Biomedical subjects

J Varani

Publications and source records attributed to J Varani.

At least 181 records · Page 10Linked to original sources

PubMed1985

Characterization of laminin-stimulated adherence and motility in tumor cells.

Laminin was shown to stimulate attachment, spreading and motility in a line of murine tumor cells. The stimulated cells began to attach and spread within 1 h and remained attached and spread throughout a 48-hour observation period. Pretreatment of the cells with laminin for 24 h beforehand did not interfere with their ability to bind laminin in a second treatment. Laminin-stimulated attachment and spreading occurred in the absence of extracellular Ca2+ or Mg2+, but not in the absence of both divalent cations. A number of cell function inhibitors blocked the adherence response, but were only partially effective even at high concentrations. These characteristics of laminin-stimulated adherence are different from those of the adherence response stimulated by peptide chemotactic factors and phorbol esters.

Animals↗

PubMed1985

Rat lung fibroblast collagen metabolism in bleomycin-induced pulmonary fibrosis.

Endotracheal bleomycin administration in rats and other animal species causes rapid development of pulmonary fibrosis, characterized by increased lung collagen synthesis and deposition. To clarify the mechanism, lung fibroblasts from bleomycin-treated rats (BRF) were isolated and maintained in tissue culture. They were then compared with those from normal untreated control animals, with respect to several key parameters of collagen metabolism. BRF synthesized collagen at a rate 35-82% above normal rat lung fibroblasts (NRF). This difference did not appear to be due to the selection of a clone by the subculture process. Furthermore, analysis of newly synthesized collagen type composition, revealed a significantly lower ratio of type III to type I collagen. Noncollagenous protein synthesis, however, was not significantly different from normal. Collagenase production and growth rate were also unaffected. BRF, however, was morphologically indistinguishable from NRF, even at the ultrastructural level. Upon further bleomycin (1 microgram/ml) exposure in vitro, BRF could be further stimulated to synthesize collagen at 82% above the rate for untreated BRF. This is comparable to the 90% increase in NRF treated in vitro (compared with untreated NRF). These results would favor the conclusion that bleomycin induces pulmonary fibrosis, by causing directly and/or indirectly lung fibroblasts (or a certain line of lung fibroblasts) to synthesize collagen at a higher rate without any associated increase in growth rate. The data, however, do not rule out the possibility that the fibroblast isolation procedure has selected for a certain population of fibroblasts that may not be typical of the in vivo situation.

Animals↗

PubMed1985

Chemotactic factor binding by metastatic tumour cells: evidence for a formyl-peptide receptor on a non-myelogenous cell.

Analysis of fMet-Leu-[3H]Phe binding to Walker 256 carcinosarcoma cells demonstrated both saturable and reversible binding, and indicated the presence of a single population of binding sites having an equilibrium dissociation constant: KD = 15.7 +/- 3.3 X 10(-9) M, and with 2425 +/- 204 binding sites per cell. The specificity of the binding site was investigated by competitive inhibition of fMet-Leu-[3H]Phe binding studies using 10 oligoformyl peptides. These results demonstrated an order of peptide reactivity with marked similarity in specificity to the leucocyte binding sites for the formyl-peptides. The most active peptides also had potent agonist activity as determined by their ability to increase the cells' adherence response to nylon-wool fibres. In addition, a competitive antagonist of the formyl-peptide receptor, tert-butoxy-Phe-Leu-Phe-Leu-Phe, completely abolished the adherence response induced by fMet-Leu-Phe, but had no inhibitory effect on the adherence response caused by the tumour-promoting agent, phorbol myristate acetate. These data demonstrate that formyl-peptide receptors may be more common than we have anticipated and may be found on cells not derived from the myeloid series. Furthermore, these studies advance our understanding of stimulus-coupled responses in tumour cells.

Animals↗

PubMed1985

Laminin inhibits the recognition of tumor target cells by murine natural killer (NK) and natural cytotoxic (NC) lymphocytes.

Tumor cells sensitive to lysis by murine natural killer (NK) or natural cytotoxic (NC) cells were shown to bind laminin. They bound 125I-labeled laminin in a time- and concentration-dependent manner, and binding of the radioactive laminin was inhibited by excess cold laminin. In the presence of laminin, cell-cell aggregation occurred. Murine tumor cells not sensitive to NK/NC-mediated killing bound much less laminin, and laminin did not induce aggregation of these cells. The addition of exogenous laminin to NK or NC cytotoxicity assays reduced target lysis in a dose-related manner. Reduction of lysis was due to an inability of NK/NC cells to bind to the targets. Target cells pretreated with laminin were reduced in their ability to cold-target compete for NK-mediated lysis of untreated target cells. These effects were unique to laminin. The control proteins (fibronectin and thyroglobulin) had no effect on NK activity. Finally, inhibition of cytolytic activity by laminin appeared to be specific for NK/NC cells. Laminin had no effect on cytolysis mediated by alloimmune cytotoxic T lymphocytes regardless of whether the targets did or did not bind laminin.

Animals↗

PubMed1985

Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical.

Human blood neutrophils stimulated by a variety of agents were shown to have cytotoxic effects on bovine pulmonary artery endothelial cells. Effective agonists included immune complexes, opsonized zymosan and 12-O-tetradecanoyl phorbol acetate. Unstimulated human neutrophils and neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine or with platelet-activating factor failed to induce significant killing even though secretory release of lysosomal enzymes occurred. In comparing the effects of the different agonists, endothelial cell killing showed a better correlation with the production of H2O2 than with the generation of O2-. Endothelial cell killing by stimulated human neutrophils was inhibited by catalase but not by soybean trypsin inhibitor or superoxide dismutase. Killing was also inhibited by two scavengers (N, N-dimethylthiourea and D-mannitol) of hydroxyl radical and by deferoxamine mesylate, an iron-chelator. Iron-saturated deferoxamine mesylate was significantly less effective in protecting the endothelial cells against killing. Agents that were protective against endothelial cell killing did not interfere with the generation of O2- in stimulated neutrophils. These results suggest that leukocyte-induced endothelial cell killing in vitro can be induced by some but not all agonists for neutrophils and that the killing is oxygen-dependent and may be directly due to hydroxyl radical production.

Animals↗

PubMed1985

A review of studies in our laboratory regarding ELLA methodology for the study of cell surface carbohydrates from tumors of varying metastatic potential.

An enzyme-linked lectin assay, termed ELLA, has been developed to investigate differences in cell surface carbohydrate groups on the surface of murine fibrosarcoma cells of varying metastatic potential. We have conjugated alkaline phosphatase to Griffonia simplicifolia I-B4 and have obtained an enzymatically-active probe for the detection of alpha-D-galactopyranosyl end groups. This probe has been used to detect different levels of expression of alpha-D-Galp end groups on the surface of unfixed murine tumor cell lines. Using the ELLA technique we have been able to demonstrate a correlation between the amount of these carbohydrate groups present on the cell surface and the degree of malignancy of the cell lines. Preliminary data suggest that the glycoprotein laminin accounts for a significant proportion of the cell surface alpha-D-Galp groups on these tumor cells and may play a functional role in their ability to metastasize. Furthermore, we have used ELLA to measure the binding of exogenous laminin to laminin-deficient cell lines and have found that this results in an increased ELLA reactivity. This increase corresponds to previously described increases in the malignant behavior of these cells after addition of exogenous laminin.

Alkaline Phosphatase↗

PubMed1985

Immunocytochemical characterization of a monoclonal antibody that recognizes mitosing cells.

The isolation and characterization of a monoclonal antibody (C5F10) which identifies dividing cells in normal and neoplastic tissues (carcinomas, sarcomas, and lymphoreticular malignancies) in formalin-fixed, paraffin-embedded sections is described. The antibody also recognizes rapidly dividing cells in normal and transformed cells in culture. A combination of autoradiography with 3H-thymidine and immunochemical localization of C5F10 showed that cells in S-phase of the cell cycle were weakly stained for C5F10, while dividing cells stained intensely with this antibody. The target structure of C5F10 appears to be different from the commonly recognized microtubule, intermediate filament and microfilament proteins and from the proliferating cell nuclear antigen (cyclin). This antibody may be a useful tool for readily detecting dividing cells in cell cultures and in tissue sections and may prove useful in studies to analyze the molecular basis of cell growth.

Animals↗

PubMed1985

Response of Walker 256 carcinosarcoma cells to 12-O-tetradecanoylphorbol 13-acetate: possible regulation by endogenous cyclooxygenase metabolites.

Walker 256 carcinosarcoma cells (Walker cells) maintained in suspension culture responded to stimulation with 12-O-tetradecanoylphorbol 13-acetate [(TPA) CAS: 16561-29-8] by becoming temporarily adherent to the substratum. Both the control and treated cells produced very low levels of cyclooxygenase metabolites as detected by radioimmunoassay procedures. Levels of prostaglandin F2 alpha, 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) (a prostacyclin metabolite), and thromboxane B2 were virtually the same as background, and prostaglandin E2 (PGE2) levels were only slightly higher. Studies employing high-performance liquid chromatography also failed to detect significant quantities of cyclooxygenase products in the supernatants from either the control or the stimulated Walker cells. Although the Walker cells maintained in culture failed to produce significant amounts of cyclooxygenase metabolites, they produced much greater amounts of these products, particularly PGE2 and 6-keto PGF1 alpha when they were maintained as an ascites tumor. Concomitant with the production of these metabolites was a loss in responsiveness to TPA in the adherence assay. Upon reestablishment in culture, the cells gradually reacquired the ability to respond to TPA. Over the same period, synthesis of cyclooxygenase products was curtailed. If the cells taken from ascites tumors were incubated with indomethacin so as to inhibit the production of cyclooxygenase metabolites, they rapidly regained responsiveness to TPA. These findings suggest that stimulus-coupled responses in the Walker cells may be regulated, at least in part, through the production of endogenous cyclooxygenase metabolites.

Animals↗

PubMed1985

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells.

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.

Animals↗

PubMed1985

DNA content of murine fibrosarcoma cell lines with varying metastatic potential.

The DNA content of murine fibrosarcoma cell lines of various metastatic potential was the subject of the current investigation. The cell lines were derived from methylcholanthrene-induced tumors as described previously (J. Varani et al., J. Natl. Cancer Inst., 71: 1281-1287, 1983). Cells were maintained in vitro and used for DNA studies no more than 48 h after passage. DNA staining was accomplished using propidium iodide and flow cytometry was used to quantitate relative amounts of DNA. Trout and chicken erythrocytes and mouse thymocytes were used as internal DNA standards for each cell line. DNA indices were calculated as the ratio of the G0-G1 peak channel number of the tumor cells to the G0-G1 peak channel number of the thymocytes. Manual chromosome counts were also obtained from each cell line using Giemsa-stained preparations. All cell lines demonstrated a single aneuploid population. The two tumor lines with the highest metastatic potential were slightly hyperdiploid whereas three low metastatic lines were near tetraploid. A sixth line of moderate metastatic potential was also found to be near tetraploid. Chromosome counts and flow cytometric analyses were in close agreement indicating that DNA content was largely due to chromosome replication. These data suggest that, in this model, metastatic potential and DNA content are inversely related once diploidy is exceeded.

Animals↗

PubMed1984

Metastatic potential of murine fibrosarcoma cells is influenced by cell surface laminin.

In order to examine the role of cell surface laminin in tumor metastasis we have utilized four well-characterized murine fibrosarcoma cell lines. Two of these lines were highly metastatic when injected into syngeneic mice while the remaining two lines were significantly less metastatic. Using indirect immunofluorescence techniques, we detected cell surface laminin on the cell surface of both highly metastatic cell lines but not on the low-metastatic cell lines. Although the low-metastatic cell lines did not possess endogeneous cell surface laminin, they had the ability to specifically bind exogenous laminin to their surface in a time- and concentration-dependent manner, indicating the presence of laminin receptors on these cells. Incubation of the low-metastatic cells with exogenous laminin prior to injection into syngeneic animals significantly increased their metastatic potential. No such increase was observed when the highly metastatic lines were preincubated with exogenous laminin. On the basis of these results, we conclude that in this fibrosarcoma model, metastatic potential is influenced by cell surface laminin and that the presence of unbound laminin receptors on the cell surface is not alone sufficient to promote metastasis of these cells.

Animals↗

PubMed1984

The Tzanck smear in the diagnosis of cutaneous herpes simplex.

We examined the effectiveness of the Tzanck preparation compared with viral isolation in 32 lesions of cutaneous herpes simplex infection in 30 consecutive patients. Seventy-eight percent had positive cultures, even though the clinical history and lesion morphological findings were typical in all situations. Other authors have also noted that viral isolation does not reach 100% even in the face of typical clinical history. Fifty-three percent of the Tzanck smears were positive. These preparations had a high correlation with positive viral cultures (94.1%). Vesicles were more likely to yield a positive culture than Tzanck smear (100% and 66.7%, respectively). Pustules yielded positive cultures in 72.8% and positive Tzanck smears in 54.5%, while crusted-ulcer lesions were usually not positive by either technique (34%, 16%). A positive Tzanck preparation is an accurate, rapid tool for the diagnosis of cutaneous herpes simplex.

Cytodiagnosis↗

PubMed1984

Contribution of alpha-D-galactopyranosyl end groups to attachment of highly and low metastatic murine fibrosarcoma cells to various substrates.

There are much greater numbers of cell surface terminal, non-reducing alpha-D-galactorpyranosyl groups in highly malignant (metastatic) cells than are found in low malignant cells derived from the same murine fibrosarcoma. We have examined the contribution of these residues to attachment of the cells to various collagens and to plastic. Removal of these carbohydrate groups with alpha-galactosidase or blocking them with lectins from Griffonia simplicifolia seeds or with anti-blood group B antiserum all dramatically inhibited the attachment of both the highly malignant and the low malignant cells. Following removal with the enzyme, the alpha-D-galactopyranosyl end groups were rapidly resynthesized. This resynthesis was inhibited by tunicamycin, an inhibitor of de novo glycoprotein synthesis. This antibiotic also impaired cell attachment and, when used in addition to treatment with alpha-galactosidase, it inhibited cell attachment more than did treatment with the enzyme alone. The effects of all treatments on cell attachment were greater for the highly malignant than for the low malignant cells. With the latter cells, inhibition by lectin was seen only in the absence of serum, whereas the adhesion of highly malignant cells was affected in both the presence and the absence of serum. On their surface membrane the highly malignant cells express much more than do the low malignant cells of a glycoprotein that cross-reacts immunologically with laminin. The basement membrane glycoprotein laminin promotes cell attachment to collagen, and both glycoproteins contain terminal, non-reducing alpha-D-galactopyranosyl groups. Attachment of cells is a requirement for the formation of a metastasis, and thus the laminin-like molecule and the alpha-D-galactopyranosyl end groups (whether on the laminin-related moiety or on other cell surface molecules) may both be important for expression of the most malignant phenotype.

Animals↗

PubMed1984

Enzyme-linked lectin assay (ELLA). II. Detection of carbohydrate groups on the surface of unfixed cells.

An enzyme-linked lectin assay (ELLA) has been developed to detect specific carbohydrate units on the surface of unfixed cells. The assay may be read in standard ELISA plate readers, since the cell-bound enzyme-lectin conjugate is specifically eluted from the cells prior to development of the conjugate. ELLA, when read in an enzyme-linked immunosorbent assay (ELISA) plate reader, allows better detection and relative quantitation of specific surface carbohydrate units than is possible by standard immunofluorescence with fluorescein-conjugated lectins.

Alkaline Phosphatase↗

PubMed1984

Functional responses of tumor cells to phorbol esters: role for prostaglandins.

The effects of phorbol esters on five murine cell lines were examined. In three of these lines, all of which were highly malignant, phorbol ester treatment induced motility and foreign surface adherence while concomitantly inhibiting proliferation. In two other, nonmalignant cell lines phorbol ester treatment stimulated proliferation but did not influence motility or adherence. Possible roles for prostaglandins in these responses were investigated. The mitogenic effects of the phorbol esters were duplicated by prostaglandin F2 alpha. In contrast, prostaglandin E1 and prostacyclin inhibited proliferation induced by the phorbol esters. These agents also inhibited phorbol ester-induced cell motility.

6-Ketoprostaglandin F1 alpha↗

PubMed1984

Identification of a laminin-like substance on the surface of high-malignant murine fibrosarcoma cells.

High- and low-malignant murine fibrosarcoma cells were stained with anti-laminin antibodies using immunoperoxidase techniques and examined by electron microscopy. With the high-malignant cells, specific staining was observed along the cell surface. Use of normal rabbit serum in place of the rabbit anti-laminin or pretreatment of the anti-laminin with soluble laminin completely eliminated this staining. No immunoperoxidase staining was observed with the low-malignant cells. In additional studies, membrane fractions were prepared from the high- and low-malignant cells and used to immunize rabbits. The animals immunized with the membrane fractions from the high-malignant cells produced antibodies that reacted by enzyme-linked immunosorbent assay (ELISA) with murine laminin obtained from the EHS sarcoma. The animals immunized with membrane fractions from the low-malignant cells did not. These studies provide strong evidence that the high-malignant cells (but not the low) express on their cell surface a substance that is immunologically cross-reactive with laminin. In addition, the high-malignant cells (but not the low) secreted a material into the cell culture fluid that could be specifically immunoprecipitated with antilaminin antibodies. The immunoprecipitated material co-migrated with purified laminin when examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis under reducing conditions. The existence of this substance associated with the surface of the high-malignant cells and its absence from that of the low-malignant cells may explain the previously noted difference between these cells in their ability to attach to type IV collagen. This difference may also contribute to the dissimilarity between these cells in their metastatic potential.

Animals↗

PubMed1984

Stimulation of murine tumour cell motility by laminin.

Several variant cell lines that are deficient in surface laminin have been isolated from heterogeneous murine tumour populations. The parent populations from which these variant lines were isolated contain cells that express high levels of surface laminin as indicated by immunofluorescence, immunoperoxidase/electron microscopy and immunoprecipitation. The laminin-deficient lines were compared with their respective parent populations for motility by both the Boyden Chamber assay and the agarose assay. In both assays, the laminin-positive populations were much more motile than the laminin-deficient lines. The addition of exogenous laminin to the laminin-deficient lines significantly increased their motility. These observations are of interest since cell motility is thought to contribute to tumour cell metastasis, and the laminin-positive cell populations are highly tumorigenic and metastatic, while the laminin-deficient cells are of low tumorigenicity and are virtually non-metastatic.

Animals↗

PubMed1984

Protein degradation following treatment with hydrogen peroxide.

Pretreatment of hemoglobin with 50-5000 nmol hydrogen peroxide (H2O2) increased its susceptibility to proteolysis by a number of purified enzymes, including trypsin, chymotrypsin, elastase, and plasmin, and by the neutral protease of rat peritoneal leukocytes. Pretreatment of the protein substrate with catalase-inactivated H2O2 had no effect. Separation of the proteolytic fragments by G-75 Sephadex gel filtration indicated no apparent differences in the size distribution of the fragments produced by treatment with the H2O2/proteolytic enzyme combination as compared with enzyme treatment alone. A partially purified preparation of rat glomerular basement membrane was also treated with proteolytic enzyme alone or in combination with H2O2. As with the hemoglobin, pretreatment of the glomerular basement membrane with H2O2 increased its susceptibility to subsequent proteolytic attack. In addition, treatment of a basement membrane glycoprotein, fibronectin, with H2O2 also increased its sensitivity to subsequent proteolysis. These results suggest that in addition to their other proinflammatory activities, oxygen-derived metabolites may contribute to tissue destruction by altering the susceptibility of proteins to hydrolytic enzymes.

Animals↗