to RT-PCR analysis. As shown in Fig. 4, expression levels of neu were substantially higher in the faster growing, more progressive 18-Hn1 tumors than in the slower growing 18-Rn1 tumors (Fig. 4, lane 5-9 vs. lanes 2- 3). LAR PTPase was observed to be elevated to about the same extent in both the rapidly growing tumors (18-Hnl) and the slower growing tumors (18-Rn1). PTPlB was elevated to a greater extent in 18-Hnl tumors than in 18-Rn1 tumors. TC-PTP was elevated comparably, in both the 18-Rn1 and 18-Hnl tumors. To assure that the mRNA expression of neu and PTPases detected using RT-PCR analysis infers the relative amount of mRNA in the cell lines studied, Northern blot analysis was performed. Since the mRNA expression of PTPases in these cell lines was extremely low, 500-1000 pg of total RNA from each cell line was required to generate sufficient mRNA to be detectable via 69 12 3 4 5 6 7 8 9 GAPDHO‘OOOOOOO “000.0000. PTP1B - TCPTP~6§‘.‘." Fig. 4. Expression of GAPDH, neu, LAR, PTPlB and TC-PTP specific RT-PCR products from neu transformed 18435 human breast epithelial cells (18-Rn1, 18-Hn1) and 18-Rn1 and 18-Hn1 human breast tumors. Lane 1, 18-Rn1 cells; lanes 2 and 3, 18-Rnl tumors; lane 4, 18-Hn1 cells; lanes 5-9, 18-Hnl tumors. PCR was performed in the presence of (a-32P)dCTP for 25 cycles and the gel separated products were analyzed using the Betascope blot analyzer. The sizes of the specific products are: GAPDH, 738 bp; neu, 266 bp; LAR, 831 bp; PTPlB, 711 bp; and TC-PTP, 579 bp. 70 Northern blot. The results were consistent with our RT-PCR data, i.e. the mRNA expression of LAR and PTPlB was elevated in the neu transformed cell lines compared with the parental 18435 cell line (data not shown). Expression of neu and PTPases in new and DMBA-induced rat mammary carcinomas. The expression of neu and PTPases was compared in rat mammary carcinomas induced in viva by neu infection or DMBA treatment (Fig. 5A and B). After RT-PCR analysis, neu expression was observed to be much higher in neu-induced rat mammary carcinomas than in DMBA-induced carcinomas. This shows that neu was successfully infected into rat mammary epithelial cells in situ. Importantly, LAR expression was also substantially higher in neu-induced rat mammary carcinomas compared to DMBA-induced carcinomas (Fig. 53). Furthermore, there was a strong correlation between the magnitude of the expression of neu and LAR (Fig. 53, insert). There was no observed difference in PTPlB expression when comparing neu- and DMBA-induced rat mammary carcinomas. Expression of P185'““, LAR and PTPlB protein in 18435 cells and in neu transformed 18435 cells. To assure that the relative levels of mRNA inferred by the above RT-PCR and Northern blot analysis were reflected in protein levels, immunofluorescent analysis was performed to determine P185"‘“, LAR and PTPlB protein expression in 18435, 18-Rnl and 18-Hn1 cells and SK-BR-3 and MCF-7 cells. Using respective primary antibodies and FITC second antibodies, the relative amounts of P185"“‘, LAR and PTPlB proteins in individual cells were determined as fluorescent units by using quantitative interactive laser cytometry. Our results show that P185’m‘ 71 Fig. 5. Expression of GAPDH, neu, LAR and PTPlB specific RT-PCR products from neu-induced and DMBA-induced rat mammary carcinomas. PCR was performed in the presence of (a-32P)dCTP for 25 cycles. A. Autoradiogram of GAPDH, neu, LAR and PTPlB specific RT-PCR products. Each lane represents an individual tumor. The sizes of these products are: GAPDH, 429bp; neu, 636bp; LAR, 890bp and PTPlB, 763bp. 3. Comparison of incorporated radioactivity (cpm) of each specific RT-PCR product measured using a Betascope blot analyzer. 3, insert. Relationship between incorporated radioactivity (cpm) of LAR and neu specific RT-PCR products in individual neu-induced rat mammary carcinomas. Regression correlation coefficient of line equals 0.834. 72 mrn_._.n_ ~24 :m c od _~_ rah fl—L\ m .m o m m o so; .0 O 1 .v w u x m bouncer/$20 0D I noozuElzmc I On... o( .m OOO 000.000.“. 12.3 I O U r O O O I I :o: D I I O O O O O O O M . ~73 tfllir 00000...- 0.5:: m m N H 2 a m A e m. a a N H pauach~N nevus-y an"... “ M erflBS: 49.5— -_" “' h.“ “ -.. . .:- . o :s'- .0... 7.. #1 l Blot Ab. oz-PY 115 Effect of LAR Introduction on Tumorigenicity of 18-Hn1 Cells in Athymic Nude Mice. The ability of five 18—Hnl-LAR cell lines (#4, 5, 7, 10 and 13) to form tumors in athymic nude mice was compared to l8-I-In1 cells transfected with the control plasmid alone. This study was conducted both without and with ZnSO4 addition to the diet (Table 2). Each cell line examined readily formed palpable tumors after s.c. inoculation into athymic nude mice regardless of the presence or absence of ZnSO4 treatment. Mean tumor weights and volumes were numerically smaller in each of 18- Hnl-LAR cell lines (#4, 5, 7, 10 and 13), compared to that observed. in 18-Hnl cells containing pTB-hyg plasmid alone. Significant (P<0.05) mean differences were observed when comparing controls (lS-Hnl-C) to cell lines lS-Hnl-LAR #4, 5, 7 and 10 in both conditions without and with ZnSO4 supplementation. 116 8.0!. .2 dove—=85 :8 “at“ 8.83 m -e 885.... 2.3 .352 .8... 82. 258.. ease as .9. 8.2.85 2.. 88 «5.2.88 2.. 22-8 . 8. H 8.3 .8. H 83 8 .8. H 8.3 8. H 82 8 283-288 .8. H 9.2 .8. H 83 8 .8. H 8.8 .8. H 8.3 2 28385.8 .:. H 8.8 .8. H 8.8 8 .8. H 83 .8. H 82 8 $5-288 .2. H and .8. H 9.2. 8 .8. H 8m... .8. H 83 8 £58.58 8. H 83 .8. H 80o em .8. H 82 .8. H 888 8 £58888 .8. H 5... .8. H 82 8 .2. H Ed .8. H 83 8 0.2.8.8 2053 mac—=3 2% H Mae 2% H w .8 2% H .5 2% H w 8 2:39, .583. £803 .885. 83:52 0833 885. £303 583- ..onfisz «on: :00 .85 .23 ea $5 “Samoa .22 8.2 .88.... 5 88 8385.8 2.. 8.0 ..:-8 8 828888 .N .38. 117 DISCUSSION The results of this study provide evidence for: (l) the construction of an effective LAR cDN A expression vector with an inducible promoter; (2) the effective introduction of the LAR gene into neu transformed human breast carcinoma cells (18-Hn1 cells) resulting in cell lines with a elevation in LAR expression; (3) a change in the morphological appearance (increased cellular size and cytoplasmic granularity) of the LAR transfected cells; and (4) a significant reduction in tumorigenicity of the LAR cells when inoculated into athymic nude mice. The human LAR cDN A expression vector (pTB-LAR) was constructed to incorporate both an inducible MT promoter region and a hygromycin resistance gene as a selection marker. The advantages of this inducible expression vector are as follows. First, the hygromycin resistance gene of PTB-LAR allowed the transfection of cells which had previously been transfected by a construct containing the neu oncogene and the neomycin resistance gene. Second, the expression level of LAR was at least partly under the control of an inducible MT promoter. This potentially facilitates a more precise control of the expression of LAR. Third, introduction of the human LAR cDNA into a LacZ based MCS allowed isolation of those colonies expressing LAR by choosing white colonies on X-Gal plates (using E. coli host IMlOQ based on a-complementation). Thus, the pTB-LAR expression vector facilitated the study of the interactions between LAR-PTPase and P185°°“-PTK activity in human breast cancer cell lines. Immunoblot analysis showed that the expression of LAR was increased in all of the 18-Hn1-LAR cell lines examined, compared to 18-Hn1 cells containing the control plasmid. Further elevation of LAR expression is detectable in 18-Hn1-LAR cell lines 118 after ZnSO4 treatment, while the presence of zinc had no effect on LAR expression in 18-Hn1 cells. These data provide evidence that the expression levels of LAR in 18-Hnl- LAR cell lines are inducible. Overexpression of LAR in 18-Hn1-LAR cells resulted in morphological changes when compared to either 18-Hn1 cells or 18-Hnl cells containing the pTB-hyg vector only. LAR overexpression increased the number of enlarged cells and increased the proportion of bi- and multi-nucleate cells. In addition, increased amounts of granules (or vesicles) were evident in the LAR transfected cells. The granules were located predominantly in the perinuclear cytoplasm. These observations are consistent with those found in other laboratories in which NIH3T3 and BHK cells showed similar morphological changes after transfection with the genes encoding the PI‘PlB PTPase (Woodford et al, 1992) or truncated TC-PTP PTPase (Cool et al, 1990, 1992). Overexpression of the carboxyl-terminal truncated TC—PTP in BHK cells resulted in cytokinetic failure and asynchronous nuclear division, leading to a multinucleate morphology (Cool et al, 1990, 1992). This suggests that the small proportion of bi- and multi-nucleate cells in the 18-Hn1-LAR clones may also have arisen from a failure of cell division as a result of overexpression of LAR. That the overexpression of LAR had no or little effect on cell growth rate in vitro, however, argues against a major defect in cell division. Alternatively, it is possible that the multi-nucleated cells were the result of differentiation of cells in response to overexpression of LAR, a phenomenon that had been described in keratinocytes (Zhao et al, 1992), neuronal cells (Aparicio et a1, 1992; den Hertog et al, 1993), and more frequently, in hematopoietic cells (Butler et al, 1990; Buzzi et al, 1992; Cohen et al, 1992; and Zafriri et al, 1993). These cells undergo 119 differentiation associated with increasing PTPase activities after treatment with maturational agents, such as lZ-O-tetradecanoylphorbol-l3-acetate (PMA) and dimethyl sulfoxide (DMSO) (Cohen et al, 1992; Butler et al, 1990, Buzzi et al, 1992), IL-6 (Zafriri et al, 1993), or after transfection of CD45 PTPase (Buzzi et al, 1992). In our studies, it will be of importance to further characterize multinucleate lB-Hn-LAR cells; such cells may represent a differentiated phenotype of cells with elevated expression of PTPases. It is necessary to consider the possibility that the morphological changes associated with the l8-I-In1-LAR cell lines may have been due to the effects of zinc cytotoxicity rather than induction of expression of an exogenous LAR gene. Among other properties, it is known that the divalent heavy metal zinc is a PTPase inhibitor (Wang et al, 1992; Tahiri-Jouti et al, 1992; Pot et al, 1991 and Itoh et a1, 1992). However, it has been demonstrated that both human and rat LAR PTPase activities were not inhibited by zinc at concentrations up to 1,000 FM in vitro, a concentration much higher than those used in our studies (Pot et al., 1991; Itoh et al, 1992). These morphological alterations were also observed in 18-I-In1-LAR cells that were not treated with zinc, albeit to lesser degree. Thus, this alternative explanation does not seem likely. Overexpression of LAR in 18-Hn1 cells resulted in significantly suppressed tumorigenicity of such cells when inoculated into athymic nude mice. This suppression occurred in spite of the fact that LAR overexpression did not affect cell growth rate in vitro. This apparent inconsistency may be interpreted as due to an increased frequency of cell-cell contact inhibition in vivo, rather than a direct inhibition of cell division (proliferation). Evidence to further support this explanation include the following. First, 120 increased LAR expression was observed as a consequence of increased cell density in both l8-Hnl and lS-Hnl-LAR cells suggesting that LAR expression in vitro was regulated in part by cell density. Second, LAR may play an important role in the regulation of cell growth via cell-cell contact due to the cell adhesion molecule (CAM) like structure on its extracellular domain (Streuli et al., 1988; 1990; 1992). This effect could occur in addition to, or in concert with, the role of LAR in the modulation of cellular phosphotyrosine. Third, Pallen et al. (1991) have demonstrated that membrane PTPase activity in Swiss 3T3 and normal fibroblasts was maintained at basal levels during cell proliferation but significantly increased in response to density-dependent cell contact arrest. Brautigan et al.(1991) reported similar results using monkey kidney epithelium CV-l cells. Furthermore, it has been shown that the treatment of cells with orthovanadate, a PTPase inhibitor, resulted in neoplastic transformation of the NRK-l cells which appeared not to be due to stimulated cell proliferation, but rather to the suppression of contact inhibition (Klarlund et al. , 1985). Based on these observations, it is proposed that the suppression of tumorigenicity resulting from LAR overexpression in lS-Hnl-LAR cells in vivo could, at least in part, be due to increased cell contact inhibition, a phenomenon that was absent or greatly reduced in vitro. Previous studies in our laboratory have indicated that the levels of endogenous expression of LAR and PTPlB PTPases in 18-Hn1 cells were not sufficient to confer a protein phosphorylation level as low as that observed in the non-neu transformed parental 184B5 cells (Zhai et al, 1993 and unpublished data). The constitutive activity of p185"“‘ protein tyrosine kinase in 18—Hn1 cells (as the consequence of introduction of activated neu oncogene) led to hyperphosphorylation of pl 85"“ and other substrates (eg. PLva) 121 in these cells and in 18-Hn1-LAR cells as well. In the current studies, immunoblot analysis shows that overexpression of LAR in l8-Hn1—LAR cells results in reduction of tyrosine phosphorylation in several cellular proteins, when compared to 18-Hn1 cells. It is conceivable that the decreased tyrosine phosphorylation observed in 18-Hnl-LAR cells (as a result of overexpression of LAR PTPase) could be the mechanism, at least in part, that explains the reduced tumorigenicity of these cells in athymic nude mice compared to control 18-Hn1 cells. It is not clear, whether LAR acts directly on p185"“‘-PTK, or on common substrates in the signaling pathway. A simple interpretation of these data would be that p185neu itself is the target substrate of LAR. It has been reported that LAR was able to dephosphorylate in vitro autophosphorylated EGFR (Hashimoto et al, 1992), a transmembrane PTK with extensive structural similarity to p185"“‘. Data from our Western blot analysis, however would not support this hypothesis, as the amounts of p185"“‘ proteins were essentially identical in 18-Hnl-LAR cells and l8-I-Inl cells despite the fact that the expression of LAR was increased in l8-Hnl-LAR cells. This would suggest that the suppressed tumorigenicity of lS-Hnl-LAR cells by LAR is not due to decreased p185"‘“ expression. Alteratively, LAR-PTPase and P185neu-PTK may not simply oppose each other’s action; rather, they may work in concert to maintain the balance of effector activation needed for the regulation of cell proliferation and differentiation. Thus, a second intriguing possibility exists that both p185"‘“ and LAR PTPase share common protein substrates. One of the substrates for p185"“‘ transforming potential appears to be phospholipase C-y (PLC-y) (Arteaga et al., 1991; Soderquist et al., 1992; Jallal et al., 1992 and Peles et al., 1991). Recent studies done by Rotin et al. 122 have demonstrated that the SH2 domain of PLC-y was able to prevent tyrosine dephosphorylation of the EGFR and other receptor linked PTKs by RP’TPy. This suggests that the PTPase and the SH2 domain of PLC-y compete for the same tyrosine phosphorylation sites in the carboxyl-terminal tail of the EGFR (Rotin et al. , 1992). Unpublished data from our laboratory suggests that LAR may form a complex with PLC- 7 in immunoprecipitation-immunoblot analysis, implying that both p185"“‘ and LAR PTPase may compete for binding to PLC-y, presumably through the SH2 domain. The regulation of PLCy by p185"‘“-PTK and LAR-PTPase, as well as the interaction between these proteins, could be a critical event in human breast cancinogenesis. Further examination of the pathways of p185"“‘ activity and its substrate dephosphorylation by LAR will no doubt contribute to defining the roles of these enzymes in the development of human breast cancer. A number of studies have explored the idea that the gene encoding LAR PTPase could be a potential candidate tumor suppressor gene because of its unique structure, tissue distribution and chromosome location (Fischer et al. , 1991; Saito and Streuli, 1991; Streuli et al., 1988; Krueger et al., 1990, 1992; Zhai et al., 1993). In contrast to certain PTPases, which have restricted tissue distribution, LAR is expressed widely in epithelial and endothelial cells of many different organs including the mammary gland (Streuli et al., 1988, 1992; Zhai et al., 1993). In addition, the overall structure of LAR, a cell adhesion molecule (CAM) homologous extracellular domain linked to two cytoplasmic PTPase domains would suggest that the activity of this enzyme may counteract the effects of certain PTKs as well as playing an important role in contact inhibition; either activity could inhibit the tumor cell growth process (Streuli et al. , 1988, 123 1989; Saito and Streuli, 1991). Furthermore, the extracellular region of LAR is structurally similar to the product of a putative colorectal tumor suppressor gene (DCC) (Streuli et al. , 1990). DCC has been shown to suffer allelic losses in approximately 40% of human breast carcinomas (Devilee et al., 1991). In addition, the human LAR gene has been mapped to chromosome 1P32-33 (Streuli et al., 1992), a region that contains a candidate breast carcinoma suppressor gene (Genuardi et al., 1989; Weinberg, 1991). These reports, as well as results from our previous studies showing that LAR is a p185"‘“-PTK responsive PTPase ( LAR became elevated in response to neu oncogene transfection), provide support for the concept that LAR may act as a tumor suppressor and that its activation or regulation could be critical in human breast cancinogenesis. 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