TGF-β1 Inhibits BRCA1 Expression through a Pathway That Requires pRb

Biochemical and Biophysical Research Communications 276, 686 – 692 (2000) doi:10.1006/bbrc.2000.3510, available online at http://www.idealibrary.com on

TGF-␤1 Inhibits BRCA1 Expression through a Pathway That Requires pRb 1 Daniel J. Satterwhite,* ,2 Nori Matsunami,† and Raymond L. White† *Department of Pediatrics and †Department of Oncological Sciences, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, Utah 84132

Received August 10, 2000

TGF-␤1 inhibits BRCA1 expression, which contradicts the model that TGF-␤1 prevents carcinogenesis by activating tumor suppressor genes. To resolve this apparent contradiction, we examined BRCA1 expression in Mv1Lu cells, a well-established model system for studying the TGF-␤1 tumor suppressor pathway. We found that inactivation of pRb by the papillomavirus type 16 E7 protein increased BRCA1 expression and abolished the ability of TGF-␤1 to inhibit BRCA1 expression. We conclude that TGF-␤1 inhibits BRCA1 expression through a pathway that requires pRb. We propose a model to explain the inhibition of BRCA1 as a target in the TGF-␤1 tumor suppressor signaling pathway. Our results suggest that the tumor suppressor functions of BRCA1 are initiated by the inactivation of pRb, and therefore that the activation of pRb by TGF-␤1 might alleviate the requirement for BRCA1 function. © 2000 Academic Press Key Words: TGF-␤1; BRCA1; retinoblastoma; papillomavirus; breast cancer; cell cycle; lung.

Transforming growth factor-␤1 (TGF-␤1) is a multifunctional growth factor that plays a pivotal role in suppressing breast carcinogenesis. The ability of TGF-␤1 to suppress breast carcinogenesis appears to be predominantly due to the growth inhibitory properties of TGF-␤1 (1– 4). Inhibition of cell growth by TGF-␤1 is cell cycle-dependent. In many cell types, TGF-␤1 reversibly arrests cell cycle progression at a point in late G 1 when added prior to the G 1/S transition (5– 8). As cells transform into cancer, they commonly lose the ability to arrest growth in response to TGF-␤1, and many of the changes in gene expression that lead Abbreviations used: TGF-␤1, transforming growth factor-␤1; Mv1Lu, mink lung epithelial cell line (ATCC: CCL-64); pRb, retinoblastoma protein; Cdk, cyclin-dependent kinase. 1 This work was funded by the Huntsman Cancer Institute. 2 To whom correspondence should be addressed. Fax: (801) 5857395. E-mail: [email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

to cancer also appear to allow cells to escape TGF-␤1 growth arrest (9, 10). Accordingly, the ability of TGF-␤1 to both arrest cell cycle progression and suppress cancer formation is thought to depend on the functions of TGF-␤1 to inhibit proto-oncogenes and activate tumor suppressor genes. In apparent contrast to this paradigm, TGF-␤1 has been shown to inhibit expression of the BRCA1 (breast cancer gene 1) tumor suppressor gene in breast epithelial cells (11). The BRCA1 gene was identified by positional cloning (12), and inherited mutations in BRCA1 account for a large fraction of hereditary breast cancers. Although the precise functions of BRCA1 are incompletely understood, available evidence indicates that BRCA1 affects the function of a number of proteins that regulate transcription, DNA repair, and cell cycle progression (reviewed in (13)). Currently, there is evidence that BRCA1 can both promote and inhibit cell cycle progression. Evidence that BRCA1 promotes cell cycle progression has come from studies of BRCA1 knockout mice and non-transformed cells in culture. The absence of BRCA1 expression in BRCA1 knockout mice was associated with impaired cellular proliferation resulting in early embryonic lethality (14). Lack of BRCA1 in the affected embryos was associated with increased expression of the growth inhibitor genes p53 and p21; however, increased apoptosis was not detected. A similar correlation between decreased BRCA1 expression and increased p21 expression was found in sporadic breast tumors (15). It is interesting to note that in a small retrospective study, female infants who were heterozygous for BRCA1 mutation were significantly smaller in birth weight and length than a set of matched unaffected relatives, suggesting that haploinsufficiency may negatively affect the growth of diverse human organ systems in utero (16). In non-transformed cells, expression of BRCA1 mRNA and protein is low or absent during the G 1 phase of the cell cycle, and expression of both the mRNA and protein is sharply induced near the G 1/S

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transition (11, 17). Recently, Wang et al. demonstrated that the induction of BRCA1 mRNA expression is regulated by the retinoblastoma tumor suppressor protein (pRb) through the E2F transcription factor (18). In addition, Chen et al. have demonstrated that BRCA1 protein is phosphorylated in late G 1 by the same cyclin dependent kinases (Cdk) that activate BRCA1 expression through pRb. The phosphorylation of BRCA1 by Cdks is thought to be an important regulator of BRCA1 activity (19). Thus, accumulating evidence suggests that BRCA1 expression is induced by positive growth signals at the point in the cell cycle where cells become committed to replicate their DNA and undergo cell division. BRCA1 expression and phosphorylation are associated with progression through the cell cycle, and the normal expression of BRCA1 appears to be necessary for the proliferation of many cell types. In contrast, the identification of BRCA1 as a tumor suppressor gene supports the general notion that loss of BRCA1 results in inappropriate cell growth (12, 20). Direct evidence implicating BRCA1 as a negative regulator of cell proliferation has come from studying the effect of altered BRCA1 expression in cultured cells. Forced overexpression of BRCA1 in MCF-7 cells inhibited cell growth, and inhibited tumor formation in nude mice (21). Conversely, reduced expression of BRCA1 from antisense mRNA in NIH3T3 cells resulted in faster proliferation and a transformed phenotype (22). Similarly, introduction of a mutant BRCA1 expression construct into 184A1 human mammary epithelial cells resulted in a faster doubling time, presumably by acting as a dominant negative (23). To begin to resolve some of the contradictory evidence regarding the function of BRCA1 in cell cycle control, as well as the role of BRCA1 in the TGF-␤1 signaling pathway, we determined the response of BRCA1 to TGF-␤1 in Mv1Lu cells. Mv1Lu is a polyclonal, non-transformed cell line derived from primary cultures of fetal mink lung. We predicted that Mv1Lu might be useful for studying BRCA1 because BRCA1 is expressed in fetal lung (24), and BRCA1 function is essential for normal development. Mv1Lu cells are a well-characterized epithelial model system for studying TGF-␤1-induced cell cycle arrest, thereby providing an established context for interpretation of results. We report here that TGF-␤1 inhibits BRCA1 mRNA expression in Mv1Lu cells, and the inhibition is largely dependent on pRb. MATERIALS AND METHODS Cell culture and synchronization. Mv1Lu cells (ATCC: CCL-64) were grown in 150 cm 2 flasks (Corning) in an incubator in 5% CO 2 at 37°C. Growth medium consisted of Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL), and 5% dialyzed fetal bovine serum (FBS, Gibco BRL). To obtain cell cycle synchronization, cells were plated at high density and allowed to grow to confluence. After 72 h at confluence,

cells reliably became arrested in the G 0 phase of the cell cycle. G 0-arrested cells were stimulated reenter the cell cycle by passaging the cells 1:3 to release form contact inhibition, and by replacing the medium with growth medium containing 20 ng/ml of epidermal growth factor (EGF, Collaborative Research) at time zero. Porcine TGF-␤1 (R&D Systems) was added to a final concentration of 10 ng/ml at the times indicated. Cell cycle distribution was determined using a standard protocol for fluorescence-activated cell-sorter (FACS) analysis following treatment with BrdU for 30 min (25). In addition, DNA synthesis was assessed in cultures by measuring [ 3H]-thymidine incorporation. Forty ␮Ci of [ 3H]-thymidine (Dupont/NEN) were added per 150 cm 2 flask and labeling was allowed to proceed for 2 h. Cells were then washed, fixed in 10% trichloroacetic acid, and lysed in 0.2 N sodium hydroxide. [ 3H]-thymidine incorporation (CPM) was measured in triplicate at time zero in quiescent cells, and after 24 h of EGFinduced growth, both with and without the addition of TGF-␤1. Creation of the Mv1Lu-LXSN and Mv1Lu-LXSN-E7 cell lines. LXSN and LXSN-16E7 retroviruses were a gift from Dr. Galloway at the Fred Hutchinson Cancer Research Center (26). Mv1Lu cells were incubated in viral supernatant plus 4 ␮g/ml polybrene (Sigma) for 24 h. Cells were then cultured in virus-free medium for 2 days, followed by selection in growth medium containing 400 ␮g/ml of G418 (Life Technologies, Inc.). Pooled populations of infected cells were generated from samples infected at 20 –50% efficiency based on the percent of cells surviving selection with G418. The concentration of G418 was reduced to 200 ␮g/ml once 100% of the uninfected control cells had died. RNA isolation and Northern analysis. Cells were harvested from 2–3 flasks and pooled for each sample at the times indicated in each figure, and polyadenylated RNA was isolated using a standard protocol (Figs. 1A and 2A) (27). RNA was resolved by electrophoresis on an agarose gel and transferred to Hybond membrane. Complete transfer was confirmed by ethidium bromide staining. [ 32P]dATP and dCTP (Dupont/NEN) labeled probes were made using a random primer DNA labeling kit (Boehringer Mannheim). The following cDNA templates were used for random primed labeling: the 0.7 kb BamHI/PstI fragment from plasmid SP65 1B15 containing the rat cyclophilin cDNA (28); the 6.2 kb Nar1 fragment from LXSN-BRCA containing the BRCA1 cDNA (21). Hybridizations were performed at 42°C using 1 ⫻ 10 6 cpm/ml of labeled DNA in hybridization buffer containing 50% formamide. Images and quantification of signal intensity were obtained using a phosphorimager (Molecular Dynamics).

RESULTS TGF-␤1 inhibits BRCA1 mRNA expression. We determined the response of BRCA1 mRNA expression to TGF-␤1 in randomly cycling Mv1Lu cells. By Northern analysis, we detected a single ⬇7.5 kb band, similar to the 7.8 kb transcript size reported for human BRCA1 (Fig. 1A). BRCA1 mRNA expression consistently began decreasing relative to control between 2 and 4 h after TGF-␤1 treatment (Figs. 1A and 1B). Based on a comparison of mRNA expression with cell cycle analysis, we concluded that BRCA1 expression began decreasing prior to the onset of G 1 arrest (Fig. 1C). BRCA1 mRNA expression continued to decrease markedly as TGF-␤1treated cells began to arrest in the G 1 phase, between 4 and 8 h after TGF-␤1 treatment (Figs. 1A and 1B). TGF-␤1 inhibits the induction of BRCA1 expression in G 1. Because the inhibition of BRCA1 appeared to be linked to events that mediate the TGF-␤1-induced

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after mitogen reproducibly inhibited S-phase entry by greater than 50% ((6) and Fig. 2C), and inhibited the accumulation of BRCA1 mRNA between 8 and 12 h (Figs. 2A and 2B). In agreement with the results in Fig. 1, we concluded that TGF-␤1 inhibits the induction of BRCA1 mRNA in G 1, prior to the onset of DNA synthesis. Interestingly, the pattern of mRNA expression during the cell cycle, and inhibition by TGF-␤1, suggested that induction of BRCA1 mRNA expression might be mediated by the inactivation of the retinoblastoma tumor suppressor protein, pRb.

FIG. 1. TGF-␤1 inhibits BRCA1 mRNA expression in randomly cycling Mv1Lu cells. (A) (⫹) indicates TGF-␤1 added to asynchronous, rapidly proliferating cells at 0 h. mRNA was isolated for Northern blot analysis from cells harvested at the times indicated. (B) Signal intensity was normalized to cyclophilin expression and represented graphically (arbitrary units). The inhibition of BRCA1 is long lasting, persisting for at least 24 h. Results shown from this single experiment are representative of multiple assays. (C) A representative analysis of cell cycle distribution by FACS indicates the typical kinetics of TGF-␤1-induced G 1 cell cycle arrest in Mv1Lu cells.

G 1 cell cycle arrest, we examined BRCA1 mRNA expression during G 1 in growth-synchronized Mv1Lu cells. Non-dividing (G 0 phase) cells were stimulated with mitogen to enter the G 1 phase of the cell cycle in a synchronized manner. We found that BRCA1 mRNA expression was virtually undetectable in non-dividing, G 0 phase Mv1Lu cells (Figs. 2A and 2B). Results of fluorescence-activated cell-sorter (FACS) analysis indicated that greater than 90% of the cells remained in the G 1 phase of the cell cycle for 8 h after mitogen (Fig. 2C). Cells began entering S-phase at about 12 h, and by 16 h after mitogen greater than 60% of the cells had entered S-phase. BRCA1 signal first became visible 8 h after mitogen, at which time most cells were in late G 1. BRCA1 expression increased sharply between 8 and 12 h, just before the majority of cells entered S-phase and began DNA synthesis. TGF-␤1 added to cells 8 h

FIG. 2. TGF-␤1 inhibits the induction of BRCA1 mRNA expression in cell cycle synchronized Mv1Lu cells. (A) Non-dividing (G 0) Mv1Lu cells were stimulated to reenter the cell cycle by addition of mitogen at 0 h (see Materials and Methods). (⫹) indicates TGF-␤1 added to cells 8 h after mitogen. mRNA was isolated for Northern blot analysis from cells harvested at the times indicated. (B) Signal intensity was normalized to cyclophilin expression and represented graphically. The induction of BRCA1 mRNA expression was inhibited in cells treated with TGF-␤1 8 h after mitogen (dashed line). Results shown from this single experiment are representative of multiple assays. (C) A representative analysis of cell cycle distribution by FACS indicates that control cells (solid line) begin S-phase about 12 h after mitogen, whereas TGF-␤1 inhibits S-phase entry (dashed line).

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TGF-␤1 inhibition of BRCA1 requires pRb. We examined whether TGF-␤1 might inhibit BRCA1 expression through a pathway that requires pRb. It has been shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) by viral oncoproteins renders cells unable to undergo growth arrest in response to TGF-␤1, yet in such cells the effects of TGF-␤1 on gene expression that do not require pRb are preserved (29). The human papillomavirus type 16 E7 oncoprotein has been shown to efficiently inactivate pRb (30), therefore we established a pooled population of Mv1Lu cells that express E7 from a retrovirus-introduced transgene (Mv1Lu-LXSN-E7). The cell cycle distribution of control cells (Mv1Lu-LXSN) cells was the same as Mv1LuLXSN-E7 cells (Fig. 3A). However, we found that Mv1Lu-LXSN-E7 cells did not undergo G 1 arrest in response to TGF-␤1, whereas Mv1Lu-LXSN responded normally (Fig. 3A). Similarly, the decrease in BRCA1 expression following TGF-␤1 was significantly attenuated in Mv1Lu-LXSN-E7 cells, indicating that the loss of pRb abolishes the ability of Mv1Lu cells to appropriately decrease BRCA1 expression (Figs. 3B and 3C). Further, BRCA1 mRNA expression was 35% higher in Mv1Lu-LXSN-E7 cells (Figs. 3B and 3D), suggesting that inactivation of pRb results in the induction of BRCA1 expression. We conclude that TGF-␤1 inhibits BRCA1 mRNA expression through a pathway that requires pRb. DISCUSSION We found that BRCA1 mRNA expression is induced in late G 1 in Mv1Lu cells, and BRCA1 expression is inhibited by TGF-␤1. Interestingly, we found that the pattern of expression of BRCA1 in Mv1Lu cells was very similar to the pattern of expression of B-myb that we published previously (6). B-myb is induced in late G 1 through a mechanism involving E2F derepression following phosphorylation of pRb family members (31, 32), and B-myb is thought to be inhibited by TGF-␤1 through a pathway that requires pRb family members. To illustrate, TGF-␤1 induces Cdk inhibitors, which block Cdk activity and thereby retain pRb family members in the growth suppressive form that binds E2F (8, 33, 34). The E2F-pRb family member complexes function to inhibit transcription from promoters containing E2F sites, including B-myb. Thus, the similar appearance of B-myb and BRCA1 during the cell cycle, and similar response to TGF-␤1, led us to test whether BRCA1 expression is regulated through the pRb pathway. Inactivation of pRb by the human papillomavirus type 16 E7 oncoprotein significantly blocked the ability of TGF-␤1 to inhibit BRCA1 mRNA expression. In addition, inactivation of pRb by E7 resulted in a 35% increase in BRCA1 mRNA expression. These findings are in good agreement with the recent report demonstrating that BRCA1 expression is regulated by pRb in

FIG. 3. Inactivation of pRb by the papillomavirus type 16 E7 oncoprotein in Mv1Lu cells alters BRCA1 expression and response to TGF-␤1. (A) (⫹) Indicates TGF-␤1 added to asynchronous, rapidly proliferating cells at 0 h. Mv1Lu cells infected with empty vector (Mv1Lu-LXSN) respond normally to TGF-␤1-induced G 1 arrest; however, Mv1Lu cells in which pRb has been inactivated by E7 expression (Mv1Lu-LXSN-E7) show no change in cell cycle distribution in response to TGF-␤1 (mean ⫾ SD, N ⫽ 3). (B) RNA was isolated for Northern blot analysis from samples harvested 24 h after TGF-␤1 treatment. mRNA signal intensity in was normalized to cyclophilin expression. A single representative experiment is shown; however, 3 independent assays were used for statistical analyses (represented graphically as mean ⫾ SD, * indicates P ⱕ 0.05). (C) The inhibition of BRCA1 mRNA expression by TGF-␤1 was significantly greater in Mv1Lu-LXSN control cells than in Mv1Lu-LXSN-E7 cells (⫺86.5 ⫾ 16.5% (SD) vs ⫺19.1 ⫾ 8.1% (SD), P ⬍ 0.01). Thus, the ability of TGF-␤1 to inhibit BRCA1 mRNA expression is significantly blocked by inactivation of pRb by E7. (D) Inactivation of pRb by E7 resulted in a 34.9 ⫾ 2.8% (SD) increase in BRCA1 mRNA expression (P ⫽ 0.087).

primary mouse keratinocytes (18). Interestingly, the increase in BRCA1 expression that we observed in Mv1Lu-LXSN-E7 cells is not due to a greater fraction of cells in the S and G 2/M phases of the cell cycle, because the cell cycle distribution was the same in Mv1Lu-LXSN and Mv1Lu-LXSN-E7 cells. Rather, be-

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FIG. 4. Schematic model of BRCA1 function as a sensor for pRb status. Inactive proteins are represented by a black diamond. A backslash indicates absence of the protein. (A) BRCA1 expression is consistent with the inactivation of pRb by mitogenic extracellular growth signals. Mitogenic extracellular growth signals (M) activate cyclin-dependent kinases (Cdks). Cdks inactivate the growth suppressive functions of pRb. Inactivation of pRb triggers BRCA1 expression, and BRCA1 is phosphorylated by Cdks. Cell growth is permitted. (B) Absence of BRCA1 expression is consistent with the activation of pRb. An absence of mitogenic extracellular growth signals, or TGF-␤1, renders Cdks inactive, and pRb active. The lack of BRCA1 expression is in agreement with the active status of pRb, and homeostasis is preserved. (C) BRCA1 expression is not consistent with the inactivation of pRb by mitogenic extracellular growth signals. pRb is inactivated by gene deletion or papillomavirus E7 protein, which triggers BRCA1 expression. The accumulation of unphosphorylated BRCA1 protein when Cdks are inactive indicates that there is a discrepancy between growth signals and pRb status. Therefore, a growth arrest signal is initiated. (D) Absence of BRCA1 expression is not consistent with activation of pRb. Loss of the BRCA1 gene results in the failure of BRCA1 protein to be expressed appropriately following inactivation of pRb by mitogenic extracellular growth signals. The inconsistency between BRCA1 expression and pRb status triggers p21 expression. p21 inactivates Cdks, thereby activating pRb and restoring consistency between pRb status and BRCA1 expression, as shown. TGF-␤1 may compensate for the loss of BRCA1 by activating pRb, thereby creating an intracellular environment in which BRCA1 expression is not normally required (as shown in B).

cause pRb is thought to be active as a tumor suppressor during the G 1 phase of the cell cycle, inactivation of pRb by E7 would be expected to increase BRCA1 expression primarily during G 1, thus accounting for the relatively modest increase in BRCA1 expression in Mv1Lu-LXSN-E7 cells. Our results support the model that BRCA1 functions, at least in part, as a sensor for appropriate pRb function (Fig. 4). One of the principal functions of pRb is to integrate extracellular growth signals during the G 1 phase of the cell cycle. Mitogenic growth signals result in inactivation of pRb in late G 1 via phosphory-

lation by Cdks. Similarly, we and others have shown that mitogenic growth signals result in the induction of BRCA1 expression (11, 17), and others have shown that mitogenic growth signals result in the phosphorylation of the BRCA1 protein in late G 1 (19). Taken together, these results suggest that the tumor suppressor functions of BRCA1 begin at the same time that the tumor suppressor functions of pRb are inactivated. In this model, the first role of BRCA1 as a tumor suppressor is to verify that the decision to exit G 1 and proceed through the cell cycle is in agreement with the appropriate inactivation of pRb by mitogenic growth signals. This model accounts for many of the apparent contradictions in the literature regarding the role of BRCA1 as a tumor suppressor in cell cycle control. The requirement for both timely expression and phosphorylation of BRCA1 by Cdks provides a doublecheck of pRb function. Our model predicts that both expression and phosphorylation of BRCA1 are required for cell cycle progression. For example, in this model, synthesis of BRCA1 indicates that pRb has been inactivated. In normal cells that are exposed to appropriate mitogenic growth signals, BRCA1 is expressed when pRb is inactivated by Cdks (Fig. 4A). The phosphorylation of newly synthesized BRCA1 by Cdks indicates, at least circumstantially, that pRb has been inactivated as a result of mitogenic extracellular growth signals mediated by Cdks. Growth is permitted in the setting where pRb has been inactivated by mitogenic extracellular growth signals (Fig. 4A). When Cdks are inactive, due to either TGF-␤1 or a lack of mitogenic extracellular growth signals, then pRb remains active as a growth suppressor and BRCA1 synthesis is blocked (Fig. 4B). The expression of BRCA1 remains in agreement with the status of pRb, and homeostasis is preserved. In contrast, if BRCA1 becomes expressed in a manner that is inconsistent with pRb inactivation by Cdks, then BRCA1 can trigger a pathway leading to growth arrest. According to our model, if pRb becomes inactivated by means other than Cdk phosphorylation, BRCA1 protein accumulates inappropriately during G 1 in the absence of active Cdks (Fig. 4C). Thus, accumulation of BRCA1 indicates that pRb has been inactivated, but the absence of BRCA1 phosphorylation by Cdks indicates that conditions are not consistent with pRb having been inactivated by mitogenic extracellular growth signals. According to our model, forced expression of BRCA1 creates a discrepancy analogous to the loss of pRb. In both cases BRCA1 accumulates inappropriately during G 1 before Cdks become active, which triggers growth arrest or apoptosis through a pathway that is as yet incompletely resolved (19), but may require pRb. Our observation that the inappropriate induction of BRCA1 expression does not trigger growth arrest in the presence of E7 is consistent with previous reports (35). Thus, in Mv1Lu cells, E7 may

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inactivate proteins in addition to pRb, perhaps including other pRb family members, that are required for BRCA1-mediated growth arrest. Conversely, in cells where BRCA1 is essential for growth, the loss of BRCA1 expression in the presence of mitogenic extracellular signals appears to trigger growth arrest through the pRb pathway (14, 15). In agreement with our model, the absence of BRCA1 expression after pRb has been inactivated by Cdks creates a discrepancy between pRb status and BRCA1 expression (Fig. 4D). This discrepancy triggers p21 expression (14, 15). p21 blocks Cdk activity (36), which restores pRb to the underphosphorylated form, thereby restoring consistency between pRb status and the absence of BRCA1 expression (Fig. 4D). Similarly, TGF-␤1 would restore consistency between pRb status and the absence of BRCA1 expression by converting the conditions illustrated in Fig. 4D to those illustrated in Fig. 4B. In this manner, TGF-␤1 might be able to restore homeostasis after the loss of BRCA1 as illustrated in Fig. 4B. On the other hand, restoring BRCA1 expression to normal levels under the conditions illustrated in Fig. 4D would also restore consistency between pRb status and BRCA1 expression, and might permit growth to resume. Our model predicts that if both BRCA1 and pRb were lost, then the ability of BRCA1 to trigger growth arrest would be impaired, and the cell would be more likely to undergo deregulated proliferation or transformation. This model resolves many of the apparent contradictions regarding the role of BRCA1 in both cell cycle control and the TGF-␤1 pathway, but will certainly require further testing and modification. In summary, we found the Mv1Lu cell line to be useful for studying the functional relationship between BRCA1 expression and epithelial cell tumor suppression by TGF-␤1. Of interest, BRCA1 mRNA expression is regulated in the same manner in Mv1Lu cells as was reported in breast epithelial cells (11, 17). We found that BRCA1 is a target in the pRb pathway, suggesting that under normal conditions TGF-␤1 activates tumor suppressors in the pRb pathway upstream of BRCA1, thereby alleviating the need for BRCA1 expression. In other words, by inhibiting pRb phosphorylation and arresting cell cycle progression in G 1, TGF-␤1 might compensate for a loss of the BRCA1 tumor suppressor functions that are required during the cell cycle subsequent to the phosphorylation of pRb. Our results imply that activating pRb in vivo, for example by increasing TGF-␤1 signaling, might be an effective strategy to prevent the formation of cancer in individuals who carry an inactivating mutation in BRCA1. ACKNOWLEDGMENTS We thank Lih-Ching Hsu, Kurt Albertine, and Kristi Neufeld for their critical review and helpful comments on the manuscript. We

thank Jeff Holt for providing the BRCA1 cDNA. We thank Denise Galloway for providing the LXSN and LXSN-16E7 retroviruses. We are grateful to Pauline Cordray for expert technical assistance.

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