The Effects Of Adenovirus-mediated Transfer Of P14～(ARF) And P16～(INK4a) On The Growth Of Human Pancreatic Carcinoma Cells

The incidence of pancreatic cancer is increasing, so far neither an early diagnosis nor a therapeutic strategy for advanced lesions has been developed yet. It is an urgent mission for both the clinicians and the scientists who are challenging pancreatic cancer to find a breakthrough using new technologies. Recent progresses indicated that cancers are a group of diseases with accumulation of genetic alternations of oncogens and tumor suppressor genes. In pancreatic carcinoma, p53,K-ras, p16INK4a,DPC4/SMAD4,BRCA2 have been considered as molecules that play key roles in tumourigenesis. In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we studied the effects of gene therapy by adenovirus-mediated transfer of p14ARF and p16INK4a into human pancreatic cancer cells.Western blot was used to determine the expression of p14ARF and p16INK4a in seven pancreatic carcinoma cell lines. Four cell lines were negative for both p14ARF and p16INK4a protein, and three of them (PC-7, Panc-1 and Maia-paca2) were used in our study of gene transfer.1. By RT-PCR technique, the wild-type cDNAs of p14ARF and p16INK4a were cloned and amplified from Hela cell. The recombinant replication-deficient adenoviral vectors Adp14 and Adp14, which contain p14ARF and p16INK4a cDNA respectively, were constructed by AdEasy System and amplified in 293 packaging cells. The pancreatic cancer cells transfected with Adp14 and Adpl6 showed expression of p14ARF and p16INK4a, respectively.2. PC-7 (p53+) and Maia-Paca2 (p53 mutant) transfected with Adp14 showed significant inhibition of cell growth and soft-agar colony formation as compared with control, G2 arrest by flow cytometry and dephosphorylation of RB protein by Western blot, but no obvious increase in apoptotic cells as detected by flow cytometry and TUNEL methods. In addition, PC-7 has elevated expression of p53 after treatment with Adp14. Panc-1, which also had mutant p53, did not show the same effects when transfected by Adp14. These results suggest that p14ARF can inhibit the growth of pancreatic cancer cells through p53 dependent as well as p53 independent pathways.