| Summary: | 碩士 === 國立清華大學 === 輻射生物研究所 === 84 === Mammalian cells response to DNA damage by blocking the cell
cycle in G1 and G2 phases. Here we studyed the upstream signal
transduction pathways by which the cell recognizes and
processes DNA damage to cell cycle regulatory mechinery. DNA-PK
consists of Ku and DNA-PKcs, and is a novel protein Ser/Thr
kinase activated by DNA double-stranded breaks (DSBs) which are
the most detrimental DNA lesion caused by IR. Ku is a
heterodimer (Ku70 and Ku80) and has the DNA end-binding (DEB)
activity. DNA-PK can phosphorylate p53 in vitro. The mutation
of phosphorylation site (Ser15) putatively by DNA-PK in human
p53 protein reduces its ability to inhibit cell growth,
suggesting the involvement of DNA-PK in p53-dependent G1
arrest. Here, by expression of anti-Ku80 antisense mRNA, we
studied whether Ku or DNA-PK is the upstream signalling
molecule that leads to cell cycle G1 arrest by IR. As expected,
expression of anti-Ku80 antisense mRNA decreased DEB acivity,
and resulted in a radio- sensitive phenotype. However, the
deficiency of Ku80 expression did not decrease the extent of X-
ray-mediated G1 and G2 arrest. Our results suggested that Ku or
DNA-PK contributes to cellular radiosensitivity but not
regulation of IR-mediated cell cycle alterations. We also asked
whether other protein phosphorylation and and dephosphorylation
contribute to regulation of IR-mediated cell cycle arrest.
Inhibitors of protein kinases and phosphatase were employed to
study their effects on cell cycle progression following X-
irradiation. Both 6-DMAP and genistein effeciently abolished X-
ray-meidated G1 arrest while did not disrupt normal cell cycle
progression through G1 phase. Both 6-DMAP and genistein did not
interfere with the p21/CIP1/WAF1 mRNA induction by X-ray. Our
preliminary results suggested the important role for protein
Ser/Thr phosphorylation in the modulation of X-ray- mediated G1
arrest.
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