Molecular characterization of the mouse Tem1/endosialin gene regulated by cell density in vitro and expressed in normal tissues in vivo.

Human tumor endothelial marker 1/endosialin (TEM1/endosialin) was recently identified as a novel tumor endothelial cell surface marker potentially involved in angiogenesis, although no specific function for this novel gene has been assigned so far. It was reported to be expressed in tumor endothelium but not in normal endothelium with the exception of perhaps the corpus luteum. Here we describe the cDNA and genomic sequences for the mouse Tem1/endosialin homolog, the identification and characterization of its promoter region, and an extensive characterization of its expression pattern in murine and human tissues and murine cell lines in vitro. The single copy gene that was mapped to chromosome 19 is intronless and encodes a 92-kDa protein that has 77.5% overall homology to the human protein. The remarkable findings are 1) this gene is ubiquitously expressed in normal human and mouse somatic tissues and during development, and 2) its expression at the mRNA level is density-dependent and up-regulated in serum-starved cells. In vitro, its expression is limited to cells of embryonic, endothelial, and preadipocyte origin, suggesting that the wide distribution of its expression in vivo is due to the presence of vascular endothelial cells in all the tissues. The ubiquitous expression in vivo is in contrast to previously reported expression limited to corpus luteum and highly angiogenic tissues such as tumors and wound tissue.

Tax oncoprotein trans-represses endogenous B-myb promoter activity in human T cells.

The B-myb gene was identified on the basis of its homology with the protooncogene c-myb, homolog of the avian myeloblastosis virus (AMV) and avian leukemia virus (E26) transforming genes. Several studies using antisense constructs or antisense oligonucleotides as well as overexpression experiments suggest that B-Myb plays an important role in the transition from G(1) to S phase of the cell cycle and that B-Myb expression is cell cycle regulated. We have previously demonstrated that the human T cell lymphotropic virus type 1 (HTLV1) trans-activator Tax is able to repress transcription from c-myb promoter reporter constructs as well as from the endogenous c-myb promoter in human T cells and that this effect is mediated through inhibition of the c-Myb trans-activating functions. Here we report that both HTLV-1 as well as HTLV-2 Tax proteins inhibit c-Myb trans-activation in mouse embryo fibroblasts (MEFs). In addition to c-Myb, B-Myb expression is also markedly downregulated in HTLV-1-transformed cells at both RNA and protein levels. Furthermore, by using a Jurkat T cell line stably transfected with a tax gene driven by a cadmium-inducible promoter (JPX9), we were able to demonstrate that Tax directly represses the endogenous B-myb promoter in T cells. Because c-Myb and B-Myb have been involved in cell cycle progression, our results suggest that Tax, by repressing both c-Myb and B-Myb endogenous promoters, may bypass their requirement for cell cycle progression in HTLV-1-transformed T cells.

HTLV-I Tax transrepresses the human c-Myb promoter independently of its interaction with CBP or p300.

The c-Myb proto-oncogene is preferentially expressed in hematopoietic lineages, and highly expressed in several leukemia types. The Human T-cell Leukemia Virus Type I (HTLV-I) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL). A previous report suggested that Tax, the viral transactivator, is able to suppress the transactivation potential of c-Myb protein by competing for recruitment of CBP. We tested whether such a competition could affect transcription from the c-Myb promoter in Tax expressing T-cells. Using several c-Myb promoter reporter constructs carrying mutations in various regions, we demonstrate that Tax suppression of c-Myb transactivation results in transrepression of the c-Myb promoter through the Myb responsive elements in Jurkat T-cells. The ability of Tax mutants M22, M47 and V89A to interact with the full-length CBP and p300 proteins in vitro, and their ability to repress the c-Myb promoter, was then evaluated. Although both M47 and M22 bind to CBP and p300 to a similar extent, only M47 was able to repress the c-Myb promoter, suggesting that competition for CBP/p300 binding was not the mechanism underlying Tax's effect. This concept was further supported by the fact that the Tax mutant V89A transrepresses the c-Myb promoter efficiently in spite of an impaired binding to CBP and p300. Therefore, Tax-mediated repression of the c-Myb promoter appears to be independent from a direct competition between c-Myb and Tax for recruitment of CBP/p300. Interestingly, a decreased transcription from the endogenous c-Myb promoter was observed in several HTLV-I transformed T-cell lines. Finally, the ability of Tax to directly repress the endogenous c-Myb promoter was demonstrated in a Jurkat cell line stably transfected with a tax gene driven by a cadmium-inducible promoter.

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.

Study of in vitro conditions modulating expression of MN/CA IX protein in human cell lines derived from cervical carcinoma.

In an effort to better understand the biological significance of MN/CA IX human tumor-associated protein, we have investigated its expression in human cervical carcinoma cell lines in vitro. SiHa cells that naturally express MN/CA IX were used as a model for expression study at the protein level. In addition, we have transfected MN/CA9 gene-negative but transcription-competent C33A cells with a plasmid carrying CAT reporter gene under a control of MN/CA9 promoter. By this way, we have generated a stable cell line C33A/MNP-CAT that was employed in analysis of MN/CA9 regulation at the level of promoter activity as estimated by CAT protein abundance. For the purpose of our study, we have chosen experimental conditions relevant to growth characteristics and phenotypic features of malignantly transformed cells. Both the level of MN/CA IX protein and the gene promoter activity were found to be substantially elevated either in culture of high density or when the adherent carcinoma cells grew in suspension, but were not markedly affected by diminished serum concentration and in the cell cycle progression. These observations support the involvement of MN/CA IX protein in aberrant cell-cell and cell-matrix interactions that facilitate loss of contact inhibition and anchorage independence of cancer cells.

Up-regulation of p53 by antisense expression of HPV18 E6 oncogene does not influence the level of MN/CA IX tumor-associated protein in HeLa cervical carcinoma cells.

Oncogenic potential of human papillomaviruses is related to capacity of HPV-encoded oncoproteins to bind and inactivate tumor suppressor proteins. Interaction of p53 with HPV E6 results in aberrant regulation of various cellular genes. We evaluated the possible involvement of MN/CA9 gene, whose expression is closely associated with cervical carcinomas, in regulatory pathways driven by p53 and E6. We demonstrated that one of the two p53 consensus sequences present in MN/CA9 promoter participates in DNA-protein interaction but it does not bind p53. Tetracycline-inducible antisense expression of HPV18 E6 in human cervical carcinoma HeLa cells resulted in increased level of p53 but did not affect expression of MN/CA IX protein. Therefore we conclude that at least in HeLa cells there is no direct relationship between expression of MN/CA IX and expression of E6 or p53.

Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.

BACKGROUND & AIMS: CA IX (formerly MN protein) is a carbonic anhydrase isoenzyme whose expression is associated with human tumors. However, it has also been found in normal gastric mucosa. The aim of this study was to determine differences in complementary DNAs (cDNAs), to obtain an overview of distribution in the alimentary tract, and to obtain data on expression in tumors. METHODS: A CA9 cDNA isolated from a human stomach library was sequenced along with the cDNA derived from HeLa cells. Western blotting and immunohistochemical analyses of human and animal tissues were performed using CA IX-specific monoclonal antibody and rabbit antiserum to human CA II. RESULTS; Sequence analysis showed no differences between the stomach- and HeLa-derived cDNAs. CA IX was detected at the basolateral surface of gastric, intestinal, and gallbladder epithelia. In stomach tumor samples, expression of CA IX was lost or reduced. CONCLUSIONS: Differential distribution of CA IX in normal and tumor tissues is not associated with cDNA mutations. Evolutionary conservation in vertebrates as well as abundant expression of CA IX protein in normal human gastric mucosa, but not in derived tumors, indicate its physiological importance.

Transient transformation of mammalian cells by MN protein, a tumor-associated cell adhesion molecule with carbonic anhydrase activity.

The MN protein is associated with certain human carcinomas, but absent in most normal tissues. It is a transmembrane protein; its extracellular part contains a domain homologous with carbonic anhydrases (CAs) and a proteoglycan-like region. In the present study, we observed that cells (human CGL1 and mouse NIH3T3 cells) transfected with MN cDNA showed morphologic transformation, but reverted to normal phenotype after 4-5 weeks. This reversion was not due to the loss, silencing, or mutations of MN insert. We also found that MN protein exerted CA enzymatic activity, but this was not relevant for morphologic transformation of cells. MN is an adhesion protein, involved in cell-to-cell contacts, this probably could explain its role in tumorigenesis.

Human endogenous retrovirus HC2 is a new member of the S71 retroviral subgroup with a full-length pol gene.

We have isolated and characterized a new human endogenous provirus, which is closely related to the human retrovirus S71, but unlike S71 has a full-length pol gene. Two degenerate oligonucleotide primers based on highly conserved motifs within the active sites of two retroviral proteins (the protease and reverse transcriptase) were designed and used for PCR. An amplified product of 847 bp in length, which showed significant homology to protease and reverse transcriptase of several retroviruses, was used for high stringency hybridization with a human genomic library. The MuLV-related endogenous retrovirus sequence, designated HC2, was isolated and completely sequenced. HC2 is a provirus with complete gag and pol genes and a 3' LTR; the 5' LTR and env gene are missing. The gag and pol genes appear complete, since they contain sequences homologous to the matrix protein, capsid protein, and nucleocapsid protein of gag and to the protease, reverse transcriptase, tether, RNase H, and integrase of pol. Phylogenetic analysis suggests that although HC2 and S71 are MuLV-related retroviruses, their characters are quite distinct, being placed outside of a clade containing most of the previously characterized MuLV-related retroviruses such as GaLV, FeLV, BaEV, and SSV/SSAV.

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.

Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.