Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

Abstract

The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262 ± 570. We describe a splice variant, BRCA1-D11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-D11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT ± PCR demonstrated that BRCA1 and D11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-D11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a D9,10 splice variant. Taken together our results suggest that fulllength BRCA1 and BRCA1-D11b may have distinct roles in cell growth regulation and tumorigenesis.

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

“…The failure of these promoter-BRCA1 constructs to support development indicates that overexpressed, or mis-expressed, BRCA1 blocks development. This result was anticipated, in part, because of studies which demonstrate very low levels of BRCA1 expression in normal cells, and other studies that show cellular growth-arrest or apoptosis in many cell lines which overexpress BRCA1 (Shao et al, 1996;Wilson et al, 1997;Zhang et al, 1998). When we transfected mammary cell lines with BRCA1 cDNAs linked to BRCA1 promoter sequences, we did not observe apoptosis or cellular arrest (T Lane and E Solomon, preliminary observations).…”

“…Testis is a site of signi®cant BRCA1 expression (Lane et al, 1995;Scully et al, 1997b) due to a presumed role for the protein in homologous strand exchange (Snouwaert et al, 1999;Zabludo et al, 1996). Antibodies were the generous gift of Drs C Wilson and D Slamon (UCLA School of Medicine, Los Angeles, CA, USA) and have been described elsewhere (Wilson et al, 1997(Wilson et al, , 1999. None of the antibodies used in this experiment cross-reacted with extracts derived from mice.…”

“…Mouse cells lacking functional Brca1 are also more sensitive to ionizing radiation and oxidative stress (Shen et al, 1998). One surprising result was that virtually all tumor-derived cell lines appeared to produce some BRCA1 (Lane et al, 1995;Scully et al, 1997a;Wilson et al, 1997). Thus, BRCA1 displays some features which are atypical for tumor suppressor genes and there remains a signi®cant need for informative and genetically tractable models of BRCA1 function.…”

“…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.…”

A role for BRCA1 in sporadic breast cancer

“…This protein is thought to accumulate in the cytoplasm (Thakur et al, 1997;Wilson et al, 1997). Synthesis of full-length BRCA1 protein is rapidly followed by translocation to the cell nucleus.…”