linoleic-acid and Fibrosarcoma

Receptor-mediated overexpression of H2O2-generating peroxisomal fatty acyl-CoA oxidase (AOX) has been implicated in peroxisome proliferator-induced hepatocarcinogenesis. To investigate the role of rat AOX generated H2O2 in transformation, we overexpressed this enzyme in a non-tumorigenic mouse fibroblast cell line (LM tk-) under control of mouse urinary protein promoter. The clones overexpressing rat peroxisomal AOX, when exposed to a fatty acid substrate (100 microM linoleic acid) for 6 to 96 h, demonstrated > 10-fold increase of intracellular H2O2. This increase in H2O2 concentration was associated with increased apoptosis as evidenced by DNA fragmentation, in situ terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL). These cell lines stably expressing AOX formed colonies in soft agar in proportion to the duration (1-7 weeks) of exposure to a fatty acid substrate (100 microM linoleic acid, erucic acid or nervonic acid) and these transformants developed into fibrosarcomas when injected in athymic nude mice. These results suggest that H2O2 generated by AOX overexpression in immortalized fibroblasts leads to apoptosis, and the extent and duration of H2O2 and possibly other DNA damaging reactive oxygen species generated by the overexpression of peroxisomal AOX can influence apoptosis and neoplastic transformation.

Topics: Acyl-CoA Oxidase; Animals; Apoptosis; Blotting, Northern; Blotting, Western; Cell Transformation, Neoplastic; Cells, Cultured; Colony-Forming Units Assay; DNA Primers; Erucic Acids; Fatty Acids, Monounsaturated; Fibroblasts; Fibrosarcoma; Gene Expression; Hydrogen Peroxide; In Situ Nick-End Labeling; Linoleic Acid; Male; Mice; Mice, Nude; Oxidoreductases; Polymerase Chain Reaction; Rats

We have demonstrated that the uptake and agonist-induced release of a pulse of arachidonate are influenced by the size and composition of preexisting endogenous fatty acid pools. EFD-1 cells, an essential fatty acid-deficient mouse fibrosarcoma cell line, were incubated with radiolabeled (14C or 3H] arachidonate, linoleate, eicosapentaenoate (EPA), palmitate, or oleate in concentrations of 0-33 microM for 24 h. After 24 h, the cells were pulsed with 0.67 microM radiolabeled (3H or 14C, opposite first label) arachidonate for 15 min and then stimulated with 10 microM bradykinin for 4 min. Because EFD-1 cells contain no endogenous essential fatty acids, we were able to create essential fatty acid-repleted cells for which the specific activity of the newly constructed endogenous essential fatty acid pool was known. Loading the endogenous pool with the essential fatty acids arachidonate, eicosapentaenoate, or linoleate (15-20 nmol of fatty acid incorporated/10(6) cells) decreased the uptake of a pulse of arachidonate from 200 to 100 pmol/10(6) cells but had no effect on palmitate uptake. The percent of arachidonate incorporated during the pulse which was released upon agonist stimulation increased 2-fold (4-8%) as the endogenous pool of essential fatty acids was increased from 0 to 15-20 nmol/10(6) cells. This 8% release was at least 3-fold greater than the percent release from the various endogenous essential fatty acid pools. In contrast, loading the endogenous pool with the nonessential fatty acids oleate or palmitate to more than 2-3 times their preexisting cellular level had no effect on the uptake of an arachidonate pulse. Like the essential fatty acids, increasing endogenous oleate increased (by 2-fold) the percent release of arachidonate incorporated during the pulse, whereas endogenous palmitate had no effect on subsequent agonist-induced release from this arachidonate pool. These studies show that preexisting pools of essential and nonessential fatty acids exert different effects on the uptake and subsequent releasability of a pulse of arachidonate.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Carbon Radioisotopes; Eicosapentaenoic Acid; Fatty Acids; Fatty Acids, Essential; Fibrosarcoma; Linoleic Acid; Linoleic Acids; Mice; Oleic Acid; Oleic Acids; Palmitic Acid; Palmitic Acids; Tritium; Tumor Cells, Cultured

We have demonstrated that icosanoid production can be inhibited by altering the distribution of arachidonate within the cell, so that it is not released from phospholipids for icosanoid synthesis. This effect was observed in a prostaglandin E2-producing cell line (HSDM1C1) by deprivation of exogenous arachidonate for 24-48 h. Icosanoid production by the cells upon bradykinin stimulation was impaired despite no change in the concentration of arachidonate within the cell and no change in the activity of cyclooxygenase, phospholipases, acyltransferases, or fatty acyl-CoA hydrolase. Associated with the decline in prostaglandin E2 production was an increase in arachidonate incorporation into ethanolamine plasmalogens and a decrease in the activity of the enzyme arachidonoyl-CoA synthetase, which may play a role in compartmentation of arachidonate within the cell. Thus, we have found that a decrease in icosanoid production can be achieved without pharmacologic intervention by a short-term restriction of exogenous arachidonate which leads to redistribution of arachidonate within phospholipids and/or subcellular membranes in the cell.

Topics: Acyltransferases; Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Coenzyme A Ligases; Dinoprostone; Fatty Acids, Unsaturated; Fibrosarcoma; Linoleic Acid; Linoleic Acids; Mice; Palmitoyl-CoA Hydrolase; Phospholipases; Plasmalogens; Prostaglandin-Endoperoxide Synthases; Prostaglandins E; Tumor Cells, Cultured

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