[Viral co-carcinogenesis and other aspects of viral interaction].
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Animals↗
Biomedical subjects
F L Kiselev
Publications and source records attributed to F L Kiselev.
[Differentiated expression of the viral genome in mammalian cells transformed by the Rous sarcoma virus].
In hybridization of 3H-DNA-product of Rous sarcoma virus with the RNA isolated from mammalian cells transformed by the virus concerned, it was found, that the virus-specific RNA, isolated from cryptovirogenic cells RVP-3, differs in nucleotide sequences from virogenic cells XC, XPO and TWERC.
Animals↗
[Identification of a thermostable antigen from normal and leukemic human tissues].
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Animals↗
[Transformation of immortalized rat embryonic fibroblasts by the E7 gene of human type 16 papillomavirus].
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Animals↗
[Cloning transforming genes from human papillomavirus type 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.
Amino Acid Sequence↗
[Status of the human DNA papillomavirus in cervical tumors].
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.
DNA, Viral↗
[Expression of the protein marker p16INK4a in the cervix uteri cancer].
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.
Adenocarcinoma↗
[Cervical carcinoma progression-associated genetic alterations on chromosome 6].
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.
Chromosomes, Human, Pair 6↗
[Interaction of viral and cellular genes in cervical tumors].
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.
DNA Methylation↗
[Methylation of the putative tumor suppressor gene, RASSF1A, in primary cervical tumors].
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.
Base Sequence↗
[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.
Adolescent↗
[A new recipient cell line for transfection of biologically active oncogenes].
The properties of the new immortalized rat cell line (REF-1) were analyzed. These cells can be used as recipient ones in transfection assays. REF-1 cells never convert spontaneously to transformed phenotype during long-term passages in vitro unlike NIH3T3 cells. This peculiarity allowed to use REF-1 cells for identification of oncogenes, which induce slow-growing tumors. The following oncogenes were used in gene transfection experiments in order to test their effects: activated human EJ-ras; N-ras; v-myc; v-mos; activated tpr-met and c-hu-met. REF-1 cells, transfected with the members of ras family; v-mos and tpr-met were found to be transformed in vitro and induce tumors in nude mice, on the contrary of c-hu-met- and v-myc-transfected cells, which are non-tumorogenic. A number of clonal cell lines carrying different oncogenes were obtained. The detailed analyses of integration and expression of exogenous sequences of different oncogenes has been presented in 18 clonal cell lines.
Animals↗
[Several virologic and biochemical aspects of vaccination with antipoliomyelitis vaccine. Fate of the parent virus].
In the present study we attempted to follow the fate of the parental poliomyelitis virus of immunological type II labeled with 3H-uridine in vaccinated animals. For this purpose, 3H-uridine labeled purified and concentrated virus was inoculated intramuscularly into cotton rats (sensitive to poliomyelitis virus) and guinea pigs (insensitive to poliomyelitis virus). The controls consisted of two groups of the same animal species: the animals of the first group were inoculated intramuscularly with pure 3H-uridine of known high activity while the animals of the second group were inoculated also intramuscularly with pure 3H-uridine and in another leg 0.5 ml of standard poliovirus vaccine of type II. Since poliomyelitis virus replicates in the cytoplasm, we studied cytoplasmic extracts of various organs (the brain, spinal cord, lungs, and spleen) of cotton rats and guinea pigs at different intervals after virus inoculation (1, 4, and 15 days). The experimental results showed the attenuated type II poliomyelitis virus to be able to persist for long periods in animals in the form of subviral particles with a higher buoyant density in cesium chloride gradient.
Animals↗
[Several virologic and biochemical aspects of vaccination with antipoliomyelitis vaccine. Characteristics of intracellular virus-specific structures].
The data are presented concerning infectious potentials of subvirus particles isolated from cytoplasmic extracts of various organs of cotton rats and guinea pigs inoculated intramuscularly with purified and concentrated poliomyelitis virus type II. As described earlier, from various organs of the animals (the brain, spinal cord, lungs, and spleen) subvirus particles were isolated which in cesium chloride gradient had a density higher by 0.05-0.1 g/ml than that of the intact virus. Determinations of the infectious titre of these subparticles in susceptible cells showed their infectivity to be lower by 4-5 lg than that of the infact virus. When DEAE-dextran was used, a titre of both subvirus particles isolated from cytoplasmic extracts of various organs of the immunized animals and of the original poliomyelitis virus increased by 3-4 lg. The subvirus particles were sensitive to pancreatic RN-ase and to the antiserum against poliovirus type II. All the foregoing permits a conclusion that attenuated type II poliovirus or a portion of its population after inoculation into the living body is capable of long-term persistence there in the form of subviral particles with high buoyant density values.
Animals↗
[Study of the synthesis of Rous virus-specific RNA in chick embryo cells].
The presence of virus-specific RNA in commercial chick embryos and its lack in chick embryos of leukemia-free chicken farm of the USSR AMS Oncological Research Center as well as in cell cultures from RIF-free chicken infected with Marek's disease virus was demonstrated by hybridizationof 3H-DNA-product of Rous sarcoma virus synthesized in vitro in the presence of actinomycin D with the total preparation of cellular RNA.
Animals↗
[Tumor cell proteins, detected using antibodies to the S- and N-terminal fragments of the fos proto-oncogene product].
Antibodies to c-fos oncoprotein were produced in rabbits by immunization with synthetic peptides, corresponding to the sequences 6-15 of N-end and 371-380 of C-end of c-fos oncoprotein. C-fos expression was tested with immunoprecipitation and immunoblotting in various transformed cell lines with antibodies to N- and C-decapeptides. It was shown that antibodies to C-terminal decapeptide revealed a c-fos gene product and also some fos-related antigens FRAs 36 kD, 46 kD, 75 kD and 90 kD in rat pheochromocytoma PC-12 cells and mouse carcinoma cell lines MAC-3 and LL. In some cell lines 46 kD FRA was expressed in the absence of p62 c-fos. Besides, different clones of the same cell line cultivated in identical conditions revealed differences in the 46 kD FRA expression. Antibodies to sequence 6-15 of N-end revealed only c-fos products and no FRAs were detected. Therefore FRAs have homology with the c-fos product in the C-terminal region and differ from it in the N-terminal region.
Animals↗
[Activity of topoisomerase I and endonucleases in cells transfected by a ras oncogene].
Activities of nuclear endonucleases and topoisomerase I were measured in rat fibroblasts which were at the stages of tumor transformation: control embryonal fibroblasts--CEF; cells immortalised by transfection of S1A segment of SA7 adenovirus--REF-1; intermedius cells transfected once by EJras oncogene--REF-1EJ; and cells transformed after the second transfection by the same oncogene--REF-2EJ. The topoisomerase I and Ca2+, Mg2+-dependent endonuclease was most decreased at the stage of immortalised cells, and the intermedius stage (REF-1EJ) was characterized by the lower activity of Ca2+, Mg2+-dependent endonuclease. The highest activity of Mn2+-dependent endonuclease is seen in REF-2EJ cells. In model experiments the ability of Ca2+, Mg2+-dependent endonuclease to split non-stochastically the EJras oncogene inserted into pBR322 plasmid was shown. The role of the investigated enzymes in the restriction of plasmid integration, cellular immortalisation and recombination of plasmids with chromosomes during cell transformation is discussed.
Adenoviridae↗
[Differences in the distribution of proviruses in low- and highly- metastatic variants of hamster cells, transformed by Rous sarcoma virus].
The structure of provirus in Syrian hamster cells, transformed by Rous sarcoma virus (RSV) varying in metastatic capability in vivo has been analysed. The original cell line and its low metastatic variants contain only one copy of the integrated RSV genome. The DNA of highly metastatic cell lines cloned from the same primary culture, contain an additional copy of provirus. This RSV copy in different highly metastatic variants has a similar integration site.
Animals↗