Effect On The Transcription Of P57～(kip2) In Lung Cancer Cell Lines By X-Irradiation

IntroductionGenomic imprinting is an epigenetic phenomenon, which, in most cases, is believed to occur in gametogenesis. Genomic imprinting occurs when both maternal and paternal alleles are present, but one allele will be expressed while the other remains inactive. As one of the imprinting genes and a cyclin — dependent kinase inhibitor silenced in a variety of human malignancies, p57 gene was discovered by Matsuoka et al in 1995. p57, belonging to CIP/KIP family, partly homologous with p21 and p27, located between D11s648 and D11s679 in chromosome 11 p15. 5, a region for about 2. 2kb, was a negative regulator for cell proliferation. Recent studies showed that the absent expression of p57 was associated with pathogenesis, differentiation and prognosis of some children's congenital tumors, especially in B - W syndrome, Wilms tumour and many adult tumors such as lung cancer, breast cancer, stomach and colorectal cancer etc. Thus p57 was regarded as a kind of tumor suppressor gene. Radiotherapy is one of the major methods for cancer treatment. The cancer cells experienced injury, repair and apoptosis after irradiation by X - ray and many factors took part in the whole process. Both whether p57 is one of these factors and what kind of role it plays still remains undiscovered. This study was designed to analyse the effect caused by X - irradiation on the expression of p57 mRNA and its clinical significance.Material and methodsMaterial1. The lung cancer cell lines A549 and NCI - H226 ( donated by Chengdu Di Ao Pharmaceutical Group Co. Ltd. ).2. RPMI 1640 cell culture medium ( GIBCO) 0. 25% trypsin and PBS buffer (TaKaRa Biotechnology Co. , Ltd, Dalian).3. Overdrive low temperature centrifuger ( Heraeus Biofuge PrimoR, America) .4. PCR Thermocycle Instrument (TaKaRa PCR Thermal Cycler)5. Electrophoresis apparatus (Type BIO -RAD PAC300, America)6. Electrophoresis horizontal chamber-. (Jiemai HM - I , Dalian)7. Gel imaging analytical system;(GIS -2020,Tanon Science & Technology Co. , Ltd, Shanghai).8. Linear accelerator (Varian 2300C/D, America).9. PCR primer (synthesized by TaKaRa Biotechnology Co. , Ltd, Dalian). RNAout;From Wuass Biotechnology (Dalian) Co. , Ltd, Shenzhen.10. RT - PCR kit and RNA polymerase ( From TaKaRa Biotechnology Co. , Ltd, Dalian).11. Polyacrylamide and agarose for electrophoresis ( Sangon Biotechnology Co. , Ltd, Dalian).Methods1. Cell culture: lung cancer cell lines A549 and NCIH 226 were cultivated under5%CO2,37tt ,15%GIBCO RPMI1640 culture medium.2. Irradiation method: logarithmic growing phase of NCI - H226and A549 were irradiated under 2, 4, 8, 12 Gy respectively by 6MV X -ray. Dose rate;200MU/min, SSD: 100cm.3. RNA extraction;NCI - H226 and A549 cell lines were incubated in cal-orstat before and after irradiation by different doses, then rinsed twice with PBS at 6, 12, 36, 48 hours after irradiation, and were scraped with cell scraper, counted 1 x 107 cells and added with 1 ml RNAout. All the equipment and reagent were dealed with DEPC water and autoclaving. Detailed RNA extraction was according to RNAout handbook. RNA extract was preserved under - 801.4. RT - PCR1). Reverse transcription to synthesize cDNA;reaction condition: 42 T!30min, 99t5min, 5Tl5min, cDNA product was preserved in -20X1.2). PCR amplification;PCR amplification was carried out in a final volume of 25|xl. The amplification conditions were as follows;an initial incubation at 94°C for 2 min, followed by 35 cycles, each at 94°C for 30s, at 55Ti for 30s and at 12X, for 45s, a final step at 72X1 was extended to 7min. The length of the PCR product is 310 bp.5. Quantitative analysis of mRNA;Agarose gel electrophoresis was performed and 8jjl1 each PCR product was run on electrophoreses at 120v for 40min (15% agarose gel, 0. 5 X TBE buffer) , then stained with ethidium bromide (EB) , finally scanned with gel imaging analytical system. OX174/Hinc II and GAPDH were served as molecular weight marker and quantitative standard marker respectively. The relative quantity of objective product;optical density of target gene/ optical density of standard marker.6. Statistical analysis;Quantitative data were expressed as mean SD. The quantitative difference between different groups was analyzed by SPSS version 13.0. P<0.05 was considered statistically significant.ResultThe level of p57 mRNA was very low before X - irradiation, and increased significantly after irradiation by different doses. In this study we found that the level of p57 mRNA in cell line A549 increased with the increased radiation dose and reached to the peak level at 24 hours after irradiation when irradiated by 2 -8 Gy. Though p57 mRNA levels of A549 cell line subjected by an irradiation dose of 12 Gy increased, yet was lower than that subjected by 2 - 8 Gy dose, and the peak time was prolonged to 36 hours. There was no obvious tendency found in the NCI - H226 cell line, but the level of p57 mRNA was increased in different radiation dose group with statistical significance.Discussionr>57 is regarded as a cancer sunnressor eene. We know that there is loss ofimprinting of p57 in human lung, breast, liver, stomach, pancreatic cancers and other malignances. As one of the CDK inhibitors, p57 makes Gl arrest and restrain the proliferation of cells by regulating the cell cycles through inhibiting the functions of the CDK components and proliferating cell nuclear antigen ( PC-NA).Sun yinping testified that irradiation alone or irradiation combined with Tax-ol induced the expression of the mRNA and protein of p57 in nasopharyngeal cancer cells. In our studies we also observed that the level of p57 mRNA increased significantly after X - irradiation. We analyzed the result and found out that the expression of p57 mRNA in lung cancer cells induced by X - irradiation may be related to the factors below.(1 ) The methylation status of the upper gene. As we know now that the transcription of p57 is regulated by its upper gene DMR - Litl, the methylation of which can down - regulate the expression of p57 , while the unmethylation can bring an increase. The methylation status of DNA can be interrupted by X - irradiation. H19, one of the imprinted genes, was found aberrant methylation after X —ray irradiation, which caused the cytosines at the CpG islands couldnt become methylated ones. So we think that the methylation status of DMR - Litl changed because of irradiation and induced the high expression of p57 mRNA.(2) The up -regulation of p73p after irradiation. p57 is proved to have a close relationship with p73p. Whether induced by MyoD, or over expression a-lone, p73(3can induce the p57 mRNA and protein expression and E2F1 Induces p57 in a p73 - Dependent Manner. Over - expression of p73beta resulted in apoptosis and an increase in the expression of p57 Ip . Recent studies showed that p73 could be induced by irradiation. In our studies there might be an up -regulation of p73 induced by X - irradiation that resulted in over - expression of p57.(3) Influence on TGF - p by X - irradiation. Studies showed that persistent , long - term, low - dose ionizing radiation exposure in humans induced the up - regulation of TGF - p, while p57 was the only CDKIs induced by TGF - p. And TGF - p — induced cell cycle arrest of human hematopoietic cells requires p57 up - regulation. But now we found no evidence to prove that TGF - pcanbe up -regulated in lung cell by X - irradiation.(4) Relationship with regulation of cell cycle. We know that radiation can result in repair of sublethal and potentially lethal damage, Gl arrest and apopto-sis, which causes redistribution of cell cycle. p57 is one of CDKIs and take part in cell apoptosis and cell cycle regulation. So we think that maybe irradiation can induce p57 directly.ConclusionX - irradiation up - regulate the level of p57 mRNA with a dose relationship, which implied that p57 took part in the course of cell cycle distribution and cell apoptosis induced by X - irradiation, and may be a predictor of radio-sensitivity.