BRCA1-associated growth arrest is RB-dependent

Abstract

BRCA1 is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells, BRCA1 inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by BRCA1, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to BRCA1 overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to BRCA1-induced growth suppression. In cells with an intact rb gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by BRCA1. In accordance with these observations, we found that BRCA1 could not suppress BrdUrd uptake in primary fibroblasts from rb؊/؊ mice and exhibited an intermediate ability to inhibit DNA synthesis in rb؉/؊ as compared with rb؉/؉ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304 -394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of BRCA1. Although it has been reported that BRCA1 interacts with p53, we find the p53 status did not affect the ability of BRCA1 to suppress colony formation. Our data suggest that the growth suppressor function of BRCA1 depends, at least in part, on Rb. G ermline mutations in BRCA1 are found in Ϸ50-60% of hereditary breast and ovarian cancers (1). Despite the unequivocal role of BRCA1 in familial breast cancer susceptibility, the biological function of the BRCA1 protein remains unclear. Experimental data suggest that BRCA1 may be a negative regulator of cell growth. Attenuation of BRCA1 synthesis by antisense oligonucleotide increased the proliferative rate of both benign and malignant mammary epithelial cells in culture (2), and BRCA1 expression decreased the capacity of MCF7 breast cancer cells to form tumors in nude mice (3). However, BRCA1 expression increases as cells progress through the G 1 and S phases of the cell cycle (4, 5). Furthermore, homozygous BRCA1 mutant mice died at day 7.5 of embryogenesis with evidence of abnormal cessation of cellular proliferation accompanied by high levels of the CDK inhibitor p21 and low levels of cyclin E and mdm2 (6, 7). Recently, colocalization and physical interaction of BRCA1 with Rad51 protein has raised the possibility that BRCA1 is involved in DNA repair (8). Moreover, the COOH terminus of the BRCA1 protein can activate transcription in in vitro experiments (9, 10) and coactivate transcription of p53-regulated genes (11,12).The experimental biology of BRCA1 therefore suggests that BRCA1 may have different functions, each best manifested in specific study systems and cell lines. Herein, we show that BRCA1 binds preferentially to the hypophosphorylated form of Rb and that the growth-suppressive phenotype of BRCA1 depends on the presence of a functional Rb protein. These data indicate the complexity of ...

BRCA1 gene in breast cancer

“…BRCA1 causes transcriptional activation of the p21 WAF1/Cip1 and Gadd45 genes (Somasundaram et al, 1997;Harkin et al, 1999;Jin et al, 2000a), which contibute to proliferation inhibition and cell cycle blockade. Several studies, including our own, document a direct interaction between the BRCA1 and RB1 proteins, through two sites within BRCA1: (1) the carboxylterminal TAD (Yarden and Brody, 1998); and (2) an amino-terminal site within aa 300-400 (Aprelikova et al, 1999;Fan et al, 2001c). Brca1 blocked entry into S-phase in Rb1 þ/þ cells but not in Rb1 À/À cells mouse embryo fibroblasts (MEFs), suggesting a requirement for Rb (Aprelikova et al, 1999).…”

“…Various classes of proteins interact with BRCA1, including: (1) components of the basal transcription machinery [e.g., RNA helicase A and RNA pol II (Anderson et al, 1998;Schlegel et al, 2000a)]; (2) generalized transcriptional coactivators [p300, CBP, Brg1 (Bochar et al, 2000;Pao et al, 2000)] and corepressors [e.g., RbAp46, RbAp48, histone deacetylases-1,2, and CtIP (Yarden and Brody, 1998;Yu et al, 1998)]; (3) tumor suppressors [e.g., p53, RB1, BRCA2 (Chen et al, 1998;Ouichi et al, 1998;Yarden and Brody, 1998;Zhang et al, 1998;Aprelikova et al, 1999;Chai et al, 1999;Fan et al, 2001c)]; (4) steroid hormone receptors, estrogen receptor-a, and androgen receptor (Yeh et al, 2000;Fan et al, 2001a); (5) DNA repair proteins [e.g., Rad51, Rad50, hMSH2 (Scully et al, 1997b;Zhong et al, 1999;Wang et al, 2001a)]; (6) other sequence-specific transcription factors [e.g., c-Myc, Oct-1, and NF-YA Fan et al, 2002b)]; and (7) cell cycle regulatory proteins [e.g., BARD1, E2F1, cyclins (Wu et al, 1996;Wang et al, 1997)]. These interactions are summarized in Figure 1; and the significance of these interactions is discussed in ''Functional Activities of BRCA1''.…”

“…BRCA1 contains the consensus RB1 family protein binding sequence LXCXE [aa 358-362; L ¼ leucine, C ¼ cysteine, E ¼ glutamic acid, and X ¼ any amino acid] and an atypical Rb binding motif LXCXXE (aa 439-444) (see Fig. 1), suggesting that a BRCA1:RB1 family protein interaction may regulate the transition from G1 into S. Recently, two protein interactions between BRCA1 and RB1 have been described, one involving the C-terminal TAD of BRCA1 (Yarden and Brody, 1998) and the other involving an N-terminal domain between aa 300 and 400 (Aprelikova et al, 1999;Fan et al, 2001c). In the latter study, the ability of BRCA1 to cause growth arrest at G1/S was dependent upon the presence of RB1 in the cell.…”

Regulation of BRCA1 by protein degradation

“…It has been shown that BRCA1 mRNA is highly expressed in late G1 phase of the cell cycle, whereas conditions that lead to cell cycle exit down-regulate the BRCA1 mRNA (Gudas et al, 1995(Gudas et al, , 1996Jin et al, 1997). However, despite the cell cycle dependence of BRCA1 mRNA expression, BRCA1 protein level does not change during the cell cycle (Aprelikova et al, 1996).…”

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

“…The process is complicated by cellular senescence or apoptosis in many cells engineered to overexpress the BRCA1 cDNA from heterologous promoters (Aprelikova et al, 1999;Shao et al, 1996;Wilson et al, 1997;Zhang et al, 1998). Since mouse embryos de®cient in Brca1 die early in development, a straightforward assay of function is to analyse human BRCA1 gene replacement vectors for their ability to rescue Brca1-de®cient mouse embryos.…”