Investigation Into The Expression Of P33/ING1 In NOK, DOK And Tca8113 Cells

In recent years, the gene has become a hot spot of the life science research.Tumor has been generally acknowledged as a kind of genopathy ,which is thegenopathy of oncogene activation and anti-oncogene inactivation. Andanti-oncogene may be the capital protective mechanism to resist tumor. Oralsquamous cell carcinoma (OSCC) is one of the most common oralcancers,which is a serious threat to human health with a very high morbidity anda low viability. Carcinogenesis must be understood in terms of accumulation ofmutation in regulatory genes, including activation of oncogenes and inactivationor loss of tumor suppressor genes. As a result, gene diagnosis and gene therapyhave been paid more attention day by day.ING1( the inhibitor of growth 1) gene , a new candidate tumor suppressorgene found in 1996, includes three exons and two introns, that Garkavtsev deploycDNA differential hybridization and combine in vivo selection technique. It ismapped at human chromosome 13q32～34,which occurs higher LOH(loss ofheterozygous) on OSCC. ING1 mRNA can form three differentspliceosomes at least, and code three proteinum, which are p33,p47 andp24.Study has shown that p33 and p47 express higher,while p24 expresses few.Now study on p33 is more, but study on p47 is few, which can't beidentified. Early study has shown that p33/ ING1 is the production ofING1 ,which locates in cellular nucleus.p33/ING1 plays an important role on theaspects of cell proliferation, apolexis and apoptosis control. When theexpression of p33/ING1 decreases cell growth can be speeded up, on thecontrary,cell growth can be suppressed and apoptosis be promoted.It's reportedthat afunction of p33/ING1 has carcinogenesis by reducing apoptosis .Manytumors have been found to have abnormal expression of p33/ING1 gene. Studieson primary gastric cancer, hematological system tumor , breast cancer,neuroglioma and squamous cell carcinoma of head and neck have shown thatp33/ING1 olig-expression must have intimate relation with them, whoseolig-expression includes gene mutation, rearrangement, deletion and loss ofexpression. OSCC is mainly close to p33/ING1 ,so the study chiefly concernswith p33/ING1.Vitro experiment has shown that either p33/ING1 or p53 inhibited canpromote cell transformation and soft agar cell colony to form . Neurogliomaapoptosis is in result of synergistic action of p33/ING1 and p53. Study hasalso shown that p33/ING1 hyper-expression can suppress cell growth andenhance apoptosis, which relate with nuclear translocation of p53 and wild typep53.Both p33/ING1 and p53 induce p21/WAF1 expression, accordingly, makecells to stagnate at G0/G1.p33/ING1 is found regulating during resting cellkaryodieresis and achieve peak during synthetic phase.Studies on some humantumor cloned strains and tumor tissue have shown that p33/ING1 mRNAexpression is lower in cancer than in according normal tissue and latero-cancertissue.And there are missense and silence mutation in gastric cancer and breastcancer, that there are deletions at 3' in fibroblastoma.It has been shown thatp33/ING1 can enhance therapeutic effect of p53 gene therapy.Recently,it hasshown that p33/ING1 is concerned with cell growth, apolexis, anchoragedependence and apoptosis. In the past ten years, we find that p53 plays animportant role on these aspects, therefore associativity of p33/ING1 and p53 hasbecome a hot spot daily. p33/ING1 also associates with mdm2 , p21/WAF1,p73 ,p63 and bcl22,etc..It's discovered that there is p33/ING1 down regulationor mutation in OSCC.Currently, studies on oral mucosa precarcinomatous process relying ongolden hamster have achieved a certain progress ,but still have shortage, suchas long experiment period, high expenses and the racial difference between theperson and the animal and so on. To discuss the expression of p33/ING1 in thecarcinomatous process of oral mucosa epithelia,we established a stable symtemof culturing human normal and abnormal oral keratinocytes, and appliedimmunofluorescence to detect p33/ING1 expression in NOK,DOK andTca8113,therefore p33/ING1 expression can be systematically observed,whichprovided theorial base. In this study ,the key issue and nodus was how toestablish a system of culturing human oral keratinocytes. To avoid the interact ofblood serum, we applied keratinocyte serum-free medium. There is a powerfulclinical meaning that p33/ING1 expressed in the system of culturing human oralkeratinocytes and we analyses the role of p33/ING1 expression in thecarcinomatous process of oral mucosa epithelia ,which hasn't been reportedhome and abroad.In experiment one ,we made use of monoclonal broad-spectrumkeratinose antibody . The dyeing results of DOK and NOK were the same ,which explained that experiment cells had keratinocyte specificity protein andcertainly origin from epithelium keratinocyte. From figure 5 , the cell growthcurve looked like"S" .Cell number decreased in the first day after passage , thenentered logarithmic growth phase in a few delitescence. Cell growth becamesteady after entering platform stage and began apolexis gradually. The growthvelocity and double time of DOK were between normal cell and malignant cell,while closed to normal cell .It proved that reproductive activity of DOK wasstronger than NOK , but the difference wasn't significant.From immunofluorescence of experiment two, it was shown that p33/ING1expression mainly located at cell nucleus and cell membrane ,seldom atendochylema . p33/ING1 protein expression had a significant difference inNOK , DOK and OSCC,which has statistical significance (P<0.05).In conclusion,loss of p33/ING1 protein expression occurs increasinglycommonly during the genesis and development of OSCC . The incidence ofpositive expression of p33/ING1 protein is higher in normal oral epitheliumcell ,and negative expression of p33/ING1 protein is higher in OSCC. Sop33/ING1 may play an important role in gene diagnosis and gene therapy of oralcarcinoma.