Overexpression of p27(KIP1) induced cell cycle arrest in G(1) phase and subsequent apoptosis in HCC-9204 cell line.

ABSTRACT

AIM:We have previously reported that inducible over-expression of Bak may prolong cell cycle in G(1) phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27(KIP1) plays an important role in this process.METHODS:In order to elucidate the exact function of p27(KIP1) in this process, a zinc inducible p27(KIP1) stable transfectant and transient p27(KIP1)-GFP fusion transfectant were constructed. The effects of inducible p27(KIP1) on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells.RESULTS:This p27(KIP1)-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27(KIP1) induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72h of p27(KIP1) expression. p27(KIP1) caused cell cycle arrest after 24 h of induction, with 40% increase in G(1) population. Prolonged p27(KIP1) expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27(KIP1) expression showed a characteristic DNA ladder on agarose gel electrophoresis.CONCLUSION:Bak may induce cell cycle arrest in G(1) phase through upregulating expression of p27(KIP1) and subsequently lead to apoptosis in HCC-9204 cells. The p27(KIP1) -GFP fusion protein can be transiently expre-ssed in HCC-9204 cells. The inducible p27(KIP1)-expressing cell line provides a model to assess p27(KIP1) function.