[Identification regulatory noncoding RNAs of human papilloma virus type 16 (Papillomaviridae: Alphapapillomavirus: Human papillomavirus) in cervical tumors].

INTRODUCTION: High carcinogenic-risk human papillomaviruses (hrHPVs) are recognized as etiological agents of cervical cancer. Constant expression of the viral oncoproteins, E6 and E7, is required for maintenance of the malignant phenotype of tumor cells. The exact mechanism of regulation of viral oncogenes expression in tumor cells is not fully elucidated. THE PURPOSE: identification of viral noncoding RNAs (ncRNAs) in HPV16-positve cervical cancer. MATERIALS AND METHODS: The reverse transcription polymerase chain reactions were used to detect viral ncRNAs in HPV16-positve primary cervical squamous cell carcinomas and SiHa and CasKi cell lines. The knockdown technique with oligonucleotides complementary to ncRNAs was used to elucidate their functions. RESULTS: We have identified ncRNAs transcribed in the upstream regulatory region of HPV16 in the cervical carcinoma cell lines and in 32 out 32 cervical squamous cell carcinomas with episomal or integrated forms of HPV16 DNA. Knockdown of sense or antisense strains of ncRNAs by oligonucleotides results in a decrease or increase of the E6 and E7 oncogenes mRNA levels in cells, respectively. These changes of oncogenes mRNA levels are accompanied by the modulation of the levels of the p53 protein, the main target of the E6 oncoprotein. CONCLUSION: The presence of regulatory ncRNAs in all examined tumors and cell lines revealed for the first time indicates their necessity for maintenance of constant expression of E6 and E7 oncogenes in them. The findings can be useful for understanding of the fundamental aspects of the viral expression regulation in HPV16-positive tumors.

[Novel human DNA viruses and their putative associations with human diseases].

In this review, we described human small DNA viruses discovered on the cusp of the 20th and 21st centuries as a result of cutting-edge technologies established in molecular biology. The problems of obtaining an evidence of the etiological role of new viruses in human diseases have been considered.

Downregulation of genes encoding for subunits of adaptor complex-3 in cervical carcinomas.

We explored the expression of four genes encoding for subunits of AP-3 in cervical tumors and cancer cell lines. Using RT-PCR we demonstrated more than twofold decrease in the levels of mRNA of AP3D1, AP3B1, AP3M1, and AP3S1 in 32, 28, 23, and 26% tumors in comparison with normal tissues of uterine cervix, respectively. The level of mRNA of at least one subunit was decreased in 28 out of 47 (60%) of tumors and in four out of five cancer cell lines in comparison to tissues adjacent to tumors. The suppression of expression of any of the subunits was revealed in 15 out of 28 cases (54%). The expression of two and more subunits was decreased simultaneously in different combinations in 13 cases (46%). This fact testifies to the lack of a common mechanism of downregulation of four subunits in tumors. There is a tendency to more frequent suppression of AP-3A expression in tumors associated with lymphatic node metastases as compared with tumors without metastases (P = 0.034). Thus, here we demonstrate for the first time the decrease in expression of genes encoding for AP-3A subunits in tumors.

DNA demethylation and carcinogenesis.

DNA methylation plays an important role in the establishment and maintenance of the program of gene expression. Tumor cells are characterized by a paradoxical alteration of DNA methylation pattern: global DNA demethylation and local hypermethylation of certain genes. Hypermethylation and inactivation of tumor suppressor genes are well documented in tumors. The role of global genome demethylation in carcinogenesis is less studied. New data provide evidence for independence of DNA hypo- and hypermethylation processes in tumor cells. These processes alter expression of genes that have different functions in malignant transformation. Recent studies have demonstrated that global decrease in the level of DNA methylation is related to hypomethylation of repeated sequences, increase in genetic instability, hypomethylation and activation of certain genes that favor tumor growth, and increase in their metastatic and invasive potential. The recent data on the role of DNA demethylation in carcinogenesis are discussed in this review. The understanding of relationships between hypo- and hypermethylation in tumor cells is extremely important due to reversibility of DNA methylation and attempts to utilize for anti-tumor therapy the drugs that modify DNA methylation pattern.

DNA methylation and carcinogenesis.

In the world of easy things truth is opposed to lie; in the world of complicated things one profound truth is opposed to another not less profound than the first. Neils Bohr The hypothesis of the exclusively genetic origin of cancer ("cancer is a disease of genes, a tumor without any damage to the genome does not exist") dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes ("cancer is a disease of gene regulation and differentiation"). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors--genetic and epigenetic--lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an "epimutation", an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.

De novo methylation of selective CpG dinucleotide clusters in transformed cells mediated by an activated N-ras.

We have explored a possible role of an activated N-ras oncogene in aberrant methylation of CpG clusters in DNAs of transformed cells. Using three lines of hamster cells transformed by Rous sarcoma virus (RSV) and the method of detection of CpG islands as clustered sites for methylation-sensitive restriction enzymes we have demonstrated that in each cell line the transcribed RSV proviruses are integrated in the vicinity of sequence containing the cluster of unmethylated CpG dinucleotides. Two out of three examined CpG clusters had hypermethylation patterns in N-ras-neo- but not in neo-transfected variants of the cell lines. De novo methylation of CpG dinucleotides correlated with transcriptional inactivation of adjacent RSV proviruses that was related neither to the lack of transcriptional factors binding RSV long terminal repeat (LTR) nor to the transcriptional incompetence of the LTR, as measured by reporter gene assays with the LTR cloned from DNA of these cells. These data suggest that activation of N-ras signal transduction pathway in transformed cells may be relevant to long-term inactivation of selective genes by hypermethylation of their CpG islands.

HPV infection in cervical-cancer cases in Russia.

The presence of human papillomavirus (HPV) sequences in 21 biopsies from cervical carcinomas, 11 specimens of tissues adjacent to tumours, 2 specimens of cervical tissues with radiation fibrosis from patients after radiation therapy of cervical cancer and 7 normal epithelial tissues from the patients with other genital tumours were examined by polymerase chain reaction (PCR) and Southern-blot analysis. All tumours were HPV-positive by type-specific PCR and 86% by Southern-blot analysis. In normal epithelial and adjacent tissues, HPV sequences were detected in 20% of samples by Southern-blot analysis and in 70% of samples by PCR, including 2 cases of tissues after radiation therapy. HPV16 was the most prevalent type in tumours (18/21) as well as in normal epithelial tissues (5/7). One HPV-positive tumour contained HPV18 DNA and 2 were doubly infected with HPVs 16 and 18 (2/21). The persistence of exclusively episomal HPV16 DNA was observed in 5 out of 11 tumours examined: 3 cases of squamous-cell carcinomas on the early stage of tumour progression and 2 advanced tumours (squamous-cell carcinoma and adenocarcinoma). The integration of HPV16 genome was detected in 6 out of 11 tumours, but most of them contained episomal forms of viral DNA simultaneously (5 out of 6). The integrative HPV18 genome was found in 2 tumours examined, and the persistence of episomal forms was also observed in one of them. Our data demonstrate that cervical tumours are associated invariably with high-risk types of HPV in Russia.

Modulation of pp60v-src and pp60c-src expression in Rous sarcoma virus-transformed hamster fibroblasts transfected with activated N-ras.

Three phenotypically different hamster cell lines transformed with Rous sarcoma virus (RSV) were transfected with plasmid DNA containing an activated N-ras oncogene, and nine clones expressing various levels of p21N-ras were characterized. We examined the effects of p21N-ras on expression and kinase activity of resident src proteins by using a variety of assays that allowed us to discriminate between viral and cellular src proteins. In eight clones with a 10- to 20-fold increase in p21N-ras levels relative to the endogenous protein, we observed a marked reduction in the synthesis and kinase activity of p60v-src. This decrease correlated with transcriptional downregulation of RSV genomic and v-src subgenomic mRNAs. In the same cells, we found a concomitant accumulation of p60c-src and, accordingly, an increase in its protein kinase activity without an apparent increase in c-src mRNA levels. Therefore, modulation of viral and cellular src proteins in cells overexpressing p21N-ras appeared to result from two distinct effects: a downregulation of long terminal repeat-driven transcription and a more complex interaction with cellular effectors that control the stability of p60c-src. Such modulation also seemed to depend on the levels of p21N-ras and, possibly, on host-cell factors, since it was not observed in the third cell line, in which the relative increase in p21N-ras was only 2.5-fold to fivefold.

Partial characterization of rat cell lines infected by a Rous sarcoma virus-33.

Rous sarcoma virus-33 (RSV-33) was obtained from a sample of chicken Rous sarcoma which had been dried and stored in 1933. RSV-33, like the RSV-29, has the minimal number of passages beyond its isolation from chicken tumour No. 1. Our experiments demonstrated that the Rous sarcoma virus-33 was replication non-defective and was pathogenic for rats. Established rat tumorigenic cell lines express the viral genome. All three species of viral RNA were detected and v-src proteins and gag polyproteins were identified as well in cells of R9 and R74 lines. The virus can be rescued from cells of R9 and R74 lines, thus indicating that the cells are virogenic. The cells of a permanent tumorigenic line RT1 are infected but not transformed by RSV-33. Although they contain a complete proviral genome, they do not express detectable virus-specific RNA. The virus is not rescuable from RT1 cells under in vivo conditions. Proviral DNA analysis showed that the RSV-33 contained a full-length genome, including the env gene, in contrast to the RSV-29 which was found replication defective.

Oncogene expression in human tumors.

The expression of 9 oncogenes in primary tumors and in human tumors passaged in nude mice was tested: a total of 28 tumor types was analyzed. Oncogenes src, fps, and mos were not expressed in any of the tumors tested but oncogene myc was transcribed in most of the tumors and myc was over-expressed in 3 tumors passaged in nude mice (Ewing sarcoma, large intestine carcinoma and kidney carcinoma) and in primary fibrous histiocytoma. Enhanced transcription of ras and fos genes was observed nonspecifically in different tumors. Oncogene sis was activated specifically in metastases of different tumors in lymph nodes.