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

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.