[Cloning of alternative isoforms of the catalytic subunit of the human thelomerase (htert)].

Most human tumors, including cervical cancer, are characterized by telomerase activation (cell proliferation activation enzyme). Such activation is implemented in the elongation of the terminal segments (telomeres) of the telomerase chromosome. The gene of the enzyme is RNA-encoded, the RNA in tumors being observed in a few isoforms. The hTERT RNA role in cell activation and control was simulated using cervical cancer, as well as its pretumoral states (CIN), as a model object. The goal of this work was to clone of the human hTERT isoforms (normal, α-, ß-, and α+ß-splice-variants). The genetic constructions containing normal hTERT sequence, α- and ß-deletion variants based on the lentivirus vector pR780 were obtained. The α- and ß-deletion variants were not obtained in this variant because of methodological problems. In further research, we plan to implement splice-variants of hTERT in eukaryotic human cells.

[MicroRNA and cancer].

Review is devoted to analysis of the role of microRNA in progression of human tumors. The following aspects of this problem are discussed: general characteristics of microRNA, expression pattern of these RNAs in human tumors and specificity of this expression, putative role of microRNAs as oncogenes and tumor suppressors for tumor growth, participation of microRNAs in induction of transformed phenotypes of tumor cells, possible role of microRNAs for early diagnosis of the disease and prognosis.

[Telomerase level versus spliced hTERT-specific RNA forms in cervical carcinoma].

An attempt was made to identify molecular markers of different clinical stages of cervical carcinoma caused by papilloma virus (HPV). Presence of viral genome, telomerase level and expression of a gene, which coded the catalytic activity of that enzyme (hTERT), were assayed in 89 patients. HPV (type 16) genome harboring tumors were detected in 73% which was in conformity with the literature and our own data. Telomerase was identified (TRAP) in all tumors and tumor cells cultured in vitro. hTERT-specific RNA was found in all tumor samples, however, increase in its expression was insignificant. As far as the three markers are concerned, no significant differences between clinical stages of tumor were reported.

The regulation of telomerase in oncogenesis.

The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones.

[Genetic and epigenetic factors of cervical tumor progression].

Infection with high risk papilloma viruses (HPV types 16, 18, and relative ones) initiates the development and progression of uterine neck cancer. The viral genome is found in pre-tumorous lesions (stage I to III intraepithelial neoplasias--CIN) and carcinomas, persisting in cells in episomal or integrated state. In all tumors, there is the expression of two viral transforming genes, E6 and E7, the main function of which is the inactivation of genes that suppress tumoral growth, p53 and retinoblastoma gene. In CIN and carcinomas, losses of heterozygosity are found in various chromosomes, mainly in the areas of suppressor genes; some of them can be specific for certain stages of the malignant process. Among epigenetic alterations, the main significance for the progress of the disease belongs to the methylation of the promoter areas of the genes involved in the process of cell division, which may be specific for each separate tumor and appears in approximately 30 to 40% of tumors. Another important epigenetic alteration is the increase in the expression of p16ink4a gene, which is the inhibitor of cyclin-dependent kinases; this appears at CIN I stage and may serve as an additional early diagnostic marker. Telomerase activity has been identified in all uterine neck tumors, but each tumor has its own spectrum of spliced RNA coding this enzyme. Expression microchip technique has shown that each tumor is individual according to the spectrum of "working" genes, and this spectrum varies in the process of tumor progression.

[Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and transcriptional level].

In this work for the first time copy number and expression changes of the tumor suppressor gene RBSP3 (3p21.3) were investigated. The study was performed on HPV-positive squamous cervical carcinoma (SCC) using real-time PCR. Deletions were detected in 42% of cases (19 of 45 studied biopsies). Frequency of deletions was significantly higher in SCC samples with metastases (64%) than in tumors without metastases (32%, P < 0.05). In a few cases amplification of RBSP3 was also found. Altogether copy number changes of RBSP3 were detected in 51% of cases (23 of 45). Expression of RBSP3 was decreased in 64% of SCC samples (21 of 33). Again decreased expression of RBSP3 was detected significantly more frequently (83%) in tumors with metastases compared with SCC without metastases (52%, P < 0.05). In several cases however increased expression was observed. Altogether changes in expression of RBSP3 were detected in 79% (26 of 33) of SCC biopsies. Comparison of copy number and expression changes showed that in 23% of SCC cases decreased expression of RBSP3 was detected in samples with deletions and in 36% cases such decrease was not associated with copy number changes. Rarely more complicated SCC cases were found. For example in some tumors increased expression of RBSP3 was detected in samples with deletions or without changes in copy number. Results of the study suggested that RBSP3 is involved in the progression of SCC and complex mechanisms for inactivation of RBSP3. We also hypothesize that these data indicate that RBSP3 in addition to dephosphorylation of pRb has other functions important for malignant transformation because pRb is almost absent in HPV-positive SCC.

[Loss of heterozygosity at chromosome 6 as a marker of early genetic alterations in cervical intraepithelial neoplasias and microinvasive carcinomas].

Oncogenic human papilloma viruses (mostly HPV types 16 and 18) are the major cause of cervical intraepithelial neoplasia (CIN) that progress into cervical cancer (CC). To reveal early genetic alterations at chromosome 6 important for CC progression we have analyzed loss of heterozygosity (LOH) in DNA from 45 CIN cases, 47 microcarcinomas and 19 invasive squamous cell carcinomas stage IB. LOH analysis of DNA samples prepared with microdissection from all CIN foci as well as from CC lesions and synchronous CIN has permitted the investigation of CIN and CC heterogeneity. 79% of CC stage 1 showed LOH with 6 microsatellite markers at chromosome 6. LOH with microsatellite markers D6S276 (6p22) and TNFalpha (6p21.3) was found in 50% of CC cases. LOH frequency in CIN lesions, synchronous with CC, was higher then LOH in CIN cases without cancer, the statistical significance (p = 0.004) was shown for marker D6S291 (6p21.2). The finding suggests that high level of LOH frequency in CIN lesions may be a marker of unfavorable prognosis for CIN. Progression from microcarcinoma to invasive CC of IB stage was associated with higher LOH frequency at D6S344 (6p25) and TNFalpha (6p21.3). The early genetic alterations were found in CIN with microsatellites D6S273 and TNFalpha located at 6p21.3. Moreover the LOH frequency at D6S273 retained the same in CIN and CC cases. Based on HPV-typing, LOH analysis and X-chromosome inactivation the polyclonality of CC lesions as well as CIN was shown in a few patients.

[Methylation of the putative tumor suppressor gene, RASSF1A, in primary cervical tumors]. / Metilirovanie predpolagaemogo gena-supressora RASSF1A v opukholiakh sheiki matki.

The methylation level of 13 CpG-dinucleotides in the promoter region of the putative tumor suppressor gene RASSF1A (3p21.31) was analyzed in HPV-positive squamous cell carcinomas of cervix using methyl-sensitive restriction endonuclease analysis followed by PCR. The methylation from 3 to 13 CpG-dinucleotides was observed in 64% (25/39) tumors, 22% (2/9) morphologically normal tissues adjacent to tumors (P = 0.0306) and in 2 from 3 leucocytes of peripheral blood of patients. The methylation of these CpG-dinucleotides was absent in DNA of healthy donor leucocytes (0/10). Methylation level of the examined fragment of the RASSF1A promoter region was significantly higher in tumors of patients with lymph node metastases in comparison to tumors of patients without metastases (P = 8.5 x 10(-12)). The methylation frequency of RASSF1A gene was in two times higher than hemi- and homozygous deletion frequency at the region of location of this gene (chromosome 3p21.31), determined earlier. These data suggest that methylation of the RASSF1A gene is one of the main ways of this gene inactivation in HPV-positive cervical squamous cell carcinomas. The methylation of the RASSF1A gene is an early event in genesis of tumor and the level of methylation increased with tumor progression.

[Interaction of viral and cellular genes in cervical tumors]. / Vzaimodeistvie virusnykh i kletochnykh genov v opukholiakh sheiki matki.

The results obtained in the Laboratory of Molecular Biology of Viruses, CRC carried out in the framework of the Human Genome program and devoted to the study of the activity of cell and viral genes in cervical cancer are summarized. DNA of human papillomaviruses persists in tumors both in episomal and integrative forms. Integration may occur in different regions of chromosomes. Viral transforming genes E6 and E7 are always present in tumor cells, while antibodies to these proteins are detected only in approximately 30% of patients. Loss of heterozygosity is detected on long and short arms of chromosome 6; some such cases are manifest already at the early stages of tumor progression, while others are typical of the late stages. Several genes that are potentially involved in tumorigenesis and are subject to hypermethylation in CpG islands were identified. Methylation of several genes is observed in approximately 30% of tumors. Tumor progression is associated with increased expression of p16ink4a, an inhibitor of cyclin-dependent kinases.

[Cervical carcinoma progression-associated genetic alterations on chromosome 6]. / Geneticheskie izmeneniia na khromosome 6, assotsiirovannye s progressiei raka sheiki matki.

To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.

[Molecular markers of the cervical cancer]. / Molekuliarnye markery raka sheiki matki.

The genome of human papilloma viruses from a high-risk group (HPV types 16 and 18) has been detected in 90% of cervical tumors and, in some cases, in the adjacent normal tissues. The presence of viral DNA is the main molecular marker of this neoplasia. HPV genome may persist in the tumors as episomal and integrative forms at early and late stages of tumor progression. The status of viral DNA and the pattern of its expression are similar in all cells of this tumor cell population and seem to be a marker of tumor cell monoclonality. Antibodies to the products of viral oncogenes E6 and E7 were found only in 35% of the patients with tumor where HPV genome is present. Thus, this criteria cannot be used for diagnostic and prognostic purposes. On chromosome 6 in the cervical tumors, the specific marker of heterozygocity on loci 6p21.3 was found. The marker appears at the precancer stage and may be regarded as a marker of tumor monoclonality. Heterozygocity loss in the specific locus in the region 6q16-21 correlates with tumor progression and suggests that there are potential tumor-suppressor genes in this region of chromosome 6. A group of HPV positive tumors with a hypermethylator phenotype is described. These tumors are characterized by the simultaneous methylation and inactivation of multiple genes, including tumor suppressor genes.

[Expression of the protein marker p16INK4a in the cervix uteri cancer]. / Ekspressiia belkovogo markera p16INK4a v rake sheiki matki.

Immunohistochemical study was carried out of 18 cervical carcinomas (13 squamous and 5 adenomatous) and of 3 cases of cervical intraepithelial dysplasia. Formalin-fixed paraffin-embedded tissue samples from biopsies as well as from surgical material were used. Staining was performed with monoclonal antibodies to protein p16INK4a. Cytologic smears of epithelial cervical cells from 7 healthy women were taken as a negative control. The reference group consisted of 5 cancer patients with other tumors (breast cancer, B-cell lymphoma). Overexpression of p16INK4a was registered in cervical cancer.

[Status of the human DNA papillomavirus in cervical tumors]. / Status DNK virusa papillom cheloveka v opukholiakh sheiki matki.

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7 proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.

[Cathepsins L and B and their endogenous inhibitors in embryonal fibroblasts transformed by different genes]. / Katepsiny B i L i ikh éndogennye ingibitory v émbrional'nykh fibroblastach, transformirovannykh razlichnymi genami.

Expression of cysteine proteinases, cathepsins L and B, and their inhibitors was studied out in three model systems of rat embryo fibroblasts, sequentially immortalized and transformed by different genes. In Model I rat embryo fibroblasts were immortalized with DNA of early region of simian adenovirus SA7 (clone REF-1) and then transformed by c-Ha-ras oncogene (REF-2EJ; malignant transformation). In Model II and III, the immortalized fibroblasts (clone IE5) were obtained by transfection with the polyoma virus LT gene and the clone IE5 used lost this gene; the malignant transformation was achieved by transfection with the E7 gene (clone trF8; Model II) and E6/E7 genes ¿clone A5E5(pC7-1); Model III]¿ of human papilloma virus types 16 and 18 respectively. In Model I, the increase in the total cathepsin L and B activity was correlated with the stages of transformation, at the same time, in Models II and III, this activity in immortalized IE5 fibroblasts was higher than at transformation stage. The activity of cathepsin L in lysates of transformed fibroblasts--REF-2EJ, significantly exceeded this activity both in transformed cells trF8 and A5E5(pC7-1)(6- and 10-fold, respectively). In cell cultures of Models I and II, the increases in secreted activity of cathepsins L and B were correlated with the stages of fibroblasts transformation, but in cultures of Model III, this activity at the stage of malignant transformation was lower than that the stage of immortalization. Therefore, the activities of cathepsins L and B were expressed to varying degrees at different stages of oncogenic transformation and the expression of their activities were dependent on type of transforming gene. It was established that changes in proteolytic potential were correlated with differences in the transforming phenotype of cell clones. An endogenous inhibitor(s) of cysteine proteinases was found in conditioned media of all type cell cultures. Expression and inhibitory properties of this inhibitor(s) were different at distinct stages of transformation.

[Cloning transforming genes from human papillomavirus type 18]. / Klonirovane transformiruiushchikh genov virusa papillom cheloveka tipa 18.

Nucleotide sequences of type 18 human papilloma virus genes E6 and E7 inserted in human DNA cloned from cervical tumor are determined. The resultant sequences are compared to the prototype variant. Five point mutations not leading to replacement of amino acid residues in polypeptide chain and 1 substituting the amino acid in codon 129 are detected in gene E6 sequence. In E7 sequence, one significant mutation in codon 92 is detected. Both substitutions of asparagine for lysine are localized in the 3'-terminal region of the genes, which may not affect the transforming potential of these sequences. DNA fragments of E6 and E7 coding regions obtained by PCR were independently cloned in bifunctional expression vector DelpC7. The identity of hybrid E6 and E7 nucleotide sequences to initial ones is verified by sequencing.

[Nuclear oncoprotein expression in lung precancer and cancer at various stages of tumor progression studies at the level of light- and electron-immunohistochemistry]. / Ekspressiia iadernykh onkobelkov pri predrake i rake legkovo na raznykh stadiiakh opukholevoi progressii (po dannym svetovoi i élektronnoi immunohistokhimii).

68 cases of lung carcinoma, 3 carcinoids and 15 fibrosing alveolitis with foci of adenomatosis and bronchiolo-alveolar carcinoma were studied. Oncoproteins c-fos, c-jun, c-ets-1, c-myc L and L-myc were identified in the tumour and surrounding tissue. Expression of c-fos was revealed in 79 of 138(59.4%) of proliferative and dysplastic changes of lung epithelium; c-jun in 40 of 61 (65.6%), c-ets-1 in 22 of 41 (53.7%), c-myc in 41 of 96(42.7%) and L-myc in 15 of 61 (24.6%), mainly in altered bronchial epithelium with a positive reaction to the antibodies against neuron specific enolase and S100 protein. More pronounced expression of nuclear oncoproteins, heterogeneity of their location in tissues, frequent cytoplasmic location in tumour cells were typical for lung carcinoma.

[Cell oncogene expression in normal, metaplastic, dysplastic epithelium and squamous cell carcinoma of the uterine cervix]. / [Ekspressiia kletochnykh onkogenov v normal'nom, metaplasticheskom, displasticheskom épitelii i ploskokletochnom rake sheiki matki.

Immunohistochemical analysis of the protein expression c-myc, ets 1, ets 2, TPR-met, c-fos, c-jun, c-ras-pan, p53, yes, src in 79 samples of normal, metaplastic squamous epithelium, intraepithelial and invasive squamous cell carcinoma of uterine cervix was performed using polyclonal rabbit antibodies to the synthetic peptides homologous active areas of corresponding oncoproteins. Higher content of myc, fos, ets2, p53, ras is noted in metaplasia, dysplasia and in tumours as compared to the normal tissues. Protein myc is revealed in the cytoplasm at a grave dysplasia and in the nucleus in the intraepithelial carcinoma: this may serve as a criterion at a differential diagnosis of these conditions. Expression of the oncoproteins fos, ets2, p53, src in the metaplastic squamous cell carcinoma was higher than in the true squamous cell (ectocervical) carcinoma. When compared to the advanced carcinomas, increase of ets2, p53, and at some degree that of myc, the increase is noted in the latter. Invasive carcinoma with a high level of oncoproteins showed a tendency to the synchronization of myc and ras expression. Poor prognosis was associated with a low level (before treatment) of the expression of the majority of the oncoproteins studied.