The Relationship Between Mutation And Protein Expression Of ING1 Gene With Protein Expression Of P53,CyclinE In Lung Cancer

The human INGl (inhibitor of growth 1, INGl) gene, located at chromosome 13q33—34, is a tumor suppressor gene. There are always genomic alteration around this site in stomach carcinoma, tumor of blood system and squamous cell carcinoma of the head and neck. This gene contains four exons, named la, lb,lc and 2, and three introns. This gene plays an important role in restriction of cell growth and proliferation, apoptosis, tumor anchorage independent growth, cellular senescence, DNA repair and chemosensitivity. Due to alternative splicing of its mRNA product, the INGl gene encodes four isoforms: p47^ING1a, p33^ING1b, p24^ING1c and p27^ING1d . The most widely expressed of these protein isforms appears to be p33^ING1b which composed of exon la and exon2. All known isoforms of human INGl have an identical region encoded by exon2. They also share a highly conserved carboxy-terminal plant homeodomain (PHD) (a C4HC3 zinc finger motif implicated in chromatin remodelling). Some study on human tumors show: the abnormal expressions of human INGl gene are mutation, rearrangement, deletion and decreased expression,etc. Its low-expression correlate significantly with the genesis and progression of the tumors. Forced overexpression of the ING1 gene couldleads to cell arrest in the Gi phase of the cell cycle and induces apoptosis in several cell types. Conversely, inhibition of INGl expression by antisense constructs could promotes the transformation of mouse breast epithelial cells and increase the frequency of focus formation with NIH3T3 cells and protect cells form apoptosis. It is perhaps the p53 gene, among the limited tumour suppressor genes, that has the closest relation to the tumor and is most popularly studied. The p53 gene is mutated in more than 50% of human cancers. So far, it is regards that there is an interrelation between p33INGlb protein and p53 protein. But there are also reports that there isn't interrelation between them in some respects. Realising the interrelation between p33INGlb and p53 plays an important role in understanding the function of INGl gene. The research has indicated that the expression of p33fNGlb is changing accordingly with the progression of cell cycle.The level of p33INGlbwas found to decrease as cells exited from Go to Gi,to increase during late Gi,and to reach a maximum in S phase.This was followed by a decrease in G2. It shows that p33INGib -s mvojve(j m ceii CyCje and growth as a potential cell cycle related protein. It is reported that p33INGlb can arrested cells in the Go / Gi phase of the cell cycle. While, CyclinE is the key cyclin in the regulation of Gi phase and the transformation of Gj/S phase. The expansion or overdue expression of the Cyclin E gene would leads to the abnormal duplicate of DNA and the maladjustment of the cell cycle, which has a intimate relation with the occurrence and development of the tumour.The tissue chip technology is a newly developing high-efficiency technology that may test several hundred, even one thousand samples simultaneously. The chips in little dimension but with high content of information can be assembled and designed in accordance with the different requirement, it also has the advantages such as time-saving, energy-saving and budget-saving as well as the strong comparability of the testing results, the low test error and the good flexibility in designing, etc. For the time being, however, the Manual Tissue Puncher costs too much and is protected by its patent right, which put the limitation in some degree to its application and development and thus it is difficult to be popularised. The tissue chips made by us manually has achieved the comparatively satisfactory results.Little is known about INGl gene's role and changes in lung cancer. In order toexplore the role of ING 1 gene's mutation on the genesis and progression of lung cancer and the relationship among the protein expression of p53 > Cyclin E and p33 .At the same time, we are also in the hope of exploring the feasibility of the manual assembling for the tissue chips in order to provide the new technology mode for the clinical pathology and, scientific, research, We used PCR-nondenaturing polyacrylamide gel electrophoresis-silver staining method, immunohistochemistry staining method and tissue chip to show the expression status of p33INGlb , p53 protein and Cyclin E protein in tumor tissues. Materials and methods:(1) Materials:Fifty-one tumor tissues were obtained from fifty-one patients with lung cancer undergoing surgical resection as primary therapy for their disease after informed consent was obtained. Histological studies were performed and all tumors were confirmed as lung cancer by two pathologists from the people' hospital of Henan province.Three types of tumor were involved namely , squamous cell carcinoma, adenocarcinoma, small cell carcinomas.The number of cases were 30, 13 and 8, respectively. Fifty-one matched normal tissues were corresponding patients' normal lung tissues (The material is sampled from the spot more than 5 cm away from the edge of the cancer tissue) .Histological studies were performed that there is no cancer tissue. Among these patients whose age ranged from 29 to 78 years,29 were males and 22 were females,and the average age was 58.4+10.5 years.All tissues were stored at -70°C after surgery for molecular analysis. The samples are obtained from the self-developed lung cancer tissue that is surgically excised in People's Hospital of Henan province in 2004.(2) Using PCR-nondenaturing polyacrylamide gel electrophoresis-silver staining method, We detected the exonla and exon2 mutation of ING 1 gene.(3) Immunohistochemistry staining method and tissue chip were used to show the expression status of pSS^0111 protein, p53 protein and CyclinE protein in 51 tumor tissues and normal lung tissues.(4) Statistics process used the SPSS 10.0 version software bag, the compartion of the sample rates used the group rates %2 test. Continuity of correction for the x2 test and therank test; while the analysis for relativity adopts the test of Pearson, check the divided data of experiment used the a=0.05 check standard. Results:(1) In SSCP, No mutation was found in exonla of ING1 gene, while abnormal single strands were found in exon2 in two cases (3.9%, 2/51) .(2)Normal lung tissues showed expression of P33INGlb m the nuclei of bronchiole epithelial cells. Reduced or in some instances complete loss of nuclear pSS11"10"5 expression, in addition to a cellular compartmental shift from the nucleus to the cytoplasm was observed in lung cancer,(3)The expression of pSS?0115 showed correlation with differentiation of cancer, metastasis of lymph node negatively, but not with the tissue type, age, sex, size of cancer. (4) There are relativity between the expressions of p53 and CyclinE protein and that of p33INGIb protein, and the reverse relativity is presented between p33INGIb and Cyclin E protein, (r = - 0.23, P<0.05)(5)The tissue chips made with our hands have thirty-six sites. It can be add the number of site according to your needs. The success ratio of the sites are 100.0%, which also presents 100.0% in its coincidence rate with that of original designing and has no site in shift or overlap, and the observation ratio in morphology is 100.0%, although the immunohistochemistry staining has been repaired by the HTHP tissue, there is no site desquamating, and the success ratio of the chromosome sites of the immunohistochemistry staining is 100.0%. In addition, the experiment has been completed with only six chips, which greatly saves the study budget and the workload. Conclusions:(l)There were mutations of ING 1 gene in exon2, which could probably interfere with the structure of individual ING1 isoforms and the functions of the PHD zinc finger motif. Gene mutation was not the main reason that ING1 loses its tumor suppressing function in lung cancer.(2)The reduction of p33INGlb expression and perhaps translocation from the nucleus to the cytoplasm may play a role in the development and progression of lung cancer. (3)The expression of pSS^0"3 protein in lung cancer was negatively correlated with that ofCyclinE protein, but related to the expression of p53 protein .(4) pSS?0113 protein's expression was related to the tumor differentiation, metastasis oflymph and can be as a useful indicator of biological behaviour for lung cancer.(5)Without special instruments such as manual tissue puncher and paraffin section aidtape transfer system(PSA),manual operations can also make high-throughput,high-density tissue microarrays.lt not only reduces the technological difficulty of tissuemicroarray making,but also preserve their quality.This method is simple and convenient,economical and easy to spread.