| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 87 === Apurinic/apyrimidinic endonuclease, APE, also termed Ref-1 (redox factor-1), is a protein with multiple functions. The roles of APE/Ref-1 in the genotoxicity, signal transductions, and cell cycle progression interfered by radio-mimetic agents were investigated by the expression of APE antisense cDNA in mammalian cells. Transient expression of APE antisense cDNA in cells markedly reduced the APE protein levels and the endonuclease activity. However, the reduced APE levels in cells transiently transfected with APE antisense cDNA were elevated by MMS treatment. The APE antisense cDNA was also persistently integrated into CHO-K1 cells. Analysis of five independent clones derived from CHO-K1 cells showed that their genome contained the APE antisense cDNA and their APE levels were reduced to 22-60% of the levels in parental cells. MTT cytotoxicity assay showed that three of the five clones were more sensitive to H2O2 than the parental cells. However, the cytotoxicities induced by MMS or CdCl2 for all the five clones were not higher than that of the parental cells. Furthermore, the APEas-H293 cells were established by persistent integration of the APE antisense cDNA into H293 cells using a retrovirus-vector; the control cell line, pBabe-H293 was also established. APEas-H293 cells had markedly lower amounts of the APE levels and lower endonuclease activity when compared with those of pBabe-H293 cells. APEas-H293 cells were more sensitive to MMS than pBabe-H293 cells using the colony-forming ability assay. The numbers of spontaneous micronuclei observed in APEas-H293 cells were higher than those in pBabe-H293 cells. However, the induction rates of micronuclei by MMS were similar in these two cell lines. Again, the reduced APE levels in APEas-H293 cells could be elevated by MMS treatment, although MMS did not induce the high APE levels in pBabe-H293 cells. MMS could induce the p38 and ERK phosphorylation at similar levels in both APEas-H293 and pBabe-H293 cells. Inhibition of p38 by SB202190 did not affect MMS-induced cytotoxicities in these two cell lines. The G2/M-arrest phenomenon can be observed in APEas-H293 cells treated with low cytotoxic dose of MMS (0.1 mM), but not in pBabe-H293 cells. High doses of MMS (0.5-1 mM) markedly induced the G2/M-arrest in both cell lines at similar levels. Thus, expression of APE antisense cDNA in cells, although did not completely inhibit APE levels, it increased the sensitive of cells to radio-mimetic agents and increased spontaneous genotoxicity. However, the biological effects of APE antisense cDNA may be compensated by the fact that MMS could induce endogenous APE gene expression in MMS-treated transfectants.
|
|---|
