Jan Zavada

Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Tumor‐associated Carbonic Anhydrases and Their Clinical Significance

Carbonic anhydrases (CAs) are physiologically important enzymes that catalyze a reversible conversion of carbon dioxide to bicarbonate and participate in ion transport and pH control. Two human isoenzymes, CA IX and CA XII, are overexpressed in cancer and contribute to tumor physiology. Particularly CA IX is confined to only few normal tissues but is ectopically induced in many tumor types mainly due to its strong transcriptional activation by hypoxia accomplished via HIF-1 transcription factor. Therefore, CA IX can serve as a surrogate marker of hypoxia and a prognostic indicator. CA IX appears implicated in cell adhesion and in balance of pH disturbances caused by tumor metabolism. Both tumor-related expression pattern and functional involvement in tumor progression make it a suitable target for anticancer treatment. Here we summarize a current knowledge on CA IX and CA XII, and discuss possibilities of their exploitation for cancer detection, diagnostics, and therapy.

Topographical analysis by monoclonal antibodies of BLV-gp51 epitopes involved in viral functions.

Abstract Mouse monoclonal antibodies, directed against eight independent antigenic sites on the bovine leukemia virus (BLV) envelope gp51 (sites A, B, C, D, E, F, G, H), were tested for their capacity to interfere with the biological activities of BLV which are associated with the gp51 molecule, as follows: the pseudotype inhibition test (PI) measures the ability of antibodies to inhibit infectivity of BLV-VSV pseudotypes; the early polykaryocytosis inhibition test (EPI) measures inhibition of syncytia-inducing activity of BLV-producing cells in the presence of these antibodies; and the complement-dependent cell lysis measures the cytoxic activity of antibodies toward BLV-producing cells in the presence of complement. Three of the eight antigenic sites (sites F, G, H) were shown to be involved in the biological activities tested for in infectivity and syncytia neutralization tests. At least one of these three regions is exposed on the surface of BLV-producing cells (site G) and can be the target for complement-dependent cytotoxicity. Limited protease digestion of gp51 showed that a peptide fragment of MW 15,000 contained at least a part of sites FGH involved in the biological activities analyzed and of site E which is inactive. The major carbohydrate residues are localized on a fragment of apparent molecular weight 35,000, also containing the antigenic sites ABCD which are not involved in the biological activities tested for.

Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection

The bovine leukaemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into the HA locus of vaccinia virus (Copenhagen strain), downstream of the vaccinia virus early-late promoter, H6, or a triple promoter element consisting of the promoter for the vaccinia virus H6 gene, the promoter for the cowpox virus A-type inclusion (ATI) gene and the promoter for the vaccinia virus HA gene. Inoculation of rabbits or sheep with the recombinant vaccinia virus coding for gp51 and gp30 or an uncleaved env precursor induced neutralizing antibodies to BLV. These antibodies competed with monoclonal antibodies directed against gp51 epitopes F, G, and H previously shown to be of crucial importance for BLV infection. Seven out of eight sheep vaccinated with the vaccinia recombinants resisted a drastic challenge (1.5 x 10(3) sheep infectious doses) with BLV-infected lymphocytes. These results show that vaccination with BLV env vaccinia recombinants protects sheep against infection with extremely high doses of BLV-infected heterologous lymphocytes.

Synthetic Peptides Approach To Identification Of Epitopes On Bovine Leukemia-Virus Envelope Glycoprotein-Gp51

Peptides corresponding to residues 21-28, 39-48, 57-67, 59-69, 78-92, 144-155, 144-157, 195-205, 255-268, and 260-268 of envelope glycoprotein gp51 of bovine leukemia virus (BLV) were chemically synthesized and coupled to keyhole limpet hemocyanin or tetanus toxoid. All peptides were immunogenic in rabbits and induced production of antipeptide antibodies. Enzyme-linked immunosorbent assays using Tween 80-purified gp51 or BLV particles showed that antibodies against peptides 78-92, 255-268, and to a lesser extent 39-48 and 144-157 were able to react with the parent glycoprotein, purified or as an integer part of BLV particles. Antisera against peptides 39-48, 78-92, and 144-157 neutralized VSV (BLV) pseudotypes in vitro, the highest neutralization titer being obtained with the 78-92 cyclized peptide. These observations confirm that the NH2 moiety of gp51 carries epitopes involved in virus infectivity.

Complementation and Phenotypic Stabilization of Vesicular Stomatitis Virus Temperature-Sensitive and Thermolabile Mutants by Avian Myeloblastosis Virus

Out of five complementation groups of vesicular stomatitis virus (VSV) type Indiana, each represented by two temperature-sensitive (ts) mutants, only representatives of group V showed genetic complementation by avian myeloblastosis virus Pseudotypes (AMV); the VSV (AMV) appeared among the progeny. Thermolabile (tl) mutants with apparently defective glycoprotein were phenotypically stabilized by growth in the presence of AMV, but a tl mutant with a defect in some other structural protein was not. Mutants with a tl glycoprotein and a ts defect corresponding to complementation group I were phenotypically stabilized by growth in the presence of AMV, but did not show genetic complementation by AMV.

Human cell surface proteins selectively assembled into vesicular stomatitis virus virions

Vesicular stomatitis virus (VSV) selectively assembled proteins from human cells into progeny virions. These proteins can be surface labeled before infection with 125I, and when purified virus was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, only two or three bands of proteins (Mr around 100K) were seen. Antisera to these proteins were produced, using as immunizing antigen VSV tsO45 mutant, defective in assembly of G protein, which had been made at the nonpermissive temperature in the three human tumor cell lines, HeLa (cervical carcinoma), T47D (breast carcinoma), and HMB2 (melanoma). After absorption with wild-type VSV, each of the antisera displayed a different pattern of reactivity; at least three antigenic specificities were detected. Two of them, corresponding to antigens selected by VSV from HeLa and T47D, were to some extent related and they showed an association mainly with epithelial cell-derived gynecological tumors, but they were absent in carcinomas of lung or of digestive tract. These (or related) antigens were expressed in a lower level in some normal tissues, mainly in ovaries. Antigen(s) assembled by VSV from the melanoma cell line was entirely different and appeared to be associated with cell growth. The grounds for selective assembly of these specific proteins by VSV are not clear; they either share with viral surface glycoproteins some physical or structural properties, which are critical for incorporation into the viral envelope, or conceivably they even may represent uncleaved precursor proteins coded by env genes of incomplete genomes of endogenous human retroviruses.

Rescue of presumptive viral information from human cells by a helper oncovirus

We have attempted to rescue presumptive human endogenous retrovirus(es) by using a competent animal oncovirus as a helper. Human melanoma cells (line HMB2) were fused, using polyethylene glycol, with mouse NIH-3T3 cells which had been infected and transformed by the Harvey murine leukaemia and sarcoma virus complex (MLV and MSV). The heteropolykaryons obtained were co-cultivated with fresh NIH-3T3 cells; filtered (Millipore 0.22 micron) medium from these was used to infect further NIH-3T3 cells. In these cells after several passages, vesicular stomatitis virus (VSV) pseudotypes could be produced. These were infectious not only for mouse cells (manifesting the helper MLV), but also for human cells (HeLa, HEC human embryo fibroblasts, HMB2); they were not infectious for CCL64 (mink) or for Vero (African green monkey) cells. The presence of such VSV pseudotypes infectious for human cells indicated that a human ecotropic virus [provisionally named rescued human virus (RHV)] had been rescued by the fusion of human melanoma cells with MLV-infected mouse cells. This was supported by the following evidence. The human-specific pseudotype was neutralized by sheep antisera raised to antigens selected by VSV from human tumour cell lines HMB2, T47D and HeLa. These antisera also aggregated NIH cells infected with MLV and RHV. Mouse antisera raised to antigens present in HIH cells infected with MLV and RHV, in contrast to sera raised to NIH cells infected with MLV only, immunoprecipitated an 85,000 mol. wt. protein band from human cells (HEC, HMB2 and HeLa) surface-labelled with 125I.

Novel gene MN is candidate oncogene

HERV-H ENDOGENOUS RETROVIRUSES: ANALYSIS OF CHIMERIC RETROVIRAL/CELLULAR TRANSCRIPTS AND DEFINITION OF LTR REGULATORY REGIONS. D. L. Mager, D. T. Nelson, P. E. Kowalski, S. Baban and J. D. Freeman HERV-H (formerly RTVL-H) human endogenous retroviral sequences are the most numerous HERV family known, being present in -1000 copies per haploid genome in humans and other Old World primates. A similar number of solitary HERV-H LTRs are also dispersed throughout the genome. HERV-H elements are highly transcribed in teratocarcinoma cell lines, some other tumor cell lines and in normal placental amnion and chorion. The LTRs of these elements not only drive expression of HERV-H elements themselves, but can also promote expression of reporter genes in transient assays. Such assays, coupled with DNA sequencing, have revealed tha t HERV-H LTRs can be grouped into at least 3 subclasses that vary in promoter function and in sequence characteristics. To unders tand HERV-H transcriptional regulation and the potential effects of these elements on the genome, we have been defining regions within the LTRs that are critical for promoter/enhancer activity. For instance, we have found that the presence of an apparent Sp-1 binding site in a subclass of LTRs with strong promoter activity contributes substantially to this activity. We have also been isolating and investigating fusion transcripts between HERV-H and heterelogous cellular sequences. We have found several examples of such chimeric transcripts in teratocarcinoma ceils, including a phospholipase A2 related gene and an abundant transcript that is promoted by HERV-H but is polyadenylated in downstream cellular sequences. We have also very recently detected a transcript that contains both HERV-H env sequences and regions with high homology to zinc finger genes. These results indicate that HERV-H elements may play a role in genetic diversity by donating regulatory elements to adjacent cellular genes.

Molecular Dissection of the Bovine Leukemia Virus Envelope Glycoprotein (BLV gp51) by a Monoclonal Antibody Study

Neoplasms of the lymphatic tissue in the bovine species can be classified into two major types according to clinical and epidemiological data: 1. The juvenile form of bovine leukosis, with three different clinical forms: the multicentric, thymic, and cutaneous forms. The juvenile form is rare and shows a random geographical distribution. It has also been called sporadic bovine leukosis. 2. The adult form of bovine leukosis leads to more diversely located lymphosarcomas, mostly of the B-lymphocyte lineage and/or to B-cell leukemias. The adult form of bovine leukosis is more common, but restricted to geographically limited regions. Although it affects only a small percentage of the cattle population, it behaves as a typical herd disease. Its distribution pattern is typically one of transmissible diseases and has allowed the classification of the adult form of lymphosarcomas as “enzootic bovine leukosis” [1].