INT . J . RADIAT . BIOL ., 1991, VOL . 59, NO . 3, 8 0 7 -814
Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
S . R . ELDRIDGE and M . N . GOULD University of Wisconsin-Madison, Department of Human Oncology, UW Clinical Cancer Center and Environmental Toxicology Centre, Madison, WI 53792, USA (Received 27 March 1990, revision received 14 September 1990 ; accepted 28 September 1990) Tissue and locus specificity of mutation induction was studied in human mammary epithelial cells (HMEC) . Primary HMEC from normal tissue, as well as immortalized HMEC (184B5) derived from normal HMEC, were cultured under identical conditions and exposed to 10 J/m2 ultraviolet (UV) radiation (254 nm peak wavelength), which produced approximately 50% mean survival in all cell strains and lines tested . UV radiation was found to induce mutations at the Na + -K + ATPase locus as determined by ouabain-resistance in both normal and immortalized HMEC . Mutation frequencies measured in these cells following UV exposure were similar to those reported for human diploid fibroblasts . In addition, mutation induction was investigated at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in normal and immortalized HMEC . Induced mutations at the HPRT locus as determined by 6-thioguanine resistance in normal primary HMEC were not observed following UV radiation . In contrast, mutation induction was observed at this locus in UV-exposed immortalized HMEC .
1 . Introduction Neoplastic transformation of cells is believed to be a multistep process, beginning with an alteration in the genetic material of a somatic cell . This somatic mutation hypothesis arises from correlations between specific locus mutation induction and cancer induction by chemical and physical agents (Committee 2 Final Report 1982, Ames et al . 1973) . Additional support for the involvement of mutations in neoplastic transformation comes from reporting an association between point mutations and the process of carcinogenesis . For example, cellular oncogenes, such as ras and neu, can be activated by single-point mutations (Barbacid 1986) . Deletion or mutational inactivation of recessive tumour suppressor genes also play an essential role in the genesis of several tumour types, including retinoblastoma and Wilms' tumour (Cavenee et al. 1983, Koufos et al . 1984) . Thus, short-term tests for specific locus mutation induction can be used to identify potential carcinogens based on their mutagenic activity . Specific locus mutation induction by chemical and physical carcinogens has been documented in human diploid fibroblasts (Jacobs and DeMars 1982, Maher et al . 1976, Myhr et al . 1979, Buchwald 1977) . However, since 85-90% of all human cancers originate from epithelial cells (Cairns 1975), it is important to determine if mutagenesis is also cell type- or organ-specific . For these reasons we examined tissue- and locus-specific mutation induction by UV radiation in human mammary epithelial cells (HMEC) . 0020-7616/91 $3 .00 © 1991 Taylor & Francis Ltd
808
S. R . Eldridge and M . N . Gould
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
2. Material and methods 2.1 . Human mammary cells Normal HMEC were derived from residual surgical material from reduction mammoplasties of eight healthy women . Procedures for the isolation of primary HMEC have been previously described (Stampfer et al . 1980, Cathers and Gould 1983, Eldridge et al . 1989) . Primary cultures of epithelial cells were maintained for 7 days with regular feedings with MCDB 170 (Hammond et al. 1984), and trypsinized with 0 .25% trypsin (Gibco, Grand Island, NY) plus 0 . 2 ng/ml EDTA (Sigma Chemical Co ., St Louis, MO) . Single-cell suspensions were obtained from primary cultures . Cultures of normal HMEC have been examined in our laboratory and exhibit limited lifespan in culture as well as diploid karyotypes (Zhang et al . 1989). In addition, we have examined early-passage cultures of normal HMEC by immunofluorescence staining for epithelial cell-specific keratin and found them to be > 90% keratin-positive (Gould et al . 1986) . Similarly cultured HMEC have been shown to express mammary cell-specific human milk fat antigen (Stampfer 1985) . Immortalized HMEC (line 184B5) obtained from Dr . Martha Stampfer were derived from normal HMEC taken from one donor and immortalized by in vitro exposure to benzo(a)pyrene (Stampfer and Bartley 1985) . Karyotypic analyses show the cells to be near diploid (Stampfer and Bartley 1985) . The cells do not grow in agar, nor do they produce tumours in nude mice (Stampfer and Bartley 1985) . These immortalized HMEC express epithelial cell-specific keratin and mammary cell-specific human milk fat antigen (Stampfer and Bartley 1985) . Nonimmortalized cells (case 184, passage 9) from the same cell lineage as the immortalized cells were also studied, as well as a late-passage HMEC strain (case B482, passage 14) that was derived in our laboratory from normal HMEC . The growth kinetics of normal and immortalized HMEC are very similar, as shown by flow cytometric analyses following propidium iodide staining (S . P . Howard and M . N . Gould, unpublished data) . Doubling time for both cell types is approximately 24 h during exponential growth . Cloning efficiencies for normal and immortalized HMEC vary between 20-50% and 60-80%, respectively . 2 .2 . Exposure to UV Exponentially growing cells were rinsed twice with phosphate-buffered saline at 37 ° C. Uncovered dishes without medium or saline were exposed to 10 J/m 2 ultraviolet (UV) radiation (254nm peak wavelength, Sylvania germicidal lamp) . The incident dose rate was 1 .3 J/m 2 per second of UV as measured with a Spectroline DMX-254X radiometer . Survival of both cell types (normal and immortalized HMEC) following 10 J/m 2 UV was 58±6% . 2.3 . Na + -K+ ATPase mutagenesis assay The general methods described by Elmore and Barrett (1982) for selection of Na+ -K + ATPase mutants in normal human diploid fibroblasts were used . At least 10 7 exponentially growing cells in secondary culture (4 x 10 5 cells per 100 mm dish ; 40 dishes per group) were exposed to 10 J/m 2 UV . After a 4-day expression time, cells were subcultured and seeded for selection of ouabain-resistant (Oua`) mutants (10 6 cells per 100 mm dish ; 50 dishes per group) in medium containing 10 -7 M ouabain . Cells were assayed for survival by also seeding into non-selective medium
UV mammary cell mutagenesis
809
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
(100 cells per 100 mm dish ; three dishes per group) following exposure and expression periods . Survival of both normal and immortalized HMEC in 10 - 'm ouabain was less than 10' . Plates were incubated at 37 ° C in 5% CO 2 for 21 days with two changes of medium. The cells were fixed in methanol, stained with a 1 :1 mixture of azure II and methylene blue, and scored for Oua` colonies containing > 100 cells . Background mutation frequencies were calculated, based on the number of colony-forming cells as determined by the corresponding cloning efficiences . For each determination, at least 10' viable cells were plated . 2 .4 . HPRT mutagenesis The methods described by Jacobs and DeMars (1982) to select for HPRT mutants in normal human diploid fibroblasts were adapted for the present study as previously described (Eldridge and Gould, submitted) . At least 2 x 10 6 actively growing cells were exposed to 10 J/m 2 UV . After treatment, cells were seeded into non-selective medium for determination of survival as described for the Na + -K + ATPase mutagenesis assay and for expression of mutations (10 5 cells per 100 mm dish ; 20 dishes per group) . Following an 8-day expression time (six population doublings), these cells were subcultured into 100 mm dishes containing 30 Ym 6-thioguanine (2-amino-6-mercaptopurine) (TG) (Sigma), which inhibited the growth of normal and immortalized HMEC by 93-96% . Background mutation frequencies were determined from unirradiated control cultures . The mutation frequencies were calculated by correcting for recovery of TG' mutants and for the corresponding cloning efficiencies as previously described (Eldridge et al. 1989, Eldridge and Gould, submitted) . At least 10 6 viable cells were plated in each experiment . 2 .5 . Characterization of mutants Oua` colonies were ring-cloned and propagated in selective medium to free the cultures of wild type Ouas cells . These cells were used to determine heritability of the mutant phenotype and uptake of rubidium ( 86 Rb) . To determine the stability of the Ouar phenotype, cells were subcultured from confluent dishes at 10 4 cells per 100 mm dish containing non-selective medium . When the 100 mm dishes were confluent, the cells were subcultured at 10 5 cells per 100 mm dish into plates containing either non-selective or selective medium . Growth of cells from the two groups was then observed . The uptake of 86 Rb, a tracer for potassium, was used to characterize Ouar mutants as described by Elmore and Barrett (1982) . The effect of ouabain on 86 Rb transport by Na + -K + ATPase was determined in Ouar mutants from both normal and immortalized HMEC . 2 .6 . Statistical analyses Statistical comparisons were done using the Mann-Whitney non-parametric two-sample test or a paired t-test . 3 . Results 3 .1 . Na + -K + ATPase mutagenesis assay +Figure 1 shows background and UV-induced mutation frequencies at the Na K + ATPase locus in normal and immortalized HMEC . Significant mutation
S . R . Eldridge and M. N . Gould
810
300
300
250
200
c
150
150
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
100
50
0
0 Normal
Immortalized
Figure 1 . UV induction of mutations at the Na + -K + ATPase locus in HMEC . Mutation frequencies at the Na + -K + ATPase locus are shown for control (open) and UV (10 J/m2 ) exposed (hatched) normal and immortalized cells (184B5) . The asterisk (*) indicates that the mean background mutation frequency in normal HMEC at this locus was not detected when > 10 s cells were screened in five independent experiments . The mutation frequency in UV-irradiated normal HMEC represents the mean ±SE of four determinations (cells from three different women) . Background (n = 4) and UV-induced (n= 5) mutations at the Na + -K + ATPase locus in immortalized cells (184B5) represent mean ±SE .
induction was observed in both normal and immortalized HMEC exposed to 10 J/m2 UV (p=0 . 01) . Uptake of 86 Rb by wild-type and Oua` mutants was measured in normal and immortalized HMEC . Representative data are presented in Figure 2 . In the presence of ouabain, uptake of 86 Rb was inhibited in wild-type HMEC as shown in Figure 2(a) . However, in Oua` mutants, 86 Rb uptake was not affected by ouabaincontaining medium (Figure 2b) . The Oua` phenotype was stable in the immortalized HMEC, as determined by growth of mutant cells in selective medium after approximately 10 populaton doublings in non-selective medium . In addition, the growth rates of wild-type and mutant HMEC were the same . 3 .2 . HPR T mutagenesis assay Background and UV-induced mutation frequencies at the HPRT locus, corrected for recovery of mutants, are presented in Figure 3 for normal and immortalized HMEC . The mutation frequency for normal HMEC exposed to UV radiation in primary culture did not differ from the background mutation frequency (p=0 . 2) . In contrast, significant mutation induction was observed in the immortalized HMEC (184B5) (p=0 . 005) . UV induction of mutations was not observed in nonimmortalized normal HMEC from the same (case 184) and different (case B482) cell lineages as the immortalized HMEC . 4.
Discussion
The quantitation of mutagenesis in mammalian cells serves as a screening assay for environmental genotoxic carcinogens . Somatic cell mutagenesis following
UV mammary cell mutagenesis
811
12000
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
10000
0
30
60
90
120 150 180
0
Time (minutes)
30
60
90
Time (minutes)
(a)
(b) Figure 2 . Characterization of Na + -K + ATPase mutants in HMEC . The transport of 86 Rb by Na + -K + ATPase in a representative variant HMEC clone was determined with or without ouabain in the medium as described in § 2 . Each data point represents the mean ± SE of three determinations . Some error bars are contained within data points . (a) Wild-type immortalized HMEC ; (b) Oua` normal HMEC . Control cultures (solid lines) ; ouabain-containing cultures (dashed lines) .
10
100 c -80
r a
- 60
V 1 0
M
Normal
Late Passage (a)
Immortalized
0
(b)
Figure 3 . UV induction of HPRT mutations in HMEC . Mutation frequencies at the HPRT locus are shown in control (open) and UV (10 J/m 2) exposed (hatched) cells . Panel (a) compares mutation frequencies in UV-exposed and control normal HMEC . Background mutation frequency for normal HMEC represents the mean ± SE of five determinations (cells from four different women) . UV-induced mutation frequency in normal HMEC represents the mean ±SE of seven determinations (cells from six different women) . Background and UV-induced mutation frequencies represent five determinations (n=2 and 3, respectively) in cases 184 (passage 9) and B482 (passage 14) . Panel (b) demonstrates the UV induction of mutations at the HPRT locus in immortalized HMEC (184B5) . The frequencies represent mean ± SE for seven paired experiments . Methods are presented in the text .
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
812
S . R . Eldridge and M . N. Gould
exposure to physical and chemical agents has been routinely quantitated in rodent cell lines and human diploid fibroblasts (Committee 2 Final Report 1982, Gupta 1984, Bradley et al . 1981) . However, human cancers are predominantly carcinomas which originate from epithelial cells . If the induction of somatic cell mutations is tissue-specific, alternative assays with the cell type of interest may be necessary . This possibility has been addressed in part by using mediated mutagenesis assays (Huberman and Sachs 1984) . In these assays an organ-specific cell, such as HMEC (Gould et al . 1986), can be used to activate test chemicals which ultimately induce mutations in a tester cell line, such as V79 hamster fibroblasts . Thus, the mediated assay examines the specificity of carcinogen activation, but not of mutation induction . In order to study the tissue-specificity of mutation induction, mutagenesis must be quantified in specific cell types . Only limited data are available for mutagenesis in human epithelial cells (Allen-Hoffmann and Rheinwald 1984) . Therefore, we investigated the specificity of mutation induction directly in HMEC using a direct-acting mutagen . + UV induced mutations at the Na -K + ATPase locus in both normal and immortalized HMEC at a frequency equivalent to that reported at this locus in human fibroblasts (Buchwald 1977) . In contrast, primary cultures of normal HMEC did not show an increased frequency of mutations at the HPRT locus when exposed to UV radiation . This unexpected observation could be due to the inability to mutate this locus or the failure to detect induced mutants . We feel that the latter possibility is unlikely, since we were able to quantitate the background mutation frequency in these cells in a range similar to that measured in several other cell types . In addition, UV-induced mutations were not found following both shorter (5 days, three population doublings) or longer (12 days, 10 population doublings) expression times at subconfluent cell densities, or with the use of different cell densities during selection of mutants . Our data suggest that UV-hypomutability at the HPRT locus can be overcome by cellular immortalization . It should be emphasized that non-immortalized cells, from the same lineage as the immortalized cells studied, were also UV-hypomutable at this locus . Arlett and Cole (1986) reported that fibroblasts from ataxia telangiectasia patients were not mutable at the HPRT locus by y-radiation, whereas their immortalized counterparts were sensitive to mutation induction . In addition, UV and y-radiation-induced mutation frequencies in normal cells immortalized by either SV40 or Epstein-Barr viruses were much higher than in their untransformed counterparts . Tatsumi and Takebe (1984) and Tatsumi et al. (1987) have reported similar findings . Collectively, these data suggest caution in the use of immortal cell lines in genotoxicity testing ; tissue-specific characteristics associated with the organ of origin may be overriden by the process of immortalization of the immortal phenotype . In summary, we have found a similar frequency of UV-induced specific locus mutations at the Na + -K + ATPase locus in human normal mammary epithetial cells as has been reported for human normal fibroblasts (Buchwald 1977) . In contrast, primary human mammary epithelial cells were UV-hypomutable at the HPRT locus when compared to human fibroblasts . Acknowledgements This work was supported by PHS NCI grants CA30295 and CA14520 . S .R .E . was supported by NIEHS grant ES07015 . Current address : Chemical Industry
UV mammary cell mutagenesis
813
Institute of Toxicology, 6 Davis Drive, Research Triangle Park, NC 27709 . The authors thank Drs Martha Stampfer for the generous gift of 184 and 184B5 cells, Cynthia J . Moore for critical review of the manuscript, Martin A . Tanner for statistical analayses, and Ms Peggy Ziebarth for preparation of the manuscript . Pituitary extract was obtained from Susan Hammond, Hammond Cell Technologies, Palo Alto, CA, USA .
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
References ALLEN-HOFFMANN, B . L . and RHEINWALD, J . G ., 1984, Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture . Proceedings of the National Academy of Sciences, USA, 81, 7802-7806 . AMES, B . N ., DURSTON, W. E ., YAMASAKI, E . and LEE, E . D ., 1973, Carcinogens are mutagens : a simple test system combining liver homogenates for activation and bacteria for detection . Proceedings of the National Academy of Sciences, USA, 70, 2281-2285 . ARLETT, C . F . and COLE, J ., 1986, Mutation studies in cells established from human cancerprone syndromes . Genetic Toxicology of Environmental Chemicals, Part A: Basic Principles and Mechanisms of Action, edited by C . Ramel, B . Lambert and J . Magnusson (Alan R . Liss, New York), pp . 237-244 . BARBACID, M ., 1986, Mutations, oncogenes and cancer . Trends in Genetics, 2, 188-192 . BRADLEY, M . 0 ., BHUYAN, B ., FRANCIS, M . C ., LANGENBACH, R . G ., PETERSON, A . and HUBERMAN, E ., 1981, Mutagenesis by chemical agents in V79 Chinese hamster cells : a review and analysis of the literature . A report of the Gene-Tox program . Mutation Research, 77, 81-142 . BUCHWALD, M ., 1977, Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts . Mutation Research, 44, 401-412 . CAIRNS, J ., 1975, The cancer problem. Scientific American, 233, 64-72 . CATHERS, L . E . and GOULD, M . N ., 1983, Human mammary cell survival following ionizing radiation . International Journal of Radiation Biology, 44, 1-16 . CAVENEE, W. K ., DRYJA, T . P., PHILLIPS, R . A ., BENEDICT, W . F., GODBOUT, R ., GALLIE, B . L ., MURPHREE, A . L ., STRONG, L . C . and WHITE, R . L ., 1983, Expression of recessive alleles by chromosomal mechanisms in retinoblastoma . Nature, 305, 779-784 . COMMITTEE 2 FINAL REPORT . (International Commission for Protection Against Environmental Mutagens and Carcinogens), Mutagenesis testing as an approach to carcinogenesis, Mutation Research, 99, 73-91 . ELDRIDGE, S . R ., MARTENS, T . W ., SATTLER, C . A . and GOULD, M . N ., 1989, Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells . Cancer Research, 49, 4326-4331 . ELMORE, E . and BARRETT, J . C ., 1982, Measurement of spontaneous mutation rates at the Na + -K + ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions . Mutation Research, 97, 393-404 . GOULD, M . N ., GRAU, D . R ., SEIDMAN, L . A . and MOORE, C . J ., 1986, Interspecies comparison of human and rat mammary epithelial cell-mediated mutagenesis by polycyclic aromatic hydrocarbons . Cancer Research, 46, 4942-4945 . GUPTA, R . S ., 1984, Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary cells : applications to mutagen screening studies . Handbook of Mutagenicity Test Procedures, edited by B . J . Kilbey, M . Legator, W. Nichols, and C . Ramel (Elsevier, Amsterdam), pp . 291-319 . HAMMOND, S . L ., HAM, R . G . and STAMPFER, M . R ., 1984, Serum-free growth of human mammary epithelial cells : Rapid clonal growth in defined medium and extended serial passage with pituitary extract . Proceedings of the National Academy of Sciences, USA, 81,5435-5439 . HUBERMAN, E . and SACHS, L ., 1984, Cell-mediated mutagenesis of mammalian cells with chemical carcinogens . International Journal of Cancer, 13, 326-333 . JACOBS, L . and DEMARS, R ., 1982, Chemical mutagenesis with diploid human fibroblasts . Handbook of Mutagenicity Test Procedures, edited by B . J . Kilbey, M . Legator, W . Nichols, and C . Ramel (Elsevier, Amsterdam), pp . 321-356 .
UV mammary cell mutagenesis
814
KouFos, A ., HANSEN, M . F ., LAMPKIN, B . C ., WORKMAN, M . L ., COPELAND, N . G ., JENKINS, N . A. and CAVENEE, W . K ., 1984, Loss of alleles at loci on human chromosome 11 during genesis of Wilms' tumour . Nature, 309,170-172 . MAHER, V. M ., OUELLETI, L . M ., CURREN, R . D . and MCCORMICK, J . J ., 1976, Frequency of ultraviolet light-induced mutations is higher in xeroderma pigmentosum variant cells than in normal human cells . Nature, 261, 593-595 . MYHR, B . C ., TURNBULL, D . and DIPAOLO, J . A ., 1979, Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts . Mutation Research, 62, 341-353 . STAMPFER, M . R ., 1985, Isolation and growth of human mammary epithelial cells . Journal of
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
Tissue Culture Methods, 9, 107-111 . STAMPFER, M . R . and BARTLEY, J . C ., 1985, Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo(a)pyrene .
Proceedings of the National Academy of Sciences, USA, 82, 2394-2398 . STAMPFER, M . R ., HALLOWES, R . C . and HACKETT, A . J ., 1980, Growth of normal human mammary cells in culture . In Vitro, 16, 415-425 . TATSUMI, K . and TAKEBE, H ., 1984, Gamma-irradiation induces mutation in ataxiatelangiectasia lymphoblastoid cells . Gann, 75, 1040-1043 . TATSUMI, K ., TOYODA, M ., HASHIMOTO, T ., FURUYAMA, J ., KURIHARA, T ., INOU, M . and TAKEBE, H ., 1987, Differential hypersensitivity of xeroderma pigmentosum lymphoblastoid cell lines to ultraviolet light mutagenesis . Carcinogenesis, 8, 53-57 . ZHANG, R ., WILEY, J ., HOWARD, S . P ., MEISNER, L . F . and GOULD, M . N ., 1989, Rare clonal karyotypic variants in primary cultures of human breast carcinoma cells . Cancer
Research, 49, 444-449 .
Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
S . R . ELDRIDGE and M . N . GOULD University of Wisconsin-Madison, Department of Human Oncology, UW Clinical Cancer Center and Environmental Toxicology Centre, Madison, WI 53792, USA (Received 27 March 1990, revision received 14 September 1990 ; accepted 28 September 1990) Tissue and locus specificity of mutation induction was studied in human mammary epithelial cells (HMEC) . Primary HMEC from normal tissue, as well as immortalized HMEC (184B5) derived from normal HMEC, were cultured under identical conditions and exposed to 10 J/m2 ultraviolet (UV) radiation (254 nm peak wavelength), which produced approximately 50% mean survival in all cell strains and lines tested . UV radiation was found to induce mutations at the Na + -K + ATPase locus as determined by ouabain-resistance in both normal and immortalized HMEC . Mutation frequencies measured in these cells following UV exposure were similar to those reported for human diploid fibroblasts . In addition, mutation induction was investigated at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in normal and immortalized HMEC . Induced mutations at the HPRT locus as determined by 6-thioguanine resistance in normal primary HMEC were not observed following UV radiation . In contrast, mutation induction was observed at this locus in UV-exposed immortalized HMEC .
1 . Introduction Neoplastic transformation of cells is believed to be a multistep process, beginning with an alteration in the genetic material of a somatic cell . This somatic mutation hypothesis arises from correlations between specific locus mutation induction and cancer induction by chemical and physical agents (Committee 2 Final Report 1982, Ames et al . 1973) . Additional support for the involvement of mutations in neoplastic transformation comes from reporting an association between point mutations and the process of carcinogenesis . For example, cellular oncogenes, such as ras and neu, can be activated by single-point mutations (Barbacid 1986) . Deletion or mutational inactivation of recessive tumour suppressor genes also play an essential role in the genesis of several tumour types, including retinoblastoma and Wilms' tumour (Cavenee et al. 1983, Koufos et al . 1984) . Thus, short-term tests for specific locus mutation induction can be used to identify potential carcinogens based on their mutagenic activity . Specific locus mutation induction by chemical and physical carcinogens has been documented in human diploid fibroblasts (Jacobs and DeMars 1982, Maher et al . 1976, Myhr et al . 1979, Buchwald 1977) . However, since 85-90% of all human cancers originate from epithelial cells (Cairns 1975), it is important to determine if mutagenesis is also cell type- or organ-specific . For these reasons we examined tissue- and locus-specific mutation induction by UV radiation in human mammary epithelial cells (HMEC) . 0020-7616/91 $3 .00 © 1991 Taylor & Francis Ltd
808
S. R . Eldridge and M . N . Gould
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
2. Material and methods 2.1 . Human mammary cells Normal HMEC were derived from residual surgical material from reduction mammoplasties of eight healthy women . Procedures for the isolation of primary HMEC have been previously described (Stampfer et al . 1980, Cathers and Gould 1983, Eldridge et al . 1989) . Primary cultures of epithelial cells were maintained for 7 days with regular feedings with MCDB 170 (Hammond et al. 1984), and trypsinized with 0 .25% trypsin (Gibco, Grand Island, NY) plus 0 . 2 ng/ml EDTA (Sigma Chemical Co ., St Louis, MO) . Single-cell suspensions were obtained from primary cultures . Cultures of normal HMEC have been examined in our laboratory and exhibit limited lifespan in culture as well as diploid karyotypes (Zhang et al . 1989). In addition, we have examined early-passage cultures of normal HMEC by immunofluorescence staining for epithelial cell-specific keratin and found them to be > 90% keratin-positive (Gould et al . 1986) . Similarly cultured HMEC have been shown to express mammary cell-specific human milk fat antigen (Stampfer 1985) . Immortalized HMEC (line 184B5) obtained from Dr . Martha Stampfer were derived from normal HMEC taken from one donor and immortalized by in vitro exposure to benzo(a)pyrene (Stampfer and Bartley 1985) . Karyotypic analyses show the cells to be near diploid (Stampfer and Bartley 1985) . The cells do not grow in agar, nor do they produce tumours in nude mice (Stampfer and Bartley 1985) . These immortalized HMEC express epithelial cell-specific keratin and mammary cell-specific human milk fat antigen (Stampfer and Bartley 1985) . Nonimmortalized cells (case 184, passage 9) from the same cell lineage as the immortalized cells were also studied, as well as a late-passage HMEC strain (case B482, passage 14) that was derived in our laboratory from normal HMEC . The growth kinetics of normal and immortalized HMEC are very similar, as shown by flow cytometric analyses following propidium iodide staining (S . P . Howard and M . N . Gould, unpublished data) . Doubling time for both cell types is approximately 24 h during exponential growth . Cloning efficiencies for normal and immortalized HMEC vary between 20-50% and 60-80%, respectively . 2 .2 . Exposure to UV Exponentially growing cells were rinsed twice with phosphate-buffered saline at 37 ° C. Uncovered dishes without medium or saline were exposed to 10 J/m 2 ultraviolet (UV) radiation (254nm peak wavelength, Sylvania germicidal lamp) . The incident dose rate was 1 .3 J/m 2 per second of UV as measured with a Spectroline DMX-254X radiometer . Survival of both cell types (normal and immortalized HMEC) following 10 J/m 2 UV was 58±6% . 2.3 . Na + -K+ ATPase mutagenesis assay The general methods described by Elmore and Barrett (1982) for selection of Na+ -K + ATPase mutants in normal human diploid fibroblasts were used . At least 10 7 exponentially growing cells in secondary culture (4 x 10 5 cells per 100 mm dish ; 40 dishes per group) were exposed to 10 J/m 2 UV . After a 4-day expression time, cells were subcultured and seeded for selection of ouabain-resistant (Oua`) mutants (10 6 cells per 100 mm dish ; 50 dishes per group) in medium containing 10 -7 M ouabain . Cells were assayed for survival by also seeding into non-selective medium
UV mammary cell mutagenesis
809
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
(100 cells per 100 mm dish ; three dishes per group) following exposure and expression periods . Survival of both normal and immortalized HMEC in 10 - 'm ouabain was less than 10' . Plates were incubated at 37 ° C in 5% CO 2 for 21 days with two changes of medium. The cells were fixed in methanol, stained with a 1 :1 mixture of azure II and methylene blue, and scored for Oua` colonies containing > 100 cells . Background mutation frequencies were calculated, based on the number of colony-forming cells as determined by the corresponding cloning efficiences . For each determination, at least 10' viable cells were plated . 2 .4 . HPRT mutagenesis The methods described by Jacobs and DeMars (1982) to select for HPRT mutants in normal human diploid fibroblasts were adapted for the present study as previously described (Eldridge and Gould, submitted) . At least 2 x 10 6 actively growing cells were exposed to 10 J/m 2 UV . After treatment, cells were seeded into non-selective medium for determination of survival as described for the Na + -K + ATPase mutagenesis assay and for expression of mutations (10 5 cells per 100 mm dish ; 20 dishes per group) . Following an 8-day expression time (six population doublings), these cells were subcultured into 100 mm dishes containing 30 Ym 6-thioguanine (2-amino-6-mercaptopurine) (TG) (Sigma), which inhibited the growth of normal and immortalized HMEC by 93-96% . Background mutation frequencies were determined from unirradiated control cultures . The mutation frequencies were calculated by correcting for recovery of TG' mutants and for the corresponding cloning efficiencies as previously described (Eldridge et al. 1989, Eldridge and Gould, submitted) . At least 10 6 viable cells were plated in each experiment . 2 .5 . Characterization of mutants Oua` colonies were ring-cloned and propagated in selective medium to free the cultures of wild type Ouas cells . These cells were used to determine heritability of the mutant phenotype and uptake of rubidium ( 86 Rb) . To determine the stability of the Ouar phenotype, cells were subcultured from confluent dishes at 10 4 cells per 100 mm dish containing non-selective medium . When the 100 mm dishes were confluent, the cells were subcultured at 10 5 cells per 100 mm dish into plates containing either non-selective or selective medium . Growth of cells from the two groups was then observed . The uptake of 86 Rb, a tracer for potassium, was used to characterize Ouar mutants as described by Elmore and Barrett (1982) . The effect of ouabain on 86 Rb transport by Na + -K + ATPase was determined in Ouar mutants from both normal and immortalized HMEC . 2 .6 . Statistical analyses Statistical comparisons were done using the Mann-Whitney non-parametric two-sample test or a paired t-test . 3 . Results 3 .1 . Na + -K + ATPase mutagenesis assay +Figure 1 shows background and UV-induced mutation frequencies at the Na K + ATPase locus in normal and immortalized HMEC . Significant mutation
S . R . Eldridge and M. N . Gould
810
300
300
250
200
c
150
150
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
100
50
0
0 Normal
Immortalized
Figure 1 . UV induction of mutations at the Na + -K + ATPase locus in HMEC . Mutation frequencies at the Na + -K + ATPase locus are shown for control (open) and UV (10 J/m2 ) exposed (hatched) normal and immortalized cells (184B5) . The asterisk (*) indicates that the mean background mutation frequency in normal HMEC at this locus was not detected when > 10 s cells were screened in five independent experiments . The mutation frequency in UV-irradiated normal HMEC represents the mean ±SE of four determinations (cells from three different women) . Background (n = 4) and UV-induced (n= 5) mutations at the Na + -K + ATPase locus in immortalized cells (184B5) represent mean ±SE .
induction was observed in both normal and immortalized HMEC exposed to 10 J/m2 UV (p=0 . 01) . Uptake of 86 Rb by wild-type and Oua` mutants was measured in normal and immortalized HMEC . Representative data are presented in Figure 2 . In the presence of ouabain, uptake of 86 Rb was inhibited in wild-type HMEC as shown in Figure 2(a) . However, in Oua` mutants, 86 Rb uptake was not affected by ouabaincontaining medium (Figure 2b) . The Oua` phenotype was stable in the immortalized HMEC, as determined by growth of mutant cells in selective medium after approximately 10 populaton doublings in non-selective medium . In addition, the growth rates of wild-type and mutant HMEC were the same . 3 .2 . HPR T mutagenesis assay Background and UV-induced mutation frequencies at the HPRT locus, corrected for recovery of mutants, are presented in Figure 3 for normal and immortalized HMEC . The mutation frequency for normal HMEC exposed to UV radiation in primary culture did not differ from the background mutation frequency (p=0 . 2) . In contrast, significant mutation induction was observed in the immortalized HMEC (184B5) (p=0 . 005) . UV induction of mutations was not observed in nonimmortalized normal HMEC from the same (case 184) and different (case B482) cell lineages as the immortalized HMEC . 4.
Discussion
The quantitation of mutagenesis in mammalian cells serves as a screening assay for environmental genotoxic carcinogens . Somatic cell mutagenesis following
UV mammary cell mutagenesis
811
12000
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
10000
0
30
60
90
120 150 180
0
Time (minutes)
30
60
90
Time (minutes)
(a)
(b) Figure 2 . Characterization of Na + -K + ATPase mutants in HMEC . The transport of 86 Rb by Na + -K + ATPase in a representative variant HMEC clone was determined with or without ouabain in the medium as described in § 2 . Each data point represents the mean ± SE of three determinations . Some error bars are contained within data points . (a) Wild-type immortalized HMEC ; (b) Oua` normal HMEC . Control cultures (solid lines) ; ouabain-containing cultures (dashed lines) .
10
100 c -80
r a
- 60
V 1 0
M
Normal
Late Passage (a)
Immortalized
0
(b)
Figure 3 . UV induction of HPRT mutations in HMEC . Mutation frequencies at the HPRT locus are shown in control (open) and UV (10 J/m 2) exposed (hatched) cells . Panel (a) compares mutation frequencies in UV-exposed and control normal HMEC . Background mutation frequency for normal HMEC represents the mean ± SE of five determinations (cells from four different women) . UV-induced mutation frequency in normal HMEC represents the mean ±SE of seven determinations (cells from six different women) . Background and UV-induced mutation frequencies represent five determinations (n=2 and 3, respectively) in cases 184 (passage 9) and B482 (passage 14) . Panel (b) demonstrates the UV induction of mutations at the HPRT locus in immortalized HMEC (184B5) . The frequencies represent mean ± SE for seven paired experiments . Methods are presented in the text .
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
812
S . R . Eldridge and M . N. Gould
exposure to physical and chemical agents has been routinely quantitated in rodent cell lines and human diploid fibroblasts (Committee 2 Final Report 1982, Gupta 1984, Bradley et al . 1981) . However, human cancers are predominantly carcinomas which originate from epithelial cells . If the induction of somatic cell mutations is tissue-specific, alternative assays with the cell type of interest may be necessary . This possibility has been addressed in part by using mediated mutagenesis assays (Huberman and Sachs 1984) . In these assays an organ-specific cell, such as HMEC (Gould et al . 1986), can be used to activate test chemicals which ultimately induce mutations in a tester cell line, such as V79 hamster fibroblasts . Thus, the mediated assay examines the specificity of carcinogen activation, but not of mutation induction . In order to study the tissue-specificity of mutation induction, mutagenesis must be quantified in specific cell types . Only limited data are available for mutagenesis in human epithelial cells (Allen-Hoffmann and Rheinwald 1984) . Therefore, we investigated the specificity of mutation induction directly in HMEC using a direct-acting mutagen . + UV induced mutations at the Na -K + ATPase locus in both normal and immortalized HMEC at a frequency equivalent to that reported at this locus in human fibroblasts (Buchwald 1977) . In contrast, primary cultures of normal HMEC did not show an increased frequency of mutations at the HPRT locus when exposed to UV radiation . This unexpected observation could be due to the inability to mutate this locus or the failure to detect induced mutants . We feel that the latter possibility is unlikely, since we were able to quantitate the background mutation frequency in these cells in a range similar to that measured in several other cell types . In addition, UV-induced mutations were not found following both shorter (5 days, three population doublings) or longer (12 days, 10 population doublings) expression times at subconfluent cell densities, or with the use of different cell densities during selection of mutants . Our data suggest that UV-hypomutability at the HPRT locus can be overcome by cellular immortalization . It should be emphasized that non-immortalized cells, from the same lineage as the immortalized cells studied, were also UV-hypomutable at this locus . Arlett and Cole (1986) reported that fibroblasts from ataxia telangiectasia patients were not mutable at the HPRT locus by y-radiation, whereas their immortalized counterparts were sensitive to mutation induction . In addition, UV and y-radiation-induced mutation frequencies in normal cells immortalized by either SV40 or Epstein-Barr viruses were much higher than in their untransformed counterparts . Tatsumi and Takebe (1984) and Tatsumi et al. (1987) have reported similar findings . Collectively, these data suggest caution in the use of immortal cell lines in genotoxicity testing ; tissue-specific characteristics associated with the organ of origin may be overriden by the process of immortalization of the immortal phenotype . In summary, we have found a similar frequency of UV-induced specific locus mutations at the Na + -K + ATPase locus in human normal mammary epithetial cells as has been reported for human normal fibroblasts (Buchwald 1977) . In contrast, primary human mammary epithelial cells were UV-hypomutable at the HPRT locus when compared to human fibroblasts . Acknowledgements This work was supported by PHS NCI grants CA30295 and CA14520 . S .R .E . was supported by NIEHS grant ES07015 . Current address : Chemical Industry
UV mammary cell mutagenesis
813
Institute of Toxicology, 6 Davis Drive, Research Triangle Park, NC 27709 . The authors thank Drs Martha Stampfer for the generous gift of 184 and 184B5 cells, Cynthia J . Moore for critical review of the manuscript, Martin A . Tanner for statistical analayses, and Ms Peggy Ziebarth for preparation of the manuscript . Pituitary extract was obtained from Susan Hammond, Hammond Cell Technologies, Palo Alto, CA, USA .
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
References ALLEN-HOFFMANN, B . L . and RHEINWALD, J . G ., 1984, Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture . Proceedings of the National Academy of Sciences, USA, 81, 7802-7806 . AMES, B . N ., DURSTON, W. E ., YAMASAKI, E . and LEE, E . D ., 1973, Carcinogens are mutagens : a simple test system combining liver homogenates for activation and bacteria for detection . Proceedings of the National Academy of Sciences, USA, 70, 2281-2285 . ARLETT, C . F . and COLE, J ., 1986, Mutation studies in cells established from human cancerprone syndromes . Genetic Toxicology of Environmental Chemicals, Part A: Basic Principles and Mechanisms of Action, edited by C . Ramel, B . Lambert and J . Magnusson (Alan R . Liss, New York), pp . 237-244 . BARBACID, M ., 1986, Mutations, oncogenes and cancer . Trends in Genetics, 2, 188-192 . BRADLEY, M . 0 ., BHUYAN, B ., FRANCIS, M . C ., LANGENBACH, R . G ., PETERSON, A . and HUBERMAN, E ., 1981, Mutagenesis by chemical agents in V79 Chinese hamster cells : a review and analysis of the literature . A report of the Gene-Tox program . Mutation Research, 77, 81-142 . BUCHWALD, M ., 1977, Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts . Mutation Research, 44, 401-412 . CAIRNS, J ., 1975, The cancer problem. Scientific American, 233, 64-72 . CATHERS, L . E . and GOULD, M . N ., 1983, Human mammary cell survival following ionizing radiation . International Journal of Radiation Biology, 44, 1-16 . CAVENEE, W. K ., DRYJA, T . P., PHILLIPS, R . A ., BENEDICT, W . F., GODBOUT, R ., GALLIE, B . L ., MURPHREE, A . L ., STRONG, L . C . and WHITE, R . L ., 1983, Expression of recessive alleles by chromosomal mechanisms in retinoblastoma . Nature, 305, 779-784 . COMMITTEE 2 FINAL REPORT . (International Commission for Protection Against Environmental Mutagens and Carcinogens), Mutagenesis testing as an approach to carcinogenesis, Mutation Research, 99, 73-91 . ELDRIDGE, S . R ., MARTENS, T . W ., SATTLER, C . A . and GOULD, M . N ., 1989, Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells . Cancer Research, 49, 4326-4331 . ELMORE, E . and BARRETT, J . C ., 1982, Measurement of spontaneous mutation rates at the Na + -K + ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions . Mutation Research, 97, 393-404 . GOULD, M . N ., GRAU, D . R ., SEIDMAN, L . A . and MOORE, C . J ., 1986, Interspecies comparison of human and rat mammary epithelial cell-mediated mutagenesis by polycyclic aromatic hydrocarbons . Cancer Research, 46, 4942-4945 . GUPTA, R . S ., 1984, Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary cells : applications to mutagen screening studies . Handbook of Mutagenicity Test Procedures, edited by B . J . Kilbey, M . Legator, W. Nichols, and C . Ramel (Elsevier, Amsterdam), pp . 291-319 . HAMMOND, S . L ., HAM, R . G . and STAMPFER, M . R ., 1984, Serum-free growth of human mammary epithelial cells : Rapid clonal growth in defined medium and extended serial passage with pituitary extract . Proceedings of the National Academy of Sciences, USA, 81,5435-5439 . HUBERMAN, E . and SACHS, L ., 1984, Cell-mediated mutagenesis of mammalian cells with chemical carcinogens . International Journal of Cancer, 13, 326-333 . JACOBS, L . and DEMARS, R ., 1982, Chemical mutagenesis with diploid human fibroblasts . Handbook of Mutagenicity Test Procedures, edited by B . J . Kilbey, M . Legator, W . Nichols, and C . Ramel (Elsevier, Amsterdam), pp . 321-356 .
UV mammary cell mutagenesis
814
KouFos, A ., HANSEN, M . F ., LAMPKIN, B . C ., WORKMAN, M . L ., COPELAND, N . G ., JENKINS, N . A. and CAVENEE, W . K ., 1984, Loss of alleles at loci on human chromosome 11 during genesis of Wilms' tumour . Nature, 309,170-172 . MAHER, V. M ., OUELLETI, L . M ., CURREN, R . D . and MCCORMICK, J . J ., 1976, Frequency of ultraviolet light-induced mutations is higher in xeroderma pigmentosum variant cells than in normal human cells . Nature, 261, 593-595 . MYHR, B . C ., TURNBULL, D . and DIPAOLO, J . A ., 1979, Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts . Mutation Research, 62, 341-353 . STAMPFER, M . R ., 1985, Isolation and growth of human mammary epithelial cells . Journal of
Int J Radiat Biol Downloaded from informahealthcare.com by Thomas Jefferson University on 01/06/15 For personal use only.
Tissue Culture Methods, 9, 107-111 . STAMPFER, M . R . and BARTLEY, J . C ., 1985, Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo(a)pyrene .
Proceedings of the National Academy of Sciences, USA, 82, 2394-2398 . STAMPFER, M . R ., HALLOWES, R . C . and HACKETT, A . J ., 1980, Growth of normal human mammary cells in culture . In Vitro, 16, 415-425 . TATSUMI, K . and TAKEBE, H ., 1984, Gamma-irradiation induces mutation in ataxiatelangiectasia lymphoblastoid cells . Gann, 75, 1040-1043 . TATSUMI, K ., TOYODA, M ., HASHIMOTO, T ., FURUYAMA, J ., KURIHARA, T ., INOU, M . and TAKEBE, H ., 1987, Differential hypersensitivity of xeroderma pigmentosum lymphoblastoid cell lines to ultraviolet light mutagenesis . Carcinogenesis, 8, 53-57 . ZHANG, R ., WILEY, J ., HOWARD, S . P ., MEISNER, L . F . and GOULD, M . N ., 1989, Rare clonal karyotypic variants in primary cultures of human breast carcinoma cells . Cancer
Research, 49, 444-449 .
