Alterations Of The P14～(ARF) Gene In Non-Small Cell Lung Cancer And The Correlation Of Its Expression With P16～(INK4a), P53 Protein

IntroductionThe generation of lung cancer is a complication course with multi - factor and multi - step. Recently some study have suggested that carcinomatous change was closely relative to cell cycle and the regulation of cell cycle refered to onco-genes and tumor suppressor genes whose aberrance could lead to cell cycle uncontrolled. As a result, the cell proliferation becomes uncontrolled. It is also considered that tumors was a group of cell cycle diseases.There are two pathways now in cell cycle; p14ARF - p53 pathway and p16INK4a - RB pathway which play key roles in Gl - S and G2- M restriction point. p14ARF and p16INK4a is located on CDKN2A locu mapping to chromosome 9p21 in which there is different reading frames. Briefly, alternative splicing of the first exon permits to encode two different transcriptor. p14ARF and pl6INK4a share common exon 2 and exon 3 , but using alone exon 1 a and exon 1 B respectively which translate p14ARF and pl6INK4a protein respectively.The mice homoprotein p19ARF often is studied to attain characteristics of p14ARF. Kamijo knocked out exon Ij3 of INK4a/ARF in mice through a technique named gene knock - out without effecting expression of p16 INK4a. The ARF~/~ juice that were dealed with DMBA in early age developed tumors soon, but those ARF+/~ and ARF+/+ mice didn' t developed tumors at the same time. It showed that p19ARF was a true tumor suppressor gene.p14ARF and p16INK4a genes located at the pathways of the p53 and the p16INK4a respectively, acted as restrictive function in the regulation of the cell cycle. p16INK4a protein is a member of the family of cyclin - dependent protein kinase inhibitor , which could competitively bind with CDK4/6 , resulting in inacti-vation of CDK4/6 kinase and dephosphorylation of Rb , maintaining the functionof detaining transcription factor E2F1, discrupted cells proliferation at the Gl stage. p14ARF protein directly combined with MDM2 and inhibit its activation of mediating degradation of p53. p53 protein promotes transcription of p21cip1, leading to a break of the restriction pont between Gl and G2 stage.The recent studies indicated that the locus of INK4a/ARF was commonly observed showing deletion and methylation. In human tumors, aberration of p14 gene played a important role in development of tumors. At the same time, the frequent losses of p14ARF, p16INK4a and p53 protein in tumor tissues were closely associated with immortalization of proliferation cells and carcinogen-esis of tumors. Studies of the aspect are becoming hot topics.Although there had been comprehensive studies about anormal expression of p16INK4d and p53 protein recently, the studies about p14ARF have been rare and mainly focus on cultured cell line in vito so far. The reports of p14ARF gene in human solid tumor, specially in lung career, were seldom seen. There have not been reports about the correlation of the three proteins so far.We investigated the expression of p14ARF, p16INK4a, p53 protein and loss of heterozygosity and homozygosityof p14ARF gene and promoter methylation p14ARF gene in NSCLC through IHC, western blot, PCR -PAGE, PCR -SSCP, MSP, RE - PCR tests. We also discussed their implication between clinical and pathological.Materials and MethodsMaterials40 samples during July 2002 to December 2002 were collected in the Department of General Thoracic Surgery, The First Affiliated Clinic Hospital of China Medical University. There were 25 male, 15 female patients with an median age of 60.5. According to the pathological subtype , there were 20 cases of squamous carcinoma , 20 cases of adenocarcinoma. According to the differentiation degree , the cases were classified as low differentiation of 17 case s , middle differentiation of 14 cases and high differentiation of 9 cases. According to 1997 UICC staging criteria , 17 casas were classified as stage I , 10 cases asstage II and 13 cases as stage M- No casas were received rediotheropy before operation. Methods1. Immunohistochemisty (IHC)IHC tests were performed by SABC...