[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.

Cellular expression of INK4a gene in cells of bladder cancer associated with human papilloma virus-16.

Hyperexpression of p16(INK4a) protein is an early marker of cervical cancer. Hyperexpression of INK4a gene encoding this protein at the level of mRNA and p16(INK4a) was detected in tumor cells of some patients with bladder cancer associated with human papilloma virus-16. However, in contrast to cervical cancer, this phenomenon in urothelial carcinomas does not correlate with expression of human papilloma virus-16 oncogenes E6 and E7.

[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.

[Detection of oncoprotein E7 HPV16 in the cancer and normal urinary bladder urothelium].

Oncoprotein E7 HPV16 was detected by immunohistochemical staining with specific polyclonal antiserum [Fiedler et al., 2004] in 7 out of the 24 (29.2%) studied bladder cancer specimens. The result is in good agreement with the hypothesis that HPVs take part in the carcinogenesis of the urothelium. However, some of the observations made seem rather hard to be interpreted at present. The latter include the detection of E7 HPV16 in a small number of cancer cells in a few bladder cancer specimens being examined; the presence of this protein in the cytoplasm, rather in the cancer cell nuclei, and its detection in some morphologically normal bladder urothelial specimens from non-cancer patients. Thus, the hypothesis that HPVs are implicated in the carcinogenesis of the bladder urothelium deserves further verification.

STAT1 gene expression in cervical carcinomas.

Expression of the STAT1 gene belonging to the group of interferon-regulated genes was analyzed in cervical tumors and cell lines harboring the genome of human papilloma viruses (HPV) of so-called high risk group. Expression of this gene in invasive carcinomas was maintained on a definite level that was not significantly distinct from that in adjacent normal (control) tissue. Tumors from different patients differ from each other by expression level of the STAT1 gene. These variations can be attributed to the heterogeneity of tumor cell population and different ratio between normal and tumor cells, as well as to putative persistence of intra-individual variability of STAT1 expression in normal cell population. It was demonstrated that viral genome status (episomal or integrative) did not influence STAT1 gene transcription. In conclusion, these data demonstrate that the STAT1 gene is expressed in an individual and specific manner both in HPV-positive cervical tumors and cell lines harboring transforming genes of these viruses.

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.

[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.

[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.

[Use of a series of synthetic peptides and purified major histocompatibility complex I (H-2K(b)) molecules for inhibiting T-suppressors, specific for the K(b) molecule, and their induction by peptides in vivo]. / Ispol'zovanie nabora sinteticheskikh peptidov i ochishchennoi molekuly glavnogo kompleksa gistosovmetimosti klassa I (H-2K(b)) dlia ingibirovaniia T-supressorov, spetsifichnykh k toi zhe molekule K(b), i ikh induktsiia peptidami in vivo.

Six synthetic peptides of the MHC class 1 molecule corresponding to individual H-2kb participants in amino acid sequences of domains alpha 1 (peptide 1 and 2) and alpha 2 (peptide 3, 4, 5, 6) were selected. Kb-specific suppressor T cells (Ts) were in vivo in mice, then pretreated with a set of peptides and assayed by proliferation decrease in the third-partial mixed lymphocyte culture (MLC). Effector function of Ts was abolished by the complex of the alpha 2-domain peptides (but not by the alpha 1-domain peptides) and decreased by each peptide (4, 5, 6) of the alpha 2-domain. Both alpha 1 and alpha 2 domain peptides, added at high concentrations, decreased the otherwise efficient enrichment of Ts during the absorption-elution procedure on the syngeneic macrophage (MP) monolayers. A similar significant effect was observed the purified Kb molecule (100 mg/ml) on the allogeneic MP monolayer. Interaction between Ts receptors with some MHC peptides indicates effector Ts activation in vivo by induction with peptides 5+6 of the alpha 2 domain. The fine mechanisms of interaction between MHC class I molecule epitopes and T cell receptors (TCR) of each of the T cell subsets separately are under study now.

Utilization of the MHC class I (H-2Kb) purified molecule and its synthetic peptides for inhibition of Kb-specific suppressor T cells and their induction in vivo by the MHC peptides.

Six synthetic peptides of the MHC class I molecule corresponding to individual H-2Kb participants in amino acid sequences of domains alpha 1 (peptide 1 and 2) and alpha 2 (peptides 3, 4, 5, 6) were selected. Kb-specific suppressor T cells (Ts) were induced in vivo in mice, then pretreated with a set of peptides and assayed by proliferation decrease in a three-cell lymphocyte culture (MLC). The effector function of Ts was abolished by the complex of the alpha 2-domain peptides (but not by the alpha 1-domain peptides) and decreased by particular peptides separately (4, 5, 6) of the alpha 2-domain. Both alpha 1- and alpha 2-domain peptides, added in high concentration, decreased otherwise efficient enrichment of Ts during the absorption-elution procedure on the syngeneic macrophage (M psi) monolayers. A similar significant effect was observed using the purified Kb molecule (100 micrograms/ml) in the allogeneic M psi monolayer. Interaction between Ts receptors and some MHC peptides indicates in effector Ts activation in vivo by induction with peptides 5 and 6 of the alpha 2-domain. The fine mechanisms of interaction between MHC class I molecule epitopes and T-cell receptors of each of the T-cell subsets separately are presently being studied.

[Measurement of the temperature of deep-seated tissues in healthy men during hypokinesia]. / Issledovanie glubinnykh temperatur tkanei zdorovogo cheloveka v usloviiakh gipokinezii.

During a head-down tilt (-5 degrees) study, in which 10 healthy male subjects took part, their body temperature variations were investigated. Skin temperature was measured using and electrothermometer with a point sensor-thermistor and core temperature was measured using a radiothermometer at the wavelength 20 cm. The study showed that the distribution profile of core temperature was close to that of skin temperature: the lowest temperatures were characteristic of the limbs and the highest of the chest, stomach and head. During head-down tilt variations in core temperatures were different from those of skin temperature. As a result, the temperature gradient between the skin and core layers increased by bed-rest day 50. In the subjects who regularly exercised during bed rest the gradient grew because their skin temperature fell while in the subjects who did not exercise the gradient increased due to both factors, i.e. skin temperature decrease and core temperature increase.

[A diagnostic method in acute pyelonephritis in pregnant women]. / Sposob diagnostiki ostrogo pielonefrita u beremennykh.

A radiothermometer has made it possible to take deep temperature directly from the kidney. Comparison of temperatures, obtained from normal nonpregnant women, normal pregnant women and pregnant patients with acute pyelonephritis has demonstrated a significant drop in renal temperature during pregnancy. In acute pyelonephritis, renal temperature drops still further, with the temperature difference between the affected kidney and the contralateral one ranging from 0.5 degrees to 1.5 degrees. Deep temperature measurements of each kidney are taken from several sites, selected at ultrasonic scanning, which makes it possible to calculate temperature variance for the kidney. Variance increases significantly (more than two-fold) in the inflamed kidney, as compared to the intact one. Therefore, the diagnosis of acute pyelonephritis can be made where deep temperature difference is between 0.5 degrees and 1.5 degrees and temperature variance is increased twofold or more in one kidney, as compared to the other, in a pregnant patient. The side of affection is also determined in this way. The proposed diagnostic method is perfectly safe for the mother and the fetus, and can be used for the diagnosis of acute pyelonephritis during pregnancy, along with other procedures.

[SHF-thermography in allergic diseases]. / SVCh-termografiia pri allergicheskikh zabolevaniiakh.

The results of combined clinical allergologic study and SHF thermography in patients with pollenosis, chronic asthmatic bronchitis, bronchial asthma, continuous and seasonal allergic rhinitis and normal controls are reported. Patients with pulmonary disorders showed a reduction by 1 degree C in mean deep temperature around the lungs, and a sevenfold increase in deep temperature dispersion, as compared to the controls. Patients with pollenosis showed a reduction of deep temperatures by 1.5-2 degrees C in the gastric area and the ascending intestinal portion. In patients with allergic rhinitis, the temperature differences between the tip of the nose and maxillary sinuses, typical for normal subjects, become insignificant. Possible mechanisms of the demonstrated temperature abnormalities are discussed. The data obtained are compared to those obtained by means of IR thermography in the same categories of patients.