Emerging roles of BRCA1 alternative splicing.

Germline mutations of the BRCA1 gene predispose individuals mainly to the development of breast and/or ovarian cancer. However, the exact function of the gene is still unclear, although the encoded proteins are involved in various cellular processes, including transcriptional regulation and DNA repair pathways. Several BRCA1 splice variants are found in different tissues, but in spite of intense investigations, their regulation and possible functions are poorly understood at the moment. This review summarises current knowledge on the roles of these splice variants and the mechanisms responsible for their formation. Because alternative splicing is now widely accepted as an important source of genetic diversity, elucidating the functions of the BRCA1 splice variants would help in the understanding of the exact role(s) of this tumour suppressor. This should help to resolve the current paradox that, despite its seemingly vital cellular functions, mutations of this gene are associated with tissue specific tumour formation predominantly in the breast and the ovary.

Constitutive intracellular expression and activation-induced cell surface up-regulation of CD44v3 in human T lymphocytes.

The cell adhesion molecule CD44 exists in multiple isoforms generated by alternative RNA splicing. Increased expression of CD44 isoforms containing exon v6 and v9 has been reported to be associated with the activated state of T lymphocytes. Using monoclonal antibodies against variant exon products we studied the expression of another variant exon, v3 on resting and in vitro activated human peripheral blood T cells. We found that CD44v3, in parallel with CD44v6, is up-regulated at the surface of normal T cells stimulated by anti-CD3 antibody or by the phorbol ester PMA, as well as on PMA-stimulated T cell leukemia lines CCRF-CEM and MOLT-4. Beside the cell surface, we demonstrated CD44v3 intracellularly in both resting and activated T cells by flow cytometry and immunomorphology. Reverse transcription-PCR and Western blot analyses confirmed the constitutive expression of CD44v3 in these cells. The increase in the cell surface expression of CD44v3 on stimulated T lymphocytes was inhibited by cycloheximide and brefeldin A, indicating the requirement of de novo protein synthesis and endoplasmic reticulum Golgi transport. Our studies establish CD44v3 as an additional activation marker for human T cells, with a yet unidentified function.

Expression profiles of BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell lines.

Disrupting the function of the BRCA1 gene by mechanisms other than germline mutations is suspected to occur in cases of sporadic breast/ovarian cancers. Using ribonuclease protection assay and multiplex RT-PCR, we examined the change of the total BRCA1 mRNA pool and the expression profile of four predominant BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell populations compared to normal breast cells. Experiments were carried out on MCF-7 and MDA-MB-231 breast cancer, OVCAR-5 ovarian cancer, and K562 leukemia cell lines. The ratio of the full length, the delta(11q), the delta(9,10), and the delta(9,10,11q) BRCA1 isoforms showed different expression patterns in the examined breast and ovarian tumor cell lines as compared to the leukemia cell line. This observation raises the possibility that the dysregulation of alternative splicing of the BRCA1 gene could be involved in tumor formation in the breast and the ovary, even in the absence of germline mutations.

Sequence alterations can mask each other's presence during screening with SSCP or heteroduplex analysis: BRCA genes as examples.

For mutation detection, various screening techniques are widely used because DNA sequencing, the gold-standard method, is still considered to be expensive and laborious for high-throughput screening. Single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis (HA) and their variant techniques are popular and frequently used for this purpose. It is widely accepted that when searching for unknown sequence variations, any revealed distinct pattern should always be sequenced. We give examples here of the BRCA1 and BRCA2 genes where the SSCP/HA techniques can produce ambiguous predictions if used to detect known genetic variants compared to positive controls. Using direct DNA sequencing, we provide evidence that in such cases, mutations or polymorphisms can mask each other's presence. This phenomenon can often influence the results of any DNA testing because genetic variations such as single-nucleotide polymorphisms occur frequently in the human genome. We suggest that even in the case of known electrophoretic patterns of well-characterized genetic alterations, every sequence alteration should be confirmed by direct DNA sequencing, especially if genetic testing is carried out for diagnostic purposes.