The Role Of Endonuclease-G In The Action Of The Antitumour Drug Paclitaxel

Abstract

Paclitaxel is an antimitotic agent used extensively for the treatment of a variety of human cancers. It causes mitotic arrest by disrupting microtubule dynamics and induces subsequent cell death through mechanisms that are not fully understood. Previous studies in this laboratory revealed that exposure of HCT116 human colon cancer cells to low concentrations of paclitaxel induced little mitotic arrest but still induced cell death associated with extensive DNA degradation but without caspase-3 activation or the induction of of reactive oxygen species. This thesis examines the hypothesis that low concentrations of paclitaxel (12.5 nM) induce the release of endonuclease-G (endo-G), which mediates the observed progressive degradation of DNA. Methods used include gene knockdown with silencing RNA, flow cytometry, cellular fractionation, reverse transcriptase polymerase chain reaction (RT-PCR), enzyme assays, fluorescence microscopy and confocal microscopy. Techniques were first developed to verify expression levels of endo-G in HCT116 colon and PC3 prostate tumour cells following transfection with silencing oligonucleotides. Transfection was optimised using fluorescence microscopy, western blotting and RTPCR. Flow cytometry with propidium iodide DNA staining was used to demonstrate a substantial decrease in the proportion of cells degrading their DNA when endo-G was knocked down prior to paclitaxel exposure. Confocal microscopy was used to track the translocation of endo-G from the mitochondria following treatment with paclitaxel, and evidence for association with nuclear DNA was also apparent. Mitochondrial fractionation and an assay of the mitochondrial marker citrate synthase were used to confirm the mitochondrial localisation of endo-G. The technique was also useful in determining mitochondrial functionality and structural integrity following drug treatment. Mitochondrial outer membrane permeabilisation (MOMP) was also investigated with fluorescence microscopy techniques. Interestingly, endo-G release appeared to be independent of MOMP as a substantial proportion of mitochondria appear structurally and functionally intact following paclitaxel exposure, implying that these cells do not die in a classical apoptotic manner. Taken together, the results support the role of endo-G in paclitaxel-induced cell death. These results suggest a model where paclitaxel induces transient or partial MOMP, allowing translocation of some endo-G from mitochondria to the nucleus, where it associates with and degrades DNA over a period of several days.