calcimycin and Fibrosarcoma

Activated platelets release angiogenic growth factors and have therefore been proposed to contribute to tumor angiogenesis within a potentially prothrombotic tumor microcirculation. The aim of the study was to investigate interactions of platelets with the angiogenic microvascular endothelium of highly vascularized solid tumors during growth and in response to endothelial stimulation in comparison with normal subcutaneous tissue. Experiments were performed in the dorsal skinfold chamber preparation of C57BL/6J mice bearing the Lewis lung carcinoma (LLC-1) or methylcholanthrene-induced fibrosarcoma (BFS-1). Fluorescently labeled rolling and adherent platelets, red blood cell velocity, and vessel diameters were assessed by intravital fluorescence microscopy on days 1, 3, 8, and 14 after tumor cell implantation. Slightly elevated numbers of rolling platelets were observed in the early stages of tumor angiogenesis at day 1 (control, 1.7 +/- 0.6; LLC-1, 3.4 +/- 1.8; BFS-1, 3.0 +/- 0.7 [1/mm/s], P <.05) and day 3 (control, 1.6 +/- 0.6; LLC-1, 4.1 +/- 1.7, P <.05; BFS-1, 2.3 +/- 0.5 [1/mm/s]) after tumor cell implantation. Endothelial stimulation with calcium ionophore A23187 at day 14 after tumor cell implantation resulted in a minor increase to 2.1 +/- 0.4 (LLC-1) and 1.8 +/- 0.8 (BFS-1) rolling platelets (1/mm/s) in tumor microvessels compared with 4.9 +/- 0.9 in controls (P <.05). Platelet adherence was not observed. We therefore conclude that in the 2 experimental tumors under study, (1) slightly increased platelet rolling is a transient phenomenon after tumor cell implantation, and (2) platelet-endothelial interaction in response to endothelial stimulation is reduced in tumor microvessels.

Topics: Animals; Blood Flow Velocity; Blood Platelets; Calcimycin; Calcium; Carcinoma, Lewis Lung; Cell Movement; Endothelium, Vascular; Fibrosarcoma; Ionophores; Male; Methylcholanthrene; Mice; Mice, Inbred C57BL; Microcirculation; Neovascularization, Pathologic; Platelet Activation; Platelet Adhesiveness; Skin Window Technique; Videotape Recording

Overexpression of membrane-type matrix metalloproteinase (MT-MMP-1) results in the activation of both endogenous and exogenous 72-kDa gelatinase. To understand the effects of MT-MMP-1 on 72-kDa gelatinase activation, we analyzed its expression in human fibroblasts and HT-1080 fibrosarcoma cells. Both cell types expressed the MT-MMP-1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2-3-fold. Concanavalin A treatment increased MT-MMP-1 mRNA levels in fibroblasts about 4-fold. Induction of MT-MMP-1 by phorbol 12-myristate 13-acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT-MMP-1 mRNA stability using actinomycin D indicated that the half-life was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT-1080 cells had significant 72-kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT-MMP-1 was present mainly in a 60-kDa form. PMA and concanavalin A caused 2-4-fold increases in its protein levels, while in HT-1080 cells PMA, concanavalin A, or overexpression of MT-MMP-1 did not significantly enhance the level of the 60-kDa protein. Instead, an immunoreactive, proteolytically processed 43-kDa form was observed, and its appearance correlated to 72-kDa gelatinase processing activity. Thus 72-kDa gelatinase activation, while enhanced by MT-MMP-1 expression, needs additional co-operating factors.

Topics: Base Sequence; Calcimycin; Cell Line; Collagenases; Concanavalin A; Cycloheximide; Cytokines; Dexamethasone; DNA Primers; Enzyme Activation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Fibrosarcoma; Gelatinases; Gene Expression Regulation, Enzymologic; Growth Substances; Humans; Immunoblotting; Interleukin-1; Lung; Matrix Metalloproteinase 1; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Animals; Arachidonic Acid; Aspirin; Calcimycin; Carbon Radioisotopes; Cell Line; Chromatography, High Pressure Liquid; Dinoprostone; Fibrosarcoma; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Interleukin-1; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The mass of total arachidonate released from phospholipids upon agonist stimulation of the cell and the fraction of released arachidonate which is converted to icosanoids are two parameters of arachidonate metabolism which have been difficult to quantitate because the mass of arachidonate released upon cell stimulation is very low. We have been able to quantitate both of these parameters under a variety of experimental conditions using a unique essential fatty acid-deficient mouse fibrosarcoma cell line (EFD-1), which when repleted with arachidonate, produces prostaglandin E2 (PGE2). Because there is no endogenous pool of arachidonate in these cells, the specific activity of exogenous arachidonate does not change upon incorporation into cells, an advantage which permits mass determination of very small quantities of arachidonate directly from radioactive counts. EFD-1 cells were incubated with various concentrations of [14C]arachidonate (for release studies) or unlabeled arachidonate (for PGE2 radioimmunoassays) for 24 h and then stimulated with bradykinin. The time courses for arachidonate release and PGE2 production demonstrated that free arachidonate was rapidly converted to PGE2 with plateau levels attained for both parameters within 240 s of agonist exposure for 2 microM and for 10 microM arachidonate-repleted cultures. There was a linear relationship (r = 0.94) between the mass of arachidonate in the cell and the mass of arachidonate released upon stimulation, up to a cellular concentration of 11 nmol of arachidonate/10(6) cells, a concentration 10-20% above normal for the parent mouse fibrosarcoma cell line (HSDM1C1) which is not essential fatty acid-deficient. Importantly, the percent of released arachidonate which was converted to PGE2 decreased from 90 to 15% with increasing concentrations of cellular arachidonate, because PGE2 production plateaued at greater than or equal to 6 nmol of arachidonate/10(6) cells, but total arachidonate release continued to rise. Finally, we demonstrated that agonist stimulation with thrombin, A23187, and bradykinin all showed the same percent conversion of released arachidonate to PGE2, implying that the determination of this fraction is not a function of the mechanism of release. These studies with our unique cell line indicate that, when the concentration of arachidonate in the cell is not elevated above amounts normally found in our HSDM1C1 cell line, released arachidonate is rapidly and almost quantitatively c

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Carbon Radioisotopes; Cell Line; Dinoprostone; Fatty Acids; Fibrosarcoma; Kinetics; Thrombin

The purpose of this investigation was to determine whether cells transformed by herpes simplex virus type 2 (HSV-2) can be stimulated to synthesize prostaglandins (PG). Stimulation was determined by measuring the release of PG into overlay fluids from cell monolayers prelabeled with [3H]arachidonic acid. Results showed that Ca2+ ionophore A-23187 markedly stimulated arachidonic acid release starting 30 min after treatment of HSV-2-transformed and nontransformed rat embryo fibroblast cells. However, only HSV-2-transformed cells were stimulated in production of PG. HSV-2-transformed, nontumorigenic, rat embryo fibroblast, line G, clone 2.0 cells synthesize nearly equal amounts of prostaglandin E2 (PGE2) and prostaglandin F2 alpha, while tumor (rat fibrosarcoma) cells synthesize primarily PGE2. Stimulation of PGE2 synthesis by Ca2+ ionophore A-23187 or 12-O-tetradecanoyl-phorbol-13-acetate decreased as rat fibrosarcoma cells were serially passaged in tissue culture. At low passage of parental rat fibrosarcoma cells, four distinct morphological clonal cell lines were isolated, which varied markedly in their capacity to be stimulated in PG synthesis by 12-O-tetradecanoyl-phorbol-13-acetate. There was correlation between the capacity of clone 1 cells to be stimulated in PGE2 synthesis by serum alone and capacity of the tumors produced by the clone 1 cells to metastasize to the lungs of syngeneic tumor-bearing rats. In summary, cell transformation by HSV-2 appears to be essential for stimulation of PG synthesis in cells. The capacity to be stimulated in arachidonic acid metabolism and PG synthesis may be important in the process of carcinogenesis by a putative human cancer virus.

Topics: Animals; Calcimycin; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Dinoprost; Dinoprostone; Embryo, Mammalian; Fibrosarcoma; Kinetics; Neoplasm Metastasis; Phorbols; Prostaglandins; Prostaglandins E; Prostaglandins F; Rats; Simplexvirus; Tetradecanoylphorbol Acetate

Mutagenesis followed by suicide with highly radioactive tritiated arachidonic acid has been used to select for mouse fibrosarcoma (HSDM1C1) cells defective in eicosanoid precursor uptake. Survivors of the selection were screened by replica plating and autoradiographic assay of [3H]arachidonate esterification; a mutant cell line, EPU-1, was established. EPU-1 cells contain one-third as much arachidonate as normal HSDM1C1 cells. The mutant lacks arachidonate-specific acyl-CoA synthetase, which accounts for decreased arachidonate uptake. EPU-1 exhibits enhanced turnover of arachidonoyl- but not linoleoyl-phosphatidylcholine. Bradykinin-induced arachidonate release and prostaglandin E2 synthesis are decreased in EPU-1. Thus, arachidonoyl-CoA synthetase is required for arachidonate homeostasis in HSDM1C1 cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Bradykinin; Calcimycin; Cell Line; Cell Survival; Coenzyme A Ligases; Fibrosarcoma; Linoleic Acid; Linoleic Acids; Mice; Mutation