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.

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.

Circulating antibodies to the feline leukemia virus and FOCMA in cats.

Presence of antibodies to the structural proteins of the feline leukemia virus and the feline oncornavirus-associated cell membrane antigen FOCMA was analyzed in sera of 120 cats from household environments. In our experiments 35% of the sera tested were positive with the viable cells of FL-74 feline lymphoma cell line. On the other hand, only 5% of the feline sera reacted with FeLV from FL-74 cells in the solid phase RIA. Selected positive sera were analyzed for the presence of antibodies to the viral structural proteins of FeLV and FOCMA in lysates of FL-74 cells by radioimmunoprecipitation. Absorption of the selected sera (reacting with viable FL-74 cells) with disrupted FeLV had a negligible effect on the precipitating activity of the 70000 protein.

Quantitative changes of tropomyosin synthesis in RSV-transformed mammalian cells in comparison with normal fibroblasts.

Protein profiles of three tumor mammalian cell lines and three control cell lines were analyzed by two-dimensional gel electrophoresis. Comparison of the cells labeled metabolically with 35S-methionine has revealed obvious differences in an area of molecular weights 34 000--36 000 daltons and pH 4.5-5.0. Two spots have been proposed to be alpha- and beta-tropomyosin subunits. Their synthesis decreases after cell transformation.

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).

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.

Using tropomyosin polymorphic phenomenon for detection of differences between rat spontaneous tumor cells and their in vitro oncovirus supertransformats.

Biochemical and immunological comparative studies of rat tumor cells of spontaneous origin and in vitro supertransformed cell populations have been done. We have focused on characterization of differences in tropomyosin molecular forms in the individual cell populations. Our experiments have shown that differences of tropomyosin forms exist not only between spontaneous transformants and supertransformants but also between supertransformants and normal rat fibroblasts. It means that superinfection of spontaneous transformants by avian sarcoma virus B77 have induced changes in tropomyosin synthesis but a pattern of tropomyosin forms in super-transformants has not been equal or similar to that of normal fibroblasts.

Surface glycoproteins of human sarcoma- and fibroblastic cells.

A comparison was made of the cell surface glycoproteins of four human cell lines, namely a giant tumor of bone cell line, an osteosarcoma line, a fibrosarcoma line and a human fibroblast line. The cells were labeled by lactoperoxidase catalyzed iodination and the glycoproteins extracted by 0.5% Triton X-100 were bound to lentil-lectin and subsequently analyzed by SDS gel electrophoresis. While the cell lines examined shared a series of common glycoproteins, it was found that the giant cell tumor line and the fibrosarcoma lines exhibited a higher degree of homology than the other cell lines.

Immunoprecipitation of membrane proteins of cultured human sarcoma cells.

Human sarcoma associated antigens (HSAA) have previously been identified by indirect immune fluorescence in human sarcoma cells in culture using sera from patients bearing different types of sarcoma. To further characterize these HSAA, surface proteins of cultured cells were labeled with 125Iodine, [3H]-glucosamine and [35S]-methionine and solubilized. After immunoprecipitation labeled proteins were detected in immune complexes by SDS polyacrylamide gel electrophoresis and autoradiography, which allowed comparison with antigens described by other groups. A surface protein (Mr 96 000) was precipitated with sera from sarcoma bearing patients, and two glycoproteins (Mr 115 000 and 85 000) were preferentially precipitated with antisera from rabbits immunized with membranes from two human sarcoma cell lines. At least two of these proteins were found in each of five human sarcoma cell lines studied (U-4SS, U-3930S, U-20S, B-5GT and B-6FS). None of the proteins were precipitated with three human control sera, and only occasionally a faint band was observed in immunoprecipitates from control cells (B-25F, B-41B, B-42FC, U-2S, and U-393S with the immune sera. These proteins are probably some of the antigens responsible for the immune fluorescence observed in determination of HSAA. However, purification of the proteins and competition experiments are needed before this can be finally established.

Two cellular proteins that are precipitated by an antitumor-RSV rat serum.

Here we report studies on non-viral proteins specific for transformation precipitated by immune sera. These sera were from rats injected by PR-RSV transformed rat XC cells and from rabbits immunized by subcutaneous injections of cytoplasmic membranes from rat cells transformed by Fujinami avian sarcoma virus. Immune complexes were analyzed in one- or two-dimensional gel electrophoresis and by autoradiography. Only by using antisera obtained from rats bearing XC-cells induced tumors, two proteins were identified with an apparent molecular weight (Mr) of 105 000 (p105) and 98 000 (p98) and an isoelectric point of 6.4 and 6.2, respectively, in extracts of mammalian cells transformed by avian sarcoma viruses (ASV). These proteins were common to all ASV-transformed cell lines. In kinase reaction, the protein p98 can be phosphorylated. Similar proteins were neither detected in normal nor in chemically transformed cells. None of the proteins were precipitated with control serum from rat, rabbit, and goat.

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.

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.

Evidence of cell-mediated antitumor immunity in rats bearing syngeneic tumors by the leukocyte adherence inhibition test.

A modified form of the leukocyte adherence inhibition (LAI) test has been used to follow cell-mediated immunity (CMI) in rats of the Lewis inbred strain (LW), F1 hybrids inbred strains LW x AVN with transplanted syngeneic tumors B77, MC-1, and the random-bred Sprague Dawley strain with spontaneous REF tumor. Peripheral blood leukocytes of animals incubated witha specific tumor extract showed an adherence capacity by 30--40% lower than the same cell population cultured without this specific extract, or with a foreign nonspecific extract. The results are commented in connection with a possible application of this modified form of LAI test in studies of the immunological state of patients with a malignant disorder.

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.

Preferential replication of murine xenotropic type-C virus in human lymphosarcoma-derived cell lines.

Murine xenotropic virus, designated 698/X, was recovered by implantation of human lymphosarcoma-derived cells U-698M into nude mice of Giovanella's colony. The budded and extracellular particles revealed typical type-C morphology, the latter possessing reverse transcriptase (RT) activity and exhibiting a buoyant density 1.17 g/ml in sucrose gradient. In competitive radioimmunoassay using iodinated p30 of Rauscher MuLV, the 698X viral concentrate and cell extracts of both implanted lymphosarcoma cells (U-698M-N-1 and U-715M-N-1) were as effective as Gross MuLV, thus indicating the murine origin of the virus. The propagation of the 698/X virus in five human, four mouse (permissive for N- and B-tropic MuLV), two rat and one bovine cell lines was followed by RT, XC syncytia assays and EM investigations. The replication of the 698/X virus seems to be restricted mainly to both human lymphosarcoma-derived cell lines U-698M and U-715M. The new recovery of the virus from the nude mouse by implantation of U-175M cells has asserted its high tropism to human lymphosarcoma cells and its murine origin. The comparative response of mouse, rabbit and rat cells exposed to both NZB and 698/X xenotropic murine viruses exhibited host range differences between these viruses. The rabbit SIRC and rat embryonic cells REC were fully permissive for the murine xenotropic NZB virus, while low viral production was detected by RT assay only in 698/X virus infected SIRC cells.

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.

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.

Differences in the organization and methylation patterns of integrated avian sarcoma proviral DNA sequences in nonpermissive and permissive mammalian cells.

Four types of avian sarcoma virus (ASV)-transformed mammalian cells were analyzed for the presence of ASV-specific sequences in their genome DNA. A great variability in the number of proviral copies and their structure within the DNA of these lines was observed. In all cells tested gag and src sequences were present in a flexible arrangement. The greatest variation was detected within proviral sequences corresponding to pol and env regions. The number of integrated ASV proviral copies do not correlate with the capability of these cells to produce viral particles. Virus-producing (K2S and K12) and virogenic (XC) cells contain in their chromosomal DNA at least one complete proviral genome, whereas proviral sequences in helper-dependent nonvirogenic cells are substantially changed. Provirus expression level does not correlate with the number of integrated virus copies. In the non-virus-producing cells the proviral sequences are hypermethylated.

The retrovirus particles in human myeloma cells RPMI8226: morphological, biochemical, immunological and infective transmission studies.

The retrovirus designated RPMI8226V (isolated from human myeloma cells RPMI8226) has been characterized with respect to its morphological, biochemical and immunological properties as well as its propagation in various animal and human cells. The myeloma cells RPMI8226 produce intracytoplasmatic A-type particles and extracellular particles. The extracellular particles have been classified as immature particles with translucent core center, typical mammalian C-type virus particles and C-type particles with intermediate membrane. However, the budded particles in secondarily infected human neoplastic cells contained complete doughnut-shaped nucleoids. This type of budding is rather characteristic for B-type particles. The 3H-uridine labeled RPMI8226 viral particles have a buoyant density 1.17 g/ml in sucrose gradient containing high molecular weight RNA and the distribution of viral structural proteins in SDS-PAGE is characteristic for oncornaviruses. The internal structural proteins according to MW are ranged from 13 000 to 30 000 daltons. The virus contains a magnesium-dependent reverse transcriptase. The cellular homogenate and viral concentrate from RPMI8226 cultures do not react with antibodies against ALSV, MuLV, FeLV, RD114, MP-MV and SiSLV. The only reaction was scored with anti BLV antibodies. However, anti BLV serum inhibiting the reverse transcriptase activity of BLV to 60% does not cross-react with the reverse transcriptase of RPMI8226V. In contrast to BLV concentrates, neither XC nor KC cells show syncytia formation by RPMI8226V. The RPMI8226V replication is restricted to human tumor and normal human glia-like cells. The possible origin of the virus is discussed.

Establishment of a human cell line (B-25F) derived from a fibroma of buccal epithelium.

A new cell line (B-25F) obtained from a benign polypoid fibrous lesion of the mucosa of oral cavity is described. The cells at first grew in suspension but after a month of cultivation they began to adhere and subsequently formed a monolayer typical for fibroblastoid cells. Population doubling time both in lower and higher passages was 60 h. Electron microscopy studies failed to detect any viral particles or mycoplasmas. A comparison of cell surface glycoproteins of fibroblasts (B-41FB), fibroma cells (B-25F), and fibrosarcoma cells (B-6FS) was made. These lines share common traits in their surface membranes although distinct differences among the individual lines could be detected. Karyological analysis showed that 64% of cells contained 46 chromosomes. This number veiled both diploid and pseudodiploid karyotypes. An aberrant chromosome, t(13;18?) was found. For isoenzyme comparison of B-41FB, B-25F, B-6FS, and HeLa cell lines the mobility of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PD) was employed. All these lines had LDH patterns of human cells, and a G-6-PD pattern of phenotype B except that of HeLa cells that had phenotype A.