BRCA1 Partially Reverses the Transforming Activity of the ras Oncogene

BRIEF ARTICLE

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BRCA1 Partially Reverses the Transforming Activity of the ras Oncogene Abhay Kumar, Christine Knott, Kristine Kuus - Reichel and Mohammad S. Saedi 1 Hybritech Incorporated, a subsidiary of Beckman Coulter Inc., P.O. Box 269006, San Diego, CA 92196 -9006 Abstract The BRCA1 gene is associated with hereditary breast and ovarian cancers. BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines. The present study was designed to test the potential of BRCA1 to reverse the transforming activity of the ras oncogene. The v - Ha ras oncogene was cloned downstream of the retrovirus LTR and stably expressed in Rat - 1 cells ( Rat - 1 / ras ) . Rat - 1 / ras ( R / R ) cells were fully transformed as indicated by change in morphology, colony formation in soft - agarose and tumor induction in nude mice. BRCA1 was stably expressed in R / R cells under the CMV promoter ( R / R - BRCA1 ) . The expression of ras and BRCA1 was confirmed by Western blot using monoclonal antibodies ( mAbs ) specific to ras and BRCA1, respectively. R / R - BRCA1 cells grew slower than the negative control, which was R / R cells transfected with vector alone ( R / R - pCEP4 ) . R / R - BRCA1 cells generated  5 to 10 times less colonies in a soft - agarose assay compared to the negative control. When injected into nude mice, R / R BRCA1 cells exhibited a delayed onset of tumorigenesis and generated smaller tumors compared to R / R or R / R pCEP4 cells. These data strongly suggest that BRCA1 partially reverses the transforming activity of the v - Ha ras oncogene indicating that BRCA1 can bypass the effects of the v - Ha ras oncogene on cell growth. BRCA1, therefore, may be used in therapy of tumors arising due to activation of v - Ha ras oncogene. Keywords: BRCA1, breast cancer, tumor suppressor, ras, oncogene.

Introduction Breast cancer is one of the most common malignancies affecting women. Each year, over 179,000 new cases and 43,500 deaths are reported in the United States alone ( American Cancer Society. Cancer facts and figures - 1999 [ web site ] Atlanta, GA, http: / / www.cancer.org ) . The cumulative risk of developing breast cancer in women is 2% by age 50 and rises to 10% by age 80 [ Ries LAG, Kosary CL, Hankey BF, Harras A, and Edwards BK (1998) , Surveillance, epidemiology and end results [ SEER ] program [ web site ] Bethesda, MD, National Institutes of Health, http: / / www - seer.ims.nci.nih.gov / ] . Breast cancer may oc-

cur in either hereditary or sporadic forms. Familial cancers are thought to represent approximately 5% to 10% of all breast cancers [ 1 ] . Two familial breast cancer susceptibility genes, BRCA1 and BRCA2 have been identified [ 2,3 ] . A number of studies suggest that mutations in BRCA1 gene account for 10% to 20% of inherited breast cancers and 45% of the families with both breast and ovarian cancers [ 4 ] . The BRCA1 gene is located on chromosome 17q21 and encodes an 1863 amino acid ( 220 kDa ) protein product which bears several well - known amino acid motifs ( reviewed in Ref. [ 5 ] ) . For example, it contains a zinc binding RING finger domain near the N - terminus, two nuclear localization signals and two copies of BRCT motif that reside near the C terminus. BRCA1 exhibits a number of biologic functions. It possesses a transactivating activity [ 6 ] , plays an important role in DNA repair [ 7 ] and participates in cell cycle control [ 8 ] . BRCA1 also has a role in development and differentiation as suggested by in utero death of the nullizygous mice [9]. Since neoplastic development in BRCA1 mutation carriers is accompanied by loss or inactivation of the wild type allele, it is suggested that the BRCA1 protein is likely to function as a tumor suppressor [ 10 ] . Several lines of evidence support this hypothesis. Overexpression of BRCA1 is toxic to 293 EBNA cells [ 11 ] and enhances the sensitivity of NIH3T3 cells to apoptosis [ 12 ] . Antisense inhibition of BRCA1 causes an increase in growth rates of normal mammary cells, MCF - 7 cells [ 13 ] , and NIH3T3 cells [ 14 ] . Antisense inhibition of BRCA1 in NIH3T3 cells results in an increased number of colony formation in soft agarose, tumorigenicity in nude mice and resistance to apoptosis [ 14 ] . Finally, transfection of wild type BRCA1 into breast and ovarian cancer cells inhibits their growth [ 15 ] . Although several lines of evidence illustrate the tumor suppressor activity of BRCA1, the mechanism of tumor suppression during normal development is not understood. To gain additional insight into the tumor - suppressor function of BRCA1, we have studied its effects on the transforming Abbreviations: mAb, monoclonal antibody; R / R, Rat - 1 / ras; kDa, kilodaltons. Address all correspondence to: Dr. Abhay Kumar, Hybritech Incorporated, a subsidiary of Beckman Coulter, Inc., P.O. Box 269006, San Diego, CA 92196 - 9006. E-mail: [email protected] 1 Requests for materials to: Hybritech Incorporated, a subsidiary of Beckman Coulter, P.O. Box 269006, San Diego, CA 92196-9006. Tel.: 858-621-3258; fax: 858-621-4610; E-mail: [email protected] Received 2 July 1999; Accepted 16 August 1999. Copyright # 1999 Stockton Press. All rights reserved 1522-8002/99/$12.00

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activity of the v - Ha ras oncogene. Ras is an essential component in the transduction of extracellular signals that induce cell proliferation and differentiation ( reviewed in Ref. [ 16 ] ) . It is a membrane - localized guanine nucleotide binding protein that is active when bound to GTP. Activating mutations ( e.g., in codons 12 and 61 ) in ras result in constitutive signaling to downstream elements and are found at a high frequency in a wide variety of tumors, including more than 50% of colon carcinomas and 90% of pancreatic carcinomas [ 17 ] . In this manuscript, we demonstrate for the first time that BRCA1 partially reversed the oncogenic effect of v - Ha ras in Rat - 1 / ras ( R / R ) cells as shown by their decreased growth rate and diminished ability to form colonies in soft agarose. In addition, BRCA1 delayed the onset of tumorigenesis by R / R cells in nude mice. While ras was still expressed at high levels in R / R - BRCA1 clones, p21WAF1 / Cip1 expression was upregulated. The data suggest that BRCA1 bypasses the effects of v - Ha ras oncogene and may act as a general tumor suppressor by perturbing the expression of proteins involved in the cell cycle. These results also suggest that BRCA1 could have therapeutic potential in many types of cancer resulting from v - Ha ras activation.

Materials and Methods Expression Vectors and Cell Lines Expression vector pLJras [ 18 ] was used to express vHa ras. In this vector, the expression of v- Ha ras ( containing G12V mutation ) is driven by the Murine Sarcoma Virus LTR. cDNA encoding the full - length BRCA1 protein ( obtained from Myriad Genetics, Salt Lake City, UT ) was subcloned into the pCEP4 ( Hyg + , Invitrogen, San Diego, CA ) under the CMV promoter, resulting in pCEP4 - BRCA1 ( Figure 1b ) . Cloning junctions of pCEP4 - BRCA1 were verified by sequencing. Rat 1 cells were cultured in RPMI 1640 + 10% fetal clone ( Hyclone, Logan, UT ) + 2 mM L glutamine and 15 mM HEPES and were first transfected with pLJras using Lipofectamine ( Life Technologies, Gaithersburg, MD ) according to manufacturer's instructions. Stable single - cell clones ( R / R ) expressing ras protein were isolated by culturing the cells in the presence of G418 ( 400 g / ml, Life Technologies ) . Stable R / R cells were then transfected with either pCEP4 - BRCA1 or with empty pCEP4 using Lipofectamine resulting in R / R - BRCA1 and R / R - pCEP4 cells, respectively. Stable single - cell clones of R / R - BRCA1 and R / R - pCEP4 were isolated by culturing cells in Hygromycin ( 2 g / ml, Calbiochem, San Diego, CA ) . To study the growth rate of R / R - BRCA1 and R / R - pCEP4, cells were seeded at 2104 cells / ml of media. Viable cells were counted by trypan blue exclusion as described previously [ 19 ] . Western Blot Cells were washed with phosphate - buffered saline ( PBS ) and lysed in lysis buffer ( 10 mM Tris pH 7.5, 1% Triton X - 100, 5 mM EDTA, 5 mM sodium azide, 10 mM

sodium pyrophosphate, 10 mM sodium fluoride, 150 mM sodium chloride, 16 g / ml benzamidine hydrochloride, 100 mM phenylmethyl sulfonyl fluoride and 10 g / ml each of phenanthroline, aprotinin, leupeptin, and pepstatin A ) for 20 minutes at 48C. Lysates were centrifuged at 10,000g for 15 minutes at 48C to remove cell debris. The protein concentration of the lysates was determined by the micro BCA assay ( Pierce Chemicals, Rockford, IL ) . Proteins were electrophoresed under reducing conditions on a 4% to 20% SDS ± polyacrylamide gel ( Novex, San Diego, CA ) and were electroblotted onto nitrocellulose membrane. Blots were probed with mouse monoclonal antibody ( mAb ) BR1H 945.2 ( anti BRCA1 amino acid 1360 ± 1555 ) [ 20 ] , rat mAb  v - H - ras, rabbit polyclonal  p21WAF1 / Cip1 and goat polyclonal  actin ( Santa Cruz Biotechnology, Santa Cruz, CA ) . Blots were developed using an enhanced chemiluminescence system ( Amersham, Buckinghamshire, England ) according to manufacturer's instructions. Soft - Agarose Colony - Formation Assay Single - cell clones of R / R - BRCA1 and R / R - pCEP4 were suspended at 103 cells / ml of tissue culture medium containing 0.3% agarose and layered in duplicate onto 0.6% agarose in 35 - mm tissue - culture wells. Cultures were incubated in a humidified incubator containing 5% CO2 at 378C. Additional 0.3% agarose containing medium was added after 7 days. Colony growth was assessed after 14 days as described previously [ 19 ] . The values are represented as percent of control ( R / R pCEP4#50 ) . Tumorigenicity Assay Single - cell clones of R / R, R / R - pCEP4 and R / R - BRCA1 were harvested in log phase and viability was determined by trypan blue exclusion. 5104 cells were injected subcutaneously into the flank of athymic nu / nu female mice ( Harlan Sprague Dawley, Indianapolis, IN ) . Ten mice were used for each cell line. Mice were examined for tumors twice a week; tumor dimensions ( lengthwidth ) were measured with calipers, and tumor volumes ( cubic centimeters ) were calculated. Statistical analysis was performed as described previously [ 21 ] .

Results To elucidate the mechanism of tumor suppression by BRCA1, the ras and BRCA1 proteins were overexpressed in Rat - 1 cells. Rat - 1 cells were chosen because of their low spontaneous transforming ability in culture. The cDNA of v Ha ras oncogene was verified to contain a mutation ( G12V ) resulting in its constitutively active, GTP - bound state [ 16 ]. v - Ha ras oncogene under the control of Harvey Murine Sarcoma virus LTR promoter, was transfected into Rat - 1 fibroblast cells, resulting in R / R cells. As expected, morphologic changes and their ability to grow in soft agarose indicated that R / R cells were transformed ( data not shown ).

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Figure 1. Expression of BRCA1 and ras proteins in R / R cells transfected with pCEP4 - BRCA1. Total cell lysates from stable single - cell clones of R / R - BRCA1 ( #2 - 14, 7 - 15, 7 - 18, 7 - 19, 20 - 3 and 20 - 10 ) were prepared as described in materials and methods. Untransfected R / R cells and R / R cells transfected with pCEP4 alone ( R / R - pCEP4#50 ) were included as negative controls. Approximately 50 g total protein was electrophoresed under reducing conditions on a 4% to 20% gel and transferred onto nitrocellulose paper. ( a ) Detection of BRCA1 with mouse mAb BR1H 945.2 ( 3 g / ml ). Arrow, position of BRCA1. ( b ) Detection of ras protein with rat mAb  v - H - ras ( 2 g / ml, Santa Cruz ) . Arrow, position of ras. ( c ) The same blot was stripped and probed with goat  actin ( 1 g / ml, Santa Cruz ) . Arrow, position of actin. Left, molecular weight markers. kDa, kilodaltons.

To express BRCA1 protein in R / R cells, cDNA encoding full length BRCA1 was cloned under the CMV promoter and the resulting plasmid, pCEP4 - BRCA1, was transfected into the R / R cells ( R / R - BRCA1 ) . Empty pCEP4 was transfected into R / R cells ( R / R - pCEP4 ) and used as the negative control in all experiments. Several stable, single -

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cell clones of R / R - pCEP4 and R / R - BRCA1 cells were isolated. Based on expression of the BRCA1, the following six clones were chosen for further studies: 2 - 14, 7 - 15, 7 - 18, 7 - 19, 20 - 3, and 20 - 10. Two clones of R / R - pCEP4 ( #50 and 52 ) and one clone of R / R were included in the experiments as controls. Stable clones of R / R - BRCA1 were

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nohistochemistry and in breast - cancer cell lines by immunoprecipitation and Western blot ( [ 20 ] , Kumar et al., manuscript in preparation ) . As shown in ( Figure 1a ) , BRCA1 migrating at 220 kDa was detected in R / R - BRCA1 cells but not in R / R or R / R - pCEP4 cells. As shown in ( Figure 1b ) , R / R, R / R - pCEP4, and R / R - BRCA1 cells continued to express significant levels of ras protein. R / R pCEP4#50 appears to express ras protein at higher level compared to R / R cells. To rule out the effect of the empty pCEP4 on ras expression, three additional independent single - cell clones of R / R - pCEP4 were analyzed and found to have variable expression of ras protein ( data not shown ) . The same blot was also probed with goat  actin polyclonal antibody to ensure equal loading of proteins in all lanes ( Figure 1c ) . These results indicate that ras and BRCA1 proteins could be stably co - expressed in Rat - 1 cells and that ras expression remains high in BRCA1 transfected clones. To study the effect of BRCA1 expression on the growth rate of R / R cells, cell growth was measured for 8 consecutive days. As shown in ( Figure 2a ) , all single - cell clones of R / R - BRCA1 showed a significantly decreased growth rate as compared to R / R - pCEP4 single - cell clones. To study the effect of BRCA1 expression on anchorage independent growth, R / R - BRCA1 cells were plated in soft agarose and their colony forming ability was assessed after 14 days. As shown in ( Figure 2b ) , the colony - forming ability of R / R - BRCA1 cells was inhibited by 75% to 90% compared to R / R - pCEP4 cells. Since no significant difference in BRCA1 protein expression, growth in culture medium or growth in semisolid medium was observed among various R / R -BRCA1 clones, two representative clones of R / R - BRCA1 ( #7.19 and #20.10 ) were chosen to study tumorigenesis in nude mice. These two clones along with R / R and R / R -pCEP4 cells were injected subcutaneously into the flank of nude mice. Ten nude mice were used for each cell line. Tumor volume Figure 2. BRCA1 slows the growth rate and diminishes the colony forming ability of R / R cells. ( a ) The R / R - BRCA1 clones ( #2 - 14, 7 - 15, 7 - 18, 7 - 19, 20 - 3 and 20 - 10 ) and R / R - pCEP4 clones ( #50 and 52 ) were seeded at 210 4 / ml in a six - well plate. Viable cells were counted daily using trypan blue dye exclusion. Data are the averages for duplicate results from a representative experiment. ( b ) Colony forming ability of R / R - BRCA1 clones ( #2 - 14, 7 - 15, 7 - 18, 7 - 19, 20 - 3 and 20 - 10 ) and R / R - pCEP4 ( #50 and 52 ) was assessed by plating the 10 3 cells in 0.3% agarose. The colonies were counted at day 14. Values are given as a percentage of control, R / R - pCEP4 #50. Data are the averages for duplicate results from a representative experiment.

morphologically indistinguishable from that of R / R - pCEP4. R / R - BRCA1 cells, however, exhibited increased adherence to culture flasks compared to R / R - pCEP4. To ascertain the expression of ras and BRCA1 proteins, lysates of single - cell clones of R / R - BRCA1 as well as R / R and R / R - pCEP4 were prepared and probed with BRCA1 - specific ( mouse mAb BR1H 945.2 ) and ras - specific ( rat mAb ) antibodies on Western blots. BR1H 945.2 mAb has been shown to detect BRCA1 in normal and cancerous breast tissues by immu-

Figure 3. Expression of BRCA1 in R / R cells delayed the onset of tumor formation in nude mice. 510 4 cells from R / R - BRCA1 clones ( #7.19 and #20.10 ) and control cell lines ( R / R and R / R - pCEP4 #50 ) were injected subcutaneously into the flank of nude mice. Tumors were measured twice a week. Data represent the mean ‹ SEM tumor volume from each group ( n = 10 ) . Data represent one of the two identical studies with similar results.

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Figure 4. The expression of p21WAF1 / Cip1 is upregulated in R / R - BRCA1 cells. Total cell lysates from stable single - cell clones of R / R - BRCA1 ( #2 - 14, 7 - 15, 7 18, 7 - 19, 20 - 3 and 20 - 10 ) were prepared as described in materials and methods. Untransfected R / R cells and R / R cells transfected with pCEP4 alone ( R / R pCEP4#50 ) were included as negative controls. Approximately 50 g total protein was electrophoresed under reducing conditions on a 4% to 20% gel and transferred onto nitrocellulose paper. Blot was probed with rabbit polyclonal antibody  p21WAF1 / Cip1 ( 2 g / ml, Santa Cruz ) . Arrow, position of p21WAF1 / Cip1. Left, molecular weight markers. kDa, kilodaltons.

was measured twice a week. As shown in ( Figure 3 ) , R / R and R / R - pCEP4 cells formed 2.2 - to 2.8 - cm3 tumors in all animals by day 14, whereas both R / R - BRCA1 clones formed very small tumors ( <0.3 cm3 ) in only 10% of the animals. On day 21, R / R - BRCA1#7.19 formed an average tumor of 3 cm3 in all animals whereas R / R - BRCA1#20.10 formed an average of 0.3 - cm3 tumors. On day 35, R / R BRCA1#7.19 and R / R -BRCA1#20.10 cells formed an average of 10 - cm3 tumors in all animals which is similar to tumor formation by R / R and R / R - pCEP4 cells on day 21 ( data not shown ) . These results indicate that expression of BRCA1 in R / R cells resulted in a delayed onset of tumor formation. These data were confirmed by a second identical study. To confirm the expression of ras and BRCA1 proteins in these tumors, cell lysates from the tumors were prepared and expression of the ras and BRCA1 proteins were determined by Western blot as described above. All tumors ( R / R -BRCA1#7.19 and R / R -BRCA1#20.10 ) continued to express ras protein while the expression of BRCA1 was lost or diminished to an undetectable level ( data not shown ) . Several recent reports show that BRCA1 can affect p21WAF1 / Cip1 expression in a p53 - dependent and p53 independent manner [ 8,22 ] . To understand whether BRCA1 exerts its growth and tumor - suppressor effect on R / R cells by modulating the levels of p21WAF1 / Cip1, the expression of p21WAF1 / Cip1 in R / R - BRCA1 cells was determined by Western blot. As shown in Figure 4, p21WAF1 / Cip1 was expressed at a higher level in all of R / R - BRCA1 clones compared to R / R and R / R - pCEP4 cells. Since all of these clones are single - cell clones, it is not surprising that the level of p21WAF1 / Cip1 was variable among different clones. These results suggest that BRCA1 may suppress growth and tumorigenicity of R / R cells at least partly by upregulating p21WAF1 / Cip1.

Discussion The BRCA1 gene and its protein product have been the subjects of intensive investigation because of their important role in hereditary and putative role in sporadic human breast, ovarian, and prostate cancers. BRCA1 has been shown to

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function as a tumor - suppressor protein. However, the mechanism of tumor suppression is not well understood. The ras oncogene has been reported to contribute to as many as one - fifth of all human cancers, including more than half of colon cancers [ 17 ] . A great deal of work has shown that ras is an essential component in the transduction of extracellular signals that induce proliferation and differentiation [ 16 ] . Additionally, ras is required for transformation by many, but not all, oncogenes [ 23 ] . The following three lines of evidence presented in this manuscript suggest that expression of BRCA1 can partially reverse the tumorigenic potential of the v - Ha ras oncogene in R / R cells: 1 ) decreased growth rate, 2 ) diminished colony forming ability in the soft agarose assay, and 3 ) delayed onset of tumor formation in nude mice. It appears that BRCA1 acts independent of v - Ha ras in regulating cell cycle. It is probable that BRCA1 may act downstream of v - Ha ras via an independent pathway. This is because BRCA1 expression in R / R -BRCA1 cells affected cell growth, even though ras protein was still expressed at high level. Furthermore, when R / R -BRCA1 cells were injected into nude mice, tumors developed with a delayed onset compared with control cells. These tumors had lost the BRCA1 expression, suggesting that BRCA1 expression initially blocked the effect of ras and loss of BRCA1 expression allowed the ras oncogene to be tumorigenic. The eukaryotic cell cycle is regulated by the coordinated activity of cyclins, cyclin - dependent kinases ( cdk ) , and cdk inhibitors. BRCA1 protein expression is closely linked to the cell cycle, both in terms of absolute level and of phosphorylation status; however, its role has not been clearly defined. BRCA1 has been postulated to inhibit cell - cycle progression into S phase by increasing the level of p21WAF1 / Cip1, a cdk inhibitor, in a p53 - dependent [ 22 ] and p53 - independent manner [ 8 ] . Ouchi et al. [ 22 ] have also demonstrated that BRCA1 can specifically stimulate p53 - responsive elements and enhance p53 - dependent gene expression by acting as a p53 co - activator. The p21WAF1 / Cip1 gene contains p53 responsive elements in its promoter and was reported to be upregulated by BRCA1 [ 22 ] . On the contrary, cells from BRCA1 null mouse embryos have increased levels of

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p21WAF1 / Cip1 mRNA, which suggests that BRCA1 may suppress p21WAF1 / Cip1 expression during development to allow cell growth [ 24 ] . In the studies presented here, we demonstrate that ectopic expression of BRCA1 in R / R cells leads to upregulation of p21WAF1 / Cip1 protein as compared to R / R and R / R - pCEP4 cells. Whether the increase in p21WAF1 / Cip1 is through a p53 - dependent or p53 - independent pathway in R / R - BRCA1 cells is not known. Our findings do not clarify whether BRCA1 is acting directly or indirectly on p21WAF1 / Cip1. A correlation between expression levels of BRCA1 and upregulation of p21WAF1 / Cip1 could not be established. Also, there did not seem to be a correlation between the levels of BRCA1 or p21WAF1 / Cip1 and growth and tumor - suppressive effects. For example, R / R - BRCA1 clone #20.10 exhibited a slow growth rate, diminished colony - forming ability and delayed onset of tumors in nude mice, yet p21WAF1 / Cip1 was only marginally overexpressed in this cell line. The above data collectively suggest that upregulation of p21WAF1 / Cip1 may not be the sole mechanism by which BRCA1 is exerting its growth - and tumor suppressor function. BRCA1 has also been shown to modulate the activity of genes that are activated during cell - cycle progression. Wang et al. [ 25 ] recently demonstrated that BRCA1 binds to and inhibits the transcriptional activity of c - myc, an early response gene that is transcriptionally activated in early G1 phase. They also showed that this inhibition affected the transforming activity caused by synergistic actions of c - myc and ras in rat - embryo fibroblasts. These observations indicate that the role of BRCA1 in cell - cycle regulation, DNA - damage repair and transcriptional regulation appears to be complicated and thus merits further research. The ras oncogene is an essential component of the receptor - mediated signal transduction pathways that control cell proliferation and differentiation. Aberrant ras signaling has been associated with a variety of human cancers. Cell lines described here provide us with reagents to further study the role ( s ) of BRCA1 in regulation of ras - mediated signaling. Based on the observations described in this manuscript, it can be postulated that BRCA1 may be able to block or reverse the effect of other oncogenes, at the very least the ones that may be acting upstream of p53 or p21WAF1 / Cip1. We ( unpublished observations ) and others [ 11,12 ] have unsuccessfully tried to overexpress BRCA1 in a number of cell lines such as NIH3T3, Rat - 1, BHK21, and Cos - 7 cells. BRCA1 overexpression may be toxic to the cells as suggested by the inability to recover any stable clones. Overexpression of BRCA1 in R / R cells provides a means to obtain a stable cell line expressing BRCA1, although the cells exhibit a slower growth rate. In summary, we demonstrate for the first time that the tumorigenicity of the v - Ha ras oncogene can be partially reversed by overexpression of the BRCA1 protein. Thus far, gene - therapy studies using BRCA1 have been limited to breast, ovarian, and prostate cancers [ 26 ± 28 ] . In light of the observations presented here, gene therapy with BRCA1 may be an option for cancers where ras is aberrantly expressed.

Acknowledgements We thank Robert Wolfert for critical reading of the manuscript and Dong To for statistical analysis.

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