[Treatment with anti-cancer agents results in profound changes in lncRNA expression in colon cancer cells].

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.

[Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer].

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.

[Expression of genes involved in retinoic acid biosynthesis in human gastric cancer].

All-trans-retinoic acid (ATRA) is the main biologically active metabolite of retinol (vitamin A) that is required for the regulation of such processes as embryogenesis, tissue differentiation, proliferation, and others. Multiple alcohol, retinol and retinaldehyde dehydrogenases (ADHs, RDHs and RALDHs) as well as aldo-keto reductases (AKRs) catalyze the biosynthesis of retinoic acid in humans. For many normal and neoplastic tissues, the key ATRA-synthesizing enzymes remain unknown. We identified ATRA-generating genes that are expressed in normal and malignant gastric tissues using the transcriptomic database analysis. Quantitative changes in the expression levels of these genes in gastric cancer were determined by semi-quantitative RT-PCR and real-time PCR. Significant decreases in the mRNA levels of genes encoding enzymes that catalyze the reversible oxidation/reduction of retinol and retinaldehyde (ADH4, ADH1B, ADH1C, RDHL, AKR1B10, AKR1B1, and RDH12), as well as the oxidation of retinaldehyde (RALDH1) were revealed in most of the tumor samples. The sharp reduction in the expression levels of genes encoding the key enzymes that convert retinol and retinaldehyde to retinoic acid could lead to a significant decrease in the content of ATRA--the transcriptional regulator of many genes, which in turn can lead to a dysregulation of cell proliferation/differentiation and initiate cancer development.

[Downregulation of AKR1B10 gene expression in colorectal cancer].

Colorectal cancer is one of the most common cancers in the world. In our work changes of AKR1B1 and AKR1B10 gene expression levels in colorectal tumors were studied. Their potential diagnostic value was previously shown for several other cancer types. These genes encode aldoso reductases, which belong to the aldo-keto reductases superfamily consisting of enzymes capable to reduce numerous aromatic and aliphatic aldehydes and ketones. They are also involved into retinoid metabolism and cancerogenesis. We have carried out comparative analysis of mRNA levels of AKR1B1 and AKR1B10 genes in paired samples of normal and colorectal tumor tissues using RT-PCR and quantitative PCR. We have shown for the first time the decrease of activity of these genes in colorectal carcinomas. Significant reduction of AKR1B10 mRNA level was detected in the most of tumor samples (88%, 65/74) even at the early stages of malignancy, and in more than 60% of cases this downregulation was much higher than 10 folds. The decrease of AKR1B1 mRNA level was shown in 10% of tumors only. Therefore, we have detected quite different mRNA expression patterns in colorectal cancer for these two structurally similar genes. These data could indicate different functional roles of these two genes in colorectum. The significant decrease of AKR1B10 mRNA in most samples of colorectal cancer could be considered as potential diagnostic marker of this type of cancer.

[Comparison of 2D analysis and bioinformatics search efficiency for colon cancer marker identification].

Modification of 2D analysis protocol was developed, based on preliminary removal of major cellular proteins by extraction with buffer saline and elimination of high molecular weight proteins by gel filtration. This approach allowed identification of 12 proteins with increased expression levels in tumors versus normal tissues. Increase in expression levels of the eight proteins in colon tumors was discovered for the first time. We performed comparison of marker search efficiency by 2D analysis and SAGE in a control panel of 19 putative colon cancer markers, discovered by us previously and at the same time independently identified by other authors. Results of 2D analysis of control panel completely coincided with published data, as compared to search in SAGE database, which allowed identification of only one third of markers.

[Proteomic expression analysis of human colorectal cancer: of soluble overexpressed proteins].

Colon cancer is one of the leading causes of cancer deaths in developed countries due to the absence of tumor specific markers for early diagnosis of the disease, providing adequate sensitivity. Search for diagnostic markers of various types of cancer by proteomic approaches has been limited by large differences in protein centration. We used preliminary extraction of major cellular proteins by 0.2 M sodium chloride in presence of nonionic detergent NP-40 in order to raise the sensitivity of the 2D PAGE detection of low-abundant soluble proteins, some of which may penetrate in blood circulation during carcinogenesis. Application of this procedure prior to 2D comparative analysis of proteomes of normal tissues and matched colon cancer specimens led to selection of ten proteins, which are frequently overexpressed in colon adenocarcinomas. Mass-spectrometric identification of selected proteins led to discovery of two novel protein markers of colon tumors--TAF9 and CISH. Low level of CISH expression in various tissues suggests that it is a novel prospective marker for diagnosis of colon cancer.

[Colorectal cancer 2D-proteomics: identification of altered protein expression].

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumor in comparison to normal tissues. Despite the wide application of proteomics in current studies, identification of proteins with stable concentration differences in normal and cancer cells remains rather difficult. The current study was directed to the search of new potential protein colorectal cancer markers using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. This widespread neoplasm is characterized by lack of evident symptoms at early stages of cancerogenesis. It is highly important to develop fast and sensitive methods of molecular diagnostics. We studied paired cancerous and normal clinical tissue samples from 11 patients with colorectal adenocarcinomas by comparative 2-D PAGE and MALDI-TOF mass-spectrometry identification. Sixteen proteins with stable differential expression were selected and identified, including 13 overexpressed and 3 downregulated proteins. In summary, we describe the discovery overexpression of GPD1 and RRBP1 proteins and lack of expression for HNRNPH1 and SERPINB6 proteins which are new candidate biomarkers of colon cancer.

[Transcription TIMP3, DAPk1 and AKR1B10 genes in squamous cell lung cancer].

Lung cancer is one of the most frequent neoplasia in the Russia, the United States and Europe. This cancer is associated with functional activity changes of many genes. In the present study TIMP3, DAPK1 and AKR1B10 genes transcription analysis of squamous cell lung cancer specimens was carried out using reverse transcription-PCR. Substantial increasing of AKR1B10 transcription level is revealed in 80% tumor samples. TIMP3 and DAPK1 transcription level is considerably decreased in 76 and 72% tumor specimens, accordingly. These results may point out that all three genes are important for squamous cell lung cancer tumorogenesis while AKR1B10 is potential oncogene whereas TIMP3 and DAPK1 are potential tumor suppressor genes. We suggest that revealed substantial transcription level-changes of investigated genes may be used for oncodiagnostics.

[Segment duplications in the human genome]. / Segmentnye duplikatsii v genome cheloveka.

The review considers the structure, evolution, and possible mechanisms of spreading of intrachromosomal and interchromosomal segment duplications (SD), which account for more than 5% of the human genome. Most SD are mosaic and consist of multiple modules, which occur in several copies in different genome regions. SD are preferentially located in pericentric and subtelomeric regions, which are least studied on the human chromosomes. Homologous recombination between SD results in various chromosome rearrangements, contributing to the genome instability and the origin of several human hereditary disorders.

[Tandem and interspersed repeats contribute to the mosaic structure of segmented duplications in the human genome]. / Mozaichnaia struktura segmentnykh duplikatsii v genome cheloveka obrazuetsia pri uchastii tandemnykh i dispergirovannykh povtorov.

Intrachromosomal and interchromosomal segmental duplications account for more than 5% of the human genome. To analyze the processes resulting in the complex mosaic structure of duplicons, a draft human genome sequence was searched for duplicated segments of a genomic fragment of the pericentric region of the chromosome 21 short arm. The duplicons found consist of modules having paralogs in various genome regions. Module ends are flanked with various tandem or interspersed repeats, which are more unstable as compared with unique sequences. In most cases, the boundaries of duplicated segments exactly coincide with or are in close proximity to hot spots of various rearrangements within repeats or boundaries between repeats and unique sequences or between two different repeats. Homologous recombination between repetitive elements was assumed to be the major mechanism contributing to the mosaic structure of duplicons.

Type D retrovirus specific sequences in lymphocytes of the children with Burkitt-type lymphoma and their parents.

Type D retroviruses cause immunodeficiency in monkey. Earlier we have revealed genetical and serological markers of type D retroviruses in children with Burkitt-type lymphoma. Using PCR/Southern blotting assay we have found sequences related to MPMV in PBMC's DNA from children with Burkitt-type lymphoma and from their parents. Moreover, the data on sequencing of virus specific sequences from one ill child and from his mother have been presented.

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites.

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich alpha21-I and the alpha21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (alpha21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.

Evidence for selection in evolution of alpha satellite DNA: the central role of CENP-B/pJ alpha binding region.

Conservation of DNA segments performing sequence-related functions is a landmark of selection and functional significance. Phylogenetic variability of alpha satellite and apparent absence of conserved regions calls its functional significance into question, even though sequence-specific alpha satellite-binding proteins pJ alpha and CENP-B have been discovered. Moreover, the function of pJ alpha is obscure and CENP-B binding satellite DNA, which is thought to participate in centromere formation, is found only in few species and not necessarily in all chromosomes. Analysis of alpha satellite evolution allows us to recognize the order in this variability. Here we report a new alpha satellite suprachromosomal family, which together with the four defined earlier, covers all known alpha satellite sequences. Although each family has its characteristic types of monomers, they all descend from two prototypes, A and B. We show that most differences between prototypes are concentrated in a short region (positions 35 to 51), which exists in two alternative states: it matches a binding site for pJ alpha in type A and the one for CENP-B in type B. Lower primates have only type A monomers whereas great apes have both A and B. The new family is formed by monomeric types almost identical to A and B prototypes, thus representing a living relic of alpha satellite. Analysis of these data shows that selection-driven evolution, rather than random fixation of mutations, formed the distinction between A and B types. To our knowledge, this is the first evidence for selection in any of the known satellite DNAs.

Cloning of a cDNA encoding a human protein which binds a sequence in the c-myc gene similar to the interferon-stimulated response element.

A human cDNA clone encoding a c-myc promoter-binding protein (IRLB) was selected by screening a human fibroblast lambda gt11 phage library with the hexamer oligodeoxyribonucleotide (oligo) 5'-GGCGGGAAAAAGAACGGA, corresponding to the protein-binding element of human c-myc similar to the interferon-stimulated response element (ISRE). The lambda gt11 phage clone, encoding a fusion protein which bound the probe oligo, was used to create an strain of Escherichia coli. The deduced amino-acid sequence of the cloned protein contains a putative alpha-helix which is expected to act as the DNA-binding domain. DNase footprinting analysis and oligo-binding specificity assays showed that the cloned factor recognizes the ISRE-like element of the P2 promoter region of human c-myc.

Genomic organization, sequence and polymorphism of the human chromosome 4-specific alpha-satellite DNA.

Two alpha-satellite fragments specific for human chromosome 4 have been cloned and characterized. Under stringent annealing conditions, they hybridized in situ only to the pericentromeric region of chromosome 4, but under non-stringent conditions they hybridized to all chromosomes containing the sequences of alpha-satellite suprachromosomal family 2 (viz., chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21 and 22). Southern blot analysis reveals the 3.2-kb higher-order repeated unit which exists in two forms: as a single MspI fragment or a combination of the 2.6-kb and 0.6-kb MspI fragments. The two chromosome-4-specific cloned sequences appear to be different parts of this repeated unit. Taken together they constitute about 60% of its length. The primary structure of the higher-order repeated unit is characterized by a dimeric periodicity of the D1-D2 type which is usual to suprachromosomal family 2. At least in one site this regularity is disrupted by monomer deletion leading to the D2-D2 monomeric order. The most likely mechanism of this monomer excision is homologous unequal crossing-over. These sequences may serve as both cytogenetic and restriction-fragment length polymorphism (RFLP) markers for the pericentromeric region of chromosome 4.

Definition of a new alpha satellite suprachromosomal family characterized by monomeric organization.

We have analyzed more than 500 alphoid monomers either sequenced in our laboratory or available in the literature. Most of them belonged to the well studied suprachromosomal families 1, 2 and 3 characterized by dimeric (1 and 2) and pentameric (3) ancestral periodicities. The sequences that did not belong to the previously known families were subjected to further analysis. About a half of them formed a relatively homogenous family. Its members were on average 80.5% identical and 89.5% homologous to the M1 consensus sequence derived from this group (39 monomers). In the genome they do not form any ancestral periodicities other than a monomeric one, and are found at least in chromosomes 13, 14, 15, 21, 22 and Y. The newly defined family was termed suprachromosomal family 4. Comparison of all 10 alphoid monomeric groups identified so far showed that the M1 sequence is closely related to the J1-D2-W4-W5 homology grouping. Notably the African Green Monkey alpha satellite, also characterized by monomeric construction, appears to be a member of the same group.

Segment substitutions in alpha satellite DNA. Unusual structure of human chromosome 3-specific alpha satellite repeat unit.

We have sequenced the full-length copy of the alpha satellite higher-order repeated unit characteristic of human chromosome 3. Its internal structure, the regular alterations of J1 and J2 type monomers, is typical of the alphoid suprachromosomal family 1. This dimeric order is disrupted by the substitution of one J1 unit by an alien dimer which is not clearly related to any of the established monomeric types. We have also observed some other similar cases of segment substitutions in alpha satellite DNA. They probably represent a special type of molecular event which could be generated by gene conversion. Segment substitutions may be one of the important factors responsible for the extreme variability of localization patterns and actual sequences of alpha satellite DNA that should be taken into account in reconstructions of alpha satellite evolution.

Chromosome-specific alpha satellites: two distinct families on human chromosome 18.

Two types of human chromosome 18-specific alpha satellite fragments have been cloned and sequenced. They represent closely related but distinct alphoid families formed by two different types of the higher-order repeated units (1360-bp EcoRI and 1700-bp HindIII fragments) that do not alternate in the genome. The individual repeats within each family are 99% identical and interfamily homology is about 78%. Sequence analysis shows that both repeats belong to alphoid suprachromosomal family 2, but their homology is not higher than that of family members located on different chromosomes. Therefore, the two repeats shared a common origin in the recent past, although they are not the direct offspring of one ancestral sequence. Our data indicate that these two 18-specific domains have appeared as a result of two separate amplification events. Despite the high degree of homology, they are not undergoing intrachromosomal homogenization, although some variation of this process might take place within each domain.