Molecular cloning and characterization of human BAG-1

Yang, Xiaolong (1999) Molecular cloning and characterization of human BAG-1. Doctoral (PhD) thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (25MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

In the past ten years, a tremendous amount of work has demonstrated that enhanced cell proliferation caused by activation of oncogenes or inactivation of tumor suppressor genes is one of the major causes of human cancer. However, recently, mounting evidence strongly suggest that decreased cell death by apoptosis via inactivation of pro-apoptotic genes or activation of anti- apoptotic genes may also play an important role in carcinogenesis. Inactivation of the pro-apoptotic gene p53 by mutations or DNA tumor viruses and overexpression of anti-apoptotic gene bcl-2 have been found in the majority of human cancers. -- In an attempt to identify the transcription factors regulating JC virus expression, our lab isolated a protein called K1, which has an amino acids sequence identical to that of BAG-1. BAG-1 is a protein originally isolated from mouse embryonic cells as a Bcl-2 interacting protein. To understand the role of BAG-1 in human cancer, I isolated a 1.3 kb full length human BAG-l cDNA by screening a human cervical cell cDNA library using mouse BAG-1 cDNA as a probe. The amino acid sequence of human BAG-1 cDNA is identical to a hormone receptor-binding protein, RAP46, and has 75% identity and 84% homology with that of mouse BAG-1. BAG-1 RNA was expressed in a variety of human tissues and at higher levels in the heart and pancreas. Characterization of BAG- 1 protein in vitro and in vivo found that human BAG-1 protein is expressed as four iso forms, p50, p46, p33, and p29, by alternative translation initiation of the start codons through a leaky mechanism. Different BAG-1 iso forms have distinct subcellular localization. -- Like mouse BAG-1, overexpression of human BAG-1 can inhibit apoptosis induced by various apoptotic stimuli such as UV irradiation, heat shock, staurosporine, cisplatin and doxorubicin. The C-terminal BAG-1 sequence is responsible for its anti-apoptotic activity. However, BAG-1 sensitizes 4-HPR-induced apoptosis by activation of caspase-3 through the BAG-l central region. This result suggests that BAG-1 can be a pro- or anti-apoptotic protein, depending on the apoptotic inducer. -- Since Bcl-2 has been shown to be overexpressed in most human cancers, I examined whether the expression of Bcl-2-interacting protein, BAG-1, was also enhanced during tumorigenesis. In this study, enhanced expression of BAG-1 protein was detected in breast and cervical cancer ceil lines and tissues. The increased BAG-1 protein in cancer cell lines was due to enhanced transcription rather than genomic rearrangement of the BAG-1 gene. Correlated with that of Bcl-2 and Bcl-XL, the levels of BAG-1 proteins increased progressively after immortalization and transformation during multistage carcinogenesis of cervical cells. The increased BAG-1 protein was correlated with progressively increased resistance to apoptosis induced by staurosporine. Moreover, the level of BAG-1 protein correlated with that of other BAG-1-binding proteins such as Bcl-2 and Bci-XL only in human breast and cervical cell lines but not tissues. -- To understand the molecular mechanism of overexpression of BAG-1 in human cancers, I cloned a 0.9 kb BAG-1 5'-flanking region from genomic DNA. Deletion analysis of this sequence localized the region of maximal BAG-l promoter activity from nucleotide positions -353 to -54, upstream of the first start codon CTG. Sequence analysis of the BAG-1 promoter region showed the absence of a TATA box but identified a CCAAT box, several GC boxes, a CpG island and several transcription factor binding sites, which may be important in the regulation of BAG-1 transcription. Most importantly, functional characterization of the BAG-1 promoter in vivo demonstrated that gain- of-function p53 mutants derived from human tumors upregulated the transcription of BAG-l RNA and the expression of a reporter gene from the BAG-1 promoter. These data indicated that overexpression of BAG-1 in some tumors may be due to upregulation of the human BAG-1 promoter by mutant p53. -- In summary, human BAG-1 cDNA and promoter/enhancer were cloned and characterized in this study. Further analysis of BAG-l RNA and protein demonstrated that enhanced expression of BAG-1 may play an important role in carcinogenesis.

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