The Initiation Of G₂/M Checkpoint By Diallyl Disulfide Associates With The Subcellular Distribution Of CyclinB1 And P21 In HL-60 Cells

Background Diallyl disulfide (DADS), an oil-soluble allyl sulfur compound found inprocessed garlic, was found to inhibit the growth of various tumors by decreasing the cell growth rate, inducing apoptosis and cell cycle arrest. The previous studies in our laboratory showed that DADS induced the growth arrest of HL-60 cells in the G2/M phase, but the detail mechanism by which DADS induced the cell cycle arrest remains obscure. CyclinBl, as a specific G2/M phase cyclin, translocates to the nucleus during prophase. it is necessary for nuclear targeting of cyclinBl to initiate the mitosis. p21, which is a cyclin-dependent kinase inhibitor, could inhibit the cdc2 activation by preventing cdc2 dephosphorylation by cdc25 and/or interfering with CAK-mediated cdc2 phosphorylation by forming p21-PCNA-cdc2-cyclinBl. But the role of the subcelullar distribution of cyclinBl and p21 expression during the initiation of G₂/M checkpoint by DADS in HL-60 cells remains to be clarified.Methods HL-60 cells were treated with 20μM DADS for the indicated time, and then cell proliferation was determined by MTT assay. Cell cycle was assayed by flowcytometry. The subcelullar distribution of cyclinBl, the expression of p21 and c-myc were measured by Western blot. The status and interaction between cyclinBl and p21 in DADS-treated HL-60 cells was measured by immunoprecipitation. The effects of c-myc on the p21 expression and the determination of HL-60 cell fate were analysed by transfecting recombinant plasmid pcDNA3-c- myc into the HL-60 cells.Results To explore the effect of DADS on cellular proliferation in HL-60 cell growth,the cells were treated with 20μM DADS, DMSO, 10nM ATRA for 24h. The results showed that DADS could inhibit HL-60 cell proliferation. The inhibitory efficiency of 20 μM DADS was similar to that of 10 nM ATRA. To determine the phase at which the cell growth arrest, the cell cycle was measured by flow cytometry. The results indicated that the percentage of G₂/M phase cells reached maxium at 12h after treated with 20μM DADS,These results suggested that DADS could inhibit the proliferation of HL-60 cells and induce the cell gowth arrest in G2/M phase.It is reported that the initiation of mitosis is regulated by the activation of cdc2-cyclinBl, and that the nuclear targeting of cyclinBl is necessary for the initiation of the mitosis. To explore the relationship between the subcelullar distribution of cyclinBl and the initiation of G2/M checkpoint by DADS, the cells were treated with 20/uM DADS. The results showed that the expression of cyclinBl increased steadily following 20/uM DADS incubation, and that the expression of cyctoplasmic cyclinBl reached the peak at 12h after treated with DADS, While the expression of nuclear cyclinBl was obviously inhibited. These results suggested that the initiation of G2/M checkpoint by DADS was associated with the cytoplasmic targeting of cyclinBl.To explore the effect of p21 on the initiation of G2/M checkpoint by DADS, HL-60 cells were treated with DADS for 0h²4h. The results indicated that DADS could induce steadily the expression of p21, and that p21 level achieved the peak at 12h after treated with DADS. To further ananlyze the status and interaction between p21 and cyclinBl, the cyclinBl from DADS-treated HL-60 cells precipitated with anti-cyclinBl antibody and followed by ProteinA/G PLUS-Agarose. The precipitation was blotted with anti-p21 monoclonal antibody. The result showed that p21 was precipitated with cyclinBl. So p21 existed in cyclinBl-bound state, and p21 conferred its biogical effects by binding to cyclinBl. These results suggested that the initiation of G2/M checkpoint by DADS was possiblly involved in p21 expression.To further confirm the possible roles of p21 in the initiation of G2/M checkpoint by DADS, recombinant pcDNA3-c-myc plasmid and pcDNA3 plasmid were transfected into HL-60 cells, respectively. The results showed that c-myc could express in c-myc -transfected-HL-60 cells, and that DADS could inhibit the c-Myc expression. Moreover, the level of p21 expression in HL-60 cells transfected c-myc gene was down-regulated by c-Myc, suggesting that the expreassion of c-myc in HL-60 cells could inhibit p21 expression induced by DADS. To explore the effects of p21 down-regulation by c-Myc on the initiation of G2/M checkpoint by DADS, the cell cycle was analyzed by flow cytometry. The result indicated that the percentage of the G2/M phase cells in c-myc-tansfected-HL-60...