OBJECTIVEBoth tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene.

METHODSAfter identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry.

RESULTSNeither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene.

CONCLUSIONp15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.