[Poised RNA polymerase II and master regulation in Metazoa].

This review is devoted to the mechanisms of transcriptional pause and poised state of RNA polymerase II. Features of poised promoters and chromatin are considered in brief. Role of regulated transcriptional pause as discrete and important stage in regulation of master genes that determine stem-cell differentiation, cell lineage and development in Metazoa is discussed. This work was supported by the Russian Science Foundation (project No. 14-50-00131).

Cancer specificity of promoters of the genes involved in cell proliferation control.

Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a "head-to-head" position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.

[Prediction of a non-small cell lung cancer sensitivity to cisplatin and paclitaxel based on the marker genes expression].

The goal of the present study was to define gene expression signatures that predict a chemosensitivity of non-small cell lung cancer (NSCLC) to cisplatin and paclitaxel. To generate set of candidate genes likely to be predictive a current knowledge of the pathways involved in resistance and sensitivity to individual drugs was used. Forty four genes coding proteins belonging to following categories: ATP-dependent transport proteins, detoxification system proteins, reparation system proteins, tubulin and proteins responsible for its synthesis, cell cycle and apoptosis proteins were considered. Eight NSCLC cell lines (A549, Calul, H1299, H322, H358, H460, H292, and H23) were used in our study. For each NSCLC cell line a cisplatin and paclitaxel chemosensitivity as well as an expression level of 44 candidate genes were evaluated. To develop a chemosensitivity prediction model based on selected genes expression level a multiple regression analysis was performed. The model based on the expression level of 11 genes (TUBB3, TXR1, MRP5, MSH2, ERCC1, STMN, SMAC, FOLR1, PTPN14, HSPA2, GSTP1) allowed us to predict the paclitaxel cytotoxic concentration with high level of correlation (r = 0.91, p < 0.01). However, none model developed was able to reliably predict a sensitivity of the NSCLC cells to cisplatin.

[Expression of FTL and FTH genes encoding ferretin subunits in lung and renal carcinomas].

The level of ferritin in serum is known to be increased frequently in most human cancers. Ferritin consists of the heavy and light chains, encoded by FTL and FTH genes. The analysis of the EST database showed that the level of FTL and FTH mRNA is decreased in lung squamous cell carcinomas as compared to the normal tissues, no change in the mRNA level was observed in clear cell renal cell carcinoma. Using real-time PCR we estimated the mRNA level of these genes in primary tumors. It was shown significant and frequent decrease of FTL and FTH mRNA level in lung squamous cell carcinoma: on the average by 11 and 9 times in 83% (33/40) and 73% (11/15) of cases, respectively. In clear cell renal cell carcinoma the changes were not so marked both with respect to the level of decrease (on the average 6 and 3 times) and to its frequency (58 and 27%). In the present work it has been shown for the first time that the FTL mRNA is frequently down-regulated even at the early stages of lung squamous cell carcinoma in all studied samples. This fact permits to consider this gene as potential oncomarker of early diagnosis. The FTL mRNA content may be quantified by non-concurrent hybridization on expression DNA microarrays. The possible causes of a serum ferritin increase in lung cancer and renal cancer are discussed.

New mutations in the human p53 gene--a regulator of the cell cycle and carcinogenesis.

Mutations in the tumor suppressor gene p53 often lead to disarrangement of the cell cycle and of genetic integrity control of cells that may contribute to tumor development. We studied p53 gene mutations in 26 primary tumors of colorectal cancer patients. Mutations in p53 were found in 17 tumors (65.4%). All point mutations affected the DNA binding domain of p53 and were localized in exons 4-8 of the gene. Mutant p53 isoforms with altered domain structure and/or with alternative C-terminus arising from frameshift mutations or abnormal splicing were found in six tumors. Mutations Leu111Gln and Ser127Phe were shown in colorectal cancer for the first time. Isoforms p53-305 with C(4) insertion in codons 300/301 and p53i9* including an additional 44 nucleotides of the 3 -end of intron 9 were discovered for the first time. Mutations of p53 were associated with lymph node metastases and III/IV stage of tumors that are signs of unfavorable prognosis in colorectal cancer.

[Detection of single base polymorphism in p53 gene by ligase detection reaction and rolling circle amplification on microarrays].

We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.

[Deletion of YNZ22 and ALU-VPA/MYCL1 loci in human colonic adenocarcinoma and postoperative prognosis]. / Deletsii lokusov YNZ22 i ALU-VPA/MYCL1 v adenokartsionomakh tolstoi kishki cheloveka i posleoperatsionnyi prognoz.

Since deletions of the short arm of chromosome 17 are the most common genetic defects in human colorectal carcinoma (CC), we tested the YNZ22 locus (D17S30, 17p13.3) for loss of heterozygosity (LH) in adenocarcinoma and in the normal colonic mucosa of 49 CC patients, and studied the association of LH with clinicomorphological features of the tumor. Allele frequency distribution of YNZ22 did not differ for the patients and healthy people. LH in YNZ22 in the tumor was found in 33% (13/39) of all informative cases, its frequency being thrice higher in men than in women (chi 2 = 5.21, p = 0.022). The defect was associated with moderate or poor histological differentiation (P2 = 0.0055) and polyploidy > 3n (P2 = 0.0035) of tumor cells and with high incidence of post-surgery relapse or metastasis. Analysis of both YNZ22 and Alu-VpA/MycL1 (1p34.3) loci in the tumor allowed reliable relapse prognosis in 76% of the CC patients. The probability of post-surgery relapse or metastasis was estimated at no less than 67% for patients with LH in at least one of the two loci in the tumor, and at somewhat more than 20% for patients without LH.

[Genetic alterations in the region of the p53 gene on human chromosome 17 in colorectal cancer]. / Geneticheskie narusheniia 17-i khromosomy cheloveka v oblasti gena p53 pri rake tolstogo kishechnika.

Most colorectal tumors are characterized, among other genetic alterations, by allele loss of the genes located on the short arm of chromosome 17 (17p13.1), including the p53 suppressor gene. In ovarian and mammary-gland tumors, deletions of another candidate tumor-suppressor gene, located in the 17p13.3 chromosome region, were observed. We analyzed allele losses in the loci of the short arm of chromosome 17 (YNZ22, MCT35.1, and the p53 gene) in colorectal-cancer patients from the former Soviet Union. Tumors with cytogenetic alterations in 17p and/or with a detected loss of heterozygosity at the YNZ22 (D17S30) locus were examined for allele losses in the p53 gene using two polymorphic sites. Different methods revealed alterations on 17p in 24 (48%) out of 50 patients with colorectal carcinomas. In all tumors with an allele loss of the YNZ22 marker (15 out of 44 informative cases), which was detected by means of PCR, allele loss of the p53 gene was found (12 out of 15 informative cases). In 5 out of 13 tumors with cytogenetic alterations in 17p, allele loss of the p53 gene was found, with the YNZ22 marker being unaffected. In one of these tumors, the i(17q) marker was found, and in the remaining four tumors, 17p translocations were detected. In 4 out of 5 tumors with translocations affecting 17p, the t(17;20)(q21;p12) translocation was detected. The informativeness of the screening for 17p translocations, using PCR for the YNZ22 locus, and the reasons for discrepancy between the data of PCR and cytogenetic analyses are discussed.

Histo-blotting: hybridization in situ detection of specific RNAs on tissue sections transferred on nitrocellulose.

A simple and rapid variant of in situ hybridization on tissue sections (histo-blotting) usable for detection of specific RNA distribution among tissues is proposed. Tissue sections prepared with a cryostatic microtome are placed on nitrocellulose and these "histo-blots" are hybridized with labelled DNA or RNA probes under conditions of Northern-blot hybridization without any particular pretreatment. Tissue specificity of the RNA distribution may be determined by comparison of autoradiograms with the histological structure of the stained section. Histological staining and light microscopy may be carried out after hybridization of histo-blots. Hybridization in situ may be easily combined with immunostaining under conditions of immunoblotting. Application of the proposed method is shown for alfa-fetoprotein (AFP) and endogenous provirus (ev-3) RNA detection in rat and chicken embryos, respectively. Histo-blotting results correlate with the distribution of given RNAs among tissues determined by independent methods. Sensitivity, specificity and resolution of histo-blotting have been evaluated and discussed.

[Tissue specificity of expression of k51 gene in rodents]. / Tkanevaia spetsifichnost' ékspressii kena k51 u gryzunov.

A new method for studding gene expression--the histoblotting was used for the detection of k51 gene expression in rodent tissues. We have demonstrated several advantages of this method over in situ hybridization for the study of gene expression in epitelial tissues in particular. The transcription of the new gene k51 was detected in the upper layers of mouse and rat skin during epidermis development before and after rat birth. These data were confirmed by Northern hybridization analysis. We found that the expression of k51 occurs mainly in flat multilayer epithelium and is not simply connected with the degree of days of rat embryo development; it is high in newborn skin and lowers during hair growth. The expression of k51 can serve as a marker of a certain stage of adult skin development.

[Detection of tissue-specific gene expression using an in situ hybridization method of histoblotting]. / Vyiavlenie tkanespetsificheskoi ékspressii genov metodom gibridizatsii in situ na otpechatkakh gistologicheskikh srezov.

A version of in situ hybridization on the histological sections that is used for screening specific mRNA in tissues in proposed. Sections of the frozen tissue samples are prepared on the cryostatic microtome, placed on nitrocellulose filters and hybridized with labelled DNA-probes under the conditions of RNA blot hybridization. The proposed method ("histoblotting") was used to study the distribution of actin and alpha-phetoprotein mRNA genes in tissues of 15 and 21-day rat embryos. The possibility of studying mRNA (by hibridizations) and protein (by immunoenzyme staining) and making histological analysis simultaneously by histoblotting is demonstrated.