[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.

[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.

[Differences in expression and functional organization of the rat tyrosine aminotransferase gene in two lines of Morris hepatoma, 8994 and 7777]. / Razlichiia v ékspressii, funktsional'no'i organizatsii gena tirozinaminotransferazy krys v dvukh liniiakh gepatom Morrisa 8994 i 7777.

Expression and structural organization of tyrosine aminotransferase (TAT) gene in Morris hepatoma cell line 7777 with active and glucocorticoid-inducible TAT gene and in hepatoma 8994, where TAT gene does not function were analysed. No differences in the number of receptor macromolecules, translocation and nuclear binding of hormone-receptor complexes in hormone sensitive (7777) and resistant (8994) cell lines were demonstrated. Dexamethasone increases TAT gene transcription in 7777 cell line but not 8994. Restriction analysis of TAT gene does not reveal any differences either in structural or in regulatory regions. Gel retardation assay with cloned TAT fragment (-400 b.p.) from normal hepatocytes showed identical shift of mobility in 7777 and 8994 cell lines. Moreover, 5'-flanking sequence (-890 b.p.) of TAT gene linked to the bacterial CAT gene is transiently expressed in both cell lines. We have shown that HpaII site (-105 b.p.) of TAT gene is methylated in those cells where TAT gene does not function (thymus, spleen, Zajdela ascites hepatoma) and is demethylated in TAT gene expressing hepatoma 7777 and normal rat hepatocytes. In hepatoma 8994 there are no DNAse I hypersensitive regions, typical to functioning TAT gene from hepatoma 7777 and normal hepatocytes.

[Tumor cell proteins, detected using antibodies to the S- and N-terminal fragments of the fos proto-oncogene product]. / Belki opukholevykh kletok, vyiavliaemye s pomoshch'iu antitel k S- i N-kontsevym fragmentam produkta protoonkogena fos.

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.

[Activation of transcription of the tyrosine aminotransferase gene in the rat McA-RN 7777 hepatoma cell line by cycloheximide]. / Aktvatsiia transkriptsii gena tirozinaminotransferazy v kletochnoi linii gepatomy krysy McA-RN 7777 tsiklogeksimidom.

The expression of the tyrosine aminotransferase (TAT) mRNA after cycloheximide treatment was analysed by Northern blotting method in Morris rat hepatoma cell lines. The level of TAT mRNA increased after 6-8 h of cycloheximide treatment only in the McA-RH 7777 cell line. McA-RH 7777 nuclear run-off assay showed that TAT transcription was induced by cycloheximide treatment. Both glucocorticoid and cycloheximide modulated TAT gene transcription in a synergistic way. There was no induction of TAT expression following cycloheximide or cycloheximide glucocorticoid simultaneous treatment in another cell line (McA-RH 8994), while c-myc and c-fos expression was superinduced by cycloheximide treatment. The possible mechanism of transcription regulation and its damage in hepatoma cells is discussed.

[Expression of oncogenes in transplantable tumors and rodent cell lines]. / Ekspressiia onkogenov v nekotorykh transplantiruemykh opuliakh i kletochnykh liniiakh gryzunov.

The expression of 8 oncogenes in transplanted rodent tumours and cell lines was tested. In 6 cases the synthesis of myc-, fos-, ras-oncogene RNA was observed. The transcription of these oncogenes was observed nonspecifically in tumours of different histological types. No difference in the set of the oncogenes expressed and the size of their transcripts was noticed between transplanted tumours and the cell lines obtained from them. The expression of myb-, sis-, Blym-, erb-B- and abl-was not observed in tested cells.

[The degree of methylation of alpha-fetoprotein in rat hepatoma cell lines and their clones producing and not producing this protein]. / Stepen' metilirovaniia gena alpha-fetoproteina v produtsiruiushchikh i neprodutsiruiushchikh étot belok kletochnykh liniiakh gepatom krysy i ikh klonakh.

The extent of methylation of MspI-HpaII sites of alpha-fetoprotein (AFP) gene in DNAs from normal rat livers, rat hepatoma cell lines and their clones was compared by Southern blot hybridization. The expression of this gene was controlled by measuring the level of AFP specific RNA and by immunoperoxidase staining with antibodies to AFP. It was found that the AFP gene contained several CCGG sites in the 5'-region methylated in the nonproductive cell lines and normal adult liver and undermethylated in productive cell lines. The same phenomena was observed for productive and nonproductive hepatoma McA-RH7777 clones. These findings suggest that specific changes in the methylation pattern are associated with changes in AFP gene expression.

[Formaldehyde-induced tight binding of protein to RNA in particles of rod-like viruses. Nature and specificity of the crosslinks]. / Prochnoe sviazyvanie RNK s belkom v chastitsakh palochkovidnogo virusa pod deistviem formal'degida. Priroda i spetsifichnost' sviazi.

A stable RNA-protein complex was obtained by treatment of a helical TMV-like virus - cucumber virus 4 (CV4) with 1.5% formaldehyde at 50 degrees C and stability and specificity of the RNA-protein bonds in the complex was studied. After alkaline of T2-RNAase hydrolysis of the complex obtained from the [32P]-labelled CV4, the RNA-protein crosslinks were identified by the presence of [32P] in the protein band during SDS electrophoresis in polyacrylamide gels. The resistance of the crosslinks to boiling in 2% SDS with 0.2% mercaptoethanol, to 0.3 N KOH and to 10% trichloracetic acid is indicative of the covalent nature of the nucleotide-protein bonds in the CV complex. Data from tryptic peptide map analysis suggest that not more than three peptides within the complex contain the [32P] label. It is concluded that formaldehyde crosslinks to the intravirus RNA only at the specific site(s) of the CV protein molecule.

[Carcinosarcomas of the lung]. / Kartsinosarkomy legkog.

The authors analysed the literature on pulmonary carcinosarcomas and described two own observations of this rare pathology. The predominant occurrence of this tumor in men of 40-65 years, rapid development of the process, frequent asymptomatic course and late surgical help are noted. Clinico-anatomical manifestations of pulmonary carcinosarcomas are nonspecific. Their histological structure is variable and depends on the nature of tissue components involved in the neoplastic process. The authors emphasize a great diagnostic value of prophylactic fluorographic examinations of the population.

[Classification of tumors of the thymus gland]. / O klassifikatsii opukholei vilochkovoi zhelezy.

The authors have analysed the data of clinical and pathoanatomical (biopsy) investigations in 41 patients, operated upon for the true tumor of the thymus (thymoma) without any signs of malignant miasthenia. Based on the literature materials and personal observations, it is considered necessary to provide for a rational clinico-anatomical classification of thymomas, which may allow certain prognostication of the results of surgical treatment. An attempt is made to work out a preliminary uniform working scheme of the histological nomenclature for thymomas.

[Identification of CD phage proteins participating in DNA-protein interaction by means of O-methylhydroxylamine]. / Identifikatsiia s pomoshch'iu o'metilgidroksilamina belkov faga CD, uchastvuiushchikh v DNK-belkovom vzaimodeistvii.

An early described hypothetical scheme is confirmed on transformation of non-covalent polynucleotide-protein interactions into covalent under the treatment of CD phage with O-methylhydroxylamine. 4 proteins are revealed, which have been covalently bound to cytosine of DNA during modification process. Fractionation of these proteins in polyacrylamide gel in the presence of sodium dodecylsulphate has revealed that three of them migrate as head structural proteins, and the rest--as internal phage CD protein. Possible mechanisms of the effect of phage protein on the reaction of DNA cytosine residues with O-methylhydroxylamine are discussed.

[Sensitivity of B- and C-DNA forms to modifications by O-methylhydroxylamine]. / O chuvstvitel'nosti B- i s-formDNK k modifikatsii o-metilgidroksilaminom

Effects of high concentrations of lithium, cesium and ammonium chlorides on the reaction ability of free CMP and cytosine in free DNA of CD phage with respect to O-methylhydroxylamine (OMHA) are studied. CMP reaction in all the cases takes place mainly for 24 hours. Like classical B-form, native DNA, having C-form in high ionic strength solution (as estimated from circular dichroism data), is not modificated. Thus, the access of some cytosine residues in intraphage DNA to OMHA is due not to the decreased DNA hydratation in situ, but to the presence of differently ordered regions in DNA.