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women living in Western society will develop breast cancer during her lifetime. Germline mutations in BRCA1, a breast cancer tumor suppressor gene, are responsible for 50% of inherited breast cancers and 90% of combined inherited breast and ovarian cancers. The BRCA1 protein, BRCA1p, is involved in many important cellular pathways, including regulation of the cell cycle, DNA repair, transcription, and cell proliferation. It has been shown to bind over 20 different proteins. One such protein is
the retinoblastoma protein, Rbp. Rbp is the product of another tumor suppressor gene, termed Rb. Like
BRCA1p, Rbp is known to be involved in regulation of the cell cycle and cell proliferation.
Regulation of cell proliferation and maintenance of genome stability are major functions of many tumor suppressors, including BRCA1p and Rbp. Rbp expression is known to decrease cell proliferation in a wide array of cells and tissues, consistent with its ubiquitous expression in mammalian cells and tissues. Loss of Rb results in rapid tumor formation and progression in mice, suggesting a direct role in tumorigenesis.
Unlike Rbp, BRCA1p's inhibition of proliferation is specific to breast and ovarian tissues, despite its ubiquitous expression pattern. Conditional BRCA1 mutations lead to mammary epithelial tumors after a long latency. This long latency suggests that loss of BRCA1 alone does not initiate tumorigenesis. Instead the loss of BRCA1 causes genetic instability, which leads to an increase in mutation rate and eventually tumor development. This evidence suggests that both BRCA1p and Rbp play fundamental roles in the controlling cell proliferation and preventing cell transformation.
BRCA1 and Rb regulate cell proliferation through many different pathways and are both known to play a role in cell cycle checkpoint regulation. Functional BRCA1p and Rbp regulate cell growth by monitoring the transition from G1 to S. phase.
It is currently believed that BRCA1p regulates cell proliferation by directly binding Rbp. This model is supported by many in vitro studies. Deletion of either BRCA1 or Rb leads to an increased growth rate of breast cancer and prostate cancer cells in culture. Over-expression of BRCA1p can suppress this increased growth only in the presence of Rbp. However, interpretation of the BRCA1p:Rbp interaction is complicated by the fact that there are two binding sites on BRCA1p for Rbp. This is analogous to the interaction of BRCA1p with p53, another tumor suppressor gene. BRCA1p has two binding sites for p53, which leads to two different effects upon binding. We hypothesize that a similar mechanism may occur in BRCA1p:Rbp
binding, and that one of the Rbp binding sites is more important for cell proliferation control. Previous work in this field has tested regulation of cell proliferation using deletions of BRCA1. We will test the role of each binding site using point mutations in the context of the full-length protein in order to retain all other functional domains. This will allow us to specifically pinpoint the role of each Rbp-binding site in cell proliferation,
Specific Aim #1: Determine the role of each BRCA1p:Rbp binding domain in regulation of cell proliferation and tumorigenesis
To reduce or eliminate binding of Rbp, point mutations will be made in each Rbp binding domain of BRCA1p.
Mutations in each domain will be assayed for their impact on growth rate in mouse embryonic fibroblasts. The differences in growth rates between cells expressing the point mutants will be compared for functional differences between binding sites. In order to assess the biological relevance of these mutations, nude mice will be injected with MCF-7 cells that over-express mutant BRCA1p, and observed for tumor development.
Specific Aim #2: Determine the downstream effectors regulated by the binding of BRCA1p and Rbp that contribute to regulation of cell proliferation.
BRCA1p and Rbp may regulate the G1/S transition by regulating the activity of E2F, a transcription factor
involved in expression of G1/S specific genes. We will use GST pull-down assays to determine if
BRCA1stabilizes the E2F interaction, and a CAT reporter assay to analyze the transcriptional repression of E2F reporter genes. This will shed light on the in vivo effects of loss of regulation of E2F transcription.
Assessing the differential roles of each Rbp-binding site in BRCA1p could provide new insight into the role of BRCA1 as a molecular switch between cell proliferation and growth arrest. In addition,
determining downstream effectors of this interaction will provide insight into the mechanism of regulation of cell proliferation by BRCA1p and Rbp in normal cells and cancer cells.
Background & Significance
Breast cancer is one of the most common cancers, affecting one in nine women in the United
States (1). Approximately 10% of all breast cancers are hereditary and are diagnosed in women with inherited mutations in known tumor suppressor genes. Over half of these cases of breast cancer are initiated by an inheritable mutation in the breast cancer susceptibility gene 1, BRCA1. Familial mutations in BRCA1 are also involved in the development of ovarian cancer and are believed responsible for 90%
of inherited breast and ovarian cancers (2, 3).
Cancer is a complex disease that results from activation and inactivation of several genes. Tumor suppressor, a class of such gene that includes BRCA1, function to maintain genome stability and to regulate cell proliferation (4). Their inactivation can lead to destabilization of the genome, allowing mutations in other genes to accumulate at a higher frequency. Accumulation of these mutations causes cellular transformation, leading to oncogene activation or tumor suppressor inactivation. Rb is another tumor suppressor gene that directly controls cell proliferation (4). Inactivation of Rb results in malignant
transformation, which can be reversed by reintroduction of Rb in null cells (4). The mechanism of transformation in cells lacking BRCA1 and Rb is not well understood. However, an understanding of their interaction with each other and with other proteins may shed light on their roles in cancer development.
BRCA1 encodes a 220 kDa phosphoprotein consisting of 1863 amino acids (1, 5). BRCA1p has many functions, including DNA repair, cell cycle control, transcription control, and regulation of cell proliferation. The protein has a ring-finger domain at the N-terminus and two breast cancer carboxyl terminal, BRCT, domains located at the carboxyl terminus (Figure 1)(1). The ring finger domain is
involved in the formation of the BRCA1p homodimer and degradation of BRCA1p (1). The BRCT
domains have been crystallized and have been identified in over 50 proteins involved in the DNA damage response or cell cycle checkpoint-mediated repair (6). The BRCT domains also contain strong
transcriptional activity (7). Histone deacetylase complexes and the RNA polymerase holenzyme bind to the BRCT
domains, suggesting this region of BRCA1p can activate as well as repress transcription (8, 27). The middle section of the protein is encoded by Exon 11, which contains 60% of BRCA1p (1). However, in some naturally occurring cytoplasmic isoforms of BRCA1p, exon 11 deleted (9).
There are two nuclear localization signals encoded by exon
11 (1). This region of the protein binds to several DNA
repair proteins and is involved in the response to cellular damage (10). In order to simplify the study of this complex protein, many scientists study BRCA1p using deletions.
These studies may not demonstrate the contribution of the full-length protein to various interactions. We therefore
Figure 1: Depction of the BRCA1p domains, along with proteins that interact with each domain (adapted from (1))
intend to study the BRCA1p and Rbp interaction by examining regulation of proliferation using point mutations in the full-length BRCA1p.
The Role of BRCA1p in Cell Proliferation
BRCA1p is known to be involved in regulation of cell proliferation. Inhibition of BRCA1p leads to an increase in proliferation of both non-tumorgenic and malignant mammary cells (11). Conversely,
expression of BRCA1p in tumor cells inhibits proliferation, but this inhibition is specific to breast and ovarian cells (12). It is currently believed that the effects of BRCA1p in cell proliferation are due to direct and indirect effects on the cell cycle. This view is supported by its interaction with the cell cycle proteins E2F, CDC2, and various cyclins (13). In addition, BRCA1p is phosphorylated in a cell-cycle dependent manner; BRCA1p is hyperphosphorylated during late G1 and S. phase and is dephosphorylated after M. phase (14). The highest level of BRCA1 mRNA occurs at the entrance to S. phase (1). In response
to DNA damage, BRCA1p activates transcription of p21 during the G1/S checkpoint. p21 protein expression leads to G1-S phase cell cycle arrest, leading to apoptosis or DNA repair (15). Loss of this arrest due to BRCA1 mutations can contribute to genomic instability and accelerate tumor formation.
Cell cycle control is a shared function of many tumor suppressors. Like BRCA1p,
Rbp is also involved in cell cycle regulation.
Its major function is to control the transition from G1 to S. phase. Rbp regulates transcription of genes needed for DNA
replication in S. phase. This protein is the target of tumorigenic mutations in many
Figure 2: Domains of Rbp are shown, as well as proteins that interact with each domain (adapted from (17))
cancers, including osteosarcomas, prostate cancer,…[continue]
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