The Expression And Localization Of XIAP And XAF1 In Human Normal Oral Keratinocytes And Tca8113 Cells

Nowadays most researchers propose that polycellular organismsmaintain homeostasis by the modulation of cell proliferation andapoptosis. If the balance of cell proliferation and apoptosis was lost,the homeostasis would be broken down and some diseases wouldoccur, such as tumor, neural degeneration and autoimmune disease. Sothe relation between apoptosis and tumor increasingly absorbs moreattention these years, especially in the field about the unbalancebetween pro-apoptotic factors and anti-apoptotic factors. The inhibitorof apoptosis proteins (IAPs) are a family of intracellular anti-apoptoticproteins with homologous structure and extensive localization in a lotof species. IAPs play important role in restriction of cell apoptosis andhave greater diversity of suppression than any other family ofapoptotic inhibitors including the bcl-2 family. IAP family currentlyincludes XIAP, HIAP-1, HIAP-2, survivin, livin, neuronal apoptosisinhibitory protein (NIAP) .In 1996,XIAP, HIAP-1 and HIAP-2 was firstly identified byRajcan-Separovic et al. Subsequent studies found XIAP have variouseffect on tumorigenesis, development and prognosis of tumor.Scholarsdiscovered small molecule XIAP inhibitors and antisenseoligonucleotides directed against XIAP would induce apoptosis ofnumerous tumor cell lines and had little cytotoxicity to normal cell.These studies are currently being evaluated in preclinical or clinicaltrials.The advance is so heart-stirring that mary scholars have paidgreat attention to this field.XIAP gene localizes in X chromatosome q25 and XIAP mRNAhas 1.5 kb coding region. The relative molecular weight of XIAP is57kD and its molecular structure is studied clearer than that of anyother member. The N-end of XIAP consists of three BaculoviralInhibitor of apoptosis Repeats (BIR), which are the essential domainsfor inhibitory action of apoptosis. The C-end of XIAP consists of aRING zinc fingers domain,which functions as ubiquitin ligase E3 andcatalyzes self or interactive proteins through ubiquitin proteasomepathway. XIAP generally expresses in most tissue of adults andinfants except for peripheral blood lymphocytes. Some experimentalresults have indicated that XIAP has subcellular localization ofperinuclear areas and cytoplasm.Caspases are a series of critical proteases regulating and executingcell apoptosis process,which initiate apoptosis signaling cascade, splitconstitutive proteins and regulatory proteins and result in cellapoptosis. Studies have shown that XIAP is most potent inhibitor ofcaspase in the members of IAP family. XIAP could bind to and inhibitcaspase-3, caspase-7 and caspase-9 depending on BIRs and catalyzecaspases degradation depending on RING zinc fingers. So XIAPcould effectively resist apoptosis. XIAP could protect impaired neuronand prevent retrogression of motoneuron.In addition, XIAP could alsopotentialize postinflammatory reparation effect of endothelial cells.Experiments have indicated XIAP abnormally expresses inmultitude neoplasmes, such as prostatic carcinoma, mammarycarcinoma, pulmonary carcinoma, esophageal carcinoma, pancreaticcarcinoma, melanoma, ameloblastoma and recurrent respiratorypapilloma etc. A finding from National cancer institute(NCI)of U.S.Ashowed that XIAP overexpressed in most of 60 carcinoma cell lines.In a study of XIAP expression involving 34 case non-small cell lungcancinoma(NSCLC),researchers found both mRNA and protein levelsignificantly increased in NSCLC tissues and concluded that XIAPmay contribute to the development of NSCLC. Krajewska et al. foundthat XIAP expression increases in prostatic carcinoma of human andmouse. Compared with normal prostatic epithelium, increased XIAPexpression was evident in prostatic intraepithelial neoplasia lesions(carcinoma in situ), suggesting that upregulation of XIAP expressionoccurs generally and early in the pathogenesis of prostate cancer. Theabnormal expression of XIAP in benign and malignant ameloblastomasuggests that XIAP contributes to the biological properties ofepithelial odontogenic neoplasms, such as cell survival, proliferation,differentiation and may be involved in tissue structuring of neoplasms.In 2000, Fong and Liston et al. found an interactive protein ofXIAP by yeast two-hybrid systemaction. Gene mapping of XAF1 is atchromatosome 17 p13.2 . The relative molecular weight of XAF1 is34 kD. The protein molecule is composed of 317 amino acid residues,of which 178 residues at N-end constitute zinc finger domain. XAF1extensively expresses in almost all normal tissues of adulta andembryos. XAF1 protein localizes in cellular nucleus and acts aspro-apoptotic factor by binding to XIAP and antagonizing theanti-caspase activity of XIAP. Overexpression of XAF1 protein caneffect a marked relocalization of XIAP from the cytoplasm to thenucleus. Recent finding shows that XAF1 can activatemitochondrial apoptotic pathway and cooperate with TNFα in theinduction of apoptosis, independent of the interaction with XIAP.XAF1 mRNA is distinctly underexpressed in most tumor tissues orcancer cell lines,such as colon carcinoma, gastric carcinoma, renalcarcinoma, prostatic carcinoma, pulmonary carcinoma, melanoma.Some results have revealed that aberrant promoter hypermethylationis primary mechanism of XAF1 mRNA downregulation.Fong et al.carried out a study with NCI-60 cell lines panel and found prevalentallele loss of XAF1 gene in cancer cell lines.Moreover theseresearchers proposed that the level of XIAP mRNA expression wasmore higher than that of XAF1 mRNA. After having finished asubcellular localization study, Ng determined that XAF1 localized innucleus of melanocytes and localized diffusely in cytoplasm andnucleus of melanoma cell.Oral squamous cell carcinoma (OSCC) is most common oralcancer,which is a serious threat to human health with a very highincidence and a low survival rate. Carcinogenesis of oral mucosa is acomplicated multiple stage involving multiple factors. Up to now,literatures have not been reported about the study of expression andsubcellular localization of XIAP and XAF1 in OSCC.The human Normal Oral Keratinocytes (hNOKs) and malignant(human tongue squamous carcinoma cell line Tca8113) keratinocyteswere cultured in vitro. Expression and subcellular localization ofXIAP and XAF1 protein were analyzed by combination of indirectimmunofluorescence and confocal laser scanning microscopy.Theeffect of XIAP and XAF1 on cancerization of oral mucosa wasexplored and new insight was provided for early diagnosis andtargeted therapy of OSCC.These future studies concerning theexpression of XIAP and XAF1 in cancer will provide insight into thesignificance of apoptotic inhibition pathways in tumorigenesis.Results : XIAP expression was weak in the hNOKs andfluorescence staining localized chiefly in the cytoplasm andperinuclear areas. In the Tca8113 cells, high level of XIAP proteincould be detected in both the cytoplasm and the nucleus, with majorityof XIAP packed in the nucleus. In the hNOKs, XAF1 distributedmostly in the nucleus, especially dense in the nuclear envelope and thenucleolus. Homogeneous nuclear and cytoplasmic distribution ofXAF1 could be visualized in the Tca8113 cells.No obvious differenceof XAF1 staining intensity was found between the hNOKs andTca8113 cells. In a few of isolated hNOKs, XIAP localized mainly inthe nucleus but underexpression in the nucleoli, while XAF1 localizedin the whole nucleus with strong positive staining. Besides, influentialfactors of cultural method of hNOKs were analysed: Bothbenzalkonium bromide and gentamicin sulfate were used forgermicidal treatment of the oral mucosa samples, then no microbialcontamination occurs in primary cultures. Under 4℃ , Completeseparation rate of epithelium of oral mucosa with 0.4% and 0.25%Dispase are 9.09% and 53.85%, respectively. The difference betweentwo groups is statistically significant(P<0.05).Conclusions : In cancerization of oral mucosa, XIAP protein couldplay important antiapoptotic role by overexpression and extensesubcellular localization, while level of XAF1 protein does not appearto be upregulated and XAF1 could not antagonize effectively the roleof XIAP. XIAP could become the new target for prophylaxis andtherapy of OSCC. When Benzalkonium bromide and gentamicinsulfate are used for germicidal treatment of the oral mucosa samples,microbial contamination of hNOKs culture in vitro will under potentcontrol. Separation of epithelium from underlying connective tissueswith 0.4% Dispase is more complete than that of 0.25% Dispase.