Effects Of DNA Damage On Cell Cycle And Apoptosis Of Mammalian Cells

This research focused on cell cycle checkpoints and apoptosis induced by DNAdamage in mammalian cells. DNA damage activates checkpoints and results in a cellcycle arrest. After DNA damage is repaired by the repair system, cells proceed into thecell cycle. It is not clear, however, whether a certain level of DNA damage is requiredfor the activation of checkpoints. Therefore, we treated Chinese hamster ovary (CHO)cells with hydroxyurea (HU), a reagent that can induce DNA strand breaks inmammalian cells. Low levels of DNA damage in CHO cells induced by short exposureto HU did not trigger checkpoints after HU was released, whereas higher levels of DNAdamage caused by longer exposure to HU resulted in S-phase delay and G2-phase arrestafter HU was released. These results argue that a threshold of DNA damage is necessaryfor activation of cell cycle checkpoints. It is considered that when DNA damage cannot be repaired, cells execute apoptosis.We used a potent anticancer reagent tripchlorolide (TC) to treat the wild type CHO cellsor the mutant cells which are deficient in DNA repair system. TC has beendemonstrated to induce apoptosis of CHO cells. TC was particularly potent in inducingapoptosis of CHO mutant cells UV41, which are defective in nucleotide excision repair.TC caused a higher level of DNA damage in UV41 cells than those in the wild typeAA8 cells or EM9 cells which are deficient in base excision repair. Further analysis 3Abstractshowed that degradation of the c-Myc protein in TC-treated UV41 cells was muchstronger than those in the AA8 cells or the EM9 cells. A proteasome inhibitor, MG132,reduced both the degradation of c-Myc and apoptosis in TC-treated UV41 cells.Overexpression of exogenous c-Myc also inhibited apoptosis of TC-treated UV41 cells.These data indicate that c-Myc degradation induced by DNA damage in the presence ofTC contributes to induction of apoptosis of UV41 cells. TC-induced apoptosis was inhibited by cycloheximide (CHX), a protein-synthesisinhibitor. We compared the protein expression profiles of TC-treated CHO cells in thepresence of CHX to that without CHX by two dimensional gel electrophoresis. Ofidentified differently-expressed protein-spots in the CHX-treated sample, only two thirdwere down-regulated, whereas one third were up-regulated, suggesting that inhibition ofTC-induced apoptosis by CHX is a more complicated process rather than simpleinhibiting the synthesis of proteins.