The Study Of P16 Gene Transfer On The Cell Cycle And Radio Sensitivity Of Colorectal Cancer Cells

It's generally accepted that some tumor was insensitivity or resistance toradiation. In the different cell cycles, sensitivity of tumor cells to radiation isdifferent. It fairly sensisitive to radiation in the G1 phase,and has invariablyresistance to radiation in the S phase. Recently p16 gene has been introducedas a gene of anti-oncogene for generation cycle regulation. p16 protein, codingby p16 gene, is the inhibitor of a cyclin-dependent kinase 4 (CDK4). In thisway, p16 protein inhibit the tumor cell generation cycle cloning from G 1 phaseto S phase,and further the growth and division of tumor cells. This studyadopts the immunohistochemistry to detect the deletion of p16 gene in thecolorectal cancer tissues. In addition, the author introduces exogenous p16gene, and makes p16 genes recover in colon cancer cells in o rder to researchthe effect of cell cycle and the change of inhibition ratio of radiation to cellsafter the introduction of p16 gene and explore the mechanism of enhance theradio sensitivity by p16 gene.Results:1. The deletion rate of p16 gene in colorectal the cancer tissue was 64.3% andthe deletion was significant statistically compared with that in the colorect altissue of normal, which was 10.7%; the deletion rate of p16 in the colorectaltissue with high differentiated carcinoma was 43.7%, while 65.2% and 82.4%with medium differentiated adenocarcinoma, and with low differentiatedadenocarcinoma separately. The differences among the groups weresignificantly in statistics (P<0.05); 41.2% in A/B phase, 68% in C phase and85.7% in D phase. The differences among the groups were significantly instatistics (P<0.05). 2. Accredit of pcDNA3+-p16 recombinant plasmidPCR product is connected with pcDNA3 + successfully. Throughdual-enzyme digestion of XbaⅠ/EcoRⅠ, recombination plasmidspcDNA3+-p16 can be confirmed to have a 478bp fragment of p16 by agarosegel electrophoresis. It is confirmed p16cDNA gene order fater sequencing.3. The transfection and screening of the expression of p16 gene in thecolorectal cancer cell.The clone took place in the control group 14 days after the culture withtransfecting 400μl/ml G418; but in the transfection group 21 days after theculture maintained by transfecting 100μl/ml G418, instead of 400μl/ml G418.Compared with those in the control group, cells in the transfection group grewslower and the sizes of cells became smaller.4. The expression of p16 gene examined by immunohistoch emical assay afterthe transfection.The results showed that partial cells of transinfected cells displayedbrownish yellow granules, the expression in the cytoplasm and the nucleus ofthem could be seen with DAB dyeing, but untransinfected cells did not.5. The cell cycle is evaluated by flow cytometry.The simple pcDNA3+-p16 plasmids transfection group compare withcontrol group, appearance G1 phase arrest evidently, the cells of G0/G1 phaseincreased obviously(P<0.01), the cells of S phase decreased obviously(P<0.05).the pcDNA3+-p16 plasmids transfection + dose irradiation in the radiationgroup compare with control group and control group+ dose irradiation in theradiation group, all appearance G1 phase arrest evidently, the cells of G0/G1 phaseincreased obviously(P<0.05 or P<0.01). The pcDNA3+-p16 plasmids transfection + dose irradiation in the radiation group compare with the simplepcDNA3+-p16 plasmids transfection group , the cells of S phase decreasedobviously (P<0.05 or P<0.01),the cells of G2/M phase increased obviously (P<0.05or P<0.01).6. The expression of p16mRNA is detected by RT-PCR.Agarose gel electrophoresis shows that:The pcDNA3+-p16 plasmids inthe transfection group and the pcDNA3 +-p16 plasmids transfection + differentdoses of irradiation in the radiation group all amplify 478bp band. And with theincrease of dose irradiation, gray level of band is enhanced. Gray level in theradiation group is stronger than in the pure pcDNA3 +-p16 plasmidstransfection group(P<0.05或P<0.01). There is no appearance of band in thepcDNA3+ plasmids transfection group and control group.7. Western blot detection for p16 protein expressThe outcome manifest that the SWWC cell transfered plasmid ofpcDNA3+-p16 after 1Gy, 3Gy and 5Gy raying, ma y be obviously expressingmore p16 protein than the cell pcDNA3+-p16 plasmids transfection withoutraying(P<0.05或P<0.01). And with the increase of irradiation dose, gray levelof protein band in radiation group decrease.8. By different dose radiation, OD value of the pcDNA3+-p16 plasmids cells inthe transfection group is much lower than that of the control group and thepcDNA3+ plasmids cells in the transfection group. According to OD value, theauthor calculates the inhibition ratio of ray on cells. The inhibition ratio of thepcDNA3+-p16 plasmid transfection group after different doses of irradiation ishigher than that of in control group and pcDNA3 + plasmids transfection group.And with the increase of radiation dose, the inhibition ratio is enhanced. The inhibition ratio of the pcDNA3+-p16 plasmids transfected after different dosesof irradiation are more than 30%.Conclusion:1. There is a depletion of p16 gene with a high frequency in colorectalcancer tissues. The depletion rate is related to the malignant de gree and clinicalstage of a tumor.2. Exogenous p16 gene could inhibit colorectal cancer cell cloning in G 1phase and induce apoptosis, only by transferring exogenous p 16 gene intocolorectal cancer cell lines.3. The introduction of Exogenous p 16 gene into colorectal cancer cellscan enhance the radio sensitivity and inhibition ratio,the inhibition ratioenhances with the increase of radiation dose.4. The mechanism of enhance the radio sensitivity by exogenous p16gene transfection into colorectal cancer cells is: exogenous p16 genetransfection into make the cell of G1 phase arrest, and made G1 phase cell adratof fairly sensisitive to radiation increased. Radiation induce express increasedof p16 gene, and the p16 gene induce arrest increased of cell cycle, andenhance the sensitivity of the colorectal cancer cells to radiation.