Role of apoptosis in multidrug resistance and tumorigenesis of human cervical cells: implication of BAG-1 and other apoptotic proteins

Ding, Zhihu (1999) Role of apoptosis in multidrug resistance and tumorigenesis of human cervical cells: implication of BAG-1 and other apoptotic proteins. Masters thesis, Memorial University of Newfoundland.

[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. Download (7MB)

Abstract

Recent studies have indicated that inhibition of apoptosis may play an important role m both multistep carcinogenesis and multidrug resistance (.MDR). Apoptosis is controlled through many cellular genes. The pattern of these apoptosis-regulating proteins varied in different cell types. The molecular mechanism of apoptosis in the multistep carcinogenesis and multidrug resistance of cervical cells is still poorly understood. -- To examine the role of apoptosis in tumorigenesis and chemoresistance of human endocervical cells, a cisplatin-resistant endocervical cell line (HEN-16-2/CDDP) was established by treating an HPV16-immortalized human endocervical cell line previously established in this lab, HEN-16-2, with cisplatin. A phenotype of MDR was identified for HEN-16-2/CDDP by clonogenic survival efficiency assay using two structurally and functionally distinct anticancer drugs: cisplatin and paclitaxel. -- The thresholds to undergo apoptosis of HEN-16-2/CDDP cells in response to various apoptotic stimuli was compared with that of its parental HEN-16-2 cells. HEN-16-2/CDDP cells were found to be significantly more resistant to cell death induced by several chemotherapeutic drugs, UV irradiation, anti-Fas antibody and heat shock. Moreover, the dysregulation of apoptosis in HEN-16-2/CDDP cells was found to confer tumorigenicity. Further characterization of HEN-16-2/CDDP cells indicated the following: 1) they displayed distinct morphologies in monolayer; 2) they had an increased rate of proliferation in medium containing physiological calcium levels; 3) they demonstrated anchorage-independent growth in vitro; 4) they expressed similar levels of pro-apoptotic genes, including p53, Bak, Bax and the anti-apoptotic gene Bcl-2, compared to the drug-sensitive cell line, HEN-16-2; and 5) they expressed significantly higher levels of the anti-apoptotic gene Bcl-XL as well as the p50 and p33 isoforms of BAG-1. Overexpression of BAG-1 in cervical carcinoma C33A cell line confers resistance to cisplatin, etoposide and doxorubicin, but not to actinomycin D and paclitaxel BAG-1 also protects C33A cells from apoptosis induced by heat shock and UV irradiation. -- The yeast two-hybrid system was established to screen BAG-1 interacting proteins from a human keratinocyte cDNA library. Eighteen positives were obtained from 2.5 x 106 clones. Further analysis of the interacting clones identified four genes: Hsp 70, Hsp70-2, Hsc70 pseudogene and a putative novel Hsp70Y. Carboxyl-terminal amino acids of BAG-1 were found to be important in the mediation of the interactions. -- Overexpression of Hsp70 or Hsp70-2 in C33A cells conferred the resistance to various apoptotic stimuli, including cisplatin, doxorubicin, etoposide, paclitaxel, actinomycin D, heat shock and UV irradiation. -- In summary, this study provided the first in vitro evidence that inhibition of apoptosis conferred MDR and tumorigenesis in endocervical cells. Increased levels of Bcl-XL and BAG-1 p50 and p33 isoforms were found to be associated with this phenotype. Hsp70s were identified as BAG-1-interacting proteins from a cDNA library using the yeast two-hybrid system, and further studies indicated that they may also contribute to the regulation of apoptosis.

Actions (login required)

View Item