Polyunsaturated fatty acids (PUFAs) inhibit proliferation and induce differentiation in leukemia cells. To investigate the molecular mechanisms whereby fatty acids affect these processes, U937 leukemia cells were conditioned with stearic, oleic, linolenic, ␣-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids. PUFAs affected proliferation; eicosapentaenoic acid (EPA) was the most potent on cell cycle progression. EPA enhanced the expression of the myeloid lineage-specific transcription factors CCAAT/enhancer-binding proteins (C/EBP␤ and C/EBP␦), PU.1, and c-Jun, resulting in increased expression of the monocyte lineage-specific target gene, the macrophage colony-stimulating factor receptor. Indeed, it is known that PU.1 and C/EBPs interact with their consensus sequences on a small DNA fragment of macrophage colony-stimulating factor receptor promoter, which is a determinant for expression. We demonstrated that C/EBP␤ and C/EBP␦ bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBP␦, which has been reported to be a tumor suppressor gene silenced by promoter hypermethylation in U937 cells. After U937 conditioning with EPA and bisulfite sequencing of the ؊370/؊20 CpG island on the C/EBP␦ promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBP␦ gene basal expression. Our results provide evidence for a new role of PUFAs in the regulation of gene expression. Moreover, we demonstrated for the first time that re-expression of the tumor suppressor C/EBP␦ is controlled by the methylation state of a site-specific CpG dinucleotide.

Randomized clinical trials have demonstrated that the increased intake of -3 polyunsaturated fatty acids significantly reduces the risk of ischemic cardiovascular disease, but no investigations have been performed in hereditary cardiomyopathies with diffusely damaged myocardium. In the present study, ␦-sarcoglycan-null cardiomyopathic hamsters were fed from weaning to death with an ␣-linolenic acid (ALA)-enriched versus standard diet. Results demonstrated a great accumulation of ALA and eicosapentaenoic acid and an increased eicosapentaenoic/arachidonic acid ratio in cardiomyopathic hamster hearts, correlating with the preservation of myocardial structure and function. In fact, ALA administration preserved plasmalemma and mitochondrial membrane integrity, thus maintaining proper cell/extracellular matrix contacts and signaling, as well as a normal gene expression profile (myosin heavy chain isoforms, atrial natriuretic peptide, transforming growth factor-␤1) and a limited extension of fibrotic areas within ALA-fed cardiomyopathic hearts. Consequently, hemodynamic indexes were safeguarded, and more than 60% of ALA-fed animals were still alive (mean survival time, 293 ؎ 141.8 days) when all those fed with standard diet were deceased (mean survival time, 175.9 ؎ 56 days). Therefore, the clinically evident beneficial effects of -3 polyunsaturated fatty acids are mainly related to preservation of myocardium structure and function and the attenuation of myocardial fibrosis. (Am J Pathol

Although the mechanisms underlying FAS overexpression in cancer are largely unknown, recent evidence (7-10) strongly suggests that, as in liver, FAS gene transcription in tumor cells is modulated by the sterol regulatory element binding protein-1 (SREBP-1).SREBP-1 is synthesized as an integral protein of the endoplasmic reticulum membranes. As its C-terminal regulatory domain interacts with SREBP cleavage-activating protein (SCAP), the SREBP/SCAP complex migrates to the Golgi membranes, where a two-step cleavage catalyzed by a serine protease [site-1 protease (S1P)] and a metalloprotease [site-2 protease (S2P)] is responsible for the release of the N-terminal sequence of SREBP-1 in the cytoplasm. At the nuclear level, mature SREBP-1, a transcription factor of the basic helix-loop-helix leucine zipper family, activates genes encoding FAS and other lipogenic enzymes by interacting with sterol response elements present in their promoter region (11). Insulin and dietary carbohydrates activate FAS gene transcription by this mechanism, whereas dietary polyunsaturated fatty acids (PUFAs) of both -6 and -3 series negatively modulate FAS expression by depressing SREBP-1 mRNA level and reducing the cleavage of the native protein (12, 13).Recently, it was found that pharmacological inhibitors of FAS are selectively cytotoxic to tumor cells in culture and in vivo (14-17). In particular, the FAS inhibitors cerulenin (2 S ,3 R -epoxy-4-oxo-7 E ,10 E -dodecadienamide) and C75 (3-carboxy-4-octyl-2-methylenebutyrolactone) induce Abbreviations: ACC, acetyl-CoA carboxylase; C/EBP, CCAAT enhancer binding protein; COX-2, cyclooxygenase-2; CPT-1, carnitine palmitoyltransferase-1; FAS, fatty acid synthetase; PPAR ␣ , peroxisome proliferator-activated receptor-␣ ; SCAP, sterol regulatory element binding protein-1 cleavage activating protein; S1P, site-1 protease; S2P, site-2 protease; SREBP-1, sterol regulatory element binding protein-1.

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine‐5′‐phosphate choline (cytidine‐5′‐phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell‐enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine‐5′‐phosphate choline and cytidine‐5′‐phosphate ethanolamine, respectively. Several kinetic properties of the choline‐phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. Km of cytidine‐5′‐phosphate ethanolamine, Km of diacyl glycerol, pH optimum, need for divalent cations), but the Km value for cytidine‐5′‐phosphate choline in glial cells was much lower (2.3 × 10−4m) than in neurons (1 × 10−3m). The Kmfor cytidine‐5′‐phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine‐dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.

To understand cardiac dysfunction in diabetes, the activity of protein kinase C (PKC) and protein contents of its isozymes (PKC-alpha, -beta, -epsilon, and -zeta) were examined in diabetic rats upon injection of streptozotocin (65 mg/kg iv). The hearts were removed at 1, 2, 4, and 8 wk, and some of the 6-wk diabetic animals had been injected with insulin (3 U/day) for 2 wk. The Ca(2+)-dependent PKC activity was increased by 43 and 51% in the homogenate fraction and 31 and 70% in the cytosolic fraction from the 4- and 8-wk diabetic hearts, respectively, in comparison with control values. The Ca(2+)-independent PKC activity was increased by 24 and 32% in the homogenate fraction and 52 and 89% in the cytosolic fraction from the 4- and 8-wk diabetic hearts, respectively, in comparison with control values. The relative protein contents of PKC-alpha, -beta, -epsilon, and -zeta isozymes were increased by 43, 31, 48, and 38%, respectively, in the homogenate fraction and by 126, 119, 148, and 129%, respectively, in the cytosolic fraction of the 8-wk diabetic heart. The observed changes in heart homogenate and cytosolic fractions were partially reversible upon treatment of the diabetic rats with insulin. The results suggest that the increased myocardial PKC activity and increased protein contents of the cytosolic PKC isozymes are associated with subcellular alterations and cardiac dysfunction in the diabetic heart.