Telomerase Activity Expression In Oral Squamous Cell Carcinomas And Its Biological Regulation

ObjectiveThe morbidity of oral mucosa carcinoma is relatively high in the population. The objective evaluation of its malignancy would help to improve the diagnosis and survival rates. Lots of researches have been done about the facts that telom-erase could be detected in some tumor cells. The correlation between telomerase and malignant tumors remains controversial: for example, what kind of relationship between tumor cells and TLMA, the activation mechanism of TLMA gene in tumor cell progression, the relationship between the telomerase activity and clinical stage and pathological classification, the corralation between telomerase activity and cell cycle, the possibility of applying telomerase inhibitors as drug to treat tumor.By PCR-ELISA and immunohistochemistry SP methods ,the telomerase activity and PCNA of oral squamous cancer and by-side tissue and normal oral mucosa were quantified. The relationship between the telomerase activity and oral squamous cancer were discussed. The correlation of cyclinA and hTRT in oral squamous cancer different stage and pre-cancer alteration were analysed. After applying the antisense oligonucleotides to human-derived oral squamous cancer cell line Tca8113, the effects on the tumor cell growth and telomerase activity were observed. This study would provide scientific foundation for the clinical application of telomerase inhibitors.Methods1. The measurement of tissue sample telomerase activity TRAP-PCR-ELISEA method by telomerase TRAP-PCR protocol. The PCRproducts were processed , denatured , hybrided, through antigen-antibody reaction to test the absorption degree A by wave length 450 nm. A >0.20 was regarded as positive.2. PCNA immunohistochemical stainingAll samples were fixed by 10% formalin, paraffin embedded and cut into thin slice. Streptomacin biological protein ultra-oxygenase immuohistochemical technology were adapted. The test was done according to the protocol, the breast cancer as positive control and PBS to substitute the first antibody as the negative control.3. The detection of the expression of hTRT gene by in-situ hybridization The digoxin-labelled probe was targeted to reverse transcription domain.The routine hybirdization were done, oral squamous cancer as positive control and the negative control without probe.4. The measurement of cyclin A expression by immunohistochemical methods Immunohistochemical SP method were adapted , PBS to substitute the first antibody as the negative control , the cancer sample as the positive control . The number of the positive cells with yellow stains ≥5% as " + ".5. The application of antisense oligonucleotides to Tca8113 cells.The test samples were classified into three groups: antisense oligonucleotides group, sense oligonucleotides group and control group in 96 well-culture plates. Each palates contained 2 × 10~5 cell, oligonucleotides concentration were 0, 1.25, 2.5, 5.0, 10.0, 20.0μmol/L, respectively. The control group contained only culture fluids. The observation time were 24, 48, 72 hours respectively.6. Cell toxicity testsThe toxicity of AS_ONS to Tca8113 cell was done by MTT method. The AS _ONS concentration were 1.25, 2.5, 5.0, 10.0, 20. 0μmol/L, respectively.The medium without serum and same volume served as control. The OD value was measured at the wave length 570 nm.7. The measurement of Tca8113 cell telomerase activityThe 5. 0μmol/L antisense oligonucleotides group , sense oligonucleotides group and control group cell telomerase activity were measured at wave length 490nm OD level according to the protocol .8. The structural observation of Tca8113The up-side-down microscope was used to observe the cell growth and structural changes two days after the antisense and sense oligonucleotides treatment.Results1. The telomerase activity expression of oral mucose squamous cancer tissueThe telomerase activity positive rate 67.6% in 46 cases of telomerase activity among 68 cases of oral mucose squamous cancer tissue, while the positive rate is 8. 8% in by-stand tissue (3 cases out of 34 cases of by - stand cancer tissue ). No telomerase activity was observed in normal tissue. The difference were statistically significant. (X2 =31.421, P < 0.001).2. The relationship between oral mocuse squamous cancer the telomerase activity expression and the clinical pathological classification . There is a relationship between oral mocuse squamous cancer the telomerase activity expression and the clinical pathological classification, that is , the telomerase activity expression rate was gradually high with the pathological classification (X2 总 =39. 695, P < 0.001;Kendall correlation cofactorr, =0.680, P < 0. 001).3. The PCNA positve materials expression in cancer tissue and by-stand cancer tissueThe brown and dark brown PCNA positive materials were mainly localized in cell nuclear, in diffused or in granule way. The positive cell with normal configuration in by-stand cancer epiderm tissue were localized in basal cell layer and thin layer of the thorn. The PCNA positve cell with abnormal hyperplasia inthe by-stand epiderm were localized in the whole layer of the epiderm. Little PC-NA positive cell in normal mucous were found only in the epiderm basal layer or deep thorn layer.4. The relationship between the PCNA positive expression and the telomer-ase activityThe PCNA positive cells densities in 46 telomerase positive cases of cancer tissue and 3 cases of by-stand cancer with abnormal hyperplasia were 165.31 ± 1. 82/mm2, while PCNA positive cells densities in 22 telomerase negative cases of cancer tissue and 10 cases of by-stand cancer with abnormal hyperplasia were 96. 77 ± 1.74/mm2. the PCNA positive cell between the telomerase positive and negative groups was statistically significant( t = 147.55, P < 0.01).5. The expression of hTRT in samples of different groups(1) The hTRT mRNA was slightly expressed in normal mucous , mainly in basal layer. The expression was high in abnormal hyperplasia to thorn cell layer. The positive signal was found in pre-cancer tissue , localized in basal cell layer and thorn layer. The hTRT positive rate was 83.1% .mainly with strong signal. The hTRT mRNA positive rate was statistically significant among groups by x2 test.(2) The hTRT expression in oral squamous carcinomasThe well-differentiate squamous hTRT positive cells distributed inside the cancer activation regions, while poorly-differentiated cells diffused in the whole tumor tissues.The hTRT expression levels between different pathological classification had no statistical significance ( P > 0.05). This test could verify the parellel relationship between the hTRT mRNA expression and oral carcinomas clinical classification.6. The expression of cyclin A in samples of different groupsNo cyclin A positive epiderm cells were found in normal oral mucous and by-side carcinomas . Cyclin A positive rate is 55.4% in the 36 cases out of 65 cases of oral mucous carcinomas.7. The correlation between the hTRT and cyclin AhTRT and cyclin A had highly positive relationship through statistical analy-sis( γs =0.9433, P < 0.01).8. The impact of antisense oligonucleotides with different levels on the Tca8113 cellAntisense oligonucleotides can apparently inhibit the Tca8113 cell growth ( P < 0. 01). Its effects were dose-dependent. Antisense oligonucleotides effects were duration-dependent.9. Cell toxicity testsThe vary AS_ONS concentration were action to Tca8113 cell . The toxicity of AS_ONS was inferior.10. The effect of antisense oligonucleotides on the cell telomerase activity Antisense oligonucleotides can apparently inhibit the telomerase activity( P < 0.01) and have duration-dependent effects.11. Antisense oligonucleotides effects on the shape of Tca8113 cell.The Tca8113 cell treated with AS_ONS was growth inhibited observed under up-side-down microscope and electronic microscope.Conclusion1. The telomerase activity expression in the oral mucous squamous carcinomas tissues has positive relationship with its clinical pathological classification.2. The telomerase activity expression in the oral mucous squamous carcinomas tissues has close correlation with PCNA expression level.3. Telomerase has very close correlation with the formation of oral squamous carcinomas. Its activation is a very important step in the development of o-ral squamous carcinomas.4. The overexpression of cyclin A plays an important role in the formation of malignant tumors.5. hTRT mRNA and cyclin A have co-effect in the formation of oral squamous carcinomas. The overexpression of cyclin A induces the malfunction of cell cycle and then activates the telomerase to promote the formation of carcinomas.6. Antisense oligonucleotides can apparently inhibit the Tca8113 cell growth. The inhibition effects are dose-dependent and duration-dependent.