Effects Of Blocking The TGFα-EGFR Autocrine Loop And Cyclin D1 Expression On The Growth Of Human Pancreatic Carcinoma Cells

In pancreatic carcinoma, EGFR and TGFα/EGF form an autocrine loop. Recent studies have demonstrated that it is TGFα but not EGF that forms the autocrine loop with EGFR, which play an important role in the progression of pancreatic tumors. The human pancreatic carcinoma cell lines established in our laboratory expressed TGFα, EGF and EGFR and our previous research showed that antisense EGFR inhibited the growth of pancreatic carcinoma cell line PC-7 cells markedly, however, antisense EGF exerted rather weak inhibition effect. The main aim of our present work was to investigate the role of TGFα, the other ligand of EGFR, in the autocrine loop of pancreatic carcinoma. The technique route of our study is introducing antisense TGFα to pancreatic carcinoma cell line cells alone and together with the antisense EGFR by gene transfer technique, and then observing their effects on cell growth and reversion of malignant phenotype of pancreatic carcinoma cells.The accomplishment of stimulating proliferation by growth factors is through the cell cycle and the regulation of the cell cycle progression is by the critical cyclins in G₁ phase. Being an important G₁ phase cyclin, cyclin D1 is the growth factor sensor. To study the influence on cell growth by blocking EGFR/TGFα autocrine loop of pancreatic carcinoma is mediated whether by the downregulation of cyclin D1 expression or not, we constructed an antisense cyclin Dl expressing vector, which was used to transfect to pancreatic carcinoma cells to inhibit directly the expression of target gene cyclin Dl, and observed the effect of antisense cyclin D1 on cell growth and the induction of cell apoptosis. Our studies included three parts:1. Constructing of the recombinant retroviral vector expressing antisense TGFα and transfecting pancreatic carcinoma PC-7 cells:A recombinant retroviral vector expressing antisense TGFα was constructed and packaged by ecotropic packaging cell line ψ-2 and amphotropic packaging cell line PA317, the replication-defective amphotropic retroviral supernatant was used to transfect pancreatic carcinoma cell line PC-7. Following puromycin selection, puromycin-resistant colonies were pooled and identified as PC-7/AS-TGFα. The integration of retrovirus vector and expression of exogenous gene in the transformant PC-7 cells were confirmed by Southern blot, Northern blot and Western blot hybridizations. The gene expression and protein...