Investigation of apoptosis-related gene expression in multi-drug resistant cells
Cronin, Deirdre (2002) Investigation of apoptosis-related gene expression in multi-drug resistant cells. PhD thesis, Dublin City University.
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The RNase protection assay (RPA) was used to identify changes in gene expression accompanying alterations in drug resistance levels in lung carcinoma cells. Based on this analysis, mcl-1 and bax were selected for transfection into drug sensitive cells to establish whether or not their expression influences drug resistance levels. The RPA was also used to investigate alterations in expression of bcl family genes as a consequence of transfection of a bcl-xL ribozyme.
Previous research in this laboratory, as well as results of the RPA analysis, indicated that caspase-3 levels were reduced in more resistant cell lines. To investigate the role of caspase-3 in drug resistant cells, the first reported ribozyme to human caspase-3 was designed and transfected into a drug-resistant variant (DLKP-A5F) of a human lung carcinoma cell line (DLKP). By both in vitro cleavage and stable and transient transfection in drug resistant DLKP-A5F cells, this ribozyme was shown to be effective at down-regulating human caspase-3 mRNA and protein levels. Initial results in stable transfectants indicated an increase in drug resistance, but on repeated subculture, these resistance levels reverted back to those of parent cells. Analysis of the multi-drug resistance protein, P-gp, revealed that its level had also decreased in some of the clones. These results indicate the importance of analysing P-gp levels in transfection studies where drug resistance is being monitored and suggest the possibility that caspase-3 expression may somehow regulate P-gp protein level.
DNA microarray technology was used to investigate differences in gene expression between two resistant variants (one high-level, one low-level) of doxorubicin-treated DLKP cells. Results indicated changes in the gene expression of some ABC transporter proteins, multi-drug resistance gene (mdr-1), apoptosis-related genes (galectin-1, apoptosis-associated protein kinase), calpain-1, retinoic acid receptor-a as well as metastases-related genes.
Finally, the expression of apoptosis-related genes was examined in a panel of archival breast tumour biopsies. The expression of these genes (bcl-1, bax, mcl-1 and bag-1) was correlated with clinicopathological parameters with the aim of identifying their prognostic significance. Bcl-2 correlated with ER status, bag-1 expression and five-year relapse-free and overall survival. Bax expression did not associate with any other parameter. Mcl-1 correlated with tumour grade and had borderline significance with lymph node status and survivin 83 expression.
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