BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor.

Mutational inactivation of BRCA1 confers a cumulative lifetime risk of breast and ovarian cancers. However, the underlying basis for the tissue-restricted tumor-suppressive properties of BRCA1 remains poorly defined. Here we show that BRCA1 mediates ligand-independent transcriptional repression of the estrogen receptor alpha (ERalpha), a principal determinant of the growth, differentiation, and normal functional status of breasts and ovaries. In Brca1-null mouse embryo fibroblasts and BRCA1-deficient human ovarian cancer cells, ERalpha exhibited ligand-independent transcriptional activity that was not observed in Brca1-proficient cells. Ectopic expression in Brca1-deficient cells of wild-type BRCA1, but not clinically validated BRCA1 missense mutants, restored ligand-independent repression of ERalpha in a manner dependent upon apparent histone deacetylase activity. In estrogen-dependent human breast cancer cells, chromatin immunoprecipitation analysis revealed the association of BRCA1 with ERalpha at endogenous estrogen-response elements before, but not after estrogen stimulation. Collectively, these results reveal BRCA1 to be a ligand-reversible barrier to transcriptional activation by unliganded promoter-bound ERalpha and suggest a possible mechanism by which functional inactivation of BRCA1 could promote tumorigenesis through inappropriate hormonal regulation of mammary and ovarian epithelial cell proliferation.

Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line.

STATEMENT OF FINDINGS: We tested the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as the inhibition of ovarian cell proliferation. Introduction of BRCA1 antisense retroviral constructs into BG-1 estrogen-dependent ovarian adenocarcinoma cells resulted in reduced BRCA1 expression. BRCA1 antisense pooled populations and derived subclones were able to proliferate in monolayer culture without estrogen, whereas control cells began to die after 10 days of estrogen deprivation. In addition, both populations and subclones of BRCA1 antisense infected cells demonstrated a growth advantage in monolayer culture in the presence of estrogen and were able to proliferate in monolayer culture without estrogen, while control cells did not. Furthermore, clonal studies demonstrated that reduced levels of BRCA1 protein correlated with growth in soft agar and greater tumor formation in nude mice in the absence of estrogen. These data suggest that reduction of BRCA1 protein in BG-1 ovarian adenocarcinoma cells may have an effect on cell survival during estrogen deprivation both in vitro and in vivo.

Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast.

The BRCA1 gene, mutations of which contribute significantly to hereditary breast cancer, was not identified in the existing YAC and BAC libraries. The gene is now available only as a set of overlapping fragments that form a contig. In this work we describe direct isolation of a genomic copy of BRCA1 from human DNA by transformation-associated recombination (TAR) cloning. Despite the presence of multiple repeats, most of the primary BRCA1 YAC isolates did not contain detectable deletions and could be stably propagated in a host strain with conditional RAD52. Similar to other circular YACs, approximately 90kb BRCA1 YACs were efficiently and accurately retrofitted into bacterial artificial chromosomes (BACs) with the Neo(R) mammalian selectable marker and transferred as circular BAC/YACs in E. coli cells. The BRCA1 BAC/YAC DNAs were isolated from bacterial cells and were used to transfect mouse cells using the Neo(R) gene as selectable marker. Western blot analysis of transfectants showed that BRCA1 YACs isolated by a TAR cloning contained a functional gene. The advantage of this expression vector is that the expression of BRCA1 is generated from its own regulatory elements and does not require additional promoter elements that may result in overexpression of the protein. In contrast to the results with cDNA expression vectors, the level of BRCA1 expression from this TAR vector is stable, does not induce cell death, maintains serum regulation, and approximates the level of endogenously expressed BRCA1 in human cells. The entire isolation procedure of BRCA1 described in this paper can be accomplished in approximately 10 days and can be applied to isolation of gene from clinical material. We propose that the opportunity to directly isolate normal and mutant forms of BRCA1 will greatly facilitate analysis of the gene and its contribution to breast cancer.

Establishment and characterization of a breast cell strain containing a BRCA1 185delAG mutation.

OBJECTIVE: The aim of this study was to examine whether cells containing the heterozygous form of a BRCA1 185delAG mutation would exhibit abnormal growth or an altered response to DNA damage. METHODS: A primary culture of human mammary epithelial cells (90P) was obtained from the nontumor breast tissue of a 35-year-old patient who had undergone a mastectomy for removal of a breast tumor. These cells were immortalized (90PE6E7) following retroviral infection with HPV-16 viral E6/E7. genes. Both the 90P cell strain and the cell line were characterized for their ability to grow in culture, form colonies in soft agar, and produce tumors in athymic nude mice compared to normal breast epithelial cells containing wild-type BRCA1. 90P cells were also analyzed for cellular response to gamma radiation and H(2)O(2). RESULTS: These cells were confirmed to contain a frameshift mutation, 185delAG, of the BRCA1 gene. Despite being heterozygous for wild-type BRCA1, the 220-kDa full-size BRCA1 protein was abundantly expressed. 90P and 90PE6E7 cells grew at a similar rate and were anchorage dependent. 90PE6E7 also failed to form tumors in athymic nude mice. Finally, 90P cells exhibited a survival response similar to that of normal mammary epithelial cells to radiation damage and exposure to oxidative stress. CONCLUSION: To our knowledge the 90P cells and the 90PE6E7 cells are the first characterized, non-tumor-derived breast epithelial cells that are heterozygous for the BRCA1 germline mutation 185delAG. Our conclusion is that these BRCA1 mutant cells appear to have growth and stress response characteristics similar to those of normal human breast cells, which is consistent with the hypothesis that loss of heterozygosity must occur to impair putative BRCA1 function.

DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene.

The promoter of the hTERT gene encoding the catalytic subunit of telomerase was recently cloned and has a dense CG-rich CpG island, suggesting a role for methylation in regulation of hTERT expression. In this study, we have initiated the analysis of the regulation of hTERT expression by examining the methylation status of up to 72 CpG sites extending from 500 bases upstream of the transcriptional start site of the hTERT gene into the first exon in 37 cell lines. These cell lines represent a variety of cell and tissue types, including normal, immortalized, and cancer cell lines from lung, breast, and other tissues. Using bisulfite genomic sequencing, we did not find a generalized pattern of site-specific or region-specific methylation that correlated with expression of the hTERT gene: most of the hTERT-negative normal cells and about one-third of the hTERT-expressing cell lines had the unmethylated/hypomethylated promoter, whereas the other hTERT-expressing cell lines showed partial or total methylation of the promoter. The promoter of one hTERT-negative fibroblast cell line, SUSM-1, was methylated at all sites examined. Treatment of SUSM-1 cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A induced the cells to express hTERT, suggesting a potential role for DNA methylation and/or histone deacetylation in negative regulation of hTERT. This study indicates that there are multiple levels of regulation of hTERT expression in CpG island methylation-dependent and -independent manners.

Estrogen upregulation of BRCA1 expression with no effect on localization.

Alterations in the expression of the breast and ovarian cancer susceptibility gene BRCA1 may contribute to the development of mammary and ovarian neoplasia. The sex-steroid estrogen modulates cell proliferation of normal and neoplastic breast and ovarian epithelial cells, but the role of estrogen regulation on the expression of BRCA1 remains to be defined. In this study, estrogen-regulated BRCA1 expression was examined in breast and ovarian cancer cells. Estrogen stimulated the proliferation of estrogen receptor (ER)-positive breast MCF-7, C7-MCF-7, and ovarian BG-1 cells as well as the expression of the estrogen-inducible pS2 gene. This was concomitant with upregulation of BRCA1 mRNA (2.5- to 5.0-fold) and a 3- to 10-fold induction of BRCA1 protein (230 kDa). Cell fractionation studies localized the BRCA1 protein to the nucleus in both unstimulated and estrogen-stimulated cells. The antiestrogen ICI-182780 inhibited estrogen-induced cell proliferation, BRCA1 mRNA induction, and BRCA1 protein expression in ER-positive cells. Conversely, estrogen did not influence expression of BRCA1 in HBL-100 cells that lacked the estrogen receptor, although the constitutive levels of BRCA1 mRNA (but not protein) in these cells were 5- to 30-fold higher than in other breast and ovarian cancer cells. Secretion of the BRCA1 protein into the cell medium did not account for the discrepancy between the mRNA and protein levels in HBL-100 cells. Proliferation of HBL-100 cells was not affected by either estrogen or ICI-182780. Taken together, these data support a role for the steroid estrogen and the involvement of the estrogen receptor pathway in the modulation of expression of BRCA1. We therefore propose that stimulation of cell proliferation may be a prerequisite for upregulation of BRCA1 in breast and ovarian cancer cells.

Evidence for two senescence loci on human chromosome 1.

Microcell-mediated introduction of a neo-tagged human chromosome 1 (HC-1-neo) into several immortal cell lines has previously been shown to induce growth arrest and phenotypic changes indicative of replicative senescence. Somatic cell hybridization studies have localized senescence activity for immortal hamster 10W-2 cells to a cytogenetically defined region between 1q23 and the q terminus. Previous microcell-mediated chromosome transfer experiments showed that a chromosome 1 with an interstitial q-arm deletion (del-1q) lacks senescence inducing activity for several immortal human cell lines that are sensitive to an intact HC-1-neo. In contrast, our studies reveal that the del-1q chromosome retains activity for 10W-2 cells, indicating that there are at least two senescence genes on human chromosome 1. Sequence-tagged site (STS) content analysis revealed that the q arm of the del-1q chromosome has an interstitial deletion of approximately 63 centimorgans (cM), between the proximal STS marker DIS534 and distal marker DIS412, approximately 1q12 to 1q31. This deletion analysis provides a candidate region for one of the senescence genes on 1q. In addition, because this deletion region extends distally beyond 1q23, it localizes the region containing a second senescence gene to approximately 1q31-qter, between DIS422 and the q terminus. STS content analysis of a panel of 11 10W-2 microcell hybrid clones that escaped senescence identified 2 common regions of loss of 1q material below the distal breakpoint of del-1q. One region is flanked by markers DIS459 and ACTN2, and the second lies between markers WI-4683 and DIS1609, indicating that the distal 1q senescence gene(s) localizes within 1q42-43.

A gene involved in control of human cellular senescence on human chromosome 1q.

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras(+)-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C. This chromosome 1 caused no change in proliferative potential of cell lines representing the other complementation groups. A derivative of human chromosome 1 that had lost most of the q arm by spontaneous deletion was unable to induce senescence in any of the immortal cell lines. This finding indicates that the q arm of human chromosome 1 carries a gene or set of genes which is altered in the cell lines assigned to complementation group C and is involved in the control of cellular senescence.

Investigation of the role of G1/S cell cycle mediators in cellular senescence.

Cellular senescence is a state of irreversible cell cycle arrest in which normal cells at the end of their lifespan fail to enter into DNA synthesis upon serum or growth factor stimulation. We examined whether proteins required for G1/S cell cycle progression were irreversibly down-regulated in senescent human fibroblasts. Both the 44- and 42-kDa forms of the MAP-kinase protein were expressed at similar levels in young and senescent cells. In contrast to young cells where both forms were phosphorylated on tyrosine in response to serum, the p42MAP-kinase was not tyrosine phosphorylated upon serum stimulation, whereas p44MAP-kinase was phosphorylated on tyrosine in serum-starved or serum-stimulated senescent cells. Examination of p53 protein in growing, quiescent, and senescent cells revealed no significant differences in levels between the different growth states. In contrast, cdk2 and cyclin A mRNAs were completely down-regulated in stimulated senescent fibroblasts, while the G1 cyclins, C, D1, and E mRNAs, were still expressed in stimulated senescent cells although at reduced levels compared to young cells. The expression of early G1 markers, but not late G1 markers, indicates that senescent cells may be blocked at a point in late G1. We investigated whether transfection of cyclin A, alone or in combination with cdc2, was sufficient for extension of lifespan or escape from senescence. Clones expressing the transfected human cyclin A or cdc2 genes senesced at a population doubling similar to controls, thereby showing that cyclin A or cdc2 expression alone was insufficient for escape from senescence.

Genetic and molecular basis for cellular senescence.

Normal human and rodent cells in culture exhibit a finite life span at the end of which they exhibit morphological changes and cease proliferating, a process termed cellular senescence or cellular aging. Many cancer cells differ from normal cells in that they do not senesce and have an indefinite life span in culture, suggesting that alterations relating to cellular senescence are involved in the neoplastic evolution of tumor cells. Recent experimental results strongly support a genetic basis for cellular senescence. Defects in the senescence program in transformed cells can be corrected by introduction of a specific chromosome from normal cells into the abnormal cells. Using this approach, possible senescence genes have been mapped to specific chromosomes. Cell cycle control genes that regulate entry into the DNA synthetic phase of the cell cycle must be altered in senescent cells. Recent findings suggest that phosphorylation of the retinoblastoma gene is altered in senescent cells. It is possible, but not yet proven, that aging at the cellular level contributes to the aging of the individual. Therefore, an understanding of cellular senescence at the genetic and molecular levels may provide new insights into both the cancer and aging processes.

Growth and transformation suppressor genes for BHK Syrian hamster cells on human chromosomes 1 and 11.

To map putative tumor suppressor genes for the near-diploid baby hamster kidney fibrosarcoma cell line BHK, we transferred five different normal human chromosomes (1, 3, 7, 11, and 12) into these tumor cells by microcell-mediated chromosome transfer. Transfer of human chromosome 1 into BHK cells resulted in suppression of cell growth both on plastic and in soft agar, indicating that chromosome 1 has a generalized effect on cell growth and thereby suppresses anchorage-independent growth. Selection against cells with an intact chromosome 1 was observed. In contrast, the introduction of chromosome 11 into BHK cells resulted in suppression of anchorage independence but not growth on plastic. Most chromosome-11 growth-suppressed BHK hybrids retained intact copies of human chromosome 11. Tumorigenic derivatives of chromosome 11 hybrids had lost this chromosome. Transfer of human chromosome 3, 7, or 12 into BHK cells did not correlate with growth suppression of BHK cells on plastic or in soft agar. Thus, we conclude that genes that suppress BHK-cell growth in general or in agar reside on human chromosomes 1 and 11, respectively.

Down-regulation of cdc2 in senescent human and hamster cells.

Senescent cells fail to respond to serum-induced signals for DNA synthesis. Because a central role for the p34cdc2 protein kinase is postulated in control of the cell cycle, we examined the status of this kinase in senescent cells and other growth-arrested cells. In growing human and Syrian hamster fibroblasts, three 35S-labeled proteins of 34-36 kDa were immunoprecipitated with p34cdc2 antiserum. Only the two slower migrating forms were phosphorylated as determined by 32P labelling. In senescent cells, which failed to incorporate [3H]thymidine, no p34cdc2 protein was synthesized and very little or no cdc2 mRNA was observed. When maintained for 48 h in 0.5% serum, young cells also retained only marginal cdc2 expression. After stimulation of low serum-arrested cells by addition of 10% serum, a time-dependent increase of cdc2 mRNA was observed, whereas serum stimulation of senescent cells did not increase cdc2 mRNA. In contrast to senescent and low serum-arrested cells, cdc2 mRNA was expressed at normal levels in cells partially growth arrested by isoleucine deficiency in G1, by aphidicolin at G1-S, by etoposide in G2, or by Colcemid in the M phase of the cell cycle, indicating that cdc2 down-regulation does not always occur upon growth arrest. Following transfection of a plasmid containing the human CDC2 gene into hamster cells, expression of human cdc2 failed to overcome the block to DNA synthesis in senescent cells. Although p34cdc2 was synthesized in the transfected cells, the multiple phosphorylated forms of the proteins were not observed. Taken together, these data support the concept that a chain of events leads to senescence. While p34cdc2 kinase may be one of the critical elements, other cell cycle controls are also involved.

Induction of cellular senescence in immortalized cells by human chromosome 1.

The control of cellular senescence by specific human chromosomes was examined in interspecies cell hybrids between diploid human fibroblasts and an immortal, Syrian hamster cell line. Most such hybrids exhibited a limited life span comparable to that of the human fibroblasts, indicating that cellular senescence is dominant in these hybrids. Karyotypic analyses of the hybrid clones that did not senesce revealed that all these clones had lost both copies of human chromosome 1, whereas all other human chromosomes were observed in at least some of the immortal hybrids. The application of selective pressure for retention of human chromosome 1 to the cell hybrids resulted in an increased percentage of hybrids that senesced. Further, the introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion caused typical signs of cellular senescence. Transfer of chromosome 11 had no effect on the growth of the cells. These findings indicate that human chromosome 1 may participate in the control of cellular senescence and further support a genetic basis for cellular senescence.

Role of a tumor-suppressor gene in the negative control of anchorage-independent growth of Syrian hamster cells.

Tumor-suppressor genes control the neoplastic phenotype of tumor cells, but the function of these genes in normal cells is unknown. In this report we show that the loss of a tumor-suppressor gene function releases negative controls on the growth of cells in agar. This conclusion is based on observations of cell hybrids and studies of cell variants that have retained or lost a tumor-suppressor gene function. Nontumorigenic cell hybrids between normal Syrian hamster embryo cells and a benzo[a]pyrene-transformed tumor-cell line (BP6T) continued to secrete autocrine and/or paracrine growth factors produced by the tumor cells but failed to respond to these factors by growing in agar. Normal diploid cells also failed to grow in agar in response to the growth factors produced by the tumor cells. Clonal variants of nontumorigenic, immortal Syrian hamster cell lines were isolated that either retained (termed supB+) or had lost (termed supB-) the ability to suppress tumorigenicity of BP6T tumor cells after cell hybridization. Neither supB+ nor supB- variants grew in agar under conditions that allowed efficient growth of the tumor cells. However, supB- cells were reversibly induced to grow in agar with high colony-forming efficiencies in the presence of tumor cell-conditioned medium or by supplementation of the medium with a combination of growth factors. Under the same conditions, the supB+ cells failed to grow in agar. This enhanced growth-factor responsiveness in agar was used to select for supB- variants existing at a low frequency in the supB+ population. These two phenotypes, loss of tumor-suppressor function and enhanced growth-factor responsiveness in agar, were seen to cosegregate. These results indicate the tumor-suppressor gene function in these cells negatively regulates the growth response of cells in agar to mitogenic stimuli. This growth regulation may depend on cell shape or adhesion because supB+ and supB- cells grown attached to plastic responded similarly to growth factors.

Longterm survival of normal, diploid Syrian hamster-cells in tumors induced by transfection with v-Ha-ras and v-myc oncogenes.

Tumors were induced following transfection of normal, diploid hamster embryo cells with plasmids containing the viral Harvey ras and viral myc oncogenes. Direct cytogenetic studies of the tumors performed at 3-7 weeks after injection of the transfected hamster cells into nude mice revealed that 100% of the hamster cells were aneuploid and no detectable diploid cells in mitosis were observed. However, when tumor explants were cultured in vitro, diploid Syrian hamster cells were frequently detected at early passages. The percentage of diploid hamster cells in the cultures varied from 2 to 94% at the first passage. After several passages in vitro, only aneuploid hamster cells were observed. The diploid hamster cells in culture had a flat morphology and senesced. The aneuploid cells in the cultures were readily cloned and formed tumors after reinjection in nude mice. Reconstruction experiments consisting of injecting cloned, aneuploid tumor cells mixed with 6 X 10(6) normal Syrian hamster embryo cells were performed. Cell cultures derived from these mixed tumors contained both normal and aneuploid hamster cells indicating that normal cells survived in vivo during the growth of the tumor cells for up to 4 weeks. During this period, some normal cells transplanted in vivo did not die or terminally differentiate. Since normal cells can persist in vivo, tumor-derived cell cultures are mixed populations and caution should be exercised in interpreting quantitative molecular or cellular studies of these uncloned populations.

Characterization of activated proto-oncogenes in chemically transformed Syrian hamster embryo cells.

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was less than 0.0008 foci/microgram DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of greater than or equal to 0.012 foci/microgram DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (less than 0.002 foci/microgram DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/microgram DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.

Evidence for multiple steps in neoplastic transformation of normal and preneoplastic Syrian hamster embryo cells following transfection with Harvey murine sarcoma virus oncogene (v-Ha-ras).

Neoplastic development of Syrian hamster embryo (SHE) cells in culture is a multistep process in which intermediate or preneoplastic cells can be identified and isolated. In an attempt to further characterize normal and preneoplastic cells, we have compared their susceptibilities to neoplastic transformation following transfection with cloned DNA of the oncogenic virus, Harvey murine sarcoma virus (HaMSV). Normal SHE cells, which are stably nontumorigenic when injected in nude mice, are competent to take up and express exogenous DNA as demonstrated by transfection experiments with pSV2-neo DNA and certain viral DNAs. SHE cells treated with 5 micrograms of HaMSV DNA per dish remained nontumorigenic. Colonies of SHE cells, isolated after cotransfection with HaMSV and pSV2-neo DNA and selection for G418 antibiotic resistance, expressed Harvey murine sarcoma virus oncogene (v-Ha-ras) RNA and were initially morphologically altered; however, all colonies senesced when subcultured. In contrast, transfection of the cells with polyoma virus DNA alone or HaMSV DNA plus MC29 viral DNA (pSVv-myc) and then injection of the cells into nude mice resulted in progressively growing tumors of hamster origin within 3 to 5 weeks. A preneoplastic cell line, DES-4, isolated after treatment of SHE cells with the human carcinogen diethylstilbestrol, was chosen for comparative analyses. These immortalized cells are nontumorigenic and excellent recipients for exogenous DNA. In contrast to SHE cells, DES-4 cells were highly susceptible to neoplastic transformation following transfection with HaMSV DNA. To further investigate the role of HaMSV DNA in the neoplastic transformation of DES-4 cells and to determine whether this occurred as a single step, clones of DES-4 cells cotransfected with pSV2-neo and HaMSV DNAs were selected by antibiotic resistance and characterized. There was a good correlation between tumorigenicity and expression of v-Ha-ras DNA; however, the clones were highly variable in terms of their latency periods in vivo and anchorage-independent growth. Neither of these two parameters correlated with the level of expression of v-Ha-ras RNA. All of the cell lines derived from tumors and reinoculated into nude mice had short latency periods in vivo, were highly anchorage independent, and had high levels of v-Ha-ras expression. These results suggest that, in these experiments, v-Ha-ras expression was necessary, but not sufficient, for the tumorigenicity of DES-4 cells and that additional changes in the cells were acquired.(ABSTRACT TRUNCATED AT 400 WORDS)