Mechanism of regulation of hepatoma cell cycle by XPD/P44 subcomplex : an in vitro experiment

Abstract

To explore the effects of xeroderma pigmentosum group D(XPD)/P44 subcomplex on the cell cycle of the hepatoma cells. Human hematoma cells of the line SMMC-7721 were cultured and transfected with human XPD gene by Lipofectamine and 2 strains with stably transfected plasmid pEGFG-N2 and stably transfected recombinant plasmid pEGFG-N2/XPD were selected. After stably transfection,the antisense oligonucleotides of P44 were added to treat the stably transfected cells. The cells were divided into 6 groups: Group (1) (control group), Group (2) transfected with the blank plasmid pEGFP-N2, Group (3) transfected with the recombinant plasmid pEGFP-N2/XPD, Group (4) transfected with ASODN complementary to the translation initiation site of pEGFP-N2/XPD, Group (5) transfected with antisense oligodeoxynucleotides (ASODN) complementary to the translation terminal site of pEGFP-N2/XPD, and Group (6) transfected with ASODN complementary to the translation exon5 site of pEGFP-N2/XPD. The expression levels of wild-type P44, XPD, cdk7, cdk2, c-myc, and cdc25A were detected by RT-PCR and Western blotting. The cell growth and the cell cycle were examined by MTT and flow cytometry (FCM). The P44 and XPD mRNA expression levels of Group (4) were significantly higher than those of Groups (1) and (2) (both P < 0.01). Western blotting indicated that the changes of P44 and XPD protein expression levels were consistent with those of their mRNAs respectively; while the mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were all decreased. MTF method showed that the hepatoma cells grew slowly, FCM showed that the number of the cells arrested at the G1 stage of Group (3) were higher than those of Groups (1) and (2). After the blockage of P44 gene expression, the expression levels of XPD mRNA and protein were decreased. The XPD mRNA and protein expression levels of Groups (4), (5), and (6) were significantly higher than those of Group (3) (all P < 0.01). The mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were upregulated. MT method indicated that cells grew fast. FCM showed that the numbers of the cells arrested at the G1 stage of Group (4), (5), and (6) were all lower than that of Group ((3) The expression levels of cell cycle regulatory genes including cdk7, cdk2, c-myc, and cdc25A were markedly decreased,the hepatoma cells grew slowly; after the blockage of P44 gene expression the expression levels of XPD mRNA and protein were decreased, whereas the expression levels of the cell cycle regulatory genes mentioned above were enhanced, and the hepatoma cells grew faster. XPD gene inhibits the proliferation and promotes the apoptosis of hepatoma cells. The expression of XPD may be regulated by its molecular partner P44. XPD/P44 subcomplex is involved in the regulation of DNA damage checkpoint.