Effect Of Transfection Of Chk1 And Chk2 On Cycle Arrest In BGC823 Cells By Diallyl Disulfide

Objective: Cell Cycle G2/M Arrest was involved in growth inhibition induced by Diallyl Disulfide on various cancer cell lines in vitro, but its mechanisms remain unclear. The present study was to construct eukaryotic expression vector pcDNA3.1(+)/Chk1, pcDNA3.1(+)/Chk2, to establish the functional expression of Chk1 and Chk2 with G418 and investigate the effect of Chk1 or Chk2 gene on the cell G2/M stage and checkpoint kinase signaling of human gastric cancer cell line BGC823.Methods: Flow cytomery was used to determine the change of BGC823 cell cycle. Construct eukaryotic expression vector pcDNA3.1(+)/Chk1, pcDNA3.1(+)/Chk2. pcDNA3.1(+)/Chk1/BGC823, pcDNA3.1(+)/Chk2/BGC823 cell lines were established by transfecting pcDNA3.1(+)/Chk1, pcDNA3.1(+)/Chk2 into BGC823 cells with G418. The transfected cell lines were verified by RT-PCR and Western blot and cell clones with higher expression of Chk1 and Chk2 were selected. Cell morphous, Growth curves and cell cycle distribution were detected after Chk1 and Chk2 gene up-regulated expression with G418 induction. Checkpoint kinase expressions on transfected cells induced by diallyl disulfide were detected by Western blot and cell cycle distribution were determined by flow cytomery.Results: Flow cytometry analysis revealed that treatment BGC823 cell with 15mg/L DADS increased in the percentage of cells in the G2/M phase. The proportion of cells in the G2/M phase with15mg/L DADS for 12h was 19.5% which was obviously increased than 9.1%; 24h was comparable (39.9%), and more than two times that occurring in untreated cells (15.5%); 36h was 32.5% to 14.8%; 48h was 19.9% to 12.7%. G2/M phase arrest lasted to 48h.The results of specific PCR showed that a 1.4 kb and 1.6kb fragment had been cloned into pcDNA3.1 (+) vector, which was further identified by sequencing and NCBI BLAST analysis. The higher expressions of Chk1 and Chk2 were detected in transfected pcDNA3.1(+)/Chk1/BGC823, pcDNA3.1(+)/Chk2/BGC823 cell lines. The ration of nucleus to cytoplasm of pcDNA3.1(+)/Chk1/BGC823, pcDNA3.1(+)/Chk2/BGC823 cells decreased with G418 induction. The growth capacity of pcDNA3.1 (+)/Chk1/BGC823 was significantly suppressed after Chk1 gene up-regulated expression with G418 induction, compared with the controls P<0.05; Chk2 gene up-regulated expression inhibited slightly the growth of capacity of pcDNA3.1(+)/Chk2/BGC823 P>0.05, compared with controls.Flow cytometry analysis revealed the proportion of pcDNA3.1 (+)/Chk1/BGC823 in the G2/M phase was significantly increased comparable with BGC823 and pcDNA3.1 (+)/BGC823 and tumor cells were arrested in G2/M phase. Chk2 gene up-regulated expression couldn't increase the proportion of G2/M phase (P<0.05).After treated with15mg/L DADS for 2,4,8,12h, Western blot analysis showed that phosphorylation of Chk1 was increased following treatment of Chk1/BGC823 cells with 15mg/L DADS. At the same time, the total Chk1 abundance did not change. Expression of phospho-Chk2 and Chk2 wasn't changed in Chk2/BGC823 by DADS. DADS treatment of pcDNA3.1(+)/Chk1/BGC823 cells for 12h decreased the level of Cdc25C in a time-dependent model (P<0.05) and this effect lasted to 48h.After treated with15mg/L DADS for 24,48h,the proportion of Chk1 transfected in BGC823 cells in the G2/M phase was significantly increased comparable with the untreated groups P<0.05.Conclusion:1. Higher expression of Chk1, Chk2 under G418 were successfully established in BGC823 cells.2. The transfection of Chk1 could obviously inhibit the prolification of BGC823 cells. Tumor cells were arrested in G2/M phase.3. DADS might phosphorylate Chk1 and then arrest Human Gastric Cancer Cells at G2/M by Chk1/Cdc25C.4. Chk2 probably didn't participate G2/M phase arrest by DADS in human gastric cancer cells.