Soluble form of carbonic anhydrase IX (CAIX) in transitional cell carcinoma of urinary tract.

We investigated the expression of cell-associated CAIX protein in histological sections of the transitional cell carcinoma (TCC) of the urinary tract and of the soluble form of CAIX (s-CAIX) shed by the tumor into the serum and urine of TCC patients. A total of 23 patients with histologically confirmed TCC or squamous cell carcinoma (SCC) were enrolled in the pilot study. Sixteen healthy individuals served as controls. Membrane-bound CAIX was present in the tumor cells near the endoluminal surface. Necrosis was observed in only 4 samples. Using Western blots, s-CAIX concentrated from urine was visualized as a double band at 50 and 54 kDa. In most cases, the presence of s-CAIX in the urine correlated with CAIX expression in the tumor. On the other hand, s-CAIX did not exceed the normal level in the serum of TCC patients. Urine from patients with TCC of the urinary bladder and renal pelvis contained s-CAIX, allowing the detection of tumors in approximately 70% of the patients. Moreover, two additional patients with suspected, but unconfirmed bladder tumor, with s-CAIX detected in urine, developed tumors identified as TCC within six months. We suggest that after a simple, rapid and sensitive test, monitoring s-CAIX levels in urine will be developed, it may be useful for early detection of relapse in patients following transurethral tumor resection.

Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients.

Tumour-associated protein carbonic anhydrase IX (CA IX) has two major forms. One is a cell-associated, transmembrane protein seen on Western blots as a twin band of 54/58 kDa, expressed in gastric mucosa and in several types of cancer. The other is a soluble protein s-CA IX of 50/54 kDa, which is released into the culture medium or into the body fluids, most likely by proteolytic cleavage of the extracellular part from transmembrane and intracellular sequences. While TC media of CA IX-positive tumour cell lines or short-term cultures of tumour explants contain a relatively high concentration of s-CA IX (20-50 ng ml(-1)), the level of this antigen in blood serum and urine of renal clear cell carcinoma patients is about 1000 x lower. The concentration of CA IX in the blood and in urine varies within wide limits and there is no obvious correlation with tumour size. After nephrectomy, s-CA IX is cleared from the blood within a few days. Only an extremely low concentration of CA IX was detectable in the sera and in urine of control individuals.

Human tumour-associated cell adhesion protein MN/CA IX: identification of M75 epitope and of the region mediating cell adhesion.

MN/CA IX is a cell surface protein, strongly associated with several types of human carcinomas. It exerts activity of carbonic anhydrase and capacity of binding to cell surface receptors. In the present work, we used affinity purified MN/CA IX protein to demonstrate that the cells adhere to immobilized MN/CA IX and that the monoclonal antibody M75 abrogates cell attachment to MN/CA IX. Using synthetic oligopeptides, we identified M75 epitope and located it in the proteoglycan domain, which contains a sixfold tandem repeat of six amino acids GEEDLP. From phage display library of random heptapeptides we identified and chemically synthesized those which compete for the epitope with M75 and inhibit adhesion of cells to MN/CA IX. These heptapeptides might serve as lead compounds for drug design.

Identification of MaTu-MX agent as a new strain of lymphocytic choriomeningitis virus (LCMV) and serological indication of horizontal spread of LCMV in human population.

In this study we elucidated the molecular character of MaTu-MX, previously described as an unusual transmissible agent. Amino acid sequencing of peptides generated from a 58-kDa MX-related protein purified from MaTu human carcinoma cells allowed us to identify it as a nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Northern blot analysis detected LCMV-specific RNAs in MaTu cells. Comparative immunoprecipitations showed cross-reactivity between NP of LCMV strain WE and MX NP. Using RT-PCR, we have cloned MX NP cDNA. According to sequence comparison, MX LCMV is as closely related to both LCMV strains WE and Armstrong as these strains are to one another. Based on this finding we propose that MX is a new strain of LCMV. We also showed that the stability of MX NP in MaTu cells is very high and that the virus is transmissible by cell-to-cell contact or by cell-free extract to human HeLa and monkey Vero cells, but not to human AGS, canine MDCK, mouse NIH 3T3, and hamster CHO cells. Finally, employing MX LCMV NP in immunoprecipitation and solid-phase radioimmunoassay, we found 37.5% prevalence of anti-LCMV antibodies in human sera, suggesting possible horizontal spread of the virus in the human population.

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.

Expression of MaTu-MN protein in human tumor cultures and in clinical specimens.

MaTu is a novel agent which may be of relevance in human oncogenesis, and has 2 components. One of them, the exogenous MX (coding for protein p58X), is transmissible to human fibroblasts, to HeLa and to HeLa x fibroblast (H/F) hybrids. The other component, MN, is a cellular gene. Its product, the protein p54/58N, is inducible by infecting HeLa cells with MX or by growing them in dense cultures. This p54/58N appears to be a tumor-associated antigen: it is expressed in HeLa and in tumorigenic cells (H/F-T), but not in fibroblasts or in nontumorigenic hybrid cells (H/F-N). Proteins related to p54/58N were also found on immunoblots prepared from human carcinomas of ovary, endometrium and uterine cervix, but not from normal tissues from corresponding organs or from placenta. Using genetically engineered MN protein, we developed a radioimmunoassay for MN-specific antibodies, and for quantitative determination of MN proteins in cell extracts. In HeLa cells infected with MX we observed conspicuous ultrastructural alterations: formation of abundant filaments on the cell surface and amplification of mitochondria. Using immunogold-staining, we visualized the p54/58N on the surface microvilli and in the nucleus, particularly in nucleoli.

A novel quasi-viral agent, MaTu, is a two-component system.

MaTu is a quasi-viral agent presumably derived from a human mammary tumor. In some respects it resembles classical viruses and in some the "slow viruses," and in others it is different from both. Using monoclonal antibodies (Mabs), we showed that it is a two-component system. One part of the complex, MX, is exogenous; it is manifested by a protein, p58X, which is a cytoplasmic antigen and it reacts with some natural sera of man and of various animals. The other component, MN, is endogenous to human cells. This is manifested by a twin protein(s), p54/58N, localized on the cell surface and in the nucleus. This protein is absent in rapidly growing, sparse cultures of HeLa, but it is inducible either by keeping the cells in dense cultures or, more efficiently, by infecting them with MX. Both inducing factors are synergistic. Only p54/58N is associated with virions of vesicular stomatitis virus (VSV), reproduced in MaTu-infected HeLa. This suggests that MN is responsible for complementation of VSV mutants and for the formation of the VSV (MaTu) pseudotype. Both p54/58N peptides are glycosylated and they form oligomers linked by disulfidic bonds; p58X is not glycosylated and it does not form S-S-linked oligomers.

An unusual transmissible agent--MaTu.

MaTu is an agent, believed to be derived from a human mammary carcinoma, which displayed several extraordinary properties. These were: RIP and PAGE revealed in MaTu-infected cells only a single protein band of Mr 58 k, the gp 58. This gp 58 was immunoprecipitated by antibodies present in some human sera as well as in some sera of rabbits, sheep, and cattle. MaTu had an extremely restricted host range: it was transmissible only to HeLa cells, but not to human embryo fibroblasts, to three human tumour cell lines (T 47 D, T 24, and HMB 2) or to monkey Vero and rabbit SIRC cells. A retrovirus with a broad host range, used as a helper (X-MLV) enabled the transmission of MaTu to human fibroblasts, but not to Vero or SIRC, which are also permissive for X-MLV. These observations, together with our previous reports, support the view that MaTu might either be a novel type of defective virus, or even a non-viral autonomous genetic element.

Rescued human virus (RHV): association with melanoma.

RHV, which is presumably a defective human retrovirus, has been recovered from the human melanoma cell line HMB2. In the presence of Moloney mouse leukemia virus (MLV), used as a helper, RHV is serially transmissible in mouse NIH-3T3 cells and can provide envelope antigens for vesicular stomatitis virus pseudotype--VSV(RHV). This pseudotype is neutralizable with an inhibitor, present in all human sera tested; the inhibitory activity is resistant to heating at 100 degrees C. To detect whether any specific response to RHV is connected with the disease, we isolated IgG from 140 human sera (70 melanoma patients and 70 control group donors) and tested it for neutralization of VSV(RHV) pseudotype in a randomized, blind experiment. Sixteen samples of IgG from the sera of melanoma patients, but only one control IgG, neutralized the pseudotype; this difference was significant (p less than 0.001). RHV thus appears to be in some way associated with melanoma.

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.

Monoclonal antibody against an antigen selectively assembled into vesicular stomatitis virus virions from HeLa cells.

A mouse hybridoma cell line IIB9, secreting IgG2b antibody specific for a HeLa cell antigen, was obtained by fusion of a mouse myeloma cell line with spleen cells from mice immunized with purified VSV tsO45 mutant (defective in assembly of G protein) which had been reproduced at a non-permissive temperature in HeLa cells. The monoclonal antibody IIB9 was strictly specific for HeLa cells in two tests: (1) reaction with VSV or Chandipura virus phenotypically mixed with host cell antigen, (2) complement-dependent cytotoxicity test (51Cr-release).

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.

Vesicular stomatitis virus phenotypically mixed with retroviruses: an efficient detection method.

Two methods of assaying vesicular stomatitis virus (VSV) particles phenotypically mixed with retrovirus-coded antigens were compared. Each of them detected phenotypically mixed particles with different minimum proportion of surface glycoprotein molecules of the donor virus, and consequently also profoundly different proportions of VSV virions containing retrovirus antigens. Only a low proportion (10(-4) of VSV virions grown in XMuLV-infected rabbit SIRC cells behaved as pseudotypes, resistant to anti-VSV serum and neutralized by anti-XMuLV serum. VSV produced in mouse L cells did not contain significant titre of pseudotype particles in the neutralization test. However, when immunoprecipitation was used with corresponding antibody and Staphylococcus aureus cells, almost 100% of the VSV virions produced in L cells and in XMuLV-preinfected SIRC cells were found to contain MuLV-related antigen molecules.

Pseudotypes of vesicular stomatitis virus with coat antigen of bovine leukaemia virus--VSV (BLV): antigenic surface mosaic and the roles of precipitating antibodies and polycations.

The pseudotype particles vesicular stomatitis virus (bovine leukaemia virus) [VSV (BLV)] contain a surface antigenic mosaic, composed of both VSV- and BLV-specific antigens, as demonstrated by increased neutralization by anti-VSV serum after addition of complement or of "second antibody". All the pseudotype infectivity was precipitated by VSV-specific antibody: it could be pelleted by low-speed centrifugation and its infectivity recovered without any loss by sonication. Polycations present during adsorption had little effect on infectivity of the pseudotype for Vero cells, but markedly increased its titre for chicken fibroblasts.

A rapid neutralization test for antibodies to bovine leukemia virus, with the use of rhabdovirus pseudotypes.

Two rhabdoviruses, vesicular stomatitis (type Indiana) and Chandipura viruses, formed pseudotype particles with envelope antigens provided by bovine leukemia virus (BLV). The pseudotypes are infectious for calf, human, mink, and rat cells, but the most sensitive indicator proved to be the Vero cells. Infectivity of the pseudotypes was increased by DEAE-dextran present during adsorption. Sera of spontaneously infected cattle contained high titers (some over 1/10,000) of antibodies neutralizing the pseudotypes, whereas sera of cattle from uninfected herds possessed no neutralizing activity in 1/10 dilution. The neutralization of these pseudotypes can serve as a rapid and sensitive test for the detection of antibodies in the cattle infected with BLV.

Pseudotype particles of vesicular stomatitis virus with surface antigens of bovine leukaemia virus--VSV (BLV) -- as a sensitive probe for detecting antibodies in the sera of spontaneously infected cattle.

Phenotypically mixed particles containing the genome of vesicular stomatitis virus (VSV) and envelope antigen corresponding to bovine leukaemia virus (BLV) -- the VSV (BLV) pseudotypes -- can be employed as a rapid, specific and sensitive probe for detecting BLV-neutralizing antibodies in bovine sera.

Comparison of the sensitivity to ultraviolet irradiation of reovirus 3 and some viruses of the Kemerovo group.

The kinetics of UV inactivation of the tick-borne Kemerovo (strain R-10) and Lipovnik (strain Lip-91) viruses which have been preliminarily classified as possible members of the Reovirus group was examined. Reovirus 3 and Sindbis virus served as reference double-stranded RNA and single-stranded RNA viruses, respectively. The parameters of UV inresembled those of Reovirus 3. This is consistent with their tentative classification as reovirus-like viruses.