| Abstract |
The etiologic correlation between cervical cancer and human papillomavirus (HPV) infection is well established. The high-risk HPV types, express the oncoproteins E6 and E7, which inactivate the tumor suppressor proteins TP53 and retinoblastoma (RB1) respectively. Thus, HPV overcomes the cell cycle control as well as apoptosis. Furthermore, the tumor suppressors TP53 and RB1 regulate directly the transcriptional activity of RNA polymerase III (RNA pol III). The changes in RNA pol III activity impact on the cellular biosynthetic capacity. The aim of the present study was the analysis of RNA pol III transcription in cervical cancer cells, infected by high-risk types of HPV, with respect to cell cycle control, cellular activation and commitment for mitosis during the G1/S phase transition. Thirty-five cervical cancer biopsies, 26 high (HSIL) and 33 low-grade squamous intraepithelial lesions (LSIL), as well as 28 normal cervical samples, as controls, were used in this study. From these samples total DNA, RNA and proteins were extracted. All the samples were tested for the HPV DNA by PCR and HPV positive cases were further analyzed for HPV genotype. All the samples of the control group were found to be HPV DNA negative. Furthermore, the HPV integration status was also examined. The HPV integration leads to the abolishment of the viral E2 gene expression, which represses the E6 and E7 oncoprotein expression. Thus, HPV integration is accompanied by E6 and E7 upregulation when compared with the expression levels of episomal HPV cases. The transcriptional levels of RNA pol III and its regulation in high-risk HPV type positive cervical cancer cells were also examined. RT-PCR analysis was performed for primary transcripts of class III genes, that transcribed by RNA pol III, like tRNAs (tRNATyr, tRNAArg, tRNALeu), 5S rRNA, MRP RNA, a RNA molecule necessary for the replication of mitochondrial DNA and the splicing of large rRNAs, and 7SK RNA, which regulates a transcription elongation factor of RNA polymerase II. It was found that the expression levels of tRNAs and 5S rRNA were upregulated in cervical cancer biopsies compared to controls. In parallel, when HPV was integrated in the host genome the RNA pol III transcription was even stronger. The 7SK and MRP transcripts presented different profiles of expression possibly as result of the distinct promoters utilized for their transcription. Analysis of the transcriptional levels of the RNA pol III transcriptional factor TFIIIC2 subunits, TFIIICα, TFIIICβ, TFIIIC90, TFIIIC102 and TFIIIC63, revealed significant differences among samples. It seems that TFIIIC2 is not restricted factor for RNA pol III transcription in cervical cells and that HPV does not induce the expression of its subunits, while HPV integration does. Analysis of the expression levels of the RNA pol III transcription factor TFIIIB subunits, TBP, Brf1 and Bdp1, revealed that Brf1 expression correlates with disease, HPV presence and integration status, as well as RNA pol III transcription levels. The increased transcriptional levels of Brf1 do not correlate with TP53 and RB1 degradation by E6 and E7 respectively. These data suggest that Brf1 is restricted factor for RNA pol III transcription in uterine cervix. Because RNA pol III transcription is significantly elevated during the G1/S phase transition of the cell cycle, the response of Brf1 was examined in G1 and S phases in HeLa cells transfected with Brf1 after synchronization. It was found that Brf1 activates the transcription of tRNAs and 5S rRNA in G1 and 5S rRNA in S phase of the cell cycle. In conclusion, three complementary mechanisms may therefore allow high-risk HPV to stimulate production of tRNA and 5S rRNA: a) E6-mediated removal of p53; b) E7-mediated neutralization of RB; and c) induction of Brf1. The resultant increase in biosynthetic capacity may contribute to deregulated cell growth. For the further investigation of G1/S transition of the cell cycle in cervical cancer cells infected by high-risk HPV types, the expression levels of 24 genes were analyzed. The genes examined were major and regulatory genes of cell cycle such as cyclins (CCNs) and cyclin-dependent kinases (CDKs), CDKs inhibitors (CDKNs) of both families Cip/Kip and ΙΝΚ4, genes of the transcription factor family E2F, genes of the RB1 (pocket proteins) and TP53 families as well as genes, necessary for the S phase of the cell cycle, that regulated by E2F. Progression of the cell cycle from G1 to S phase depends on the presence of two holoenzymes, Cyclin D/Cdk4 or Cdk6, and Cyclin E/Cdk2. Regulation of this cell cycle checkpoint is based on the TP53 (TP53-MDM2-CDKN1A-CDKN2D) and RB1 (RB1-E2F-CCND1-CDK4-CDK6-CDKN2A) pathways. Inactivation of TP53 and pocket proteins, RB1, RBL1 and RBL2, by the viral oncoproteins of high-risk HPV types, E6 and E7, is a prerequisite for oncogenesis but is not sufficient to convert normal cells into an immortalized or malignant state. A number of secondary factors, are thought to influence the likelihood that a high-risk type HPV infection will persist and progress to cervical cancer, possibly though genetic and epigenetic alterations, activation of proto-oncogenes and loss of tumour suppressors as well as immunological responses. Squamous intraepithelial lesions (SIL) infected by high-risk HPV types and expressing E6 and E7 do not exhibit such a proliferative or invasive phenotype as tumours. Furthermore, cervical carcinoma develops infrequently even after infection with high-risk types of HPV, and it typically occurs years to decades after the initial infection. These observations suggest that other regulatory proteins of the G1/S checkpoint may act to compensate for aggressive growth. Five out of 24 genes tested, RBL2, E2F2, CDK6, CCNE1 and MYC, found to be upregulated in cervical cancer in 80%, 80%, 97%, 74% and 74% of cases respectively. Eight genes found to be upregulated in both cervical cancer and HSILs, E2F1 in 84% and 71%, E2F3 in 81% and 74%, E2F5 in 86% and 81%, CCND1 in 77% and 52%, CDK2 in 88% and 62%, CDKN1B in 86% and 77%, PCNA in 89% and 69%, as well as POLA in 62% and 52%. Only 5 out of 24 genes tested found to be in elevated levels in all three groups of cases, cervical cancer, HSIL and LSIL, when compared with controls, TP53 is elevated in 80% of tumours, 69% of HSILs and 64% of LSILs, E2F4 in 89%, 81% and 76%, CDKN1A in 82%, 73% and 70%, CDKN2A in 86%, 82% and 77% as well as DHFR in 75%, 70% and 62%, respectively. The MDM2 gene presents a unique profile with overexpression in 88% and 73% of HSILs and LSILs, respectively. The RBL1 found to be downregulated in 97%, 88% and 73% of tumours, HSILs and LSILs. TP73 presented lower levels in 60% of cervical cancers. However, TP73 isoforms containing exon 13 is significant increases in 73% and 69% of tumours and HSILs, while isoforms containing exon 2 found to be downregulated in 97% and 73% of tumours and HSILs respectively. Three genes, RB1, CDK4 and CDKN2D, did not exhibit any significant alteration in gene expression. Our data suggest that deregulation of G1/S phase transition in cervical carcinogenesis is a progressive process. Certain clusters of genes are activated very early in pre-cancerous SILs while others are activated later, during malignant transformation. The ability of this array of markers to identify disease status suggests that it could be used in clinical arena. Hierarchical clustering of gene expression was able to classify the cervical tissue samples according to disease status, with few discrepancies (6.6%). The examined genes were clustered in three groups of low, TP73 exon 2, RBL1 and TP73; moderate, POLA, CCND1, CDK2, E2F4, CDK6, MDM2, B2M, CDKN2D, MYC, CDKN1A, RBL2, E2F2, TP73 exon 13, CCNE1, and CDKN2A; and high, CDK4, E2F3, E2F5, E2F1, RB1 PCNA, CDKN1B, TP53 and DHFR, expression. These data suggests a possible diagnostic importance.
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Μελέτη του ενζύμου της RNA πολυμέρασης III σε δείγματα καρκίνου του τραχήλου της μήτρας που έχουν μολυνθεί από τον ιο του θηλώματος του ανθρώπου (HPV)
