Search Results

The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34%, 0.025 < P < 0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42%, significantly lower than those of the cells transfected with chkl sense chain (54.64%, 0.005 < P < 0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.
*Apoptosis/radiation effects ; Cell Cycle/radiation effects ; HL-60 Cells ; Oligonucleotides, Antisense/*genetics ; Protein Kinases/*genetics ; Protein Kinases/metabolism ; Radiation Tolerance/*genetics ; Transfection

6.Failure to inactivate CDK activity is responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene.

Jinniu, DENG ; Jianfeng, ZHOU ; Fankai, MENG ; Dengju, LI ; Hanying, SUN

Journal of Huazhong University of Science and Technology (Medical Sciences) 2002;22(3):193-6

Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and irradiation-like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937-ASPI3K (ATM, negative) and U937-pZeosv2(+) (ATM, wild-type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2, (Thr 161) or p34cdc2 (Thr 14, Tyr 15). RT-PCR was used to estimate CDC25 transcript levels. U937-ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-threonine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937-pZeosv2(+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937-ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post-transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In summary, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.