Cloning and characterization of genes differentially expressed in human pancreatic carcinoma

Introduction: Identification of ‘novel’ genes involved in pancreatic tumor progression is essential as a basis for the development of new treatment or diagnostic modalities. This study was about the cloning and characterization of several undefined clones, which were obtained by DDRT-PCR on RNA from patients with pancreatic cancer, chronic pancreatitis and normal pancreas from a former study of our group. Methods: RACE (Rapid Amplification of cDNA Ends) was performed for amplifying the 5’ end of clone 14/6 and 14/1. Panc–1 pancreatic cancer cells were stably transfected by antisense constructs of genes of interest (ANK-B and SSR3) upregulated in pancreatic carcinoma. Down-regulation of the respective genes was comfirmed by RT-PCR, real time PCR and western blot. Cell proliferation was determined by WST–1 and soft agar assay. Cell migration and invasion capability was checked by Matrigel coated transwells. Effect on tyrosine phosphorylation was performed on 10% FCS stimulated cells by immunoblot, focal adhesion kinase (FAK) phosphorylation was carried out by immunoprecipitation and western blot. Results: Clone 14/6 and 14/1 were identified having 98% (446/453) and 100% (399/ 399) alignment with gene ANK-B and SSR3 respectively. Transfection of Panc–1 cells with antisense constructs of the two genes decreased their expression on RNA and protein level, exhibited a reduction of cell growth rate of about 30%, showed colony forming efficacy of 60% for ANK-B and 76% for SSR3 compared to the parental cells, attenuated the ability of invasion of 45.9% (ANK-B) and 30.4% (SSR3), migration of 64.6% (ANK-B) and 52.4% (SSR3). Reduced phosphorylation of tyrosine and FAK was observed in transfected cells. FCS activated the phosphorylation of tyrosine in both transfected and non- transfected cells. Conclusion: Our findings document that ANK-B and SSR3 are overexpressed in pancreatic cancer and that the suppression of these genes expression attenuates the growth potential of pancreatic cancer cells and some tumor characteristics. The identification of pancreatic cancer related ’novel’ genes which have never been studied in pancreatic carcinoma before may contribute new targets to the gene therapy of pancreatic disease.

Keywords: ANK-B, SSR3, antisense transfection, gene cloning, pancreatic cancer