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Molecular cloning and functional characterization of BRCA1 splice variants in human mammary cells.

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Call No.	RC268.43 .L8 1999
Author	Lu, Meiling
Title	Molecular cloning and functional characterization of BRCA1 splice variants in human mammary cells.
Date	1999
Abstract	BRCA1 has been identified as a tumor suppressor gene that is mutated in many cases of inherited breast and ovarian cancer. Recent data suggest that multiple splice forms of BRCA1 exist, but the structure and function of these alternative transcripts have not been elucidated. By sequence analysis of RT-PCR products, we have determined that a major splice form of BRCA1 expressed in malignant and non-malignant breast epithelial cells contains an in-frame deletion of 3309 nucleotides from exon 11. A second alternative splice event results in the in-frame deletion of the 123 nucleotides that make up exons 9 and 10. These splice variants are associated with polysomes, and are therefore predicted to encode 80-85 kDa BRCA1 -derived proteins lacking approximately 60% of the internal amino acids that constitute full-length BRCA1. Western analysis confirmed the existence of BRCA1 splice variants in human mammary cells. Using mammary cell line systems as a model, we demonstrated that the full-length and the exon 11-lacking BRCA1 isoforms could transactivate the human p21/WAF1 promoter in a dose-dependent manner. The transactivation activity of BRCA1 is dependent on a functional p53. Co-transfection with a combination of the full-length BRCA1 and the exon 11-lacking BRCA1 leads to more than an additive effect on the p21/WAF1 promoter activity. Western immunoblot analysis documented a correlation between the expression levels of BRCA1 proteins and its transactivation activity. The increased expression levels of BRCA1 proteins, to some extent, are attributed to the increased expression levels of BRCA1 mRNA. Finally, we examined the effect of mutant BRCA1 on its transactivation activity. We found that the full-length mutant BRCA1 functions similarly as wild type BRCA1, however, the exon 11-lacking mutant BRCA1 is deficient for the transactivation of the p21/WAF1 promoter. Taken together, we have identified multiple splice variants of BRCA1 in mammary epithelial cells. In addition, we have demonstrated that the exon 11-lacking BRCA1 isoform alone is also transcriptionally active. The results of this thesis research have provided insight into the molecular mechanism of BRCA1 tumor suppressor function in mammary carcinogenesis, as well as its potential therapeutic application.
Description	1 online resource (xii, 145 leaves) : illustrations (some color)
Format	application/pdf
Advisor	Arrick, Bradley A.
Subject	Antioncogenes. ; Breast -- Cancer -- Genetic aspects.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D)--Dartmouth College.
Copyright	In Copyright. This material is protected by copyright and/or related rights. You are free to use this material in any way that is permitted by copyright law that applies to your use. For other uses, you need to obtain permission from the rights-holder(s). http://rightsstatements.org/page/InC/1.0/
DOI	https://doi.org/10.1349/ddlp.3237

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Molecular cloning and functional characterization of BRCA1 splice variants in human mammary cells.

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