Expression of p53 protein in rat colon cancer cell lines transfected with Rous sarcoma virus-33.

Expression of the cytoplasmic soluble form of p53 protein in the different rat colon cancer cell lines transfected and non-transfected with Rous sarcoma virus-33 was studied. Concentrations of the p53 protein were detected by commonly used immunochemical methods after its isolation by affinity chromatography columns with the gel fiberglass membranes. The main component of tumor-associated antigens (TAA) eluted from virus-transfected cells was the 53 kDa protein in its cytoplasmic soluble fraction. The non-virogenic colon cancer cells contain a few proteins and concentration of 53 kDa protein was low. Western immunoblotting revealed that the 53 kDa protein isolated from the cell lyzates studied was distinctly recognized by the p53 MAb. ELISA showed that its concentration was markedly higher in the lyzate obtained from the highly virogenic and tumorigenic R9 cell line compared with the non-virogenic cell line RT1. We concluded that the expression of the p53 protein is related to the viral transfection of cancerous cells. The possible role of this phenomenon in the etiology of cancer is discussed.

Active transforming growth factor-beta in human melanoma cell lines: no evidence for plasmin-related activation of latent TGF-beta.

Cultured human melanoma cells were found to secrete TGF-beta mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-beta in the range from 370 to 610 pg per 10(6) cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-beta activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-beta present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-beta 1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-beta produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas.

Cell surface phenotype and increased penetration of human multidrug-resistant ovarian carcinoma cells into in vitro collagen-fibroblasts matrix.

Human multidrug resistant ovarian carcinoma cells (A2780/ADR) exhibited increased in vitro penetration into the collagen-normal human fibroblasts matrix, increased cell surface expression of alpha 6 integrin (CD49f antigen) and slightly increased expression of alpha 2 (CD49b) integrin compared with that of parental drug-sensitive A2780 cells. Both, multidrug-resistant and parental, drug-sensitive, cell lines did not express the 67 kDa non-integrin high affinity laminin receptor on their cell surfaces. As there were no marked differences between metalloproteinase activity of both A2780 cell sublines (with similar intensity of 72 kDa and 92 kDa lysis bands in zymograms), the increased penetration of the drug-resistant subline into the collagen-fibroblast gel matrix might be associated with the increased expression of adhesion proteins (including collagen-binding alpha 2 integrin), or cell surface-associated collagenase-stimulating protein(s). This multidrug resistant ovarian carcinoma cell line might serve as an in vitro model of neoplastic cells with increased biological aggressiveness, molecular mechanisms of which require further analysis.

Interaction of avian sarcoma/leukemia viruses with heterologous hosts: inference for host-range and some pathogenic properties of human immunodeficiency viruses.

Although there are substantial differences between retroviruses originating from avian and primate species, a comparison of these two different biological systems reveals that interaction of these retroviruses with heterologous hosts involves similar biological principles. Retroviral isolates with high replicative capacity in natural targets (e.g. CD4+ lymphocytes and macrophages for human immunodeficiency viruses (HIVs) can infect other cell types [e.g. CD- astrocytes, follicular dendritic cells (FDC) in vivo and/or CD4+ neoplastic T cells in vitro] as well. These viral isolates may have a potential of infecting heterologous cells in vitro and can enlarge their host-range by establishing infection in other species, distantly related. Strains of avian sarcoma/leukemia viruses (ASLV) originating from their natural hosts, chickens, and infectious for other avian species, ducks, can frequently infect mammals (rodents). Similarly, HIV-1 strains infectious for chimpanzees possess capacity of establishing chronic infection in pig-tailed macaques. The broad host-range of retroviral isolates in both viral systems is accompanied by presence of additional structures in viral envelope. These novel or additional envelope structures may recognize alternate viral receptor(s). Moreover, the enlarged host range of primary HIV-1 isolates is evaluated by infection of neoplastic CD4+ permanent cell line, MT2, and serves as a predictive marker of progression of the viral infection toward AIDS.

Expression of the alpha 2-macroglobulin receptor on human neoplastic fibroblastoid cells.

The alpha 2-macroglobulin membrane-associated receptor (alpha 2MR) has been previously detected on hepatocytes, fibroblasts, macrophages, syncytiotrophoblasts and recently on human malignant blood cells of myelomonocytic leukemia. In cells growing in vitro from human germ cell tumors alpha 2MR mRNA was detected by Northern blotting. Endocytosis of alpha 2M from culture medium was detected in these cells by indirect immunofluorescence. In cell extracts alpha 2M and its degradation products were detected by immunoblotting. The cells expressing alpha 2MR and internalizing alpha 2M were identified as fibroblasts both by their morphology and expression of vimentin intermediate filaments. The role and function of alpha 2MR receptor in the analyzed neoplastic cells of teratomatous origin is discussed.

Determination of the synthesis and uptake of alpha 2-macroglobulin by cultured human glioma cells.

Using immunological techniques, the synthesis of alpha 2-macroglobulin was studied in established cell lines derived from human glioblastomas multiforme. alpha 2-Macroglobulin was detected in cytoplasm and in the culture medium of the analyzed cell lines. Radioimmunoprecipitation revealed a protein with M(r) corresponding to alpha 2-macroglobulin in the medium conditioned by U-118MG and U-343MG cells. On the other hand, using immunoblot analysis, alpha 2-macroglobulin was detected in all of the analyzed lines. In immunofluorescence test, alpha 2-macroglobulin was determined also in all four cell lines, but with different staining pattern. Conditioned culture medium (CCM) of U-536MG cells with the lowest level of alpha 2-macroglobulin exerted the lowest mitogenic activity for human fibroblasts.

Modulation of cell surface EGF receptor and HLA expression on glioma cell lines induced with cytokines and differentiation promoters.

Alterations of cell surface expression of HLA (class I, class II DR, DP and DQ) and EGF-receptor on two malignant glioma cell lines (U-343MG and U-563MG) induced with cytokines (IFN-gamma, TNF-alpha, IL-1 alpha) and differentiation promoters (all-trans retinoic acid, phorbol ester TPA) were analyzed with the aid of flow cytometry. IFN-gamma induced a 10-15fold increase of HLA class I. TNF-alpha alone induced a two- to fivefold increase of HLA class I cell surface density and increased the IFN-gamma induced upregulation of HLA class I to approximately 20-24 times the antigen density of uninduced cells. TNF-alpha was able to increase HLA class II DR and DP cell surface expression on glioma lines, but it enhanced only the IFN-gamma-induced HLA class II DR upregulation. All-trans retinoic acid and TPA regulated in the opposite way the EGF-receptor cell surface expression on U-563MG cells.

Binding of tissue-type plasminogen activator to human melanoma cells.

We have shown (Bizik et al., Cell Regul 1:895-905, 1990) that tPA can activate plasminogen on the surface of human melanoma cells in the presence of alpha 2-macroglobulin (alpha 2M) secretion. In the present study, we investigated the binding of tPA on the surface of Bowes melanoma cells, selected since they lacked production of PAI-1 and alpha 2M. Elution of tPA from the cell layers indicated that polylysine (5 micrograms/ml) and tranexamic acid (10 mM), an analog of lysine, were the most efficient agents for disrupting the interaction between tPA and cell surface component(s). Using a panel of monoclonal antibodies against individual domains of tPA revealed that an antibody directed to the kringle-2 domain of tPA interfered most significantly with cell-surface plasmin generation. As tPA is a glycoprotein, interactions between the tPA sugar moieties and cell surface were also tested by the use of a series of monosaccharides. N-acetyl-D-glucosamine (100 mM) was the most potent sugar to release tPA from melanoma cells, but the results indicated that the oligosaccharides of tPA play only a supportive role in the binding of tPA to the cell surface. Quantitative comparison of the cell surface localized tPA, which was eluted by tranexamic acid, with the total cellular tPA showed that cell surface bound tPA could represent up to 10%. We conclude that tPA interacts with the melanoma cell surface in a similar manner as has been described for binding of tPA to fibrin and to the putative endothelial cell surface receptor.

Modulation of protectin (CD59 antigen) cell surface expression on human neoplastic cell lines.

The ability of cytokines (IFN alpha, IFN gamma, TNF alpha, IL-1 alpha, IL-6), all-trans retinoic acid, 1,25(OH)2-vitamin D3 and the tumor promoting phorbol ester TPA to regulate cell surface expression of protectin (CD59 antigen) on human hematopoietic and non-hematopoietic neoplastic cell lines was examined with the aid of immunocytofluorometric measurements. The tumor promoting phorbol ester TPA induced a marked up-regulation of protectin in all examined cell lines with the exception of promyelocytic leukemia HL-60, where TPA significantly decreased protectin cell surface expression. All-trans retinoic acid weakly down-regulated cell surface protectin on K-562, while 1,25(OH)2-vitamin D3 produced such effect on HL-60 cells. None of the examined cytokines induced a significant protectin down-regulation in the examined cell lines.

Partial characterization of rat cell lines infected by a Rous sarcoma virus-33.

Rous sarcoma virus-33 (RSV-33) was obtained from a sample of chicken Rous sarcoma which had been dried and stored in 1933. RSV-33, like the RSV-29, has the minimal number of passages beyond its isolation from chicken tumour No. 1. Our experiments demonstrated that the Rous sarcoma virus-33 was replication non-defective and was pathogenic for rats. Established rat tumorigenic cell lines express the viral genome. All three species of viral RNA were detected and v-src proteins and gag polyproteins were identified as well in cells of R9 and R74 lines. The virus can be rescued from cells of R9 and R74 lines, thus indicating that the cells are virogenic. The cells of a permanent tumorigenic line RT1 are infected but not transformed by RSV-33. Although they contain a complete proviral genome, they do not express detectable virus-specific RNA. The virus is not rescuable from RT1 cells under in vivo conditions. Proviral DNA analysis showed that the RSV-33 contained a full-length genome, including the env gene, in contrast to the RSV-29 which was found replication defective.

A new variant of Rous sarcoma virus-33 nondefective, pathogenic for rats.

Rous sarcoma virus-33 (RSV-33) belongs to RSVs that have the least number of passages beyond its isolation from chicken tumor No. 1 among all current strains of RSV. Biological characterization indicated that it was pathogenic for rats. The results of the proviral restriction enzyme analysis showed that the established rat tumorigenic cell lines were the most likely infected by the same virus having a full-length genome.

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.

Coexpression of tumor-associated alpha 2-macroglobulin and growth factors in human melanoma cell lines.

alpha 2-Macroglobulin (alpha 2M) is known as an inhibitor of various proteinases and to bind several of the growth factors. We previously demonstrated that clonal variation exists in the production of alpha 2M in a human melanoma and that this variation may be associated with growth stimulation. We have now analyzed six human melanoma cell lines for the simultaneous expression of TGF-alpha, TGF-beta, PDGF-A chain, PDGF-B chain, and tumor-associated alpha 2M. In Northern blot analysis TGF-alpha was detected in four of the cell lines, TGF-beta in all, PDGF-A chain in three, and PDGF-B chain in none of the cell lines. alpha 2M, detected by immunoblotting, varied significantly between the different melanoma cell lines and only one cell line was found to be negative. Evaluation of growth-promoting activity in conditioned media suggested that alpha 2-macroglobulin, secreted by these tumor cell lines, is a significant modulator of melanoma cell growth.

Immunofluorescent detection of actin cytoskeleton in spontaneously transformed and B77-supertransformed cells using antibody to tropomyosin.

Differences in immunofluorescence of actin cytoskeleton between normal rat fibroblasts and two transformed cell lines SAMIV and SAMB77 were detected by antiserum to tropomyosin. In both transformed cell lines reduction in number and shortening of microfilament bundles (stress fibers) were observed. In some transformed cells ruffling membranes (peripheral ruffles) were seen. Differences were found in actin cytoskeleton among individual cells of spontaneous transformants (SAMIV) and supertransformants (SAMB77).

Alpha-2-macroglobulin is not synthesized by human urothelial cell lines in vitro.

We have analyzed 14 human urothelial cell lines in two different transformation grades for the production of a tumor-associated-alpha-2-macroglobulin. It has been shown in radioimmunoprecipitation and immunoblotting tests that human urothelial cells in vitro do not synthesize the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Four of the tested cell lines were additionally tested for the expression of specific mRNA which resulted in negative findings. The significance of the absence of alpha-2-macroglobulin in the human urothelial cells is discussed.

Clonal variation in the production of tumor-associated alpha 2-macroglobulin in a malignant human melanoma and association with growth stimulation.

alpha 2-Macroglobulin (alpha 2-M) is known as a wide-spectrum proteinase inhibitor and to bind covalently certain growth factors. We have previously characterized tumor-associated alpha 2-M synthesized and secreted by human tumor cell lines. Of the cell lines studied, the melanoma cell line HMB-2 produced the largest amount of this glycoprotein. Immunofluorescence staining of cultured HMB-2 cells suggested that the cell population is heterogeneous with respect to alpha 2-M production. We have now isolated clones from the parental HMB-2 cells and characterized eight representative ones in detail. They varied considerably in the quantity of alpha 2-M secreted, from 4.2 to 46.5% of total protein. No relationship between the production of alpha 2-M by these clones and their pigmentation or tumorigenicity in nude mice was found. However, statistically there was a strong correlation between the modal chromosome number and population doubling time (r2 = 0.88, P less than 0.001) and also between the modal chromosome number and alpha 2-M production (r2 = 0.73, P less than 0.01). The growth rate of the clones correlated with the level of alpha 2-M in culture medium (r2 = 0.69, P less than 0.01). Clones with lower alpha 2-M production had a proportionally shorter population doubling time than the clones with intermediate or high production. Northern hybridization indicated quantitative variation in the alpha 2-M mRNA expressed by the parental cells and the clones, that was comparable but not identical with the quantity of alpha 2-M in the respective culture media; both parental cells and clones expressed platelet-derived growth factor A-chain mRNAs with little difference in levels. Serum-free medium from low alpha 2-M producer clones stimulated normal stationary fibroblasts significantly more than clones producing intermediate or high amounts of alpha 2-M. alpha 2-M decreased and anti-alpha 2-M IgG increased the stimulation. These results suggest that production of tumor-associated alpha 2-M is related to both autocrine and paracrine growth-stimulating activity of the tumor cells.

Localization of alpha 2-macroglobulin in human primary sarcomas and synthesis in established cell lines.

The presence of alpha 2-macroglobulin (alpha 2M) was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas. alpha 2M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein, which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the detection of alpha 2M in situ and in vitro an antiserum to tumor-associated alpha 2-macroglobulin was used.

Tumor-associated alpha-2-macroglobulin in human melanomas.

We and others have previously shown that human melanoma cell lines in culture synthesize alpha-2-macroglobulin (alpha 2M). We have now studied melanomas from 30 patients for the presence of alpha 2M using the peroxidase anti-peroxidase technique on histologic sections from paraffin-embedded tissues and primary antibody raised against tumor-associated alpha 2M in rabbits. alpha 2M was detected in 10 of the 30 melanomas studied. In all but 2 cases the presence of alpha 2M was restricted to solitary tumor cells or to solitary foci of tumor tissue. In one case of melanoma almost all tumor cells were positive for alpha 2M, while in the others between 20% and 50% of tumor cells were positive. In all but one of the melanomas, the positivity was characteristic of epithelioid or large-cell type or was confined to this component in melanomas with more than one cell type. In 4 positive cases, differences in the extent of alpha 2M-containing tumor tissue were observed between primary tumor and metastases or metastases from different localizations, with equivocal trend. Clinical follow-up of the melanoma patients suggested that alpha 2M-positively tends to correlate with an unfavorable prognosis.

Monoclonal antibody to a human osteogenic sarcoma cell line.

Monoclonal antibodies against a human osteogenic sarcoma cell line were prepared by production of a somatic cell hybrids between the spleen cells from U-393OS--immunized mice and the mouse myeloma cells SP2/0. From 7 producing and well-growing clones only one--B-0S12--produced antibodies, reactive preferentially with osteosarcoma cells as identified by binding second antibodies and 125I-labeled Protein A. This antibody was tested against a panel of normal and tumor cell targets to determine the pattern of the antigen detected. The monoclonal antibody reacted strongly against U-3930S cells and another human sarcoma in vitro and more weakly against human fibroblasts, peripheral lymphocytes, red blood cells and was negative against mouse fibroblasts. When tested against a panel of unrelated human tumor cell lines, B-0S12 antibody was positive with melanoma cells and negative with cells from bladder, cervix and mammary carcinoma. These cross reactions suggested, that the antibody is reactive with a protein, expressed on different tumor types. This protein is not expressed on the cell surface and is probably associated with cytoskeleton, as revealed by immunofluorescence experiments. Western-blot analysis of a cytoskeletal preparation of U-3930S cells suggests, that B-0S12 antibody recognizes a protein with Mr 55 kD. Further studies are needed to characterize the molecules, carrying the epitope, identified by this monoclonal antibody.

BrdU-induced changes in the provirus of PR-RSV in mammalian cells.

Previously we demonstrated differences in the organization and methylation pattern of integrated Rous sarcoma proviral sequences in helper-dependent virogenic rat TWERC cells. In the present study we attempted to induce changes in the integrated viral genome of TWERC cells using 5-bromodeoxyuridine (BrdU). Four clones (A, B, C, and F) derived from the parental cell line were treated for 10 months with different concentrations of BrdU. Restriction enzyme analysis of the parental cell line and its clones showed that the cells contained in their genomic DNA two copies of deleted provirus. In TWERC cells, the PR-RSV provirus lost the whole env gene and part of the 3' end of the pol gene. The proviral sequences in DNA from the TWERC-derived clones were found to be hypermethylated. A slightly different situation was seen in clone C where demethylation of the provirus in the 3' region and in the 5' end of the genome was found. The level of mRNA expression both in the parental cells and in the clones correlated with the methylation pattern of the PR-RSV provirus. Clone C was less methylated and expressed more virus-specific RNA. The possible role of BrdU in these events is discussed.