Anti-apoptotic and apoptotic effects of Xrel3 in human cervical cancer cells

Shehata, Marlene Fouad Amin (2003) Anti-apoptotic and apoptotic effects of Xrel3 in human cervical cancer cells. Masters thesis, Memorial University of Newfoundland.

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Abstract

Cervical cancer is considered a common yet preventable cause of death in women. It has been estimated that about 420 women out of the 1400 women diagnosed with cervical cancer will die during 5 years from diagnosis (National Cancer Institute of Canada, 2003). This study addresses the role of a member of the Rel/NF-κB family of genes, Xrel3, in the growth of the malignant HeLa cervical cells and its effect on chemotherapeutic treatment. -- The hypothesis of this study is that Rel/NF-κB promotes chemotherapeutic resistance in cancer cells. In order to study this problem, the amphibian Xrel3, a Rel/NF-κB homologue, was used because it was previously shown to be constitutively active when transfected into HeLa cells, which normally do not express Rel/NF-κB. This allowed the opportunity to test whether Xrel3 expression imbues chemoresistant properties to these cells. -- The expression of Xrel3 slowed the growth of HeLa cervical cells associated with an increase in expression of the cell cycle inhibitor p21. The expression level of the apoptosis pathway protein caspase-8, its activated product and cleaved poly (ADP-ribose) polymerase, PARP, were also elevated 6-fold relative to the non-induced state, perhaps associated with the initiation of apoptosis consequent to cell growth arrest. -- To determine the effect of chemotherapeutic agents on HeLa cells expressing Xrel3, cells were treated with the DNA-crosslinking and chelating agent, cisplatin. At 1 J.LM cisplatin, the expression of Xrel3 initiated an anti-apoptotic effect after 24 hours of treatment, based on the expression of significantly lower levels of the apoptotic proteins, Bax (P<0.05), caspase-8 (P<0.05) and MDM-2 (P<0.01). Furthermore, the level of the tumor suppressor protein p53 was suppressed by 3-fold, along with a reduction of caspase-3 and p21. The expression of the anti-apoptotic BAG-1 isoforms (P<0.01) was also increased. The expression of Xrel3 sensitizes HeLa cells to 1 μM cisplatin after a lag period of three days, however, as assessed by a decrease in numbers of viable cells. -- After 24 hours of 5 J.LM cisplatin treatment, there was a significant increase in the levels of the pro-apoptotic proteins relative to controls, including Bax (P<0.05), MDM-2 (P<0.05), the cell cycle inhibitor p21 (P<0.01), cleaved PARP, caspase-8, and caspase-3. However, p53 was significantly decreased (P<0.05) while BCL-2 (P<0.01) and BCL- XL (P<0.01) levels were elevated significantly. The level of the anti-apoptotic protein BAG-I remained constant (P<O.O 1) in cisplatin-treated Xrel3- expressing cells. Despite the increased expression ofBAG-1, the balance between pro- and anti-apoptotic factors shifted towards increased expression of pro-apoptotic proteins. The Human Papillomavirus (HPV) oncoprotein E6 expression was not affected by the transfected Xrel3 gene. -- These results suggested that Xrel3, a homolog of c-Rel, causes an anti-apoptotic effect (chemoresistance) when cells are initially exposed to low cisplatin concentrations (1 μM). This result supports the hypothesis of this study. However, with prolonged contact to 1 μM cisplatin or upon 24 hours exposure to 5 μM cisplatin, Xrel3 had a synergistic apoptotic effect when coupled with cisplatin, thus promoting chemosensitivity and cell death. These results support the previously proposed idea that Rel/NF-κB proteins modulate their activity depending on cellular stress.

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