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A Raz

Publications and source records attributed to A Raz.

At least 145 records · Page 8Linked to original sources
PubMed1989

Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation.

Regulation of cyclooxygenase expression was studied in homogenous preparations of epithelial cells isolated from sheep tracheal mucosa. Cellular capacity to generate cyclooxygenase-derived arachidonate metabolites (predominantly prostaglandin E2) increased markedly in cultured compared to freshly isolated cells. A 70 kDa cyclooxygenase protein and corresponding 2.8 kb mRNA were coordinately expressed but their levels did not increase proportionately to the increase in cyclooxygenase activity. Rehybridization of Northern blots at lower stringency revealed the presence of a new tissue-specific 4.0 kb mRNA species exhibiting increased expression during cell culture. Hybridization of the 4.0 kb mRNA with two nonoverlapping cDNA probes at only low stringency conditions suggests that it is derived from a distinct gene. Its relatedness to cyclooxygenase and its increase in parallel with enzymatic activity further suggest that the larger mRNA may encode for a cyclooxygenase.

Animals↗
PubMed1989

Identification of the metastasis-associated, galactoside-binding lectin as a chimeric gene product with homology to an IgE-binding protein.

We report the complete primary and secondary structures of a metastasis-associated Mr 34,000 galactoside-binding lectin. The polypeptide sequence (264 amino acids) was derived from the nucleotide sequence of three overlapping complementary DNA clones isolated from lambda gt11 and lambda gt10 phage libraries of UV-induced murine fibrosarcomas. Striking features of the polypeptide sequence are two distinct regions of beta-sheet and globular structures at the amino and carboxy terminals, respectively. Homology search suggests that the polypeptide is a chimeric gene product formed by fusion of the 5'-end of an Mr approximately 14,000 galactoside-binding lectin with an internal domain of the collagen alpha gene. Enzymatic treatment with collagenase confirmed the presence of a collagen-like structure in the polypeptide. Unexpectedly, the entire sequence is greater than 85% homologous to a rat low affinity IgE-binding protein.

Amino Acid Sequence↗
PubMed1989

Differential expression of cell adhesion molecules in variants of K1735 melanoma cells differing in metastatic capacity.

We have investigated the expression of 2 neural-cell adhesion molecules, NCAM and LI, in K1735-C116 and -MI melanoma cells which differ qualitatively in their metastatic potential, i.e., MI cells are metastatic whereas C116 cells are not. We have found that NCAM in C116 cells are expressed as 2 quantitatively major glycosylated polypeptides with Mr of 145,000 and 120,000 and a minor 190,000 Mr polypeptide, whereas MI cells expressed NCAM as 3 glycosylated polypeptides with MR of 200,000, 140,000 and 120,000. The amount of NCAM in MI cells constituted only 60% of the amount observed in C116 cells. In C116 cells, the 145,000 and 120,000 Mr NCAM polypeptides were sulphated whereas NCAM did not appear to be sulphated in MI cells. No phosphorylation of NCAM in the 2 cell lines was observed. LI was expressed as a phosphorylated glycoprotein with Mr of 210,000 in MI cells whereas no LI expression was observed in C116 cells. LI was not sulphated in MI cells.

Animals↗
PubMed1989

Biochemical and immunological characterization of K-1735P melanoma galactoside-binding lectins and their modulation by differentiation inducers.

Lectins purified by affinity chromatography on immobilized asialofetuin from extracts of mouse K-1735P melanoma cells appeared as two polypeptides [L-14.5 (Mr 14,500) and L-34 (Mr 34,000)] in one-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. However, in two-dimensional electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis) the L-14.5 polypeptide was resolved into three acidic forms of pI 4.6, 4.9, and 5.8, whereas the L-34 was resolved into two polypeptides of pI 4.9 and 5.3. Antibodies directed against galactoside-binding lectins from rat and bovine lungs, mouse 3T3 fibroblasts, and mouse UV-2237 fibrosarcoma cells reacted with the K-1735P lectins in immunoblots, and normal mouse lung extracts were found to contain cross-reactive proteins that comigrated with the two melanoma lectins. Indirect immunofluorescence staining using the above antibodies demonstrated that both L-14.5 and L-34 were expressed on the surface of viable K-1735P cells. Treatment of these cells with 1 microM beta-all-trans-retinoic acid or 1 mM N6,O2'-dibutyryl cyclic AMP for 5 days induced morphological differentiation, inhibition of anchorage-dependent and anchorage-independent growths, and a selective decrease in the L-34 lectin level. Growth inhibition by starvation for serum factors, which did not induce differentiation, had no effect on the level of L-34. These results demonstrate that the melanoma lectins are immunologically related to normal cell lectins and that the two polypeptide species are expressed on the cell surface. Further, they demonstrate that the L-34 lectin level can be modulated by agents that suppress the transformed phenotype by enhancing differentiation.

Animals↗
PubMed1989

The cell biology of fibroblast cyclooxygenase.

We have prepared polyclonal antisera against sheep seminal vesicles cyclooxygenase (COX) which cross-reacted with human COX. We employed this antisera in studies with human dermal fibroblast cultures to immunoprecipitate selectively the COX enzyme. Labeling of the cells with [35S]-methionine, solubilization of cellular COX followed by its immunoprecipitation, SDS-PAGE electrophoresis and fluorography enabled us to determine directly the synthetic rate of COX protein and its modulation by the monokine interleukin-1 (IL-1). The immunoprecipitated [35S]-labeled COX, as judged from SDS-PAGE electrophoresis, has a molecular size of approximately 73,000 daltons, similar to that of native sheep COX and [3H]-acetyl COX. IL-1 stimulation of enhanced COX synthesis was time and dose dependent; as little as 0.03 units/ml of IL-1 produced significant stimulation of [35S]-labeled COX synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with IL-1 for 12-16 hours. IL-1 treatment of cells in serum-free media yielded parallel dose response curves for stimulation of PGE2 formation, cellular solubilized COX activity and synthesis of newly formed COX, suggesting that this IL-1 effect is mediated solely via induction of new COX protein synthesis. In contrast, IL-1 effect on cells incubated in the presence of fetal calf serum is more complex. Serum synergistically augments the IL-1 effect on PGE2 synthesis in intact cells but concurrently blunts IL-1 induction of COX synthesis, thus suggesting that a factor (or factors) in serum may stimulate PGE2 production by activating cellular phospholipase(s).

Animals↗
PubMed1989

Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases.

We have recently shown that the synthesis of cyclooxygenase [also called prostaglandin (PG) synthase or PG endoperoxide synthase; 8,11,14-icosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1] in human dermal fibroblasts is markedly stimulated by the cytokine interleukin 1 (IL-1). We now show that the temporal sequence of the induced synthesis of PG synthase can be separated into an early transcriptional (i.e., actinomycin D inhibitable) phase and a subsequent translational (cycloheximide but not actinomycin D inhibitable) phase and that IL-1 exerts its effect during the transcriptional phase. Phorbol 12-myristate 13-acetate also stimulates synthesis of PG synthase and, together with IL-1, produces a synergistic stimulatory effect. Inhibitors of protein kinase C activation abolished the stimulatory effect of IL-1, suggesting that protein kinase C activation is a critical event in the signal-transduction sequence of the IL-1-induced increase of PG synthase synthesis. The antiinflammatory glucocorticosteroids dexamethasone and triamcinolone, but not progesterone or testosterone, were potent inhibitors of PG synthase synthesis (complete inhibition at 20 nM; IC50, 1 nM) when added during the translational phase of the synthesis sequence. The glucocorticosteroid effect was blocked by RNA and protein synthesis inhibitors. This report suggests that glucocorticosteroids exert their effect via a newly synthesized protein, causing a profound translational control of PG synthase synthesis. This novel mechanism of suppression of arachidonate metabolism is distinct from any influence of steroids on phospholipase A2 activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Loss of metastatic responsiveness to cell shape modulation in a newly characterized B16 melanoma adhesive cell variant.

Repeated selection of an adherent subpopulation from B16-F1 melanoma cells growing in suspension culture on poly(hydroxyethylmethacrylate) [poly(HEMA)] coated plates resulted in the isolation of an adherent variant designated B16-A10. B16-A10 cells are more adherent to poly(hydroxyethylmethacrylate) coated plates than are B16-F1 cells and express an organized actin structure characteristic of highly adherent low metastatic cells as opposed to the poor cytoskeletal organization of B16-F1 cells. Upon growth in suspension, B16-A10 cells do not acquire the enhanced metastatic capability characteristic of B16-F1 cells and they express similar lung colonizing ability irrespective of the culture conditions. The increased metastatic ability of B16-F1 cells in suspension culture has previously been associated with the decreased accessibility of surface proteins to lactoperoxidase catalyzed iodination and with the increased expression of sialylated peanut agglutinin-binding oligosaccharides on these proteins. B16-A10 cells which show no cell shape induced increase in metastatic ability do not undergo alteration in either of these two properties in suspension culture. The absence of these two phenomena on B16-A10 cells grown in suspension indicates that they are interrelated and involved in the increased metastatic ability of B16-F1 cells grown in suspension.

Animals↗
PubMed1988

Regulation of fibroblast cyclooxygenase synthesis by interleukin-1.

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.

Animals↗
PubMed1988

Interferon-gamma treatment of B16 melanoma cells: opposing effects for non-adaptive and adaptive immune defense and its reflection by metastatic spread.

The impact of interferon-gamma (IFN) treatment of tumor cells on non-adaptive and adaptive immune defense and its reflection by metastatic spread were evaluated using a weakly metastasizing variant of B16 melanoma (B16-FI). Treatment of B16-FI with IFN resulted in a decrease in binding structures for NK cells and concomitantly in augmented metastasizing capacity. In line with this, activation of NK cells and Mo, which led to reduction of metastatic nodes, was less efficient with IFN-treated B16-FI, while after elimination of non-adaptive immune defense, the number of metastases increased significantly, but irrespective of IFN treatment. On the other hand, IFN-treated B16-FI cells become more prone to killing by cytotoxic T-cells (CTL). This was due to increased lysability by CTL and to increased immunogenicity; i.e., a higher frequency of B16-specific CTL was observed after immunization with IFN-treated than with untreated B16-FI. The reverse phenomenon was observed with anomalous and/or lymphokine-activated killer cells (AK/LAK). The common cause of increased antigenicity and immunogenicity may reside in increased expression of class-I and de novo expression of class-II MHC antigens after IFN treatment. Increased antigenicity and immunogenicity of IFN-treated B16-FI was reflected by significant reduction of metastatic nodes, prolonged survival and increased TD100 in animals immunized with IFN-treated vs. untreated melanoma cells. Comparison of the divergent effects of IFN treatment on B16-FI melanoma cells showed that the benefit of increased antigenicity/immunogenicity clearly outweighed the disadvantage of reduced susceptibility to non-adaptive immune defense.

Animals↗
PubMed1988

Expression of two different endogenous galactoside-binding lectins sharing sequence homology.

The endogenous beta-D-galactoside-binding lectins of UV-2237-IP3 fibrosarcoma cells consist of two polypeptides with molecular weights of 14,500 (L-14.5) and 34,000 (L-34). Recently we constructed complementary DNA (cDNA) libraries from these cells in the expression vector lambda gt11 and isolated and characterized cDNA clones encoding the two lectin species (A. Raz et al., Exp. Cell Res., 173: 109, 1987). We report here on the similarity in structure and gene number in genomic DNA of the fibrosarcoma cells and syngeneic normal mouse lung DNA. The expression of mRNAs hybridizing to these cDNAs was evaluated in three pairs of normal versus their transformed cell variants. Messenger RNA corresponding to the Mr 34,000 lectin was present more abundantly in the transformed cells than in their normal counterparts, while no difference was detected between the cell pairs with respect to mRNA levels corresponding to the Mr 14,500 lectin. We have established the nucleotide sequences of the two cDNA clones. The deduced amino acid sequence of L-14.5 lectin cDNA clone (L-3) is significantly homologous to the sequence of six other L-14 vertebrate galactoside binding lectins. The sequence of the Mr 34,000 lectin cDNA clone (M5) revealed that it shares a stretch of 39 amino acids with all of the L-14 galactoside binding lectins, irrespective of origin of the species, suggesting both that they are all derived from a common ancestral gene, and that this domain is necessary for similar sugar binding properties.

Amino Acid Sequence↗
PubMed1988

Adhesive properties of metastasizing tumour cells.

Cancer metastasis depends on a functional property which enables tumour cells to depart from the primary site of growth, to disseminate to distant organs and to establish secondary growth. The acquisition of a metastatic phenotype by neoplastic cells most probably involves alterations in their adhesive properties as the migrating cells continuously break and establish cellular contacts throughout the process. In vitro, normal cells of either mesenchymal or epithelial origin usually depend on adhesion to and spreading on a solid substratum (anchoring) for cell division. Neoplastic cells, however, are free of dependence on the support of solid substrata for cell proliferation (anchorage independent). The search for the characteristic alterations in cell adhesion, spreading and morphology which may accompany neoplastic transformation in general and cancer metastasis in particular has engendered a wide range of research activities. These studies have led to the identification of various membrane receptors that mediate cell-cell and cell-extracellular matrix recognition and adhesion on normal and tumour cells. Central to this is the effect of cell adhesion on cell shape and cytoskeleton organization in relation to metastasis. The use of specific antibodies, ligands, drugs and culture conditions permits exploration and identification of some of the macromolecules involved in tumour cell adhesion in vitro and metastasis in vivo. Nevertheless the specificity of the interactions which might determine organ-specific metastasis remains to be elucidated. This paper discusses the interrelation between cell adhesion, cell shape, cytoskeleton and metastasis.

Animals↗
PubMed1988

Endogenous lectins as mediators of tumor cell adhesion.

Endogenous carbohydrate-binding proteins have been found in various normal tissues and cells. Although lectins with different sugar-binding specificities have been described, the most prevalent ones are those that bind beta-galactosides. The ability of some normal and malignant cells to bind exogenous carbohydrate-containing ligands suggested that lectinlike activity is associated with the cell surface and that carbohydrate-binding proteins might mediate intercellular recognition and adhesion. We found that extracts of various cultured murine and human tumor cells exhibit a galactoside-inhibitable hemagglutinating activity. This activity was associated with two proteins of molecular weights of 34,000 and 14,500 daltons, which were purified by affinity chromatography by using immobilized asialofetuin. That these lectins are present on the cell surface was indicated by the binding of monoclonal antilectin antibodies to the surface of various tumor cells and by the immunoprecipitation of 125I-labeled lectins from solubilized cell-surface iodinated cells by polyclonal antilectin antibodies. That these cell surface lectins are functional was demonstrated by the ability of the galactose-terminating asialofetuin to enhance cell aggregation and of asialofetuin glycopeptides to block this homotypic aggregation as well as to suppress cell attachment to substratum, and by the inhibition of both asialofetuin-induced cell aggregation and cell attachment to substratum by the binding of monoclonal antilectin antibodies to the cell surface. These findings implicate cell surface lectins as mediators of cell-cell and cell-substratum adhesion. Some of these cellular interactions might be important determinants of tumor cell growth and metastasis.

Animals↗
PubMed1988

Lectins in cancer cells.

Studies carried out over the last few years have demonstrated that tumor cells and malignant tissues contain lectins that are similar in sugar-binding specificity, molecular size, and antigenicity to the lectins found in normal cells and tissues. Lectins from tumor cells also share marked sequence homology with lectins from normal tissues. Lectins were purified from various tumor cells by affinity chromatography and monoclonal and polyclonal antilectin antibodies were prepared against them. These enabled us to establish the following: (1) Lectins are present on the surface of all the tumor cells that were examined, albeit at varying levels. (2) The level of cell surface lectins increases after normal cells are transformed by transfection with certain oncogenes or by retroviruses, or when cells transformed with a temperature-sensitive viral mutant are switched from growth at the nonpermissive to the permissive temperature. (3) Among tumor cells differing in metastatic propensity, those exhibiting a higher potential express higher levels of surface lectins. (4) Tumor cell surface lectins might be involved in cell-cell adhesion, cell attachment to substratum, the expression of the transformed phenotype (anchorage-independent growth), and blood-borne metastasis. (5) The levels of the lectins in tumor cells are modulated by agents that suppress the transformed phenotype (as represented by anchorage-independence) or enhance differentiation. Numerous studies by others have shown that cell surface carbohydrate-containing molecules are modified after transformation, and our findings demonstrate that the expression of cell surface carbohydrate-binding proteins is also altered by transformation. Obviously, any of these changes may result in alterations in cellular interactions. All the above findings implicate tumor cell lectins in cellular interactions (adhesion, attachment, possible binding of exogenous soluble glycoconjugates), cell growth and anchorage-independent growth, malignant transformation, tumor cell differentiation, and metastasis. It is clear that even if these lectins are involved in only a few of these fundamental processes, it is important to elucidate their functions and the mechanisms by which their expression is regulated during neoplastic transformation and tumor progression and the suppression of the transformed phenotype.

Antibodies, Monoclonal↗
PubMed1987

Cell shape modulation alters glycosylation of a metastatic melanoma cell-surface antigen.

B16-F1 melanoma cells cultivated in vitro as spheroids on a non-adhesive substrate acquire in a reversible fashion an increase in lung colonization in vivo as compared to cells cultured as a monolayer. After neuraminidase treatment of protein blots, the spheroidal cells expressed an increased binding of 125I-labelled peanut lectin (PNA) to a unique glycoprotein of Mr 78,000 (gp78) which after desialylation migrated in SDS-polyacrylamide gels as an Mr 86,000 protein. Antibodies were generated against this glycoprotein purified on PNA-Sepharose and its expression on the surface of viable B16-F1 cells was demonstrated. Growth of B16-F1 melanoma cells in suspension is associated with the altered glycosylation of gp78 which may be related to the increased metastatic ability of these cells. In vitro treatment of B16-F1 cells with anti-gp78 Fab fragments prior to their injection into the tail veins of syngeneic mice resulted in a 2-fold increase in the appearance of tumor lung colonies.

Animals↗
PubMed1987

Transformation-related changes in the expression of endogenous cell lectins.

Endogenous lectins purified from UV-2237-IP3 murine fibrosarcoma cells by affinity chromatography consisted of two polypeptide species of Mr 14,500 (L-14.5) and Mr 34,000 (L-34). Antibodies against this material immunoprecipitated the lectins from cells radiolabelled on the cell surface as well as in the cytoplasmic compartment. Similar analyses of normal rat embryonal fibroblasts revealed the presence of only the L-14.5. In contrast, both L-14.5 and L-34 were found in oncogene-transfected, immortalized cell clones derived from the normal rat cells, as well as in untransformed BALB/c-3T3 clone A31 and its descendant subclones selected for expression of the transformed, the tumorigenic and the metastatic phenotypes. Among the cells constituting the latter system, a marked increase in the amount of cellular and cell-surface lectins was observed upon progression to the metastatic phenotype. These results suggest that the expression of endogenous tumor-cell-surface lectins is associated with transformation and metastasis.

Animals↗
PubMed1987

Cell-contact and -architecture of malignant cells and their relationship to metastasis.

The interaction of metastatic cells with the host environment occurs, to a large extent, through the cell surface, and the cell cytoskeletal system controls the distribution and motility of cell surface receptors. During metastasis, tumor cells migrate from one organ to another, and the dynamic properties and mechanochemical deformability of disseminated cells play a central role in the process. The studies described here under suggest an interrelationship between the cytoskeleton and cell adhesion, which can control and augment the expression of the metastatic phenotype of neoplastic cells.

Actins↗