.25.}: , .. WV 33%. , ‘ . V . . . : .Ill..~.u.<.k :. . :1 :x.)).t llllili\\\\\\\l\\lllllll\\\\\\\\\\\\\\\\\ 31293 This is to certify that the thesis entitled DEVELOPMENT OF HUMAN CELL LINES CONTAINING CYTOCHROME P4501181 presented by Sheri Jeanine Batterman has been accepted towards fulfillment of the requirements for M.S. degree in Human NUtY‘ition awe. #4414} I Major professor William G. Helferich Date 11-12-93 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE ll 4]: ' lJ ulna-9.1 MSU Is An Affirmative Action/Equal Opportunity lnditution 7 mm DEVELOPMENT OF HUMAN CELL LINES CONTAINING CYTOCHROME P4SOIIBl BY Sheri Jeanine Batterman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1993 ABSTRACT DEVELOPMENT OF HUMAN CELL LINES CONTAINING CYTOCHROME P450IIBl BY Sheri Jeanine Batterman Cytochrome P-450 enzymes detoxify foreign chemicals in the body. Unfortunately, during detoxification, harmful intermediate compounds may be formed, some of which have been shown to bind to and mutate DNA, and therefore may play a major role as cancer initiators. This project was designed to develop a human cell line which could be used to evaluate the presence of mutagenic compounds in foods. Two plasmids PMTP4SOIIB1 and PMMTVP4SOIIB1, which encoded for cytochrome P—4SOIIBl (under the control of either a metallothionein or glucocorticoid promoter) and hygromycin resistance were constructed and transfected separately into MSU 1.1 cells. Hygromycin resistant cells were selected, established as cell lines, and tested. for' plasmid. integration, cytochrome .P-4SOIIBl expression and enzymatic activity. Both cell lines showed integration of the transfected plasmids into the genomic DNA. Expression of cytochrome P-4SOIIBl at the RNA level and the protein (enzymatic) level was undetectable. This thesis is dedicated to those at Luther College who inspired me, especially Wendy Stevens, who was influential in my decision to study nutrition, and also to Dr. John Neill at Pioneer Hi-bred International for his generosity and patience while teaching me many useful molecular biology techniques. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Bill Helferich for all his understanding and direction during my Master's degree program. I would also like to thank the other members of my committee, Dr. Ian Gray and Dr. Won Song for their guidance and suggestions“ IMuch.thanks goes to Dr. Christine Mehigh for her assistance with many of the laboratory techniques used in this project. A great deal of thanks is extended to Ann Ryan and Quingping Wang from the MSU Carcinogenesis laboratory. I would also like to extend my gratitude to Elizabeth Shipp for her generous help with the enzymatic assay, as well as the use of one of her figures for this manuscript. I would also like to extend my appreciation to Valerie Elias for her help with various lab procedures. Thanks 106 goes to my office-mate Ross Santell for all his kidding, humor and support for the past two years. A very special thanks goes to Dr. Juan Azcona-Olivera for his help with the RNA isolation, and the preparation of this manuscript, as well as his genuine concern, encouragement and support. Finally, I would like to thank others in the Food Science and Human Nutrition department for their assistance during my program. iv TABLE OF CONTENTS LIST OF FIGURES............. ...... ................... Vi LIST OF ABBREVIATIONS..... ..... ...................... viii INTRODUCTION......................................... 1 LITERATURE REVIEW.......... ................... ....... 4 Xenobiotic metabolism........................... 4 Cytochrome P—450................................ 5 Bioactivation................................... Induction....................................... Nomenclature for cytochrome P-450............... 12 Cytochrome P-4SOIIBl............................ Mutagenic Assays................................ 14 MSU 1.1 cells......... ........... . ...... ........ 18 Advantages of MSU 1.1........................... 20 MATERIALS AND METHODS. ............ . ........ .. ...... .. 22 Cell Culture.................................... 22 Plasmid Construction.. ...................... .... 22 Ligation....................... ...... ........... 24 Transfection..... ........... .... ............. ... 25 Hirt Lysates.................................... 26 Selection for hygromycin resistance............. 27 Southern Analysis............................... 28 Northern Analysis... ...... ...................... 30 Enzyme assays....... ............... ... ...... .... 31 RESULTS AND DISCUSSION..... .............. ............ 33 Transfection Assessment... ............ ... ..... .. 33 Southern Analysis.......... ............ ......... 35 Plasmids......................... ..... .......... 41 Northern Analysis......... ......... ............. 43 Fluorometric Assay... ..................... ...... 48 Recommendations.................... ........ ..... 49 SUMMARY AND CONCLUSIONS.............................. 50 LISTOFREFERENCESOOOOOOC......OOOOOOOOOOOOO0.0.0.... 53 V Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES The mechanism of action of P450 (Coon eta10’1992) O..........OOOOOOOOOOOO-OOOOOO 7 The detoxification and activation of aflatoxin B1 and benzo[a]pyrene (adapted from Cheeke and Shull, 1985; Bailey and Williams, 1993) ........................... 10 Induction of cytochrome P450 proteins by polycyclic aromatic hydrocarbons as mediated by the Aromatic Hydrocarbon Receptor (Denison, 1991) . ..... ...... ...... 13 Plasmid map of PMMTVP4SOIIBl . . . . . . ........ 23 Slot blot analysis of low molecular weight (plasmid) DNA from MSU 1.1 cells transfected with PMTP4SOIIB1 and PMMTVP4SOIIBl.. ..... .............. ........ 34 Southern Analysis of high molecular weight DNA from PMTP4SOIIB1 transfected MSU 1.1 cells hybridized with 1.9 Kb hygromycin resistance fragment ............ 36 Southern Analysis of high molecular weight DNA from untransfected MSU 1.1 cells and PMTP4SOIIBl transfected MSU 1.1 cells hybridized with SV 40A+ fragment ..... . .......... ........ .......... 39 Southern Analysis of high molecular weight DNA from three plates of PMMTVP4SOIIBl transfected MSU 1.1 cells hybridized with 1.9 Kb hygromycin resistance fragment ....................... 40 Northern Analysis of RNA from induced PMTP4SOIIB1 transfected MSU 1.1 cells, and induced PMMTVP4SOIIBl transfected MSU 1.1 cells hybridized with 2 Kb cytochrome P4501181 fragment .......... ... 45 vi Figure 10. Northern Analysis of RNA from induced PMTP450IIB1 transfected MSU 1.1 cells and 3 plates of induced PMMTVP4SOIIBl transfected MSU 1.1 cells hybridized withGAPDH ..... ..................... ...... 47 vii LIST OF ABBREVIATIONS AhR: aromatic hydrocarbon receptor AFBI: aflatoxin B1 depc water: diethyl pyrocarbonate water DMSO: dimethyl sulfoxide EDTA: ethylenediaminetetraacetic acid GAPDH: glyceraldehyde phosphate dehydrogenase GGP: glucose-G-phosphate GGPDH: glucose-6-phosphate dehydrogenase HEPES: N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid LB broth: Luria-Bertani broth HEM: modified Eagle's media PBS: phosphate buffered saline pMMTV: mouse mammary tumor virus promoter pMT: metallothionein promoter SDS: sodium dodecyl sulfate SSC: standard saline citrate TE: 10 mM Tris/1 mM EDTA Tris: tromethamine viii INTRODUCTION Humans are constantly exposed to foreign compounds either through. their' diet or from environmental exposure. Approximately 80% of all human cancers are linked to both dietary and environmental factors (Doll and Peto, 1981). A careful review of the Doll and Peto manuscript by Bailey and Williams (1993) indicated that 35% of all cancers are influenced by dietary factors. Some examples of these dietary factors linked to cancer are natural components of foods, products resulting from food processing and metabolites from microbial growth. Significant environmental factors include pollutants, as well as products of medical, agricultural and industrial practices (Doll and Peto, 1981). Many of these foreign.compounds (xenobiotics) which.enter the body, areiable to bind to DNA. This binding to DNA, if not repaired correctly, will mutate DNA, which increases the risk of cancer. Although numerous environmental and dietary contaminants may enter our bodies, there are many protective mechanisms ‘which. prevent ‘these contaminants from eliciting’ a toxic response such as DNA mutation. It has been demonstrated that minor dietary food constituents play a role in inhibiting or 2 preventing cancer through a variety of mechanisms. Some are blocking agents which prevent carcinogens from reaching or reacting with critical target sites. Others are suppressors which prevent evolution of neoplastic processes in cells that would otherwise become malignant (Wattenberg, 1992). Another example of a protective mechanism against contaminants is through metabolism. There are a variety of metabolic reactions in animals which function to detoxify and enhance the excretion of foreign substances from the body. One key reaction for metabolism and detoxification of foreign substances is the cytochrome P-450 system. The details of this system are explained in the literature review section. The overall goal of this project was to incorporate an easily inducible cytochrome P-450 metabolic system into a human cell line that could be used as a bioassay to evaluate which dietary contaminants and cytochrome P-450 isozymes are involved in the bioactivation of promutagenic compounds such as aflatoxin B1 (AFBQ and benzo[a]pyrene to mutagenic compounds. The specific objectives of the project were: (1) Subclone the cytochrome P-450IIB1 cDNA into an expression vector. (2) Expand the newly constructed plasmid and purify it. (3) Transfect MSU 1.1 cells with PMTP4SOIIBl and PMMTVP4SOIIBl plasmids and select stable clones. 3 (4) Confirm that the PMTP4SOIIBl (metallothionein promoter) and PMMTVP450IIBl (glucorcorticoid promoter) plasmids had been incorporated into the genomic DNA of MSU 1.1 cells. (5) Confirm that the cells lines express the full length, active cytochrome P4501IBl protein. LITERATURE REVIEW Xenobiotic metabolism. When foreign chemicals enter the body, they are subjected to various metabolic reactions. These metabolic processes are called biotransformation and usually involve both cytosolic and microsomal enzymes. In general, the overall purpose of biotransformation is to alter the parent xenobiotic to a less toxic form that is more polar, and more easily excreted from the body. There are numerous enzymes located in different cellular compartments which can biotransform xenobiotics in such a way as to make them more water soluble. These enzymatic reactions have been classified by pharmacologists and toxicologists as Phase I and Phase II metabolism (Dauterman, 1984; Hodgson and Dauterman, 1984; Nebert, 1979; Parke and Williams, 1969). Phase I metabolism is characterized by oxidation/reduction and hydrolysis reactions which play an important role in forming oxidative products or addition of hydroxyl groups. Phase II is involved in taking the foreign compound or Phase I-derived metabolites and linking it to an endogenous molecule to produce a polar conjugate. 5 Cytochrome P-450. The most important enzyme systems involved in Phase I reactions are the cytochrome P-450 containing monoxygenases. The cytochrome P-450 system is a complex membrane bound metabolic system that has been found to exist in most living organisms sudh as bacteria, yeasts, plants, insects, fish and mammals (Ioannides and Parke, 1990). In mammals, the cytochrome P-450 is found in nearly all tissues except striated muscle and erythrocytes (Guengerich, 1988). In humans, cytochrome P-450 appears to be concentrated at specific sites which are involved in protecting the body from the effects of foreign compounds (Parke and Williams, 1969). For example, significant.amounts.of cytochrome P-450 are found in internal organs such as the kidney, lung, small intestine, which function to excrete foreign compounds from the body. The highest concentration of cytochrome P-450 is located in the endoplasmic reticulum in the cells of the liver. The liver also functions to detoxify or get rid of xenobiotics from the body. It has also been shown that skin contains moderate amounts of cytochrome P-450. The skin covers such a large area on the human body, that the total amount of cytochrome P-450 contained within it has potential to greatly contribute to the overall biotransformation processes of the body (Klaassen et al., 1986). This is a fact of particular importance to us since human skin cells are used in this research project. 6 The cytochrome P-450 monooxygenases are complex multiple component systems requiring molecular oxygen and nicotinamide- adenine dinucleotide phosphate (NADPH). The cytochrome P-450 system is made up of the two enzymes: (1) NADPH cytochrome P- 450 reductase, and ( 2) a heme containing enzyme, cytochrome P- 450. . These enzymes are embedded in the phospholipid matrix of the endoplasmic reticulum. More than 75% of the reductase molecule is positioned above the lipid bilayer, while P-450 molecules are closely embedded in the endoplasmic reticulum membrane. Therefore electrons are easily transferred from NADPH via the reductase to cytochrome P-450 (Nebert, 1979, Guengerich, 1991). .After the transfer of electrons, one atom of molecular oxygen combines with the substrate (drug, xenobiotic or endogenous molecule such as a steroid) through a heme molecule, and the other atom of oxygen forms water (Guengerich, 1993). The oxygenated substrate then dissociates, regenerating an oxidized form of cytochrome P- 450. A general schematic diagram of this system is shown in Figure 1 (Coon et al., 1992). The oxygenated substrate is less harmful to the body and more easily eliminated. Thus, the ultimate function of this monooxygenase system or initial (Phase I) metabolism is to make foreign compounds more polar and therefore more water soluble so they can be readily metabolized by Phase II enzymes and excreted from the body (Dauterman, 1984; Parke and Williams, 1969). ROH RH Fe3* ' (Bonn-'33. (D (RH)F91’3;’e—:- R LOOH ® @ (Ro)(Fe-OH)3' (RH)Fe2* X R'H + LO 7, 2e- ‘tCD H20 _._.Ofiuoo can COHDMOAHAxODoo one .N ouzmam 58:. 6802086 .o 3833023 32an 883 a 58: 050596 .s \ O. i m .i m A F 969a 059586 n3=0n3o8 33366»: '7. c8890 58: 05028.6 .m 5655. n . o :00 \ _ /_\o/—V 3269i othvoenu nxoo . o o . 0 Li . x x O _ ZO_._.<0_u=XO._.mo 11 Induction. .Another feature of the cytochrome P-450 system is that it is induced or "turned on" by a wide variety of chemicals to increase its specific enzymatic activity. Certain chemicals can induce many isoforms of cytochrome P-450 at once, while suppressing other forms at the same time» The mechanism of induction of most of the cytochromes P-4505 is not well understood. Most induction processes are regulated at the transcriptional level, but it has also been observed that some chemicals have the ability to physically block degradation of certain P-4505. Also, some hepatic cytochrome P-4505 are under strong hormonal regulation.and.are changed by alterations in ‘the levels of androgens, which are also influenced by peptide hormones (Guengerich, 1988). The mechanism of induction of cytochrome P-4SOIA1 has been extensively worked out. Briefly, cytochrome P-4501A1 is induced by halogenated aromatic hydrocarbons such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) which are known to bind to the soluble, cytosolic receptor called the aromatic hydrocarbon receptor (AhR) . The ligand-receptor complex translocates into the cell nucleus. This ligand-receptor complex binds to enhancer sequences upstream of the cytochrome P-4SOIA1 gene and increases pre-mRNA transcription from the gene. This pre-mRNA is processed to the mature mRNA and transported out to the cytoplasm where it is translated into new P4SOIA1 protein. Increased amounts of newly synthesized cytochrome P-4501A1 ultimately increases metabolism. A 12 diagram of the mechanism of induction of cytochrome P—4SOIA1 can be seen in Figure 3 (Denison, 1991). Some examples of inducers which "turn on" the P-450 system, include phenobarbital (PB), 3-methylcholanthrene (3- MC), polychlorinated biphenyls (PCBs), isosafrole, isoniazid, ethanol and clofibrate; animals treated with these compounds show' several fold, increases in ‘various cytochrome 1P-450 isoforms in liver'microsomes (Ryan and.Levin, 1990). Overall, certain chemicals can induce the cytochrome P-450 system, which increases the rate of metabolism and elimination of the specific compounds from the body. Nomenclature for cytochrome P-450. To date, more than 200 distinct isoforms of cytochrome P-450 have been discovered (Coon et al., 1992; Nebert, 1993). Because there are so many cytochrome P-450s, the nomenclature used by researchers can be very confusing. Therefore, attempts have been made to categorize cytochromes based on their evolutionary relationships. Roman numerals placed directly after the P-450 represent distinct gene families. A family consists of those cytochrome P-450 who have protein sequences that are >40% homologous. Capital letters signify subfamilies which contain sequences that are >59% identical. Finally, Arabic numbers are used for individual genes (Nebert et al., 1989). As stated above, more than 200 cytochrome P-450 isoforms exist (Nelson et al., 1993). Many of these isoforms have not 13 NUCLEUS mood- / De and Activation toxitication —L Figure 3. Induction of cytochrome P450 proteins by polycyclic aromatic hydrocarbons as mediated by the Aromatic V lToxicitil \ J Metabolism ‘ ~ [New Polypeptides H. Translation I 7 i Increased Cytochrome P7450|Al Hydrocarbon Receptor (Denison, 1991). 14 been studied, characterized or even named yet. In this project we have specifically chosen to study cytochrome P- 4SOIIBl because it is one of the most well characterized isoforms with regard to its metabolic activity and detoxification of xenobiotics. Cytochrome P-4SOIIBI. The cytochrome P-4SOIIBl used in this study was from rat origin. This was chosen because humans are not known to have the IIB1 isoform of cytochrome P-450. Cytochrome P-4SOIIBl is known to be induced by phenobarbital, which occurs primarily through an increase in transcription rate in the corresponding genes. It has also been speculated that cytochrome P-4SOIIB1 is capable of catalyzing the monooxygenation of a wide variety of foreign organic chemicals such as plant toxins and pesticides (Juchau, 1990). Overall, as stated previously, cytochrome P-450 is a metabolic system which breaks down foreign compounds in order to eliminate them from the body. Unfortunately, in some instances, the metabolism of a foreign compound leaves harmful intermediates, which can damage or mutate DNA. Therefore it is critical to test foreign compounds or chemicals to see how mutagenic or damaging they are in a living system. Various assays have been designed for this purpose. Nutagenic Assays. The Salmonella/mammalian microsomal mutagenic assay developed by Ames et al. (1975) has been the 15 most widely used assay for research in the food industry as a means of determining the mutagenic potential of many environmental and dietary compounds (Maron and Ames, 1982). This assay, referred to as either the Ames assay or Ames test, is a bacterial based assay which uses a modified Salmonella strain (TA98, TA100). The modified strain has a mutation on the gene that codes for enzymes involved in the synthesis of histidine. Therefore, these Salmonella strains require histidine in order to grow. Salmonella are plated with minimal histidine to allow a lawn of bacteria. Only those bacteria that contain a mutation in the gene will revert back to wild type (reversion) and allow cell growth in the absence of histidine. Thus, only the Salmonella which have had DNA damage mediated by the test compound will form colonies. The greater the number of revertant colonies, the greater the mutagenic potential of the chemical (Hoffmann, 1982). Salmonella is a prokaryote, and therefore has many unique cellular characteristics such as: 1) no nucleus 2) no introns 3) permeable cell membrane 4) poor repair mechanisms. These characteristics show that Salmonella DNA is unprotected, which allows for it to be easily damaged, and may give rise to false positives in the Ames assay (Ames and.Gold, 1992). The accuracy of the.Ames assay has been questioned.due to the great number of conflicting results when compared to rodent in vivo carcinogenic assays. For example, chemicals that have been shown not to be carcinogenic in a number of 16 animal models, show up as being mutagenic in the Ames assay. It has been estimated that 50% of compounds testing positive in the Ames assay are not actually carcinogens (Ames and.Gold, 1990b). Additionally, compounds found to be non-mutagenic in the Ames assay are carcinogenic in rodent models (Ames and Gold, 1990a). Although the Ames assay has been used extensively to evaluate mutagenic potential of a variety of compounds, the assay' has certain limitations because it utilizes bacterial cells. Due to the limitations of the Ames assay, it was thought that a eukaryotic system may be a more accurate indicator of mutagenicity of foreign compounds. Eukaryotic systems have greater means of protection such as a strong cell wall and a nucleus, as well as efficient DNA.repair mechanisms, which.may be more analogous to repair mechanisms in humans. Systems utilizing the enzyme hypoxanthine phosphoribosyl-transferase (HPRT) are commonly used to determine mutagenicity of foreign compounds in eukaryotic cells. Briefly, the HPRT enzyme is coded for on the HPRT gene located on the X chromosome. Normal cells possessing HPRT activity will convert purine analogues substrates to their respective ribotides. When 6-thioguanine (6-TG) and 5- bromodeoxyuridine (5-BDURD) are used in the media and incubated with normal cells, death occurs. 'The reason for the cytotoxicity is because 6-TG and 5-BDURD are metabolized by the enzyme HPRT and these metabolites are incorporated into 17 the DNA. Incorporation of these nucleotides results in a high degree of DNA mutation and subsequent cell death. On the other hand, if the normal cells are incubated in media containing 6-TG and 5-BDURD with the addition of a mutagenic compound, the HPRT locus is mutated, which stops production the functional HPRT enzyme. If the HPRT enzyme is not functional, the 6-TG and 5-BDURD are not metabolized and can not be incorporated into DNA. Cell death does not occur, resulting in formation of colonies. The greater the mutagenicity of the compound being tested, the greater the number of colonies that will be formed (Albertini et al., 1982). Although the utilization of the HPRT gene within a cell line is a common method for testing mutagenicity of foreign compounds, it has an important limitation: this system only detects mutations on one gene (HPRT) or locus. Toxic chemicals have the potential to mutate a‘wide variety of genes in the human body, which may go unmeasured by this system. Although the Ames assay and the HPRT are two commonly used systems for evaluating mutagenic potential of foreign compounds, they both have limitations. These systems are not extremely accurate and they are not directly applicable to humans. Therefore a more accurate, reliable system is needed by researchers to better assess the potential of foreign compounds to alter DNA and cause cancer in humans. 18 The optimal cell system to test for mutagenic compounds should incorporate the following characteristics: 1) The cell system should be comprised of eukaroytic, mammalian cells, preferably human cells. 2) The cells should be easy to maintain in culture. 3) The cells need to have the ability to normally express cytochrome P-450 under certain growing conditions. 4) After coming into contact with mutagenic compounds, these cells should give a clearly visible, accurate indication of DNA damage in the form of a forward mutation. All of these characteristics are found in MSU 1.1 cells, which is why this particular cell line was chosen for the following study. MSU 1.1 cells. MSU 1.1 is a human fibroblastic cell line obtained from Dr. J. McCormick at the Carcinogenesis Laboratory at Michigan State University (McCormick, and Maher, 1981; McCormick and Maher, 1985; Hurlin et al., 1989). The MSU 1.1 cells arose from a diploid neonatal foreskin derived cell line transfected with a plasmid containing a v-myc oncogene and a selectable marker for neomycin resistance (Yang et al. , 1992) . These cells have fibroblastic morphology and a stable, near diploid karyotype composed of 45 chromosomes, including two marker chromosomes (Fry et al., 1990). Except for their infinite life span, MSU 1.1 cells do not possess traits commonly associated with tumor derived malignant cells. MSU 1.1 cells have normal, unaltered morphology, and they are 19 dependent.on.growth factors. .Additionally,1MSU 1.1 cells will not grow large colonies in soft agar and they will not form tumors when injected into the nude mouse model (Wilson et al., 1990). Like many other continuously growing cell lines, MSU 1.1 has low endogenous expression of cytochrome P-4505, thus they do not effectively metabolize xenobiotics (Doehmer et al., 1988). Therefore cytochrome P-4505 need to be added to this system in order to activate metabolism. This can be accomplished by using molecular biology techniques to construct a plasmid which contains specific cytochrome P-450 DNA coding sequences. After this plasmid is transfected into the cells, the cytochrome P-450 mRNA can be expressed, and translated into active protein. Expression of the cytochrome P-450 mRNA can be controlled by an inducible promoter, which is induced or "turned on" by the addition of certain chemicals. Once the MSU 1.1 cells are transfected with plasmids, such as those used in this study, potentially toxic compounds can be added to the cells. In the presence of mutagenic compounds, MSU 1.1 cells are transformed into rapidly growing cells which will form foci. These foci can easily be stained and counted. The number of foci formed should directly relate to the mutagenicity of the compound being tested. Foci that grow in soft agar, grow well in media without growth factors or form tumors after being injected into nude mice, are 20 thought to be malignant cells (McCormick and Maher; 1988, Yang et al., 1992). Advantages of M80 1.1. The development of a mammalian based mutagenic assay system such as the MSU 1.1 cell line has several advantages over both the Ames assay and the HPRT system. Often times the Ames assay results in false positives because the Salmonella bacteria have weak cell membranes and they lack a nucleus, which makes them extremely susceptible to mutagens. MSU 1.1 is a eukaroytic system, which means each cell has a nucleus, a strong cell membrane, and its genes have introns, all of which.protect the DNA from the harmful effects of xenobiotics. Eukaroytic cells such as MSU 1.1 have better mechanisms by which to repair DNA than prokaryotes. Therefore, a toxic compound in the MSU 1.1 cell would have to pass through the plasma membrane, nuclear membrane, interact with chromatin structure and escape repair mechanisms before any effect could be observed in foci formation. Such defense mechanisms against xenobiotics exhibited by the MSU 1. 1 system would mimic those occurring in other eukaryotic systems such as animals and humans. The transfected MSU 1.1 cell line is also advantageous over the HPRT system because it does not require a toxic compound to damage only one specific gene, as in the HPRT system. The DNA within the MSU 1.1 cells can potentially be damaged at numerous undesignated places and the effects can 21 still be detected by the number of foci formed. Overall, the data obtained from the MSU 1.1 cells would be analogous to the detoxification system in the human body. MATERIALS AND METHODS Cell Culture. MSU 1.1 cells were routinely grown in modified Eagle's medium (MEM) (Sigma, St. Louis, MO) prepared with 10 % supplemented, defined bovine serum (Hyclone, Logan, Utah), 5 units/m1 penicillin] 5 ug/ml streptomycin (Sigma), and 5 ug/ml hydrocortisone (Sigma). Cells were maintained at 37°C in.a humidified incubator with 5% C0,. Cells were examined on a daily basis. Media were replaced every 48 hours, and when cells were confluent, the cultures were split. Plasmid Construction. Plasmid PMMTVP450IIB1 (Figure 4) construction was done in the following manner: pSP450 oligo plasmid was cut with restriction enzymes NheI and SmaI to release a 2 Kb insert which contains the complete cytochrome P-4SOIIB1 cDNA sequence. After the plasmid was cut, it was run on an agarose gel to separate the bands. The band in the 2 kb region was removed and purified. Purification was done as follows: The agarose containing the band was put into a 1 ml syringe with a yellow pipet tip affixed to the end. The contents were pushed through into a 1.5 ml eppendorf tube and 200 pl TE was also pushed through to remove :remaining'.agarose. ‘Next, 0.5 ml of water saturated 22 23 <88 I 0.00 si 1 0.80 M A am H' 1.00 P450 "31 ‘“ pMMTVP450Il81 ;; é 2: 3,, 9.20 Kb ,. 9M '2; 5 "'7'" Barn H! 2.90 AMP Not I 5.40 Figure 4. Plasmid map of pMMTVP4SOIIBl The solid black area of the map is the 2000 Kb base pair insert (cytochrome P450 portion) from the pSP P450 oligo vector. The hyg portion represents the hygromycin B resistant .gene, which allows for the selection of transfected cells. The AMP area represents the ampicillin resistant gene, which allows for this plasmid to be grown up in E. Coli. The pMMTV portion is the glucocorticoid promoter which turns on expression of cytochrome P-450 with the addition of glucocorticoids to the cell media. The other plasmid used, called PMTP450IIB1 is identical to the.above figure, except it has a PMT promoter (which turns on expression of cytochromes with the addition of metals) in place of the PMMTV. 24 phenol (equilibrated in 0.1 M TE) was added, the tube was vortexed, and put into the -80°C freezer overnight. The following day, the tube was centrifuged at 12 K x g for 10 minutes“ 'The top aqueous layer was collected and.divided into 2 eppendorf tubes. Then 0.5 ml phenol/chloroform (50:50 v/v) was added to each tube, vortexed and centrifuged at 12 K x g for 3 minutes. The top layer was collected and ethanol- precipitated. Tubes were centrifuged at 12 K x g for 10 minutes and then washed with 70 % ethanol. After the wash, the tubes were dried in a Spin Vac (Savant, Farmingdale, NY), resuspended in water and quantitated at Am” on spectrophotometer (Cary 3E Varian Instruments, Sunnyvale, CA). The cytochrome P-45011B1 NheI and SmaI 2 kb insert was ligated into PMMTV-hyg vector (gift from Dr. J. McCormick). This ligation was performed by cutting the PMMTV-hyg vector with NheI and NaeI enzymes to generate ends which were compatable with the P45011B1 insert. Briefly, this ligation was possible due to the fact that the cytochrome P-45011B1 2 kb NheI end attaches to the NheI site of the PMMTV-hyg vector. The SmaI and NaeI generate blunt ends, which attach during the ligation procedure. Ligation. The ligation of the cytochrome P-4SOIIBI 2 kb NheI and SmaI insert to the PMMTV-hyg vector involved using: 500 ng P4501131 insert and 200 ng PMMTV vector were mixed in 10.5 ul deionized water and heated at 65°C for 5 minutes. This 25 generated a 10:1 molar ratio of insert to vector. Then 1.5 ul 10x ligation buffer (0.66 M Tris pH 7.5, 50 mM MgCl2 and 50 mM dithiothreitol) , 1 pl 2 mM ATP and 1 ul T4 ligase (5 units/ml) (Boehringer Mannheim, Indianapolis, IN) were added. This mixture was mixed and incubated at room temperature for 16 hours. The newly sub-cloned PMMTV-P45011B1 expression vector was then transformed into competent Escherichia coli cells (DHS-a) (Seidman, 1989) . The bacterial cells were then propagated overnight in Luria-Bertani (LB) broth at 37°C with vigorous shaking. The plasmid was purified using the alkaline lysis/ cesium chloride gradient procedure (Maniatis et al., 1989). Construction of the other plasmid PMTP45011B1 used in this study, was done previously by Dr. C.S. Mehigh. The PMTP45011B1 plasmid was also propagated overnight in LB broth, but it was purified using Nucleobond Ax columns (The Nest Group, Southboro, MA). Transfection. Transfection of the MSU 1.1 cells was done using the polybrene/dimethyl sulfoxide (DMSO) (Sigma) method (Morgan et al., 1985). Twenty-four hours before the transfection, MSU 1.1 cells were plated at 2 x 10’ cells/100 mm diameter plate. The following day, normal growth media was removed from the plates. It was replaced with transfection media which consisted of 2 ml of MEM containing 30 ug/ml polybrene (hexadimethrine bromide) and 2 pg of plasmid DNA 26 (linearized with Not 1). Plates were incubated at 37°C for 6 hr. in a 5 % CO2 humidified environment. Plates were gently agitated each hour during the 6 hour incubation to insure even dispersion of transfection media. Control transfection plate was prepared in the same manner, except no DNA was added to cells. Following the incubation, transfection media was removed and the cells were subjected to a 4 minute shock treatment using MEM containing 30 % DMSO (freshly prepared) (Sigma). Cells were then rinsed twice with 0.15 M phosphate buffered saline (PBS) pH 7.4, and normal growth media was replaced. Hirt Lysates. Forty-eight hours after transfection, Hirt lysates (Hirt, 1967) were collected in order to do a slot blot. Media were removed from the plates and cells were rinsed three times with PBS. One ml Hirt lysis buffer (10 mM Tris pH 7.5, 10 mM EDTA, 0.6% SDS) was added to each 100 mm plate of cells. Twenty minutes later, 250 pl 5 M NaCl was added to plates. Cells were then scraped from plates, pipetted into 1.5 ml eppendorf tubes and incubated at 4°C for 16 hours. The tubes were then centrifuged at 12 K x g for 20 minutes at 4°C. The supernatant was collected and extracted once with phenol/chloroform (50:50 v/v), followed by chloroform/isoamyl alcohol (24:1 v/v) extraction. The aqueous phase was collected, ethanol-precipitated, and centrifuged at 27 12 King for 10 minutesc The resulting pellet which contained the low molecular weight DNA, was resuspended in 50 pl water. Varying amounts of the low molecular weight DNA, along with 20 pl each of NaOH (2 M) and ammonium acetate (2 M) were added to the slot blotter (Schleicher and Schuel, Keene, NH) which contained. Hybond. N, a non charged nylon. membrane (Amersham, Arlington Heights, IL) The blot was probed with a 32P (Dupont, Boston, MA) random prime labeled (Feinberg and Vogelstein, 1983) 1.9 Kb fragment which expresses hygromycin resistance from the PMMTVP45011B1 plasmid. Varying amounts of the entire original PMMTVP4SOIIBI vector were also used as positive controls and for quantitation purposes. Selection for hygromycin resistance. Transfected MSU 1.1 cells were allowed to grow for 72-96 hours. The cells were then split 1:5 into 100 mm diameter plates. After the cells had attached to the plates (z 5 hrs.), hygromycin B (Sigma) was added to the media for a final concentration of 300 pg/ml. Throughout the rest of this thesis, hygromycin B will simply be referred to as hygromycin. Fresh media and hygromycin were replaced on the plates every three days. After 12—15 days, plates were carefully checked for the presence of hygromycin resistant cell colonies. Hygromycin resistant colonies that were found were moved to 35 mm plates. Moving the colonies consisted of removing the media and wiping an area (z 3 cm. in diameter) around the 28 colony with a sterile cotton swab. Approximately 20 pl of 1x trypsin (Sigma) was pipetted directly onto the cluster, and left there for 30-45 seconds, or until the cells were round in shape instead of elongated. After the trypsin was removed, the cells were quickly resuspended in 50 p1 MEM and transferred to 35 mm plates. Cells were grown in selective media containing 300 pg/ml hygromycin (final concentration) until they were confluent, upon which they were transferred.to 60 mm plates and finally to 100 mm plates. The total amount of selection time in which hygromycin was in the media was approximately 3-5 weeks. Cells from several 100 mm plates were frozen in liquid nitrogen as described below. Freezing cells consisted of removing media from the plates and placing 3 ml 1x trypsin directly onto the 100 mm plate. After approximately 45 seconds, when the cells became rounded, trypsin was removed, and the cells were resuspended in 1 ml of MEM containing 10% DMSO, 10 % supplemented defined bovine serum, 5 units/ml penicillin/ 5 pg/ml streptomycin and 5 pg/ml hydrocortisone. The 1 ml cell suspension was pipetted into a sterile cryogenic vial (Corning, Corning, NY) and placed in the -20°C freezer for 1 hour. The vial of cells was then placed in the -80°C freezer for 15 minutes and finally submerged into liquid nitrogen storage freezer. Southern Analysis. Genomic (high molecular weight) DNA was extracted from transfected and non transfected cells by 29 dissolving them in a lysis buffer (10 mM tris pH 7.5, 50 mM EDTA, .1% SDS and 50 pg/ml proteinase K), and incubated overnight at 30°C (Maniatis et al., 1989). Solubilized DNA was extracted twice with phenol and once with chloroform/isoamyl alcohol (24:1 v/v) and dialyzed overnight at 4°C against 2 liters of 10 mM Tris/1 mM EDTA (TE). DNA yield was quantitated at A260 on a spectrophotometer. Approximately 15 pg DNA was cut with restriction enzymes, separated on.a 0.7% agarose gel and.transferred to non-charged nylon membrane (Hybond N-Amersham) using standard procedures (Maniatis et al., 1989). Three Southern blots were done using this genomic DNA. Two Southerns, were hybridized with a 32P random prime labeled (Boehringer Mannheim) 1.9 Kb fragment which expresses hygromycin resistance from the original PMMTVP45011B1 plasmid. The third blot was probed with a 32P random prime labeled SV 40 A+ region of the PMMTVP4501181 plasmid. The prehybridization of the blot was done using 50% formamide, 3x SSC, 5x Denhardts (Maniatis et al., 1989) and 50 mM phosphate buffer. The hybridization was done in 50 % formamide, 3x SSC, 5 % dextran sulfate, 50 mM phosphate buffer, 5x Denhardts and .1% SDS. After the hybridization, the membrane was washed using a high stringency wash: 2x SSC, .1 % SDS for 1 hr. at 65°C and .1x SSC, .1% SDS for 15 minutes at 65 °C. The membrane was exposed to x-ray film (X-OMAT AR, 30 Eastman Kodak Co. Rochester, NY) with two intensifying screens at -80°C. Nerthern Analysis. RNA expression from either the MMTV or MT promoter was induced 24 hours before harvesting the cells. This was accomplished through the addition of 75 pm ZnSo, (Sigma) to the PMTIIB1P450 transfected cells, and by the addition of 2.55 pH dexamethasone (Sigma) to the PMMTVIIBIP450 transfected cells. Total RNA was isolated by a single step method (Chomczynski and Sacchi, 1987) using the RNA STAT-60 kit (Tel Test-"B", INC. Friendswood, TX). Cells were scraped from plates, and centrifuged at 1000 X‘g for 6 minutes. The pellet ‘was resuspended in a