| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 96 === Human MMS21 (hMMS21) is a SUMO ligase required for DNA damage repair. In our previous study, we found that the hMMS21 played an important role in cell growth. The growth rate of I15, an hMMS21-depleted stable clone, was much slower than MCF-7 and ectopic expression of hMMS21 rescued the growth rate of I15 cells depending on its SUMO ligase activity. Flow cytometric analysis of cell cycle of these asynchronous cells indicated that the growth of I15 cells was delayed at G1→S progression. However, when cells were synchronized by serum starvation and re-stimulated by serum, I15 showed a significant lag in cell cycle entry. Thus depletion of hMMS21 from MCF-7 cells appears to affect both G0→G1 and G1→S progression. To explore further the mechanism involved, we compared the levels of various G1 phase-specific cyclins, cyclin kinases and inhibitors along the cell cycle between MCF-7 and I15 cells. Both cyclin D1 and D2 levels were lower in I15, but the cyclin kinase inhibitor (CKI) p21 remained higher at late G1 phase. In addition, cyclin D2 was not detectable in nuclear fraction throughout the whole G1 phase in I15, whereas there was a manifested and transient increase in D2 in early G1 in MCF-7 cells. Moreover, immunofluorescence analysis demonstrated that there was a period of co-localization of D2 and hMMS21 in nucleus during the early G1 phase in MCF-7 cells. On the other hand, ectopic expression of hMMS21 but not the mutant C215A in I15 cells resulted in an increase in nuclear D2 and some of which were also co-localized with hMMS21 at early G1 phase. Further investigation into the effect of hMMS21 on growth factor influence on cell growth, we found that EGF, IGF-1 and Insulin-stimulated p42/p44 MAPK or PKB/Akt activation were decreased in I15 cells. Ectopic expression of hMMS21 but not SUMO ligase-deficient mutant C215A, rescued the cell response to EGF, Insulin and IGF-1. In summary, this study demonstrated that hMMS21 may regulate MCF-7 growth by controlling growth factor-induced activation of MAPK and PKB/Akt activities and cell cycle entry as well as the G1-S transition, probably by a mechanism involving sumoylation and the stability of cyclin D1, D2 and CKI p21.
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