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Overcoming drug resistance of cancer cells is the major challenge in molecular oncology. Here, we demonstrate that long non-coding RNA LINC00973 is up-regulated in normal and cancer cells of different origins upon treatment with different chemotherapeutics. Bioinformatics analysis shows that this is a consequence of DNA damage response pathway activation or mitotic arrest. Knockdown of LINC0973 decreases p21 levels, activates cellular proliferation of cancer cells, and suppresses apoptosis of drug-treated cells. We have found that LINC00973 strongly increases p21 protein content, possibly by blocking its degradation. Besides, we have found that ectopic over-expression of LINC00973 inhibits formation of the pro-survival p53-Ser15-P isoform, which preserves chromosome integrity. These results might open a new approach to the development of more efficient anti-cancer drugs.

Using RNA-seq, RT-qPCR, and bioinformatics we have studied the influence of a wide spectrum of chemotherapeutic drugs on transcription of AKR1B10 , AKR1C1 , ALDH1A1 , and ALDH1A3 genes, which encode the major aldehyde-metabolizing enzymes. The strongest alterations were detected in case of AKR1B10 mRNA that was significantly upregulated in wild type p53 cancer cells, but downregulated in mutant p53 cancer cells. Subsequent experiments demonstrated the significant and consistent decrease in the AKR1B10 mRNA content in sera of colon cancer patients, as compared to sera of healthy donors (p<0.0001, SPE=92.9%, SNE=79.3%, AUC=0.889), which implies that this RNA is a valuable marker for serological diagnosis of colorectal cancer. Moreover, we have found that ALDH1A3 protein is a key inactivator of ROS-generated aldehydes, which is a perspective target for the development of new chemotherapeutic drugs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Early prediction of tumor relapse depends on the identification of new prognostic cancer biomarkers, which are suitable for monitoring tumor response to different chemotherapeutic drugs. Using RNA-Seq, RT-qPCR, bioinformatics, and studies utilizing the murine tumor xenograft model, we have found significant and consistent changes in the abundance of five lincRNAs (LINC00973, LINC00941, CASC19, CCAT1, and BCAR4) upon treatment of both HT-29 and HCT-116 cells with 5-fluorouracil, oxaliplatin, and irinotecan at different doses and durations; both in vitro and in vivo. The most frequent changes were detected for LINC00973, whose content is most strongly and consistently increased upon treatment of both colon cancer cell lines with all three chemotherapeutic drugs. Additional studies are required in order to determine the molecular mechanisms by which anticancer drugs affect LINC00973 expression and to define the consequences of its upregulation on drug resistance of cancer cells., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Using real-time RT-PCR in combination with bioinformatics, we have shown for the first time that the treatment of HCT-116 and HT-29 colon cancer cells with two anti-cancer agents (doxycycline or 3,3'-diindolylmethane) results in profound changes in the intracellular content of several lncRNAs (by up to 100 times). Since many of these RNAs are secreted by tumors into the bloodstream, the obtained results provide a basis for developing more sensitive protocols for serological monitoring of tumor relapse and metastasis, as well as for search of new anti-cancer drugs.

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

The review describes the changes observed in long noncoding RNA (lncRNA) content and function at various stages of carcinogenesis, as well as the prospects of lncRNA application in cancer prognosis.

This review describes current methods of protein immunoanalysis: radioimmunoanalysis, ELISA, immuno-PCR, electrochemical analysis and chromatin immunoprecipitation, as well as main areas of their application.

The aim of this study was to identify new protein markers of the intestinal and diffuse type gastric adenocarcinoma and to determine their relation to local relapses and distant metastasis. Using two-dimensional gel electrophoresis, we searched for proteins that are overexpressed in the intestinal and/or diffuse type gastric adenocarcinoma, as compared to matched normal mucosa samples with further change confirmation by Western blot. Expression of the selected proteins was further assessed by immunohistocemistry in a large panel of gastric adenocarcinoma with various clinicopathological features. Expression level of cyclophilin A measured with western blot appeared to be increased on average ten times in 63 % of gastric adenocarcinoma vs. paired samples of normal mucosa. The frequency of immunihistochemistry detected cyclophilin A protein expression was found to be equal in tumor of both histotypes, but staining intensity was higher in intestinal versus diffuse types of gastric adenocarcinoma. cyclophilin A protein expression appeared to be lower in deeply invading glandular and cribriform structures of intestinal tumors, as well as in discretely placed groups of the intestinal tumor cells. Local relapses as well as distant metastases registered within 3 year follow up were observed to occur much less frequently in patients with positive cyclophilin A immunostaining in gastric tumors. Analysis of cyclophilin A expression has a potential value for prognosis of gastric adenocarcinoma recurrence and distant metastasis.

Anew immuno-PCR format is described that is based on detection of membrane protein CDH17 in serum exosomes. Format application allows distinction between sera samples of healthy donors and colon cancer patients. Obtained results open a possibility of serological colon cancer diagnosis in high risk groups.

This review summarizes current knowledge on the role of tumor exosomes and microvesicles in progression, metastasis, and angiogenesis of tumors, as well as in suppression of adaptive and innate immunity.

This review describes the most popular methods of search for serological markers of tumors that are used in clinical setting, provided with comparison of their efficiency.

It was demonstrated that enteric alpha-defensin 5 is undetectable in five blood serum samples of healthy donors, whereas its processed form is present in two out of five serum samples of colon cancer patients. Obtained results open a possibility of serological diagnosis of colon tumors in high risk cancer patients.

A new construct of DNA reporter has been designed for protein quantification by immuno-PCR. It has been shown that amplification efficiency of a reporter that contains a fragment of human adenovirus 2 flanked by homoprimer sequences is much higher vs. standard PCR format based on use of two different primers. Application of a new construct and its homoprimer-based detection opens a way to a significant increase in the immuno-PCR sensitivity and the efficiency of single molecule PCR.

Microbes living in the mammalian gut exist in constant contact with immunity system that prevents infection and maintains homeostasis. Enteric alpha defensins play an important role in regulation of bacterial colonization of the gut, as well as in activation of pro- and anti-inflammatory responses of the adaptive immune system cells in lamina propria. This review summarizes currently available data on functions of mammalian enteric alpha defensins in the immune defense and changes in their secretion in intestinal inflammatory diseases and cancer.

This review summarizes currently available data on enteric alpha defensins structure, their functions in the innate and adaptive immunity systems and the role in development of intestinal illnesses.

Comparison of protein expression in intestinal and diffuse stomach tumors by 2D gel electrophoresis led to identification of three proteins (SOD2, S100A6, and TXN), which are overexpressed in tumors as compared to normal controls. It was shown, that overexpression of proteins SOD2 and TXN occurs much more frequently in diffuse tumors than in intestinal ones. A control panel of eleven proteins overexpressed in stomach tumors has been selected based on the data of comparative 2D analysis described in the literature. Bioinformatics search for mRNAs encoding proteins from the control panel in Oncomine database (which contains the results of determination of mRNA transcription level in tumor vs. normal samples) demonstrated the coincidence of proteomic and transcriptomic data for seven out of 11 proteins.

A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.

LRP1B is a novel candidate tumor suppressor gene that is inactivated by genetic and transcript alterations in nearly 50% of non-small-cell lung cancer cell lines. The gene-encoded protein is highly homologous to the gigantic lipoprotein receptor-related protein 1 (LRP1) that belongs to the family of low-density lipoprotein receptors. Using a combination of PCR-based genome walking and long-distance interexon PCR, we have determined the genomic organization of LRP1B and built a contiguous array of BAC clones spanning this gene. A total of 91 exons, varying in size from 77 bases (exon 87) to 1899 bases (exon 91), were identified in the more than 500-kb-long gene sequence. Comparative analysis of the genomic structures of LRP1B and the homologous LRP1 gene revealed a striking similarity in the location and sizes of their exons., (Copyright 2000 Academic Press.)

The family of tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptors, including the pro-apoptotic DR4 and p53-regulated KILLER/DR5, as well as the decoys TRID and TRUNDD, are all located on human chromosome 8p21-22. This region of the genome is frequently altered in head and neck cancer. We previously reported that KILLER/DR5 can be mutationally inactivated in head and neck cancer. Here, we report that the FaDu nasopharyngeal cancer cell line contains an abnormal chromosome 8p21-22 region. In addition, there appears to be a homozygous deletion involving DR4 but not KILLER/DR5 in FaDu cells. The homozygous loss within the DR4 gene encompasses its death domain, which is required for apoptotic signaling. The deletion of DR4 in FaDu cells is associated with resistance to the cytotoxic effects of TRAIL. Re-introduction of wild-type DR4 leads to apoptosis and restores TRAIL sensitivity of FaDu cells. These observations suggest that the death inducing DR4 receptor gene may be a rare target for inactivation in human cancer and that DR4 loss may contribute to resistance to TRAIL therapy.

A variety of studies suggest that allelic losses at chromosome 2q are associated with aggressive behavior of various forms of human neoplasia. Using a probe to detect homozygous deletions on chromosome 2q21.2 in kidney and bladder cancer cell lines, we identified a new candidate tumor suppressor gene, lipoprotein receptor-related protein-deleted in tumors (LRP-DIT). The predicted LRP-DIT product of 4599 amino acids has extensive homology to a gigantic receptor, LRP1, which mediates endocytosis of multiple proteins from the cell surface. Homozygous deletions in LRP-DIT were detected in 17% (4 of 23) of non-small cell lung cancer (NSCLC) cell lines. The expression of only abnormal transcripts missing portions of the LRP-DIT sequence was demonstrated in an additional 30% (11 of 36) of NSCLC lines. Finally, a missense mutation at codon 3157 was detected in one of four NSCLC lines tested for the large open reading frame. In contrast, no LRP-DIT alterations were identified in a major fraction of SCLC cell lines, indicating that this gene is preferentially inactivated in one histological type of lung cancer. Our data suggest that inactivation of LRP-DIT occurs in at least 40% of NSCLC lines and thus may play an important role in tumorigenesis of NSCLCs.

Representational difference analysis (RDA) was used to generate Y-specific probes by enriching for and cloning the differences between the male (XY) and the female (XX) C57BL/6J mouse genomes. Characterization of 35 clones revealed 12 families related by sequence similarity. One clone from each family was chosen for detailed analysis by Southern blot hybridization, polymerase chain reaction (PCR) on normal and aberrant genomes (Sxr), and fluorescence in situ hybridization. From one difference product we have characterized 12 Y-specific probes for hybridization, created seven male-specific PCR assays, mapped all repeat families, and identified one repeat with a distinct XY homology. We report the first cloning of a Y-specific long interspersed repeat element (LINE) fragment. In total, RDA has identified six novel Y Chromosome repeat families and allowed us to extend the characterization of six known Y repeats. We conclude that this novel use of RDA for whole chromosome subtraction successfully enriches chromosome-specific sequences and is suitable for the rapid generation of new Y Chromosome-specific probes.

A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.

Representational difference analysis (RDA) is an efficient method for finding the differences between complex genomes. The numerous applications of RDA include the cloning of probes for the detection of genetic lesions in cancer, the identification of sequences from the genomes of unknown pathogens and the rapid isolation of polymorphic markers linked to a trait without the use of pre-existing genetic maps.

We demonstrate the use of representational difference analysis for cloning probes that detect DNA loss and amplification in tumors. Using DNA isolated from human tumor cell lines to drive hybridization against matched normal DNA, we were able to identify six genomic regions that are homozygously deleted in cultured cancer cells. When this method was applied in the reverse way, using normal DNA to drive hybridization against tumor cell DNA, we readily isolated probes detecting amplification. Representational difference analysis was also performed on DNAs derived from tumor biopsies, and we thereby discovered a probe detecting very frequent homozygous loss in colon cancer cell lines and located on chromosome 3p.

We describe a technique, genetically directed representational difference analysis (GDRDA), for specifically generating genetic markers linked to a trait of interest. GDRDA is applicable, in principle, to virtually any organism, because it requires neither prior knowledge of the chromosomal location of the gene controlling the trait nor the availability of a pre-existing genetic map. Based on a subtraction technique described recently called representational difference analysis, GDRDA uses the principles of transmission genetics to create appropriate Tester and Driver samples for subtraction. We demonstrate the usefulness of GDRDA by, for example, successfully targeting three polymorphisms to an interval of less than 1 cM of the mouse nude locus of chromosome 11.

We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver). Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin. After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer. The method has been applied to purification of fragments from a 2.9 kb plasmid added to E. coli DNA at equimolar quantity. Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR.

A differential library enriched in rapidly evolving human genomic sequences was obtained by phenol-enhanced hybridization of human genomic DNA with an excess of chimpanzee DNA. A DNA fragment 110 bp in length that did not hybridize to either chimpanzee or other primate DNA was identified in this library. It was shown to be a substantially diverged member of the human beta satellite family of tandem repeats. The genomic sequences homologous to the fragment were located on the short arms of human acrocentric chromosomes by in situ hybridization. The human-specific fragment failed to hybridize with RNA from different human tissues. The human-specific fragment exhibits a remarkable level of DNA polymorphism in humans and may be used in the identification of human tissue samples, in the selection of human/rodent somatic cell hybrids containing human acrocentric chromosomes, and in the mapping of these chromosomes.

The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion G----T, leading to amino acid substitution Val146----Phe. This mutational change marks the second domain of the beta subunit involved in rifampicin binding.

Three new rif-r-mutations, obtained independently, were localized in the rpoB gene coding for the beta-subunit of DNA-dependent RNA polymerase of E. coli. Two of them led to identical Asp(516)-Asn amino acid substitution with relatively low resistance of corresponding E. coli strains to rifampicin. The third mutation affected the His 526 residue transforming it into Tyr and endowed the E. coli cells with a high resistance against rifampicin.

For the first time a mutation of streptolydigin resistance was localized. It was discovered to be a double substitution, namely Gly544----Asp, Phe545----Ser, in the region where most rif-r mutations are located. One may suppose that this region takes part in the formation of both elongation NTP binding site, blocked by streptolydigin, and RNA chain binding and translocation site that is blocked by rifampicin.

BglII fragment of S. typhimurium DNA, containing rpoB gene coding for the RNA polymerase beta-subunit, was cloned. The nucleotide sequence of the rpoB gene EcoRI-C fragment (2873 b.p.) was determined.

A bank enriched in sequences specific for the human genome was obtained. In course of the analysis, a clone containing an Alu family repeat was identified and its primary structure determined.

The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its beta subunit.