Studies Of Tumor Suppressor Gene P33～(ING1b) In Exocrine Pancreatic Carcinoma

Background: Tumor suppressor gene negatively control cell growth, proliferation and differentiation. Loss of its functions caused by gene deletion and/or mutation contribute to cell transformation and tumor formation. ING1, a recently identified candidate tumor suppressor gene, is downregulated and is deleted and mutated in a variety of primary tumors and established cell lines. In exocrine pancreatic carcinoma, inactivation of wild-type p53 is an important molecular event in tumorigenesis. Gene net related with p53 functions plays a major role in cell progression, differentiation and sustaining normal morphologic structure. p33ING1bprotein encoded by ING1 gene was closely physically related with p53 protein. p33ING1bprotein may be a member of the family of p53 protein. Some of the functions of ING1, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. To investigate the alterations and expressions of p33ING1bgene, as well as its relationship with p53, may be efficiently demonstrate the molecular mechanism of pancreatic carcinogenesis.Methods: Two tissue arrayers were constructed which contain 167 cases of pancreatic carcinomas and 106 cases of matched normal tissues. Immunohistochemistry was applied to detect the expression of p33ING1b and p53 and to observe the relationship between them. 23 cases of pancreatic carcinomas were analyzed for heterozygosity(LOH) at ING1 locus, using four narrow spaced microsatellite markers. The mutation of p33ING1b gene was examined by PCR-SSCP technique and DNA sequencing in 40 cases of pancreatic carcinomas. Semi-quantitative RT-PCR wasemployed to detect the reduced expression of p33ING1bmRNA in 9 cases of cancer tissues compared with matched normal tissues. The p33DK51b cDNA fragment was cloned into PCDNA3 eukaryotic expression vector and the recombinant plasmid was transfected into pancreatic cell line SW1990 and the changes of SW1990 cell growth and apoptosis rate were analysized. To further observe the effects of p33INGlb and p53 cooperation function on pancreatic cancer cell, pSS1*"1 and wild-type p53 cDNA were transfected into SW1990 cell simultaneously. After transfection, the changes of SW1990 cell apotosis rate^ Go/Gj arrest and the expression of p53 and p33INGlb proteins were analysized, using FCM and Western blot. Results:1. Two tissue arrayers contain 171 and 190 sites respectively. The positive expression of p33INGlband p53 protein by IHC were 77.9% and 59.8%, respectively. Both of them were located in nucleus and had the same positively present areas. The expression of p33INGlb and p53 protein had a closely relation in PCs (P<0.01).2. The results of PCR-SSCP and DNA sequencing demonstrated that INGlb gene did not have a fragmental frame shift or deletion in 40 cases of tumor and surrounding tumor tissues. Only one case of PC present abnormal band shifting by SSCP which had a point mutation in p33ING1bgene exon 2. In all surrounding pancreatic tumor tissues p33ING1b gene did not exhibit mutation. p33ING1b gene had a low mutation rate in PCs, which suggested that gene mutation was not the main reason that p33ING1b lost its tumor suppressing function in pancreatic carcinomas.3. There are loss of heterozygosity on ING1 locus of chromosome 13q33-34. Fourteen (60. 9%) of 23 tumor samples showed LOH in all of the informative markers tested, including D13S261, D13S1047, D13S1315 and DS42490. The rates of the four microsatellite markers were 8. 7%, 21.7%, 13. 0% and 26. 1%, respectively. But only 2 of them show no protein expression. This suggest that genetic alterations that abrogate normal function of ING1 may contribute to pancreatic carcinoma cell carcinogenesis, but not contribute to the loss expression of P33ING1b protein.4. Results from semi-quantitative RT-PCR reveal the different expression of p33ING1bmRNA in 9 cases of cancer tissues and matched normal tissues. The expression of p33 mRNA were reduced in 6 of 9 pancreatic carcinoma, but only one of which show no p33...