The Expression Of Polι And PolιS5 In Transitional Cell Carcinoma And The Difference Of Their Function In Replication

Background and objective:The dangerous effects of transitional cell carcinoma such as smoking, chemical industry(aminobenzene), medicine(cyclophosphamide, phenacetin) are all DNA damage agents. Although DNA lesions can be removed by nucleotide excision repair and base excision repair processes, many lesions escape repair and present a block to continued transcriptional elongation by RNA polymerases and to replication by DNA polymerases. Human DNA polymeraseι(Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity. Due to the mismatch of the nucleotide, it suggests us that Polιprobably easily lead to genetic mutation. One of the hallmarks of cancer cells is genetic instability. In addition to large chromosomal changes involving thousands of bp, another form of genetic instability results in an increased mutation rate at the nucleotide level due to a perturbation in nucleotide synthesis or in cellular processes such as DNA repair and replication。So we think that the occurrence and development of transitional cell carcinoma maybe associate with the DNA translesion replication of Polιwhich is a mismatch repair polymerase different from other translesion synthesis DNA polymerase. Many studys have showed us Polιprobably easily lead to genetic mutation. Hypoxia-inducible factor-1-mediated Polιgene expression may be involved in the generation of translesion mutations during DNA replication. The Polιactivity was observed in seven out of eight malignant tumors, while it was absent in the normal uveal tract cells of the same patients. These findings serve as an additional confirmation of the Polιoncogenic potential.Yang J, et al found a Polι's homologous isomer which is shorted of the fifth exon of Polιcompaired with Polι, and named it PolιS5, then, they cloned the Polιand PolιS5 gene and build their eukaryotic expression vector, but didn't study the specific function of PolιS5. Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan organisms. Different splice variants of a given protein can display different and even antagonistic biological functions. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, the specific roles of such variants in normal and disease physiology, and how alternative splicing is coordinated on a global level to achieve cell- and tissue-specific functions.We also detected the expression of PolιS5 in BIU87 and T24, so it is very important to study the function of PolιS5 futher and discuss the significance of the expression of Polιand PolιS5 in transitional cell carcinoma.Methods:1,Expression of Polιand PolιS5 in cell lines of transitional cell carcinomaCurrently, there are no reports describing the expression of Polιin cell lines of transitional cell carcinoma. To develop a more accurate picture of the abundance of Polιexpressed in these cells, we analyzed a series of transitional cell carcinoma cell lines. The cell lines of transitional cell carcinoma used in this study include T24 and BIU87. The clinical tissue samples, bladder normal membrana mucosa, as the control group, were obtained from the Institute of Urological Research of the Third Military Medical University. Expression of Polιand PolιS5 in cell lines of transitional cell carcinoma were detected by RT-PCR .2,The identify of pEGFP-C1-Polιand pEGFP-C1-PolιS5pEGFP-C1,pEGFP-C1-Polι,pEGFP-C1-PolιS5 were presented by Dr. Yang Jin. Firstly, pEGFP-C1,pEGFP-C1-Polι,pEGFP-C1-PolιS5 were transformed into Escherichia coli DH5αand were mini-extracted, then they were sequenced and identified by Blat (www.genome.ucsc.edu).3,The diference of function between Polιand PolιS5In order to improve our understanding of the biological function of Polιand PolιS5, we have analysed its cellular localization. The cDNA encoding enhanced green fluorescent protein(EGFP) was fused in-frame to the N-terminus of Polι(EGFP-C1-Polι) and PolιS5 (EGFP-C1-PolιS5), and then pEGFP-C1 vector, pEGFP-C1-Polιand pEGFP-C1-PolιS5 were transfected into HEK293 cell respectively. Due to the nuclear localization signal consensus motif in its amino acid sequence, EGFP were localized within the nucleus and appeared homogeneously distributed. The distribution of EGFP-Polιand EGFP-PolιS5 in the nuclear localization were detected by by Laser scanning confocal microscope. We irradiated the transfected cells with UVC(15 J/m2) and observed change of distribution of EGFP-C1-Polιand EGFP-C1-PolιS5 in the nuclear localization 12 h later.4,Expression of Polιand PolιS5 in tissue of transitional cell carcinoma.The clinical tissue samples were obtained from the Institute of Urological Research of the Third Military Medical University, including bladder normal membrana mucosa(15), bladder tumor(28), renal pelvic carcinoma (11),which include transitional cell carcinoma grade I (13), grade II (16) and grade III (10). Expression of Polιand PolιS5 were detected by RT-PCR .Results:1,Expression of Polιand PolιS5 in cell lines of transitional cell carcinomaIt was observed that the expression of Polιappears to be low in bladder normal membrana mucosa but significantly elevated in transitional cell carcinoma cell lines(P<0.01). The expression of PolιS5 was also detected in BIU87 and T24, but not in bladder normal membrana mucosa, and the expression of Polιwas much higher than that of PolιS5 in BIU87 and T24(P<0.01). The expression of Polιwas significantly elevated in transitional cell carcinoma cell lines and the over expression of Polιin cell probably lead to high genetic mutation when DNA was damaged. The function of PolιS5 will be still studied.2,The identify of pEGFP-C1-Polιand pEGFP-C1-PolιS5pEGFP-C1,pEGFP-C1-Polι,pEGFP-C1-PolιS5 were transformed into Escherichia coli DH5αand were mini-extracted, then they were sequenced and identified by Blat (www.genome.ucsc.edu).It was determined that the cDNA of PolιS5 in pEGFP-C1-PolιS5 is shorted of the fifth exon of Polιcompaired with the cDNA of Polιin pEGFP-C1-Polι. The next experience will be based on the result.3,The diference of function between Polιand PolιS5EGFP were localized within the nucleus and appeared homogeneously distributed. The EGFP-C1-Polιand EGFP-C1-PolιS5 were predominantly localized within the nucleus and appeared homogeneously distributed. In 2% of EGFP-C1-Polιtransfected cells, and in 4% of EGFP-C1-PolιS5 transfected cells, EGFP- C1-Polιand EGFP-C1-PolιS5 were localized in many intranuclear foci. This localization pattern was strikingly similar to observations with pol eta .Consequently, we asked whether the distribution of EGFP-C1-Polιand EGFP-C1-PolιS5 changes after DNA damage. We irradiated the transfected cells with UVC(15 J/m2) and observed the pattern of EGFP-C1-Polιand EGFP-C1-PolιS5 12 h later by Laser scanning confocal microscope. Percentage of EGFP-C1-Polιtransfected cells and EGFP-C1-PolιS5 transfected cells increased to 6% and 20% respectively. It were suggested that PolιS5 probably has the similar function with Polιin translesion sythesis and PolιS5 may be more sensitive to DNA damage that lead by UVC than Polι, but the more specific function will still be studied.4,Expression of Polιand PolιS5 in tissue of transitional cell carcinoma.It was observed that the expression of Polιappears to be low in bladder normal membrana mucosa but significantly elevated in tissue of transitional cell carcinoma(P<0.01). The level of expression of Polιwas associated with the grade of the transitional cell carcinoma. The expression of PolιS5 was not detected in tissue of transitional cell carcinoma. It was suggested that the over expression of Polιin tissue of bladder tumor and renal pelvic carcinoma may be associated with the occurrence of transitional cell carcinoma and the heterogenicity of transitional cell carcinoma.Conclusion:1. The expression of PolιS5, Polι's splice isomer, was detected in BIU87 and T24;2. PolιS5 probably has the similar function with Polιin translesion sythesis and may be more sensitive to DNA damage that lead by UVC than Polιin function, but the more specific function will still be studied;3. The over expression of Polιin transitional cell carcinoma cell lines and tissue of bladder tumor and renal pelvic carcinoma may be associated with the occurrence of transitional cell carcinoma;4. The over expression of Polιin tissue of bladder tumor and renal pelvic carcinoma may be associated with the heterogenicity of transitional cell carcinoma;5. PolιS5 was not detected in clinical tissue of transitional cell carcinoma, but it could not exclude the expression of PolιS5 in other specific tissue.