[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.

[THE CHARACTERISTIC OF SOMATOTYPE AND FUNCTIONAL STATE OF CIRCULATORY SYSTEM OF STUDENT YOUTH OF THE NORTHEAST OF RUSSIA].

The article considers results of single-step study in random sampling of female students of the M.K. Ammosov north-east federal university (n=456). The study was carried out to investigate somatotype and functional state of circulatory system. The standard technique was applied to measure height, body mass, chest circumference, level of arterial pressure and rate of heart beats. The type of somatotype was established using Pignet index. The tone of vegetative system was determined using Kérdö index. The adaptation potential of circulatory system was determined using functional changes index. The results of study established that in 61% of examined female students the type of constitution corresponds to normosthenic one. The percentage of persons with asthenic and hypersthenic type of constitution amounted to 27% and 12% correspondingly. The signs of increasing oftone ofsympathetic nervous system are observed in 89% of girls. The functional condition of circulatory system is evaluated as "tension of adaptation mechanisms" that is apparently related to period of adaptation to new conditions. The prolonged preservation of such states results in exhaustion offunctional resources of organism and can promote development of diseases. In conditions of impacting of unfavorable ecological factors the deconditionning disorders can significantly contribute to health disturbances and decreasing of life quality. To preserve youth's health during period of education the comprehensive strategy is to be implemented such components as dynamic monitoring of health, organization of adequate diet, explanation of necessity of observance of sleep and rest pattern, development of conditions for active aerobic physical exertion and activities on correction of risk factors of development of diseases are to be included.

[HPV-associated carcinomas of the female genital tract. Molecular mechanisms of development]. / HPV-assoziiertes Karzinom des weiblichen Genitaltrakts. Molekulare Mechanismen der Entstehung.

Infections with human papillomaviruses (HPV) are a common occurrence in both men and women. In contrast HPV-associated neoplasias are relatively rare and occur only in certain areas of the body. The virus has obviously developed efficient mechanisms for its persistence without inducing too much damage to the host. The formation of neoplasia seems to be more an exception. Epigenetic mechanisms play an important role in the regulation of viral gene expression. Investigations have indicated that exactly the transition from the permissive infection stage to a transformation stage, where neoplastic alterations can occur due to expression of the viral oncogenes, is associated with certain methylation patterns of the viral genome which promote the expression of the oncogenes E6 and E7. The transforming stage is seen as the actual carcinogenic event and can be immunohistochemically detected by the biomarker p16(INK4a).

High-risk human papillomavirus in non-melanoma skin lesions from renal allograft recipients and immunocompetent patients.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) can be detected in a proportion of non-melanoma skin cancers. Data on prevalence are inconclusive, but are essential to estimate the relevance of HR-HPV, particularly with regard to prophylactic HPV vaccines for skin cancer prevention. METHODS: High-risk human papillomavirus DNA was investigated in 140 non-melanoma skin lesions from 54 immunocompetent patients and 33 immunosuppressed renal allograft recipients. Expression of p16(INK4a), a marker for HR-HPV oncogene expression in the uterine cervix, and of p53 and pRB was evaluated immunohistochemically. RESULTS: The highest prevalence of HR-HPV was found in squamous cell cancer (SCC) (46.2% (6 out of 13) in immunosuppressed and 23.5% (4 out of 17) in immunocompetent patients). High-risk human papillomavirus positivity was accompanied by diffuse p16(INK4a) expression in most SCC (P<0.001) and basal cell cancers (P=0.02), while almost all SCC in situ were p16(INK4a) positive irrespective of HR-HPV presence (P=0.66). Diffuse p16(INK4a) expression was associated with lack of pRB expression (P=0.001). p53 was strongly expressed in 40.0% (56 out of 140) of the lesions irrespective of HR-HPV presence. CONCLUSION: High-risk human papillomavirus can be detected in lesions of keratinised squamous epithelia. The association of HR-HPV with diffuse p16(INK4a) expression might indicate HR-HPV oncogene expression in a proportion of lesions. Overexpression of p53 suggests p53 pathway alterations in HR-HPV-positive and -negative lesions.

[Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts].

Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The aim of this study was to elucidate peculiarity of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF).The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papilloma virus (HPV-16). Papilloma virus type 16 and 18 are etiological factors of cervical cancer. The primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF involved: determination of MMP-2 and MMP-9 activity by hydrolysis of specific substrate--radioactive collagen type IV; obtaining of MMP spectra by zimographic assay and estimation of the mRNA expression (by RT-PCR) of MMP-2, MMP-9, MT1- MMP and TIMP-2. It was found: 1) collagenolytic activity of MMP was increased only in TF and was dependent on the degree of cell tumorogenity; 2) the study of MMP spectra was shown that MMP-9 was found in TF only but MMP-2 was found in all investigated clones; 3) The mRNA expression of MMP-9, MT1-MMP and TIMP-2 was increased in all TF while the MMP-2 expression was increased in TF only after TF cell selection on rats; 4) The collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 themselves and of MMP-2 endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to PF. The data obtained indicate changes in the enzyme/inhibitor/activator ratio and also suggest of a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV-16 gene in cell culture.

[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.

Expression of cathepsin L and its endogenous inhibitors in immortal and transformed fibroblasts.

METHODS: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. RESULTS: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. CONCLUSIONS: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.

[Type I and IV collagenases and their endogenous regulators in immortalized and transformed fibroblasts]. / Kollagenazy I i IV tipov i ikh éndogennye reguliatory v immortalizirovannykh i transformirovannykh fibroblastakh.

To elucidate the role of matrix metalloproteinases (MMP) in carcinogenesis, the expression of collagenases of types I (MMP-I) and IV (MMP-2 and MMP-9) as well as the behaviour of urokinase-like plasminogen activator (uPA) and of tissue MMP inhibitors (TIMP) in immortalized (IF) and transformed (TF) fibroblasts were investigated. The study was carried out using embryo rat fibroblasts, sequentially immortalized with the LT gene of human papilloma virus and transformed with the E7 gene of human papilloma virus (HPV-16). As control was used the primary fibroblast (PF) culture of Fisher rats. In IF, the collagenase activity was at the same level as it was in PF. The activity of uPA in IF was increased by 2-2.5-fold; the titrated amount of free endogenous inhibitors in IF and PF was at essentially the same level while being markedly higher than in TF. At the stage of fibroblast transformation with the E7 gene of HPV-16, there was seen an increase of Type IV collagenases and a decrease of Type I collagenase, both these indices being most pronounced in the cells with most developed tumorigenic properties. In TF there occurred a decrease of free endogenous MMP inhibitors relative to the enzyme activity and, at the same time, a decrease in uAF activity, indicating the changes occurring in the enzyme/inhibitor/activator ratio and hence the enhancement of the destructive potential of the cells (in this case, at the cost of Type IV collagenase activity).

Cloning of E6 and E7 genes of human papilloma virus type 18 and transformation potential of E7 gene and its mutants.

E6 and E7 genes of human papilloma virus type 18 have been subcloned from plasmid pC7, carrying an insert of DNA from squamous cell carcinoma of cervix. Both genes in comparison to prototype variant contain one mutation that changes asparagine to leucine. In the case of E6 gene this mutation is mapped in codon 129, in the case of E7 the same change AAC to AAA mapped in codon 92. In addition both genes contain few point mutations that do not change the aminoacid sequences of the protein. Two mutants of E7 gene have been constructed by site directed mutagenesis based on PCR technology-one in codon 10 (change Asp to Asn) and one in codon 24 (change Asp to Gly). The first type of mutation did not influence the transformation potential of the E7 gene in comparison to the parental one with mutation in codon 92. The mutation in codon 24 (region responsible for the interaction with Rb protein) eliminate the transformation potential of the gene. The cells transformed with E7 mutants in codons 10 and 92 were tumorigenic for syngenic rats.

[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.

Integrin expression and collagenase activity of RSV-transformed Syrian hamster fibroblasts, differing in spontaneous metastasizing.

The expression of extracellular matrix (ECM) specific receptors, integrins, and the activities of type IV collagenase and interstitial collagenase were investigated in two strains of oncogenically transformed fibroblasts, drastically differing in spontaneous metastasizing. Both strains were shown to express quite limited patterns of integrins. Of those, alpha 5 beta 1 integrin is greatly reduced on highly metastatic (HM) cells, while the expression of alpha v beta 3 is strongly suppressed on lowly metastatic (LM) fibroblasts. No differences between the strains were found in either intracellular or secreted activities of type IV collagenase, while the activity of interstittial collagenase, secreted by HM cells, was twice as much as secreted by LM cells. The results imply the role of cooperated modifications of integrin-directed cell-ECM interaction and collagenase activity in establishing a metastatic phenotype.

[Tears in the clothing after being run over by an automobile wheel]. / Razryvy odezhdy pri pereezde kolesom avtomobilia.

Clothes taken from 120 bodies of victims in case of death due to car wheel overrunning were examined and in 10 cases shred-shaped tissue ruptures in the form of an angle were detected. They proved to appear in case of oblique direction in overrunning and the angle of a shred turned back was directed to the side of wheel rotatory movements, i.e. contrary to wheel translational movement.

[Determining direction in cases of bodies run over by automobile wheels]. / Ustanovlenie napravleniia pereezda tela kolesom avtomobilia.

Investigation of injuries sustained by 110 pedestrians during fatal traffic accident and observed in 8 biomannequins during experimental overrun by car wheels resulted in detection of morphological features indicating overrun direction and posture of a victim. It was stated that in case of multiple trauma the areas of wheel rolling and primary force application in collision with a car are located at opposite sides.