[12]
V79 CELLSEXPRESSINGCYTOCHROMEP450
117
[12] V79 C h i n e s e H a m s t e r Cells G e n e t i c a l l y E n g i n e e r e d for Stable E x p r e s s i o n of C y t o c h r o m e s P 4 5 0 B y JOHANNES DOEHMER
and FRANZ OESCH
Introduction Cultivated mammalian cells genetically engineered for stable expression of cytochromes P450 and other xenobiotic metabolizing enzymes will serve as an efficient analytical tool in toxicology and pharmacology. 1-8The usefulness and applicability of such cell lines for mutagenicity and drug metabolism studies depends on cultivation conditions, growth parameters, karyotype stability, cellular integrity during cultivation, and stability and level of cytochrome P450 activity. Therefore, an experimental strategy aimed at the construction of a constantly cytochrome P450-expressing cell line involves careful and critical consideration of various aspects, and especially some experience in handling cells and genes. The main aspects that need consideration are (1) the choice of eukaryotic expression vector, (2) the way the cytochrome P450 cDNA is inserted into the vector, (3) the choice of mammalian cell serving as a recipient for the cytochrome P450 recombinant eukaryotic expression vector, and (4) a quick means to determine and to follow cytochrome P450 expression in a culture at the cellular level. Materials and Methods
Our experimental strategy for the construction of stable cytochrome P450-expressing cells involves the following steps. Step 1: Cloning of Cytochrome P450 Full-Length cDNA. Cloning inl H. Autrup, Carcinogenesis 11, 707 (1990). 2 N. Battula, J. Biol. Chem. 264, 2991 (1989). 3 C. L. Crespi, D. T. Steim¢l, T. Aoyama, H. V. Gelboin, and F. Gonzalez, Mol. Carcinog. 3, 5 0 9 9 0 ) .
4 R. L. Davies, C. L. Crespi, K. Rudo, R. T. Turner, and R. Langenbach, Carcinog. 10, 885 (1989). 5 S. K. Hansen, J. A. Ross, J. M. Siegfried, S. Leavitt, K. Rudo, R. Langenbach, and S. Nesnow, Mol. Carcinog. 2, 261 (1989). 6 L. H. Thompson, Environ. Mol. Mutagen. 14, 264 (1989). 7 j. Doehmer, S. Dogra, T. Friedberg, S. Monior, M. Adesnik, H. R. Glatt, and F. Oesch, Proc. Natl. Acad. Sci. U.S.A. 85, 5769 (1988). 8 S. Dogra, J. Doehmer, H. R. Glatt, H. M61ders, P. Siegert, T. Friodberg, A. Seidel, and F. Oesch, Mol. Pharmacol. 37, 608 (1990).
METHODS IN ENZYMOLOOY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
HETEROLOGOUS EXPRESSION
[ 12]
volves the generation of a eDNA library with expression of the cDNA in the bacterial host in case an antibody is to be used to search for the wanted cDNA, and without expression in case an oligonucleotide can be used. The procedure routinely applied in our laboratory 8 has been developed by Keith Stanley and co-workers. 9 In order to obtain full-length cDNA, an oligo(dT)-primed eDNA library is screened. Missing 5' ends may be searched for among shorter and randomly primed eDNA overlapping with the oligo(dT)-primed cDNA in order to construct a full-length cDNA. ~° Usually, it is of advantage, if the full-length eDNA has retained its own polyadenylation signal. Step 2: Eukaryotic Expression Vector. The full-length cDNA has to be provided with expression signals by recombination with an expression vector. The stability and level of expression of a cDNA may depend on the choice of expression vector, the construction of the recombinant expression vector, and the kind of cell functioning as the recipient in the DNA-mediated gene transfer. Based on previous experience, some assumptions can be made. For example, the thymidine kinase promoter of herpes virus (TK promoter) usually resulted in low levels of expression, whereas the metallothionein promoter, cytomegalovirus promoter, SV40 early promoter, or retroviral LTR promoters yielded high levels of expression. In our hands, a recombinant plasmid containing the simian virus 40 (SV40) early promoter reading into the eDNA encoding cytochrome P450IIB1, 7 cytochrome P450IA1, 8 and cytochrome P450IA21° was sufficient to yield high and stable expression in V79 Chinese hamster cells. The original recombinant plasmid, p S V 4 5 0 , 7 is now being routinely used as a master plasmid for the construction of all other cytochromes P450 eDNA to be expressed in V79 cells by substituting the cytochrome P450IIB1 eDNA. 8,1° Viral expression vectors may have some advantages. The circular DNA of bovine papilloma virus (BPV) is episomally maintained in a cell at a high copy number. However, it has frequently been observed that BPV recombinant vectors are unstable and lose inserted cDNA. 1~Recombinant retroviral vectors as infectious particles can be used to transfer eDNAs at 100% efficiency as each cell can be infected, and the eDNA is inserted into the chromosomal DNA without loss of sequences via the retroviral enzymatic insertion mechanism. 2 However, obtaining infectious recombi9 H. Haymede, J. Herz, M. Bressan, R. Frank, and K. Stanley, Nucleic Acids Res. 14, 8615 (1986). l0 C. W61fel, K. L. Platt, S. Dogra, H. R. Glatt, F. W~ichter, andJ. Doehmer, Mol. Carcinog., in press (1991). 11 M. Edigkaufer, S. Dogra, F. Oesch, and J. Doehmer, in "Cytochrome P-450: Biochemistry and Biophysics" (I. Schuster, ed.), p. 560. Taylor & Francis, London, 1989.
[12]
V79 CELLSEXPRESSINGCYTOCHROMEP450
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nant retroviral particles requires insertion of the cDNA into the retroviral genomic DNA followed by DNA transfer into a packaging cell line. This may turn out to be cumbersome, as the insertion of cDNA into retroviral genomic DNA may interfere with the functional organization of the retroviral genomic DNA. Furthermore, the intact insertion of the recombinant retroviral genomic DNA into the chromosomal DNA of the packaging cell line is an essential prerequisite for obtaining recombinant retroviral particles and may occur at an extremely low frequency. Using the classic Ca/P coprecipitation technique may still be the fastest and most efficient way to get a stable cell clone.12 Step 3: DNA-Mediated Gene Transfer. For gene transfer, preparation of the recombinant vector DNA and the choice of recipient cell are the most important points to consider. The classic Ca/P coprecipitation technique is routinely used.~2 The recombinant vector DNA is linearized with a unique cutting restriction enzyme, whenever possible, that leaves stretches of DNA on either end of the cDNA construct for protection against exonucleases in the recipient cell. This increases the chance for integration of an intact functional cDNA into the chromosomal DNA of the recipient cell. V79 Chinese hamster cells are chosen to serve as recipient cells. These cells are derived from lung fibroblasts. 13 Usually, 20 ttg of recombinant cytochrome P450 vector DNA is mixed for coprecipitation with 1/xg of recombinant vector DNA containing the neomycin phosphotransferase gene under control of the metallothionein promoter. DNA is mixed in 1.0 ml of HEPES-buffered saline (137 mM NaC1, 6 mM dextrose, 5 mM KC1, 0.7 mM Na2HPO4, 20 mM HEPES, adjusted to pH 6.95-7.05 with 0.5 M NaOH). The DNA is precipitated by the addition of 52.6/zl of 2.5 M CaCI2 (final concentration 125 mM), and the mixture is left at room temperature for 25 min. The DNA precipitate is added to 1 x 10 6 V79 cells in a 60-mm petri dish. Best results are obtained with a fine rather than a bulky or cloudy precipitate. After a 4-hr incubation, cells are subjected to treatment with 15% (v/v) glycerol in Dulbecco's modified Eagle's medium (DMEM) for 2 min ("glycerol shock"), washed twice gently with fresh medium, and refed with 10 ml of growth medium. After overnight incubation, cells are split 1 : 5 in 60-mm petri dishes. The neomycin derivative G418 is added to the medium for selection on the third day after transfection, at a concentration of 800 /~g/ml of growth medium, because it takes at least two mitotic rounds to get a reasonable number of clones that have the transferred DNA stably integrated into their chromosomal DNA. G418-resistant colonies 12 F. Graham and A. van der Eb, J. Virol. 52, 456 (1973). 13 E. H. Y. Chu and H. V. Mailing, Proc. Natl. Acad. Sci. U.S.A. 61, 1306 (1968).
120
HETEROLOGOUSEXPRESSION
[12]
appear I0 days after transfection and are picked 3 weeks later by cloning cylinders and grown in mass culture for further studies. Special attention is given to the morphological appearance of the cell clones. Only those clones with a mitotic index of at least 5%, and in which the shape of the cell and nucleus resembled that of the parental V79 cell most, are picked. Step 4: Characterization of Selected Cell Clones. G418-resistant colonies are expanded to mass culture over a period of 2 to 3 months in order to obtain enough material for Southern, Northern, and Western blotting by standard techniques. These analyses at the DNA, RNA, and protein level demonstrate that the cDNA is stably integrated into the chromosomal DNA, that the cDNA is expressed, and that the cDNA-encoded mRNA yields an authentic protein as compared to in vivo material. Over the course of cell propagation an aliquot of 2 to 5 x 104 cells of each selected clone are checked weekly for expression by immunofluorescence staining as a quick means to determine expression level, homogeneity of expression, and stability of expression. Cells are grown on special glass plates in small chambers (Chamber Slides, Nunc, Inc., Naperville, IL). The staining procedure involves a wash with phosphatebuffered saline (PBS; 15 mM sodium phosphate pH 7.4, 0.15 M NaC1), fixation in a cold ( - 2 0 °) mixture of acetone/methanol (I:1) for 5 to 10 min, followed by drying at room temperature for 5 min. Fixed cells are incubated in 400/~1 of DMEM plus 10% fetal calf serum (FCS) containing 5/~g//zl of a cytochrome P450-specific monoclonal antibody for 35 min at room temperature in a humidified chamber. Excess first antibody is removed by three cycles of washing in water for 10 min each cycle with slow shaking. Thereafter, glass plates are incubated in 400/~1 DMEM plus 10% FCS containing goat anti-mouse IgG coupled to Texas Red or fluorescein isothiocyante (FITC) at a dilution of 1 : 30 or 1 : 50 for 35 min at room temperature in a humidified chamber. Excess second antibody is removed as described above. Cells are embedded in a solution of 20% (v/v) Mowiol (Hoechst AG, Frankfurt, Germany), 3% (v/v) glycerol in PBS and carefully covered by sliding a coverslip over the cells, avoiding air bubbles. The solution becomes solid by polymerization within 1 hr. Embedded cells are kept in the dark and are observed and photographed within a week.
Results and Comments Here, emphasis is placed on the construction of stably cytochrome P450-expressing V79-derived cell lines. Cell lines expressing rat cytochrome P450IIB1 and rat cytochrome P450IA1 have been validated in
[12]
V79 CELLSEXPRESSINGCYTOCHROMEP450
121
cytotoxicity, mutagenicity, and metabolism studies, s,14-16V79-derived cell lines stably expressing rat cytochrome P450IA2 have been established recently. ~° Cells of the V79 Chinese hamster cell line have been used for mutagenicity testing for more than 20 years, because they grow fast (doubling time 12 hr) and have a high cloning efficiency (better than 90%), stable karyotype, and a convenient marker gene for mutagenicity studies (the HPRT locus on a single X chromosome). However, V79 cells either do not express or express cytochromes P450 at an extremely low levels.17 It became obvious that lack of cytochrome P450 activity in parental V79 cells presented a unique situation as the V79-derived cell lines are defined for the genetically engineered cytochrome P450 isoenzyme, which makes these cell lines a valuable analytical tool in toxicological and pharmacological studies. Expression of the apoprotein is not sufficient for enzymatic activity, however, as the protein has to be inserted into the membrane of the endoplasmic reticulum, has to complex with a heme group, and has to be associated with the cytochrome-P450 reductase in order to become operative. Picking V79 cells as the recipient for cytochrome P450 cDNA was largely a matter of luck, because the amount of cytochrome-P450 reductase was sufficient, even though the level of cyrochrome-P450 reductase is the lowest compared to 20 other cell lines.~8 In addition, enough heme is made in V79 cells, and there is enough endoplasmic reticulum to anchor the genetically engineered cytochrome P450 isoenzyme (Fig. 1). The cytochrome P450 content in SD1 cells was estimated by Western blotting to be 50 pmol/107 cells. ~5 The application of Ca/P coprecipitation for DNA-mediated gene transfer implies some uncertainties, because the integrity and chromosomal integration site of transferred cDNA is unpredictable. However, it is possible to obtain a cell clone that stably expresses cytochrome P450. Nevertheless, some precautions on the preparation of cDNA for gene transfer and selection of cell clones as described above should be taken, in order to increase the chance for obtaining a stably expressing clone. Usually, clones that maintain cytochrome P450 expression over a period of 3
14 j. Doehmer, A. Seidel, F. Oesch, and H. R. Glatt, Environ. Health Perspect. 88, 63 (1990). 15 D. Waxman, D. P. Lapenson, J. J. Morrissey, S. S. Park, H. V. Gelboin, J. Doehmer, and F. Oesch, Biochem. J. 260, 81 (1989). 16 K. L. Platt, E. Molitor, J. Doehmer, S. Dogra, and F. Oesch, J. Biochem. Toxicol. 4, 1 (1989). 17 F. Kiefer and F. J. Wiebel, Toxicol. Left. 48, 265 (1989). 18 H. R. Glatt, I. Gemperlein, G. Turchi, H. Heinritz, J. Doehmer, and F. Oesch, Mol. Toxicol. 1, 313 (1987).
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HETEROLOGOUSEXPRESSION
[12]
FIG. 1. Immunofluorescence staining of SDI cells. The first antibody was rabbit anticytochrome P450IIBI, and the second antibody was anti-rabbit IgG coupled to FITC. The nuclear membrane and the endoplasmic reticulum are fluorescent. There is no diffuse staining in the cytoplasm, no staining in the cell nucleus, and no staining in the cellular membrane. The two cells at center had completed mitosis and started to spread again. (Cells were prepared and picture was taken by Dr. Brian Storrie, Virginia Polytechnic Institute and State University, Blacksburg, Virginia.) months, as judged b y i m m u n o f l u o r e s c e n c e staining, will continue to produce c y t o c h r o m e P450. B e t w e e n 10 and 30 V79-derived cell clones should be initially screened in order to get a b o u t three clones that produce cytoc h r o m e P450 constantly and h o m o g e n e o u s l y . The v e r y first cell clone, SD1, that we picked in 1987 still p r o d u c e s c y t o c h r o m e P450IIBI. Cell lines should be kept in growth m e d i u m in the p r e s e n c e of 400/zg/ ml G418. G418 m a y be left out for a few w e e k s and during testing. Optimal e x p r e s s i o n rates are obtained w h e n cells are grown u n d e r optimal conditions. This d e s e r v e s special attention as V79 cells grow fast and m a y e x h a u s t the growth m e d i u m within a day. M a x i m u m c y t o c h r o m e P450 production is obtained at a confluence o f 60 to 80% with feeding e v e r y second day. Acknowledgments We are indebted to our colleagues at the Institut ffir Toxikologie, in particular, Hansruedi Glatt, Karl Platt, Helmut Thomas, Albrecht Seidel, Satish Dogra, Michael Edigkaufer,
[13]
P 4 5 0 EXPRESSION I N H U M A N C E L L L I N E
123
Catherine W6lfel, Thomas Friedberg, Angelika Martin, Sabine Borg, Elvira Molitor, Sonja Reichert, Karin Pauly, Ute Armbrust, and Andrea Pi6e. Drs. Felix W~ichter(CIBA-GEIGY, Basel), David Waxman (Harvard Medical School, Boston), and Roland Wolf (University of Edinburgh) helped us with purified cytochrome P450 and cytochrome P450-specific antibodies. The project is funded by the Deutsche Forschungsgemeinschaft, SFB 302, "Kontrollfaktoren der Tumorentstehung" and the BGA, Berlin.
[13] E x p r e s s i o n o f C y t o c h r o m e P 4 5 0 c D N A s in H u m a n B L y m p h o b l a s t o i d Cells: A p p l i c a t i o n s to T o x i c o l o g y a n d Metabolite Analysis By CHARLES L. CRESPI
Introduction
Our laboratory has focused on the development of human cell systems for toxicological applications in general and gene locus mutation assays in particular. It is our belief that, compared to nonhuman in vitro systems, human cell systems have the potential to be better models of human susceptibility to the toxic effects of environmental compounds. The key to the development of such systems has been the expression of xenobiotic metabolism in human cell culture. After attempting a variety of traditional approaches to increasing cellular capacity to metabolize xenobiotics and achieving limited success with these methods, we have developed an approach based on cDNA transfection for the introduction of specific human xenobiotic-metabolizing enzymes into human cells. The results of such modifications have been dramatic. Introduction of the appropriate P450 enzyme can increase the sensitivity of the cell line to a particular procarcinogen by more than 100,000-fold. In addition, the levels of P450 expression were sufficiently high to allow analysis of metabolites either in intact cells or in microsomal preparations from the cells. Host Cell Line The AHH-1 TK+/- human B lymphoblastoid cell line was isolated from the RPMI 1788 cell line. 1 AHH-1 TK÷/- cells grow rapidly in suspension culture and form colonies in 96-well microtiter plates with an efficiency of 30-50%. This cell line contains native CYP1A1 activity. The basal level of this activity was quite low and seldom interfered with the measurement I C. L. Crespi and W. G. Thilly, Mutat. Res. 128, 221 (1984).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
V79 CELLSEXPRESSINGCYTOCHROMEP450
117
[12] V79 C h i n e s e H a m s t e r Cells G e n e t i c a l l y E n g i n e e r e d for Stable E x p r e s s i o n of C y t o c h r o m e s P 4 5 0 B y JOHANNES DOEHMER
and FRANZ OESCH
Introduction Cultivated mammalian cells genetically engineered for stable expression of cytochromes P450 and other xenobiotic metabolizing enzymes will serve as an efficient analytical tool in toxicology and pharmacology. 1-8The usefulness and applicability of such cell lines for mutagenicity and drug metabolism studies depends on cultivation conditions, growth parameters, karyotype stability, cellular integrity during cultivation, and stability and level of cytochrome P450 activity. Therefore, an experimental strategy aimed at the construction of a constantly cytochrome P450-expressing cell line involves careful and critical consideration of various aspects, and especially some experience in handling cells and genes. The main aspects that need consideration are (1) the choice of eukaryotic expression vector, (2) the way the cytochrome P450 cDNA is inserted into the vector, (3) the choice of mammalian cell serving as a recipient for the cytochrome P450 recombinant eukaryotic expression vector, and (4) a quick means to determine and to follow cytochrome P450 expression in a culture at the cellular level. Materials and Methods
Our experimental strategy for the construction of stable cytochrome P450-expressing cells involves the following steps. Step 1: Cloning of Cytochrome P450 Full-Length cDNA. Cloning inl H. Autrup, Carcinogenesis 11, 707 (1990). 2 N. Battula, J. Biol. Chem. 264, 2991 (1989). 3 C. L. Crespi, D. T. Steim¢l, T. Aoyama, H. V. Gelboin, and F. Gonzalez, Mol. Carcinog. 3, 5 0 9 9 0 ) .
4 R. L. Davies, C. L. Crespi, K. Rudo, R. T. Turner, and R. Langenbach, Carcinog. 10, 885 (1989). 5 S. K. Hansen, J. A. Ross, J. M. Siegfried, S. Leavitt, K. Rudo, R. Langenbach, and S. Nesnow, Mol. Carcinog. 2, 261 (1989). 6 L. H. Thompson, Environ. Mol. Mutagen. 14, 264 (1989). 7 j. Doehmer, S. Dogra, T. Friedberg, S. Monior, M. Adesnik, H. R. Glatt, and F. Oesch, Proc. Natl. Acad. Sci. U.S.A. 85, 5769 (1988). 8 S. Dogra, J. Doehmer, H. R. Glatt, H. M61ders, P. Siegert, T. Friodberg, A. Seidel, and F. Oesch, Mol. Pharmacol. 37, 608 (1990).
METHODS IN ENZYMOLOOY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
HETEROLOGOUS EXPRESSION
[ 12]
volves the generation of a eDNA library with expression of the cDNA in the bacterial host in case an antibody is to be used to search for the wanted cDNA, and without expression in case an oligonucleotide can be used. The procedure routinely applied in our laboratory 8 has been developed by Keith Stanley and co-workers. 9 In order to obtain full-length cDNA, an oligo(dT)-primed eDNA library is screened. Missing 5' ends may be searched for among shorter and randomly primed eDNA overlapping with the oligo(dT)-primed cDNA in order to construct a full-length cDNA. ~° Usually, it is of advantage, if the full-length eDNA has retained its own polyadenylation signal. Step 2: Eukaryotic Expression Vector. The full-length cDNA has to be provided with expression signals by recombination with an expression vector. The stability and level of expression of a cDNA may depend on the choice of expression vector, the construction of the recombinant expression vector, and the kind of cell functioning as the recipient in the DNA-mediated gene transfer. Based on previous experience, some assumptions can be made. For example, the thymidine kinase promoter of herpes virus (TK promoter) usually resulted in low levels of expression, whereas the metallothionein promoter, cytomegalovirus promoter, SV40 early promoter, or retroviral LTR promoters yielded high levels of expression. In our hands, a recombinant plasmid containing the simian virus 40 (SV40) early promoter reading into the eDNA encoding cytochrome P450IIB1, 7 cytochrome P450IA1, 8 and cytochrome P450IA21° was sufficient to yield high and stable expression in V79 Chinese hamster cells. The original recombinant plasmid, p S V 4 5 0 , 7 is now being routinely used as a master plasmid for the construction of all other cytochromes P450 eDNA to be expressed in V79 cells by substituting the cytochrome P450IIB1 eDNA. 8,1° Viral expression vectors may have some advantages. The circular DNA of bovine papilloma virus (BPV) is episomally maintained in a cell at a high copy number. However, it has frequently been observed that BPV recombinant vectors are unstable and lose inserted cDNA. 1~Recombinant retroviral vectors as infectious particles can be used to transfer eDNAs at 100% efficiency as each cell can be infected, and the eDNA is inserted into the chromosomal DNA without loss of sequences via the retroviral enzymatic insertion mechanism. 2 However, obtaining infectious recombi9 H. Haymede, J. Herz, M. Bressan, R. Frank, and K. Stanley, Nucleic Acids Res. 14, 8615 (1986). l0 C. W61fel, K. L. Platt, S. Dogra, H. R. Glatt, F. W~ichter, andJ. Doehmer, Mol. Carcinog., in press (1991). 11 M. Edigkaufer, S. Dogra, F. Oesch, and J. Doehmer, in "Cytochrome P-450: Biochemistry and Biophysics" (I. Schuster, ed.), p. 560. Taylor & Francis, London, 1989.
[12]
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119
nant retroviral particles requires insertion of the cDNA into the retroviral genomic DNA followed by DNA transfer into a packaging cell line. This may turn out to be cumbersome, as the insertion of cDNA into retroviral genomic DNA may interfere with the functional organization of the retroviral genomic DNA. Furthermore, the intact insertion of the recombinant retroviral genomic DNA into the chromosomal DNA of the packaging cell line is an essential prerequisite for obtaining recombinant retroviral particles and may occur at an extremely low frequency. Using the classic Ca/P coprecipitation technique may still be the fastest and most efficient way to get a stable cell clone.12 Step 3: DNA-Mediated Gene Transfer. For gene transfer, preparation of the recombinant vector DNA and the choice of recipient cell are the most important points to consider. The classic Ca/P coprecipitation technique is routinely used.~2 The recombinant vector DNA is linearized with a unique cutting restriction enzyme, whenever possible, that leaves stretches of DNA on either end of the cDNA construct for protection against exonucleases in the recipient cell. This increases the chance for integration of an intact functional cDNA into the chromosomal DNA of the recipient cell. V79 Chinese hamster cells are chosen to serve as recipient cells. These cells are derived from lung fibroblasts. 13 Usually, 20 ttg of recombinant cytochrome P450 vector DNA is mixed for coprecipitation with 1/xg of recombinant vector DNA containing the neomycin phosphotransferase gene under control of the metallothionein promoter. DNA is mixed in 1.0 ml of HEPES-buffered saline (137 mM NaC1, 6 mM dextrose, 5 mM KC1, 0.7 mM Na2HPO4, 20 mM HEPES, adjusted to pH 6.95-7.05 with 0.5 M NaOH). The DNA is precipitated by the addition of 52.6/zl of 2.5 M CaCI2 (final concentration 125 mM), and the mixture is left at room temperature for 25 min. The DNA precipitate is added to 1 x 10 6 V79 cells in a 60-mm petri dish. Best results are obtained with a fine rather than a bulky or cloudy precipitate. After a 4-hr incubation, cells are subjected to treatment with 15% (v/v) glycerol in Dulbecco's modified Eagle's medium (DMEM) for 2 min ("glycerol shock"), washed twice gently with fresh medium, and refed with 10 ml of growth medium. After overnight incubation, cells are split 1 : 5 in 60-mm petri dishes. The neomycin derivative G418 is added to the medium for selection on the third day after transfection, at a concentration of 800 /~g/ml of growth medium, because it takes at least two mitotic rounds to get a reasonable number of clones that have the transferred DNA stably integrated into their chromosomal DNA. G418-resistant colonies 12 F. Graham and A. van der Eb, J. Virol. 52, 456 (1973). 13 E. H. Y. Chu and H. V. Mailing, Proc. Natl. Acad. Sci. U.S.A. 61, 1306 (1968).
120
HETEROLOGOUSEXPRESSION
[12]
appear I0 days after transfection and are picked 3 weeks later by cloning cylinders and grown in mass culture for further studies. Special attention is given to the morphological appearance of the cell clones. Only those clones with a mitotic index of at least 5%, and in which the shape of the cell and nucleus resembled that of the parental V79 cell most, are picked. Step 4: Characterization of Selected Cell Clones. G418-resistant colonies are expanded to mass culture over a period of 2 to 3 months in order to obtain enough material for Southern, Northern, and Western blotting by standard techniques. These analyses at the DNA, RNA, and protein level demonstrate that the cDNA is stably integrated into the chromosomal DNA, that the cDNA is expressed, and that the cDNA-encoded mRNA yields an authentic protein as compared to in vivo material. Over the course of cell propagation an aliquot of 2 to 5 x 104 cells of each selected clone are checked weekly for expression by immunofluorescence staining as a quick means to determine expression level, homogeneity of expression, and stability of expression. Cells are grown on special glass plates in small chambers (Chamber Slides, Nunc, Inc., Naperville, IL). The staining procedure involves a wash with phosphatebuffered saline (PBS; 15 mM sodium phosphate pH 7.4, 0.15 M NaC1), fixation in a cold ( - 2 0 °) mixture of acetone/methanol (I:1) for 5 to 10 min, followed by drying at room temperature for 5 min. Fixed cells are incubated in 400/~1 of DMEM plus 10% fetal calf serum (FCS) containing 5/~g//zl of a cytochrome P450-specific monoclonal antibody for 35 min at room temperature in a humidified chamber. Excess first antibody is removed by three cycles of washing in water for 10 min each cycle with slow shaking. Thereafter, glass plates are incubated in 400/~1 DMEM plus 10% FCS containing goat anti-mouse IgG coupled to Texas Red or fluorescein isothiocyante (FITC) at a dilution of 1 : 30 or 1 : 50 for 35 min at room temperature in a humidified chamber. Excess second antibody is removed as described above. Cells are embedded in a solution of 20% (v/v) Mowiol (Hoechst AG, Frankfurt, Germany), 3% (v/v) glycerol in PBS and carefully covered by sliding a coverslip over the cells, avoiding air bubbles. The solution becomes solid by polymerization within 1 hr. Embedded cells are kept in the dark and are observed and photographed within a week.
Results and Comments Here, emphasis is placed on the construction of stably cytochrome P450-expressing V79-derived cell lines. Cell lines expressing rat cytochrome P450IIB1 and rat cytochrome P450IA1 have been validated in
[12]
V79 CELLSEXPRESSINGCYTOCHROMEP450
121
cytotoxicity, mutagenicity, and metabolism studies, s,14-16V79-derived cell lines stably expressing rat cytochrome P450IA2 have been established recently. ~° Cells of the V79 Chinese hamster cell line have been used for mutagenicity testing for more than 20 years, because they grow fast (doubling time 12 hr) and have a high cloning efficiency (better than 90%), stable karyotype, and a convenient marker gene for mutagenicity studies (the HPRT locus on a single X chromosome). However, V79 cells either do not express or express cytochromes P450 at an extremely low levels.17 It became obvious that lack of cytochrome P450 activity in parental V79 cells presented a unique situation as the V79-derived cell lines are defined for the genetically engineered cytochrome P450 isoenzyme, which makes these cell lines a valuable analytical tool in toxicological and pharmacological studies. Expression of the apoprotein is not sufficient for enzymatic activity, however, as the protein has to be inserted into the membrane of the endoplasmic reticulum, has to complex with a heme group, and has to be associated with the cytochrome-P450 reductase in order to become operative. Picking V79 cells as the recipient for cytochrome P450 cDNA was largely a matter of luck, because the amount of cytochrome-P450 reductase was sufficient, even though the level of cyrochrome-P450 reductase is the lowest compared to 20 other cell lines.~8 In addition, enough heme is made in V79 cells, and there is enough endoplasmic reticulum to anchor the genetically engineered cytochrome P450 isoenzyme (Fig. 1). The cytochrome P450 content in SD1 cells was estimated by Western blotting to be 50 pmol/107 cells. ~5 The application of Ca/P coprecipitation for DNA-mediated gene transfer implies some uncertainties, because the integrity and chromosomal integration site of transferred cDNA is unpredictable. However, it is possible to obtain a cell clone that stably expresses cytochrome P450. Nevertheless, some precautions on the preparation of cDNA for gene transfer and selection of cell clones as described above should be taken, in order to increase the chance for obtaining a stably expressing clone. Usually, clones that maintain cytochrome P450 expression over a period of 3
14 j. Doehmer, A. Seidel, F. Oesch, and H. R. Glatt, Environ. Health Perspect. 88, 63 (1990). 15 D. Waxman, D. P. Lapenson, J. J. Morrissey, S. S. Park, H. V. Gelboin, J. Doehmer, and F. Oesch, Biochem. J. 260, 81 (1989). 16 K. L. Platt, E. Molitor, J. Doehmer, S. Dogra, and F. Oesch, J. Biochem. Toxicol. 4, 1 (1989). 17 F. Kiefer and F. J. Wiebel, Toxicol. Left. 48, 265 (1989). 18 H. R. Glatt, I. Gemperlein, G. Turchi, H. Heinritz, J. Doehmer, and F. Oesch, Mol. Toxicol. 1, 313 (1987).
122
HETEROLOGOUSEXPRESSION
[12]
FIG. 1. Immunofluorescence staining of SDI cells. The first antibody was rabbit anticytochrome P450IIBI, and the second antibody was anti-rabbit IgG coupled to FITC. The nuclear membrane and the endoplasmic reticulum are fluorescent. There is no diffuse staining in the cytoplasm, no staining in the cell nucleus, and no staining in the cellular membrane. The two cells at center had completed mitosis and started to spread again. (Cells were prepared and picture was taken by Dr. Brian Storrie, Virginia Polytechnic Institute and State University, Blacksburg, Virginia.) months, as judged b y i m m u n o f l u o r e s c e n c e staining, will continue to produce c y t o c h r o m e P450. B e t w e e n 10 and 30 V79-derived cell clones should be initially screened in order to get a b o u t three clones that produce cytoc h r o m e P450 constantly and h o m o g e n e o u s l y . The v e r y first cell clone, SD1, that we picked in 1987 still p r o d u c e s c y t o c h r o m e P450IIBI. Cell lines should be kept in growth m e d i u m in the p r e s e n c e of 400/zg/ ml G418. G418 m a y be left out for a few w e e k s and during testing. Optimal e x p r e s s i o n rates are obtained w h e n cells are grown u n d e r optimal conditions. This d e s e r v e s special attention as V79 cells grow fast and m a y e x h a u s t the growth m e d i u m within a day. M a x i m u m c y t o c h r o m e P450 production is obtained at a confluence o f 60 to 80% with feeding e v e r y second day. Acknowledgments We are indebted to our colleagues at the Institut ffir Toxikologie, in particular, Hansruedi Glatt, Karl Platt, Helmut Thomas, Albrecht Seidel, Satish Dogra, Michael Edigkaufer,
[13]
P 4 5 0 EXPRESSION I N H U M A N C E L L L I N E
123
Catherine W6lfel, Thomas Friedberg, Angelika Martin, Sabine Borg, Elvira Molitor, Sonja Reichert, Karin Pauly, Ute Armbrust, and Andrea Pi6e. Drs. Felix W~ichter(CIBA-GEIGY, Basel), David Waxman (Harvard Medical School, Boston), and Roland Wolf (University of Edinburgh) helped us with purified cytochrome P450 and cytochrome P450-specific antibodies. The project is funded by the Deutsche Forschungsgemeinschaft, SFB 302, "Kontrollfaktoren der Tumorentstehung" and the BGA, Berlin.
[13] E x p r e s s i o n o f C y t o c h r o m e P 4 5 0 c D N A s in H u m a n B L y m p h o b l a s t o i d Cells: A p p l i c a t i o n s to T o x i c o l o g y a n d Metabolite Analysis By CHARLES L. CRESPI
Introduction
Our laboratory has focused on the development of human cell systems for toxicological applications in general and gene locus mutation assays in particular. It is our belief that, compared to nonhuman in vitro systems, human cell systems have the potential to be better models of human susceptibility to the toxic effects of environmental compounds. The key to the development of such systems has been the expression of xenobiotic metabolism in human cell culture. After attempting a variety of traditional approaches to increasing cellular capacity to metabolize xenobiotics and achieving limited success with these methods, we have developed an approach based on cDNA transfection for the introduction of specific human xenobiotic-metabolizing enzymes into human cells. The results of such modifications have been dramatic. Introduction of the appropriate P450 enzyme can increase the sensitivity of the cell line to a particular procarcinogen by more than 100,000-fold. In addition, the levels of P450 expression were sufficiently high to allow analysis of metabolites either in intact cells or in microsomal preparations from the cells. Host Cell Line The AHH-1 TK+/- human B lymphoblastoid cell line was isolated from the RPMI 1788 cell line. 1 AHH-1 TK÷/- cells grow rapidly in suspension culture and form colonies in 96-well microtiter plates with an efficiency of 30-50%. This cell line contains native CYP1A1 activity. The basal level of this activity was quite low and seldom interfered with the measurement I C. L. Crespi and W. G. Thilly, Mutat. Res. 128, 221 (1984).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
