Int. J. Cancer: 50,321-324 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union InternationaleConire le Cancer
COMPARISON OF SPONTANEOUS MUTAGENESIS IN EARLY-PASSAGE HUMAN MAMMARY CELLS FROM NORMAL AND MALIGNANT TISSUES Sandra R. ELDRIDGE' and Michael N. GOULD' Department of Human Oncology, University of Wisconsin Clinical Cancer Center and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WZ 53792, USA. were used for normal and carcinoma HMEC. Briefly, tissue was grossly dissected, and skin and apparent fat discarded. The remaining tissue containing mammary parenchyma interspersed with stromal tissue was minced, then enzymatically digested overnight in a mixture containing collagenase and hyaluronidase (for details, see Eldridge et al., 1989). After dissociation, the epithelial cells in the form of ductal fragments (> 10 cells per fragment) were collected. Primary cultures, started from either fresh or c o reserved cells, were plated at an approximate density of 10Ycells P per 100-mm plastic tissueculture dish (Lux, Miles, Naperville, IL). Epithelial cells grew out from the attached ductal fragments. These cultures were maintained for 7 days with regular feedings with MCDB 170 medium, and trypsinized with 0.25% trypsin (GIBCO, Grand Island, NY) plus 0.2 ng/ml EDTA (Sigma, St. Louis, MO). Single-cell suspensions were obtained from primary cultures. The process by which a normal cell is converted to a Immortalized HMEC (line 184B5) obtained from Dr. M. malignant one is complex and includes cellular accumulation Stampfer were derived from normal HMEC from one donor of multiple genetic alterations. A multi-stage genetic process, and immortalized by in vitro exposure to benzo[a]pyrene such as that recently reported for the stepwise development of (Stampfer and Bartley, 1985). Non-immortalized cells (case colon cancer in humans (Vogelstein, 1989), has been docu- 184, passage 9) from the same cell lineage as the immortalized mented for many cancer types. Cancer as a genetic disease can cells were studied, as well as an HMEC strain (case B482, be driven either by a differentially increased mutation rate in passage 14) derived in our laboratory from normal HMEC. malignant and pre-malignant cells or by a process of clonal All cells used in this study were cultured under identical selection of mutated cells. It is not known which mechanism is predominant, although it has been suggested by Nowell (1986) conditions. Cells were maintained in 100-mm plastic tissuethat tumor progression occurs in genetically unstable cells. culture dishes in serum-free MCDB 170 medium prepared in This hypothesis has been both supported and refuted by our laboratory, as described by Hammond et al., (1984). The various experimental findings. Using a murine melanoma growth kinetics of all 3 cell types (normal, carcinoma and model, Cifone and Fidler (1981) have shown that metastatic immortalized HMEC) were very similar, as shown by flowtumors exhibit a higher mutant frequency than their non- cytometric analyses following propidium iodide staining (S.P. metastatic counterparts. This observation was not confirmed in Howard and M.N. Gould, data not shown); doubling time was a murine mammary model (Yamashima and Heppner, 1985). approximately 24 hr. Cloning efficiencies for normal, carciIt has also been suggested that increased mutation rates play a noma and immortalized HMEC varied between 20 and 50%, 5 role in tumor instability and heterogeneity, and in the emer- and 30% and 60 and SO%, respectively. gence of drug-resistant clones during therapy (Pratt and HPRT mutagenesis assay Raddon, 1979). Damen et al. (1989) have failed to demonstrate The methods described by Jacobs and DeMars (1982) to an increase in metastatic potential in hypermutable variants of select for HPRT mutants in normal human diploid fibroblasts Chinese-hamster ovary-cell lines. were adapted for the present study. At least 2 x lo6 exponenThe Nowell hypothesis (1986) predicts that tumor cells tially growing primary cells were seeded into non-selective would be hypermutable when compared to the normal cellular medium (lo5cells per 100-mmdish, 20 dishes per group). After counterpart. However, it is not clear whether tumor cells have 8 days (6 population doublings), these cells were subcultured a higher rate of mutagenesis than their normal equivalents, or into selection medium containing 30 FM TG (2-amino-6whether human tumors of a specific type or from a specific mercaptopurine, Sigma), which inhibited the growth of norindividual vary in their spontaneous mutant frequencies. To address these questions, we have developed methods to quantify specific locus mutations in HMEC cultures. Here we 'Present address: Chemical Industry Institute of Toxicology, 6 Davis report a quantitative comparison of spontaneous mutations at Drive, Research Triangle Park, NC 27709. the HPRT locus in primary human epithelial cells derived from normal and malignant breast tissues.
Spontaneous mutant frequencies were determined in earlypassage epithelial-cell strains derived from either normal or malignant human breast tissues, as well as a non-tumorigenic immortalized human mammary epithelial cell line (I8465) derived from normal cells. Mutations at the hypoxanthineguanine phosphoribosyltransferase (HPRT) locus were quantified by determining the 6-thioguanine resistance. Mutant frequencies in human mammary epithelial cells (HMEC) from 4 normal and 5 carcinoma tissue samples did not differ significantly. In contrast, the mutant frequency in the immortalized HMEC was approximately 10 times higher than in average normal HMEC and normal non-immortalized cells from earlypassage cultures of the same cell lineage. Our data suggest that the progression of normal breast cells to invasive carcinoma cells does not necessarily involve the establishment of a general genetic instability during this progression.
MATERIAL AND METHODS
Cells Normal HMEC were derived from residual surgical material from reduction mammoplasties of 4 healthy women. Carcinoma HMEC were derived from 5 samples of malignant human breast tissue. Procedures for the isolation of primary HMEC have been described (Stampfer et al., 1980), and identical techniques
'TO whom correspondence and reprint requests should be addressed, at University of Wisconsin-Madison, Department of Human Oncology, K4/332,600 Highland Avenue, Madison, WI 53792. Abbreviations: XP, xeroderma pigmentosum; UV, ultraviolet light; HMEC, human mammary epithelial cells; HPRT, hypoxanthineguanine phosphoribosyltransferase; TG, 6-thioguanine; TG', 6-thioguanine-resistant; TG', 6-thioguanine-sensitive. Received: July2, 1991 and in revised form August 30,1991.
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mal, carcinoma and immortalized HMEC equally well. Then, 5 x lo4normal or immortalized H M E C were plated per 100-mm dish into at least 200 or 100 dishes per experiment, respectively. Carcinoma H M E C were plated at a density of lo4 cells per 100-mm dish, into at least 1,000 dishes per experiment. In addition, 100 cells per 100-mm dish were plated into nonselective medium in order to determine cloning efficiencies. Cells were plated in such a way that saturation density was never achieved. Plates were incubated at 37°C in 5 % C 0 2 for 21 days with 2 changes of medium. The cells were fixed in methanol, stained with a 1:l mixture of azure I1 and methylene blue, and scored for TG' colonies containing > 100 cells. At least 10' cells were at risk in each experiment. Cell yields from carcinoma tissues were such that only 1 determination could be made per sample.
Reconstruction experiments Due to the possibility of cross-feeding of the toxic metabolite of T G from HPRT' wild-type cells to HPRT- mutant cells via gap junctions, the number of cells per dish which yielded optimal recovery of mutant cells during selection was determined for each of the 3 cell types. One hundred TG'immortalized H M E C were co-cultured with various numbers of TG' normal, carcinoma or immortalized H M E C in the presence of TG. Reconstruction experiments using TG' normal or carcinoma H M E C were not done due to the inability to expand mutant clones of these cell types with finite lifespans. However, we have previously shown that gap-junction intercelM a r communication is qualitatively similar between homologous normal or carcinoma H M E C cultures, and co-cultures of normal or carcinoma H M E C with immortalized H M E C (Eldridge et al., 1989). Survival of immortalized TG' cells cocultured with 5 x lo4 immortalized, normal or carcinoma wild-type HMEC, or lo4 carcinoma wild-type H M E C was loo%, 67%, 8.5% and 77%, respectively. These correction values, using immortalized T G cells as the target cells, accurately estimated recovery of mutants of each cell type; corrected mutation frequencies measured for different cell densities were equivalent (Eldridge et al., 1989). The mutation frequencies were thus calculated by correcting for recovery of TG' mutants and for the corresponding cloning efficiencies. Characterization of mutants TG' colonies wcre ring-cloned and propagated in selective medium to free the cultures of parental T G cells. These cells were used to determine heritability of the mutant phenotype and H P R T activity. T o determine the stability of the TG' phenotype, cells were subcultured from confluent dishes at lo4 cells per 100-mm dish containing non-selective medium. When the 100-mm dishes were confluent, the cells were subcultured at 10' cells per 100-mm dish into plates containing either non-selective or selective medium. Growth of cells from the 2 groups was then observed. H P R T activity was measured as described by Jacobs and DeMars (1982). Cells were plated in 50-mm X 20-mm chamber slides, grown to conflucnce, and assayed in sctu for conversion of ''C-hypoxanthine to inosinic acid and inosine. Protein determinations were made using the fluorescamine assay (Undenfriend et al., 1972). For each mutant, 2 chamber slides were assayed for enzyme activity and 2 others were assayed for protein. Lesch-Nyhan fibroblasts with no H P R T activity were used as negative controls, and wild-type normal, carcinoma and immortalizcd HMEC served as positive controls. Due to the limited lifespan of normal and carcinoma HMEC, it was not possible to propagate enough clonally-derived mutant cells to determine H P R T activity in TG' mutants from these 2 sources. H P R T activity was therefore measured only in mutant immortalized HMEC.
Statistical anabses Statistical comparisons were done using the Mann-Whitney non-parametric 2-sample test.
RESULTS
Mutagenesis in normal, carcinoma and immortalized HMEC Individual mutant frequencies at the H P R T locus in normal, carcinoma and immortalized H M E C are presented in Table I. Inter-individual variation was comparable to experimental variation (see case A935). Spontaneous mutant frequencies in normal and carcinoma H M E C did not differ significantly (p = 0.09). A 95% chance that there was no difference between the 2 means was determined after considering the variation between individuals. Immortalized H M E C had a higher background mutant frequency at the HPRT locus than either normal or carcinoma H M E C strains (p < 0.05). However, spontaneous mutant frequencies in non-immortalized passage 9 from the same individual as the immortalized H M E C (case 184) as well as in passage 9 of an independent (case B482) cell strain were similar to those of other early-passage normal H M E C strains (Table I). Characterization of mutants The H P R T activities from wild-type H M E C and TG' mutants selected from immortalized H M E C were measured. Only one of the 8 independently derived mutant clones tested had measurable H P R T activity, with enzyme levels 9% of those measured in wild-type immortalized HMEC (Table 11). In addition, wild-type normal, carcinoma and immortalized H M E C had similar levels of H P R T activity (Table 11). The T G phenotype was stable in the immortalized HMEC, as determined by growth of mutant cells in selective medium after approximately 10 population doublings in non-selective medium. In addition, the growth rates of wild-type and mutant H M E C were the same. TABLE I -BACKGROUND MUTANT FREQUENCIES IN HUMAN MAMMARY EPITHELIAL CELLS
Cell strain
Normaf early passage A935 A935 B269 B290 B499 Average (n = 5) Normal late passage 184' B482 Carcinoma 3401 3528 3546 3685 3603 Average (n = 5 ) Immortalized 184B5
Mutants per 19" surviving cells
-
6.0 2.3 0.9 2.6 0.4 2.44 2 0.98 0.5
0.03 (2)' 3.8
2
3.3 11.8 4.8 6.0 1.9 5.56 2 1.70 25
?
8.5 (7)'
'Mutant frequencies at the HPRT locus were determined and corrected for mutant cell recovery as described in "Material and Methods".-'Case 184 is from the same cell lineage as the immortalized cell line, 184B5.-'Mean 5 standard error for the number of independent experiments indicated in parentheses.
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MUTAGENESIS IN HUMAN BREAST CELLS TABLE I1 - HPRT ACTIVITY I N TG‘ MUTANTS AND WILD-TYPE HUMAN MAMMARY EPITHELIAL CELLS
Cell type
HPRT activity’ (pmol product/&gcell oroteinl30 min)
Immortalized HMEC Normal HMEC Carcinoma HMEC Immortalized TG‘ clone 1 Immortalized T G clone 2 Immortalized TG‘ clone 3 Immortalized T G clone 4 Immortalized TG‘ clone 5 Immortalized T G clone 6 Immortalized TG‘ clone 7 Immortalized TG‘ clone 8
333 f 3782 550 f 78 209 f 27 31 f 23 0 0 0 0 0 0 0
HPRT activity was quantified on the basis of radioactive hypoxanthine converted to inosinic acid and inosine per pg protein as described in “Material and Methods”. Normal and carcinoma HMEC data were obtained from 2 separate donors each, with duplicate experiments for each donor (n=4). HPRT activity was determined in an immortalized HMEC line 18485 (n=4).‘Corrected for background counts; total protein ranged from 24 to 72 pg per chamber slide.-*Mean k SE.
DISCUSSION
In the current study, mutant frequency was quantified in 3 different types of HMEC: normal, carcinoma and immortal celIs. Cultures of these 3 HMEC types have been characterized in our laboratory as well as in others under similar culture conditions. W e have examined early-passage cultures of normal and carcinoma HMEC by immunofluorescence staining for epithelial cell-specific keratin and found them to be > 90% keratin-positive (Gould et al., 1986). In addition, similarly cultured H M E C express mammary-cell-specific human milk fat antigen as shown by indirect immunofluorescence (Stampfer, 1985). Both normal and carcinoma H M E C examined in our laboratory exhibit limited lifespans in culture, and diploid or near-diploid karyotypes (Zhang et al., 1989). However, only carcinoma-derived strains contain clonal karyotypic changes such as translocations and deletions (Zhang et al., 1989). Cell strains derived from normal and carcinoma cells also differ greatly in their capacity for intercellular communication (Eldridge et al., 1989). H M E C from malignant tumors grow in agar, whereas normal HMEC d o not (Besch et al., 1983). Immortalized H M E C (line 184B5) do not grow in agar, nor do they produce tumors in nude mice. They express epithelialcell-specific keratin and mammary-cell-specific human milk fat antigen. Karyotypic analyses show the cells to be near-diploid (Stampfer and Bartley, 1985). While tumorigenic cell lines have been derived from many different immortalized cell lines in culture (Gruenert, 1987; Heidelberger et al., 1983), it is unclear whether the immortal phenotype is a prerequisite for neoplastic transformation. This association is not required in the clinical situation, since 70 cell doublings are more than sufficient to cause death of a patient. Immortalized cells appear to represent a very limited proportion of malignant breast cancer cells and occur at a late stage in the progression of human breast cancer (Smith et al., 1987). Specific locus mutation frequencies in mammalian cells have usually been quantified in either extensively passaged rodent
and human cell lines or early-passage normal human diploid fibroblasts. Limited mutagenesis data have also been reported for several organ-specific human cell strains, including lymphocytes (Seshadri et al., 1987), endothelial cells (Reznikoff and DeMars, 198l), and skin keratinocytes (Allen-Hoffmann and Rheinwald, 1984). Attempts at direct comparisons of mutant frequencies in normal and tumor-derived cells of similar cell types have been limited. Seshadri et al. (1987) have reported a higher background mutant frequency in malignant lymphoid cells than in norma1 lymphocytes. However, the malignant cells were immortalized cell lines, whereas the normal lymphocytes were early-passage cell strains. In contrast, Elmore and Barrett (1982), comparing mutant frequencies in human early-passage fibroblast strains and in a chemically transformed cell line, reported no differences between these cells in background mutant frequencies at 2 loci. Kaden et al. (1989) showed that, while the mutation frequency in normal and malignant hamster fibroblast cell lines was similar, a high frequency of large deletions was observed among the H P R T mutants recovered from the tumor cell lines. In our study, we compared background mutant frequencies in early-passage H M E C derived from normal breast tissue and breast carcinomas obtained from several individual women, as well as an immortalized H M E C line derived from normal HMEC. No significant difference in background mutant frequencies at the H P R T locus in normal o r carcinoma-derived HMEC was found. The molecular basis of the rare mutant phenotypes observed is unknown. It is thus not known whether these HPRT-locus data can be generalized to other loci that are mechanistically related to the carcinogenesis process. The mutant frequencies found were similar to those reported for early-passage human diploid fibroblasts. In contrast, immortalized H M E C had a higher background mutant frequency at this locus than either normal or malignant HMEC strains. This increase in mutagenesis observed in the immortalized H M E C line was not a characteristic of the individual from whom the immortalized cells were derived, since nonimmortalized, early-passage H M E C from this donor had a background mutant frequency similar to that of other normal HMEC. This suggests that the increased background mutant frequency observed in this cell line is probably a result of immortalization. These data, if generalizable to other loci, suggest that the progression of normal human breast cells to invasive carcinomas is not driven by the induction of genetic instability. ACKNOWLEDGEMENTS
We thank Drs. M. Stampfer for the generous gift of 184 and 184B5 cells, S. Howard for providing primary carcinoma cell cultures, C.J. Moore for critical review of the manuscript, and M.A. Tanner for statistical analyses. We also thank Ms. P. Ziebarth for preparation of the manuscript. Pituitary extract was obtained from Ms. S. Hammond, Hammond Cell Technologies, Palo Alto, CA. This work was supported by PHS NCI grants CA30295 and CA14520. S R E was a predoctoral trainee supported by NIEHS grant ES07015.
REFERENCES
ALLEN-HOFFMANN, B.L. and RHEINWALD, J.G., Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in cub ture. Proc. nat. Acad. Sci. (Wash.), 81,7802-7806 (1984). BESCH,G.J., WOLBERG, W.H., GILCHRIST, K.W., VOELKEL, J.G. and GOULD,M.N., A comparison of methods for the production of monodispersed cell suspensions from human primary breast carcinomas. Breast Cancer Res. Treat., 3, 15-22 (1983).
CIFONE, M.A. and FIDLER,I.J., Increasing metastatic potential is associated with increasing genetic instability of clones isolated from murine neoplasms.Proc. nut. Acad. Sci. (Wash.), 78,6949-6952 (1981). DAMEN, J.E., TAGGER, A.Y., GREENBERG, A.H. and WRIGHT,J.A., Generation of metastatic variants in populations of mutator and amPlificatormutants. Carzcer Insf.2 81,628-631 (1989). ELDRIDGE, S.R., MARTENS,T.W., SATTLER, C.A. and GOULD, M.N.,
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Association of decreased intercellular communication with the immor- PRATT,W.B. and RADDON, R.W., The anticancer drugs, Oxford 1Jniv. tal but not the tumorigenic phenotype in human mammary epithelial Press, New York (1979). cells. CancerRes., 49,43264331 (1989). REZNIKOPF, C.A. and DEMARS,R., In virro chemical mutagenesis and ELMORE, E. and BARRETT, J.C., Measurement of spontaneous muta- viral transformation of a human endothelial cell strain. Cancer Res., 41, 1114-1126 (1981). tion rates at the Na'/K' ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions. Mutation Rex, 97, SESHADRI, R., KUTLACA, R.J., TRAINOR, K., MATTHEW, C. and MOR393404 (1982). LEY, A.A., Mutation rate of normal and malignant human lymphoGOULD, M.N., GRAU,D.R., SEIDMAN, L.A. and MOORE,C.J., Interspe- cytes. Cancer Res., 47,407-409 (1987). cies comparison of human and rat mammary epithelial cell-mediated SMITH,H.S., WOLMAN, S.R., DAIRKEE, S.H., HANCOCK, M.C., LIPPmutagenesis by polycyclic aromatic hydrocarbons. Cancer Res., 46, MAN, M., LEFF,A. and HACKETT, A.J., Immortalization in culture4942-4945 (1986). occurrence at a late stage in the progression of breast cancer. J. nut. GRUENERT, D.C., Differential properties of human epithelial cells CancerInst., 78,611-615 (1987). transformed in vitro. Biotechniques, 5,740-749 (1987). STAMPFER, M.R., Isolation and growth of human mammary epithelial HAMMOND, S.L., HAM,R.G. and STAMPFER, M.R., Serum-free growth cel1s.J. Tiss. Cult. Meth., 9, 107-115 (1985). of human mammary epithelial cells: rapid clonal growth in defined STAMPFER, M.R. and BARTIEY,J.C., Induction of transformation and medium and extended serial passage with pituitary extract. Proc. nut. continuous cell lines from normal human mammary epithelial cells after exposure to benzo[a]pyrene. Proc. nat. Acad. Sci. (Wash.), 82, Acad. Sci. (Wash.), 81,5435-5439 (1984). 2394-2398 (1985). HEIDEIBERGER, C., FREEMAN, A.E., PIENTA,R.J., SIVAK,A., BERM.R., HALLOWES, R.C. and HACKETT,A.J., Growth of TRAM, J.S., CASTRO, B.C., DUNKEL, V.C., FRANCIS, M.W., KAKUNAGA, STAMPFER, T., LITTLE, J.B. and SCHECHTMAN, L.M., Cell transformation by normal human mammary cells in culture. In Vifro, 16,415425 (1980). chemical agents-a review and analysis of the literature. A report of UNDENFRIEND, S., STEIN,S., BOHIEN,P., DAIRMAN, W., LEIMGRUBER, the US Environmental Protection Agency Gene-Tox Program. Muta- W. and WEIGELE,M., Fluorescamine: a reagent for assay of amino tion Rex, 114,283-285 (1983). acids, peptides, proteins, and primary amines in the picamole range. JACOBS,L. and DEMARS,R., Chemical mutagenesis with diploid Science, 178,871-872 (1972). human fibroblasts. In: B. Kilby (ed.), Handbook of mutagenicity test VOGEISTEIN,B., FEARON, E.R., K ~ R NS.E. , and HAMILTON, S.R., procedures, pp. 321-356. Elsevier, Amsterdam (1982). Allelotypes of colorectal carcinomas. Science, 244,207-21 1 (1989). KADEN, D.A., BARDWEIL,L., NEWMARK, P., ANISOWICZ, A., SKOPEK, YAMASHINA, K. and HEPPNER,G.H., Correlation of frequency of T.R. and SAGER,R., High frequency of large spontaneous deletions of induced mutation and metastatic potential in tumor cell lines from a DNA in tumor-derived CHEF cells. Proc. nut. Acad. Sci. (Wash.), 86, single mouse mammary tumor. Cancer Res., 45,4015-4019 (1985). 2306-2310 (1989). ZHANG,R., WILEY,J., HOWARD, S.P., MEISNER,L.F. and GOLILD, NOWEIL, P.C.. Mechanisms of tumor progression. Cancer Res., 46, M.N., Rare clonal karyotypic variants in primary cultures of human breast carcinoma cells. Cancer Rex, 49,444-449 (1989). 2203-2207 (1986).
Publication of the International Union Against Cancer Publication de I'Union InternationaleConire le Cancer
COMPARISON OF SPONTANEOUS MUTAGENESIS IN EARLY-PASSAGE HUMAN MAMMARY CELLS FROM NORMAL AND MALIGNANT TISSUES Sandra R. ELDRIDGE' and Michael N. GOULD' Department of Human Oncology, University of Wisconsin Clinical Cancer Center and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, WZ 53792, USA. were used for normal and carcinoma HMEC. Briefly, tissue was grossly dissected, and skin and apparent fat discarded. The remaining tissue containing mammary parenchyma interspersed with stromal tissue was minced, then enzymatically digested overnight in a mixture containing collagenase and hyaluronidase (for details, see Eldridge et al., 1989). After dissociation, the epithelial cells in the form of ductal fragments (> 10 cells per fragment) were collected. Primary cultures, started from either fresh or c o reserved cells, were plated at an approximate density of 10Ycells P per 100-mm plastic tissueculture dish (Lux, Miles, Naperville, IL). Epithelial cells grew out from the attached ductal fragments. These cultures were maintained for 7 days with regular feedings with MCDB 170 medium, and trypsinized with 0.25% trypsin (GIBCO, Grand Island, NY) plus 0.2 ng/ml EDTA (Sigma, St. Louis, MO). Single-cell suspensions were obtained from primary cultures. The process by which a normal cell is converted to a Immortalized HMEC (line 184B5) obtained from Dr. M. malignant one is complex and includes cellular accumulation Stampfer were derived from normal HMEC from one donor of multiple genetic alterations. A multi-stage genetic process, and immortalized by in vitro exposure to benzo[a]pyrene such as that recently reported for the stepwise development of (Stampfer and Bartley, 1985). Non-immortalized cells (case colon cancer in humans (Vogelstein, 1989), has been docu- 184, passage 9) from the same cell lineage as the immortalized mented for many cancer types. Cancer as a genetic disease can cells were studied, as well as an HMEC strain (case B482, be driven either by a differentially increased mutation rate in passage 14) derived in our laboratory from normal HMEC. malignant and pre-malignant cells or by a process of clonal All cells used in this study were cultured under identical selection of mutated cells. It is not known which mechanism is predominant, although it has been suggested by Nowell (1986) conditions. Cells were maintained in 100-mm plastic tissuethat tumor progression occurs in genetically unstable cells. culture dishes in serum-free MCDB 170 medium prepared in This hypothesis has been both supported and refuted by our laboratory, as described by Hammond et al., (1984). The various experimental findings. Using a murine melanoma growth kinetics of all 3 cell types (normal, carcinoma and model, Cifone and Fidler (1981) have shown that metastatic immortalized HMEC) were very similar, as shown by flowtumors exhibit a higher mutant frequency than their non- cytometric analyses following propidium iodide staining (S.P. metastatic counterparts. This observation was not confirmed in Howard and M.N. Gould, data not shown); doubling time was a murine mammary model (Yamashima and Heppner, 1985). approximately 24 hr. Cloning efficiencies for normal, carciIt has also been suggested that increased mutation rates play a noma and immortalized HMEC varied between 20 and 50%, 5 role in tumor instability and heterogeneity, and in the emer- and 30% and 60 and SO%, respectively. gence of drug-resistant clones during therapy (Pratt and HPRT mutagenesis assay Raddon, 1979). Damen et al. (1989) have failed to demonstrate The methods described by Jacobs and DeMars (1982) to an increase in metastatic potential in hypermutable variants of select for HPRT mutants in normal human diploid fibroblasts Chinese-hamster ovary-cell lines. were adapted for the present study. At least 2 x lo6 exponenThe Nowell hypothesis (1986) predicts that tumor cells tially growing primary cells were seeded into non-selective would be hypermutable when compared to the normal cellular medium (lo5cells per 100-mmdish, 20 dishes per group). After counterpart. However, it is not clear whether tumor cells have 8 days (6 population doublings), these cells were subcultured a higher rate of mutagenesis than their normal equivalents, or into selection medium containing 30 FM TG (2-amino-6whether human tumors of a specific type or from a specific mercaptopurine, Sigma), which inhibited the growth of norindividual vary in their spontaneous mutant frequencies. To address these questions, we have developed methods to quantify specific locus mutations in HMEC cultures. Here we 'Present address: Chemical Industry Institute of Toxicology, 6 Davis report a quantitative comparison of spontaneous mutations at Drive, Research Triangle Park, NC 27709. the HPRT locus in primary human epithelial cells derived from normal and malignant breast tissues.
Spontaneous mutant frequencies were determined in earlypassage epithelial-cell strains derived from either normal or malignant human breast tissues, as well as a non-tumorigenic immortalized human mammary epithelial cell line (I8465) derived from normal cells. Mutations at the hypoxanthineguanine phosphoribosyltransferase (HPRT) locus were quantified by determining the 6-thioguanine resistance. Mutant frequencies in human mammary epithelial cells (HMEC) from 4 normal and 5 carcinoma tissue samples did not differ significantly. In contrast, the mutant frequency in the immortalized HMEC was approximately 10 times higher than in average normal HMEC and normal non-immortalized cells from earlypassage cultures of the same cell lineage. Our data suggest that the progression of normal breast cells to invasive carcinoma cells does not necessarily involve the establishment of a general genetic instability during this progression.
MATERIAL AND METHODS
Cells Normal HMEC were derived from residual surgical material from reduction mammoplasties of 4 healthy women. Carcinoma HMEC were derived from 5 samples of malignant human breast tissue. Procedures for the isolation of primary HMEC have been described (Stampfer et al., 1980), and identical techniques
'TO whom correspondence and reprint requests should be addressed, at University of Wisconsin-Madison, Department of Human Oncology, K4/332,600 Highland Avenue, Madison, WI 53792. Abbreviations: XP, xeroderma pigmentosum; UV, ultraviolet light; HMEC, human mammary epithelial cells; HPRT, hypoxanthineguanine phosphoribosyltransferase; TG, 6-thioguanine; TG', 6-thioguanine-resistant; TG', 6-thioguanine-sensitive. Received: July2, 1991 and in revised form August 30,1991.
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mal, carcinoma and immortalized HMEC equally well. Then, 5 x lo4normal or immortalized H M E C were plated per 100-mm dish into at least 200 or 100 dishes per experiment, respectively. Carcinoma H M E C were plated at a density of lo4 cells per 100-mm dish, into at least 1,000 dishes per experiment. In addition, 100 cells per 100-mm dish were plated into nonselective medium in order to determine cloning efficiencies. Cells were plated in such a way that saturation density was never achieved. Plates were incubated at 37°C in 5 % C 0 2 for 21 days with 2 changes of medium. The cells were fixed in methanol, stained with a 1:l mixture of azure I1 and methylene blue, and scored for TG' colonies containing > 100 cells. At least 10' cells were at risk in each experiment. Cell yields from carcinoma tissues were such that only 1 determination could be made per sample.
Reconstruction experiments Due to the possibility of cross-feeding of the toxic metabolite of T G from HPRT' wild-type cells to HPRT- mutant cells via gap junctions, the number of cells per dish which yielded optimal recovery of mutant cells during selection was determined for each of the 3 cell types. One hundred TG'immortalized H M E C were co-cultured with various numbers of TG' normal, carcinoma or immortalized H M E C in the presence of TG. Reconstruction experiments using TG' normal or carcinoma H M E C were not done due to the inability to expand mutant clones of these cell types with finite lifespans. However, we have previously shown that gap-junction intercelM a r communication is qualitatively similar between homologous normal or carcinoma H M E C cultures, and co-cultures of normal or carcinoma H M E C with immortalized H M E C (Eldridge et al., 1989). Survival of immortalized TG' cells cocultured with 5 x lo4 immortalized, normal or carcinoma wild-type HMEC, or lo4 carcinoma wild-type H M E C was loo%, 67%, 8.5% and 77%, respectively. These correction values, using immortalized T G cells as the target cells, accurately estimated recovery of mutants of each cell type; corrected mutation frequencies measured for different cell densities were equivalent (Eldridge et al., 1989). The mutation frequencies were thus calculated by correcting for recovery of TG' mutants and for the corresponding cloning efficiencies. Characterization of mutants TG' colonies wcre ring-cloned and propagated in selective medium to free the cultures of parental T G cells. These cells were used to determine heritability of the mutant phenotype and H P R T activity. T o determine the stability of the TG' phenotype, cells were subcultured from confluent dishes at lo4 cells per 100-mm dish containing non-selective medium. When the 100-mm dishes were confluent, the cells were subcultured at 10' cells per 100-mm dish into plates containing either non-selective or selective medium. Growth of cells from the 2 groups was then observed. H P R T activity was measured as described by Jacobs and DeMars (1982). Cells were plated in 50-mm X 20-mm chamber slides, grown to conflucnce, and assayed in sctu for conversion of ''C-hypoxanthine to inosinic acid and inosine. Protein determinations were made using the fluorescamine assay (Undenfriend et al., 1972). For each mutant, 2 chamber slides were assayed for enzyme activity and 2 others were assayed for protein. Lesch-Nyhan fibroblasts with no H P R T activity were used as negative controls, and wild-type normal, carcinoma and immortalizcd HMEC served as positive controls. Due to the limited lifespan of normal and carcinoma HMEC, it was not possible to propagate enough clonally-derived mutant cells to determine H P R T activity in TG' mutants from these 2 sources. H P R T activity was therefore measured only in mutant immortalized HMEC.
Statistical anabses Statistical comparisons were done using the Mann-Whitney non-parametric 2-sample test.
RESULTS
Mutagenesis in normal, carcinoma and immortalized HMEC Individual mutant frequencies at the H P R T locus in normal, carcinoma and immortalized H M E C are presented in Table I. Inter-individual variation was comparable to experimental variation (see case A935). Spontaneous mutant frequencies in normal and carcinoma H M E C did not differ significantly (p = 0.09). A 95% chance that there was no difference between the 2 means was determined after considering the variation between individuals. Immortalized H M E C had a higher background mutant frequency at the HPRT locus than either normal or carcinoma H M E C strains (p < 0.05). However, spontaneous mutant frequencies in non-immortalized passage 9 from the same individual as the immortalized H M E C (case 184) as well as in passage 9 of an independent (case B482) cell strain were similar to those of other early-passage normal H M E C strains (Table I). Characterization of mutants The H P R T activities from wild-type H M E C and TG' mutants selected from immortalized H M E C were measured. Only one of the 8 independently derived mutant clones tested had measurable H P R T activity, with enzyme levels 9% of those measured in wild-type immortalized HMEC (Table 11). In addition, wild-type normal, carcinoma and immortalized H M E C had similar levels of H P R T activity (Table 11). The T G phenotype was stable in the immortalized HMEC, as determined by growth of mutant cells in selective medium after approximately 10 population doublings in non-selective medium. In addition, the growth rates of wild-type and mutant H M E C were the same. TABLE I -BACKGROUND MUTANT FREQUENCIES IN HUMAN MAMMARY EPITHELIAL CELLS
Cell strain
Normaf early passage A935 A935 B269 B290 B499 Average (n = 5) Normal late passage 184' B482 Carcinoma 3401 3528 3546 3685 3603 Average (n = 5 ) Immortalized 184B5
Mutants per 19" surviving cells
-
6.0 2.3 0.9 2.6 0.4 2.44 2 0.98 0.5
0.03 (2)' 3.8
2
3.3 11.8 4.8 6.0 1.9 5.56 2 1.70 25
?
8.5 (7)'
'Mutant frequencies at the HPRT locus were determined and corrected for mutant cell recovery as described in "Material and Methods".-'Case 184 is from the same cell lineage as the immortalized cell line, 184B5.-'Mean 5 standard error for the number of independent experiments indicated in parentheses.
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MUTAGENESIS IN HUMAN BREAST CELLS TABLE I1 - HPRT ACTIVITY I N TG‘ MUTANTS AND WILD-TYPE HUMAN MAMMARY EPITHELIAL CELLS
Cell type
HPRT activity’ (pmol product/&gcell oroteinl30 min)
Immortalized HMEC Normal HMEC Carcinoma HMEC Immortalized TG‘ clone 1 Immortalized T G clone 2 Immortalized TG‘ clone 3 Immortalized T G clone 4 Immortalized TG‘ clone 5 Immortalized T G clone 6 Immortalized TG‘ clone 7 Immortalized TG‘ clone 8
333 f 3782 550 f 78 209 f 27 31 f 23 0 0 0 0 0 0 0
HPRT activity was quantified on the basis of radioactive hypoxanthine converted to inosinic acid and inosine per pg protein as described in “Material and Methods”. Normal and carcinoma HMEC data were obtained from 2 separate donors each, with duplicate experiments for each donor (n=4). HPRT activity was determined in an immortalized HMEC line 18485 (n=4).‘Corrected for background counts; total protein ranged from 24 to 72 pg per chamber slide.-*Mean k SE.
DISCUSSION
In the current study, mutant frequency was quantified in 3 different types of HMEC: normal, carcinoma and immortal celIs. Cultures of these 3 HMEC types have been characterized in our laboratory as well as in others under similar culture conditions. W e have examined early-passage cultures of normal and carcinoma HMEC by immunofluorescence staining for epithelial cell-specific keratin and found them to be > 90% keratin-positive (Gould et al., 1986). In addition, similarly cultured H M E C express mammary-cell-specific human milk fat antigen as shown by indirect immunofluorescence (Stampfer, 1985). Both normal and carcinoma H M E C examined in our laboratory exhibit limited lifespans in culture, and diploid or near-diploid karyotypes (Zhang et al., 1989). However, only carcinoma-derived strains contain clonal karyotypic changes such as translocations and deletions (Zhang et al., 1989). Cell strains derived from normal and carcinoma cells also differ greatly in their capacity for intercellular communication (Eldridge et al., 1989). H M E C from malignant tumors grow in agar, whereas normal HMEC d o not (Besch et al., 1983). Immortalized H M E C (line 184B5) do not grow in agar, nor do they produce tumors in nude mice. They express epithelialcell-specific keratin and mammary-cell-specific human milk fat antigen. Karyotypic analyses show the cells to be near-diploid (Stampfer and Bartley, 1985). While tumorigenic cell lines have been derived from many different immortalized cell lines in culture (Gruenert, 1987; Heidelberger et al., 1983), it is unclear whether the immortal phenotype is a prerequisite for neoplastic transformation. This association is not required in the clinical situation, since 70 cell doublings are more than sufficient to cause death of a patient. Immortalized cells appear to represent a very limited proportion of malignant breast cancer cells and occur at a late stage in the progression of human breast cancer (Smith et al., 1987). Specific locus mutation frequencies in mammalian cells have usually been quantified in either extensively passaged rodent
and human cell lines or early-passage normal human diploid fibroblasts. Limited mutagenesis data have also been reported for several organ-specific human cell strains, including lymphocytes (Seshadri et al., 1987), endothelial cells (Reznikoff and DeMars, 198l), and skin keratinocytes (Allen-Hoffmann and Rheinwald, 1984). Attempts at direct comparisons of mutant frequencies in normal and tumor-derived cells of similar cell types have been limited. Seshadri et al. (1987) have reported a higher background mutant frequency in malignant lymphoid cells than in norma1 lymphocytes. However, the malignant cells were immortalized cell lines, whereas the normal lymphocytes were early-passage cell strains. In contrast, Elmore and Barrett (1982), comparing mutant frequencies in human early-passage fibroblast strains and in a chemically transformed cell line, reported no differences between these cells in background mutant frequencies at 2 loci. Kaden et al. (1989) showed that, while the mutation frequency in normal and malignant hamster fibroblast cell lines was similar, a high frequency of large deletions was observed among the H P R T mutants recovered from the tumor cell lines. In our study, we compared background mutant frequencies in early-passage H M E C derived from normal breast tissue and breast carcinomas obtained from several individual women, as well as an immortalized H M E C line derived from normal HMEC. No significant difference in background mutant frequencies at the H P R T locus in normal o r carcinoma-derived HMEC was found. The molecular basis of the rare mutant phenotypes observed is unknown. It is thus not known whether these HPRT-locus data can be generalized to other loci that are mechanistically related to the carcinogenesis process. The mutant frequencies found were similar to those reported for early-passage human diploid fibroblasts. In contrast, immortalized H M E C had a higher background mutant frequency at this locus than either normal or malignant HMEC strains. This increase in mutagenesis observed in the immortalized H M E C line was not a characteristic of the individual from whom the immortalized cells were derived, since nonimmortalized, early-passage H M E C from this donor had a background mutant frequency similar to that of other normal HMEC. This suggests that the increased background mutant frequency observed in this cell line is probably a result of immortalization. These data, if generalizable to other loci, suggest that the progression of normal human breast cells to invasive carcinomas is not driven by the induction of genetic instability. ACKNOWLEDGEMENTS
We thank Drs. M. Stampfer for the generous gift of 184 and 184B5 cells, S. Howard for providing primary carcinoma cell cultures, C.J. Moore for critical review of the manuscript, and M.A. Tanner for statistical analyses. We also thank Ms. P. Ziebarth for preparation of the manuscript. Pituitary extract was obtained from Ms. S. Hammond, Hammond Cell Technologies, Palo Alto, CA. This work was supported by PHS NCI grants CA30295 and CA14520. S R E was a predoctoral trainee supported by NIEHS grant ES07015.
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