miR-155 as a diagnostic and prognostic marker in hematological and solid malignancies.

MicroRNAs (miRNAs) are small RNAs that have emerged as potent regulators of the target genes messenger RNAs expression in the response of cell to both physiological and pathophysiological conditions. Reflecting pathological processes today, miRNAs are widely validated for their potential role in diagnostic, prognostic and novel therapeutic targeting for cancerous and other diseases. miR-155 is considered as a typical multifunctional miRNA including its role as oncomiR (cancer-associated miRNA). Expression of miR-155 is upregulated in cells with high proliferative activity and decreased apoptotic capability. It belongs to cluster of well-characterized tumor associated miRNAs detectable also in the peripheral blood. In this review we summarize the principles of miR-155 host gene expressional regulation, as well as its role in regulation of the target genes mRNAs. Altered expression of miR-155 has been described in multiple cancerous and other diseases, reflecting staging, progress and treatment outcomes. Therefore, miR-155 became a potential biomarker and candidate for clinical utilization as predictor of the presence of cancer, its staging and prognosis.

S100P, a peculiar member of S100 family of calcium-binding proteins implicated in cancer.

S100P belongs to several members of the S100 family of calcium-binding proteins, associated with malignant phenotype. Altered levels of S100P expression have been described at different stages and types of cancer. Transcriptional regulation involves different pathways activated by glucocorticoids, growth factors and bone morphogenic factor via the corresponding receptors. Signals coming from these pathways appear to be transmitted through ERK1/2 (extracellular-signal regulated kinase) and mediated presumably by STAT, SMAD, NFkB transcription factors. The secreted form of S100P can bind to extracellular ligand-binding site of RAGE (receptor for advanced glycation end-products), and via activation of ERK/MAPK pathway can influence gene expression, cell proliferation and survival. In addition, S100P interacts and modulates the activity of several targets with multiple binding modes and simultaneous coordination of further target proteins in larger multiprotein complexes, e.g. scaffolding proteins -IQGAP1 and ezrin, known to promote and regulate signal transduction pathways. The majority of S100P binding partners are proteins involved in cytoskeletal dynamics, and their physical interactions with S100P lead to defects in cellular morphogenesis and tissue disruption, the acquisition of uncontrolled migratory and invasive features. Finally, the evidence for S100P role in cancer metastasis opens a new direction for the future research efforts.

Expression of S100P gene in cervical carcinoma cells is independent of E7 human papillomavirus oncogene.

High-risk human papillomaviruses (HPV) significantly contribute to development of cervical cancer. HPV E7 oncoprotein interferes with the control of cell growth via functional inactivation and/or regulation of multiple molecular targets. Induction of ectopic E7 in breast carcinoma cells has been proposed to decrease transcription of S100P gene, which encodes a calcium-binding protein associated with different types of tumors. We examined a possible relationship between E7 and S100P genes in cervical cell lines. RT-PCR analysis revealed that all HPV-positive cell lines expressed approximately equal levels of E7. Out of them, HeLa, CGL3 and SiHa carcinoma cells as well as HCE16/3 immortalized cells expressed also S100P gene. Inhibition of a DNA methylation by 5-aza-2'-deoxycytidine (5-aza-dC) in S100P-negative cell lines CGL1 and Caski resulted in induced transcription of S100P, but the normal S100P level in SiHa cells was not further increased. Our results suggest that S100P gene expression is independent of E7 in cervical cell lines and that at least in some cases it is subjected to regulation by DNA methylation.

Interferon alpha 2b but not interferon alpha 2a detected in human genomic DNA.

The presence of the genes for human interferon (IFN)-alpha 2a and IFN-alpha 2b subvariants in human genomic DNA was studied using polymerase chain reaction (PCR). The respective genes differ in the coding sequence only at the position 137, adenine (2A) being substituted by guanine (2B). IFN-alpha 2-sequences were selectively amplified from the placental genomic DNA using specific primers. When sequencing the mixture of PCR-derived clones, at the position 137 guanine, specific for subvariant 2b could be detected. This indicates that, at least in the genome analyzed, only sequence coding for IFN-alpha 2b was found. Our finding supports the view, that genes for IFN-alpha 2a and IFN-alpha 2b seem to be rather allelic than occurring at distinct loci.

Interferon alpha2b is the predominant subvariant detected in human genomic DNAs.

Chromosomal DNAs isolated from eight individuals from the Slovak population and from lymphoblastoid Namalwa cells were analyzed for the presence of genes coding for three subvariants of human interferon-alpha 2 (IFN-alpha 2), namely a, b, and c. The respective genes are regarded allelic, because they differ in the coding nucleotide sequence only at the position 137 (a:A, b/c:G) and/or at the position 171 (a/b:A, c:G). IFN-alpha 2 sequences in genomes were selectively amplified using polymerase chain reaction (PCR). Resulting "consensus" PCR-product (the total mixture of PCR-derived clones) was sequenced and the subvariant-specific nucleotides at position 137 and 171 were determined. In one placental genomic DNA and in a mixture of genomic DNAs from leukocytes of seven donors only nucleotides specific for subvariant IFN-alpha 2b could be detected. This suggests that the placental DNA contained only genes coding for IFN-alpha 2b and these alleles were at least prevailing in donor's genomes. On the other hand, the majority of genomic alpha 2-sequences in Namalwa cells (from which IFN-alpha 2c was originally derived), seems to be corresponding to subvariant IFN-alpha 2c.

Sequence and characterisation of the Z gene encoding ring finger protein of the lymphocytic choriomeningitis virus MX strain.

We have cloned and characterised a cDNA encoding Z protein of recently identified MX strain of lymphocytic choriomeningitis virus (LCMV) persistently infecting human MaTu cells. Deduced amino acid sequence of LCMV MX Z protein showed 88.9% identity with that of the LCMV Armstrong (ARM) strain and 80.9% identity with that of the LCMV Traub (TRA) strain. It contained conserved zinc-binding RING finger domain and C-terminal proline-rich region. Northern blot analysis of total RNA from MaTu cells revealed presence of abundant truncated forms of L RNA. Z protein-specific rabbit antibodies were produced to glutathione S-transferase (GST)-Z fusion protein expressed in E. coli and used for the detection of Z protein in MaTu cells. Western blot and immunofluorescence analyses detected relatively high levels of Z protein indicating its role in maintenance of persistent LCMV.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8-9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Human MN/CA9 gene, a novel member of the carbonic anhydrase family: structure and exon to protein domain relationships.

We have isolated, sequenced, and characterized a human MN/CA9 gene. This gene is a novel member of the carbonic anhydrase (CA) family, which codes for widely distributed catalysts of the reversible conversion of carbon dioxide to carbonic acid. So far, MN/CA IX is the only tumor-associated CA isoenzyme. The entire genomic sequence of MN/CA9, including the 5'-flanking region, encompasses 10.9 kb. The coding sequence is divided into 11 exons, whose organization and relationships to predicted protein domains suggest that the gene arose by exon shuffling. Exon 1 encodes a signal peptide and a proteoglycan-related region. Exons 2-8 code for a CA domain with a highly conserved active site. The exon/intron pattern of the CA coding region is similar but not identical to other described animal kingdom alpha-CA genes. Exons 10 and 11 encode a transmembrane anchor and an intracytoplasmic tail, respectively. We have also determined the transcription initiation and termination sites by RNase protection assay and analyzed the 3. 5-kb region upstream of the MN/CA9 gene. Sequence of the proximate 5' end of the flanking region shows extensive homology to the long terminal repeats of HERV-K endogenous retroviruses. The putative MN/CA9 promoter immediately preceding the transcription start site does not possess a TATA box, but contains consensus sequences for the AP1, AP2, p53, and Inr transcription factors. This study will allow further investigations of the molecular events regulating expression of MN/CA IX as well as elucidation of its biological function.

Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element.

The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3. 5-kilobase pair MN 5' upstream region by deletion analysis led to the identification of the -173 to +31 fragment as the MN promoter. In vitro DNase I footprinting revealed the presence of five protected regions (PRs) within the MN promoter. Detailed deletion analysis of the promoter identified PR1 and PR2 (numbered from the transcription start) as the most critical for transcriptional activity. PR4 negatively affected transcription, since its deletion led to increased promoter activity and was confirmed to function as a promoter-, position-, and orientation-independent silencer element. Mutational analysis indicated that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were found to be in direct contact with PR4 by UV cross-linking. Increased cell density, known to induce MN expression, did not affect levels of PR4 binding in HeLa cells. Significantly reduced repressor level seems to be responsible for MN up-regulation in the case of tumorigenic CGL3 as compared with nontumorigenic CGL1 HeLa x normal fibroblast hybrid cells.