Inhibition of DNA replication by fish oil-treated cytoplasm is counteracted by fish oil-treated nuclear extract

2002 ◽  
Vol 283 (5) ◽  
pp. C1365-C1375 ◽  
Author(s):  
Sybille Rex ◽  
Maria A. Kukuruzinska ◽  
Nawfal W. Istfan

We have recently noted that cells treated with fish oil and n–3-fatty acids show slower DNA replication rates than cells treated with a control emulsion or corn oil only. However, it is not clearly understood how such an effect is induced. Fish oil and its metabolites are known to have several modulating effects on signal transduction pathways. Alternatively, they may influence DNA replication by interacting directly with nuclear components. To investigate this problem in greater detail, we have studied the kinetics of DNA synthesis in a cell-free system derived from HeLa cells. Nuclei and cytosolic extract were isolated from cells synchronized in early S phase after treatment with control emulsion, corn oil, or fish oil, respectively. The nuclei were reconstituted with cytosolic extract and a reaction mixture containing bromodeoxyuridine (BrdU) triphosphate to label newly synthesized DNA. The rate of DNA synthesis was measured by bivariate DNA/BrdU analysis and flow cytometry. We show that fish oil-treated cytosol inhibits the elongation of newly synthesized DNA by ∼80% in control nuclei. However, nuclei treated with fish oil escape this inhibitory effect. We also show that addition of nuclear extract from fish oil-treated cells reverses the inhibitory effect seen in the reconstitution system of control nuclei and fish oil-treated cytosol. These results indicate that polyunsaturated fatty acids can modulate DNA synthesis through cytosolic as well as soluble nuclear factors.

1985 ◽  
Vol 162 (4) ◽  
pp. 1336-1349 ◽  
Author(s):  
C A Leslie ◽  
W A Gonnerman ◽  
M D Ullman ◽  
K C Hayes ◽  
C Franzblau ◽  
...  

B10.RIII and B10.G mice were transferred from a diet of laboratory rodent chow to a standard diet in which all the fat (5% by weight) was supplied as either fish oil (17% eicosapentaenoic acid [EPA], 12% docosahexaenoic acid [DHA], 0% arachidonic acid [AA], and 2% linoleic acid) or corn oil (0% EPA, 0% DHA, 0% AA, and 65% linoleic acid). The fatty acid composition of the macrophage phospholipids from mice on the chow diet was similar to that of mice on a corn oil diet. Mice fed the fish oil diet for only 1 wk showed substantial increases in macrophage phospholipid levels of the omega-3 fatty acids (of total fatty acid 4% was EPA, 10% docosapentaenoic acid [DPA], and 10% DHA), and decreases in omega-6 fatty acids (12% was AA, 2% docosatetraenoic acid [DTA], and 4% linoleic acid) compared to corn oil-fed mice (0% EPA, 0% DPA, 6% DHA, 20% AA, 9% DTA, and 8% linoleic acid). After 5 wk this difference between the fish oil-fed and corn oil-fed mice was even more pronounced. Further small changes occurred at 5-9 wk. We studied the prostaglandin (PG) and thromboxane (TX) profile of macrophages prepared from mice fed the two diets just before being immunized with collagen. Irrespective of diet, macrophages prepared from female mice and incubated for 24 h had significantly more PG and TX in the medium than similarly prepared macrophages from male mice. The increased percentage of EPA and decreased percentage of AA in the phospholipids of the macrophages prepared from the fish oil-fed mice was reflected in a reduction in the amount of PGE2 and PGI2 in the medium relative to identically incubated macrophages prepared from corn oil-fed mice. When this same fish oil diet was fed to B10.RIII mice for 26 d before immunization with type II collagen, the time of onset of arthritis was increased, and the incidence and severity of arthritis was reduced compared to arthritis induced in corn oil-fed mice. The females, especially those on the fish oil diet, tended to have less arthritis than the males. These alterations in the fatty acid pool available for PG and leukotriene synthesis suggest a pivotal role for the macrophage and PG in the immune and/or inflammatory response to type II collagen.


2006 ◽  
Vol 26 (5) ◽  
pp. 1997-2011 ◽  
Author(s):  
Paris Petersen ◽  
Danny M. Chou ◽  
Zhongsheng You ◽  
Tony Hunter ◽  
Johannes C. Walter ◽  
...  

ABSTRACT We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.


2018 ◽  
Vol 44 (1) ◽  
pp. 9
Author(s):  
Amir Moslehi ◽  
Ali Asghar Sadeghi ◽  
Parvin Shawrang ◽  
Mehdi Aminafshar

Background: Liver plays an important role in energy homeostasis. Intense liver diseases are accompanied with lower concentrations of n-3 and n-6 poly unsaturated fatty acids (PUFA). It has been found that n-3 PUFA play importantly protective roles in the liver. There was limited information about the effects of lipid sources on serum lipid components and liver sterol regulatory element binding-1 (SREBP-1) gene expression. Therefore, the aim of this study was to evaluate and compare the effects of fish oil, corn oil, olive oil and tallow, respectively, as dietary sources of n-3, n-6, n-9 and saturated fatty acid on serum lipid compound and liver SREBP-1 gene expression in broiler chicks.Materials, Methods & Results: In a completely randomized design, 240 one-day-old broiler chicks were allocated to five dietary treatments, four replicates and 12 chicks per each. Dietary treatments included of: control (diet without lipid supplementation) and diet supplemented with fish oil as a n-3 fatty acid source, corn oil as a n-6 fatty acid source, olive oil as a n-9 fatty acid source and tallow as a saturated fatty acid which were added to diets at 1.5, 3 and 4% in the starter, grower and finisher, respectively. At days 28 and 42 of age, liver tissue was dissected out and samples were placed in liquid nitrogen, also blood samples were collected. The SREBP-1 mRNA expression in liver tissue was quantitated using RT-PCR. Broilers fed diets containing fish oil, corn oil and olive oil as unsaturated fatty acid sources had lower concentrations of triacylglycerol than those fed other diets. Also, diets containing fish oil and tallow showed the lowest low density lipoprotein (LDL) levels at day 28 of age. Diets containing corn oil and olive oil showed higher levels of cholesterol and high density lipoprotein (HDL) than other diets but, these differences were not significant at days 28 and 42 of age  (P ≥ 0.05). In both sampling periods, the chickens fed diets containing tallow expressed higher (P < 0.05) SREBP-1 gene as compared to those fed other dietary lipids. Broilers fed diet containing corn oil had higher (P < 0.05) SREBP-1 gene expression than those fed diet containing fish oil and olive oil.Discussion: Serum lipids were affected by dietary fat source. Lower concentration of serum triacylglycerols was found in broilers fed fish oil and corn oil diets as dietary n-3 and n-6 fatty acid sources. It was shown that dietary PUFA, especially n-6 and n-3 fatty acids could reduce hepatic fatty acid and triacylglycerol synthesis. Broilers fed corn oil diet showed elevated LDL levels respect with those fed fish oil or tallow at day 28 of age. The results showed that dietary polyunsaturated fatty acids and saturated fatty acids substantially affected on SREBP-1 gene expression at days 28 and 42 of age. The birds fed fish oil as n-3 PUFA source expressed lower SREBP-1 gene compared with other groups at day 28 of age. Longer chain length, high number of double bonds, and the presence of the first double bond gives these fatty acids distinct and unique properties that separate them and their metabolic products from the more common n-6 and n-9 fatty acids. The finding of this study indicated lipogenic effects of tallow and then corn oil. The results confirmed that each of dietary lipid sources had different effect on serum lipid components. It was also found that SREBP-1 gene expression is age-dependent and it increased as age of broilers increased.


1999 ◽  
Vol 82 (11) ◽  
pp. 1522-1527 ◽  
Author(s):  
Antonio Carletto ◽  
Patrizia Guarini ◽  
Simonetta Galvani ◽  
Domenico Biasi ◽  
Paolo Bellavite ◽  
...  

SummaryTo investigate the possible regulating role of ω-6 and of ω-3 fatty acids on platelet adhesiveness, we randomised 60 volunteers into three groups to take 20 ml (equivalent to 0.3 g ω-6, 3.6 g ω-3; ω-6/ω-3 ratio 0.1) per day of a fish oil supplement, or to take 25 g (equivalent to 1.5 g ω-6, 0.5 g ω-3; ω-6/ω-3 ratio 3) per day of a soy lecithin supplement, or to continue on their usual diet without any supplement (control group) for a period of 15 days. Platelet adhesion on fibrinogen-coated 96-well microtitre plates was evaluated in the resting condition and after stimulation with 2 ωM ADP or 0.02 U/ml thrombin. Compared to the values before the experimental period, the fish oil group showed a significant reduction in stimulated adhesion (with ADP: from 18.8% to 15.6%, p <0.01; with thrombin: from 24.4% to 20.8%, p <0.005), whereas no difference was noted in the resting condition (from 3.6% to 3.5%, NS). In the soy lecithin group, platelet adhesion was increased in all test conditions (with ADP: from 18.7% to 23.2%, p <0.001; with thrombin: from 24.0% to 29.9%, p <0.001; resting: from 3.5% to 6.6%, p <0.001). No significant changes were observed in the control group. A good correlation was found between platelet adhesion data and the changes in the platelet fatty acid ω-6/ω-3 ratio caused by the different supplementations. Our results indicate an inhibitory effect of fish oil rich in ω-3 fatty acids on stimulated human platelet adhesiveness and a stimulatory effect of soy lecithin rich in ω-6 fatty acids on resting and stimulated adhesion. They suggest moreover that the ω-6/ω-3 ratio is a determinant of platelet adhesion.


2001 ◽  
Vol 357 (2) ◽  
pp. 481-487 ◽  
Author(s):  
Xiaozhong ZHENG ◽  
Michael AVELLA ◽  
Kathleen M. BOTHAM

The effects of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (derived from fish or corn oil respectively) on the secretion of very-low-density lipoprotein (VLDL) lipid and apolipoprotein B (apoB) by rat hepatocytes in culture was investigated. Remnants were prepared in vivo from chylomicrons obtained from rats given an oral dose of fish or corn oil and incubated with cultured hepatocytes for up to 16h. The medium was then removed and the secretion of cholesterol and triacylglycerol into the whole medium or the ρ < 1.050g/ml fraction during the following 7–24h was determined. After exposure of the cells to fish-oil as compared with corn-oil remnants, secretion of both cholesterol and triacylglycerol into the whole medium was decreased by 25–35%, and secretion into the ρ < 1.050g/ml fraction was decreased by 20–25%. In addition, the levels of apoB48 found in the ρ < 1.050g/ml fraction were significantly lower in cells treated with fish-oil rather than corn-oil remnants, although the levels of apoB100 remained unchanged. The expression of mRNA for apoB, as determined by reverse-transcriptase PCR, however, was not significantly changed after exposure of the cells to both types of remnants. These results demonstrate that the effects of dietary n-3 polyunsaturated fatty acids in depressing hepatic VLDL secretion occur directly when they are delivered to the liver from the intestine in chylomicron remnants, and that the secretion, but not the synthesis, of apoB is targeted.


1991 ◽  
Vol 112 (4) ◽  
pp. 557-566 ◽  
Author(s):  
G H Leno ◽  
R A Laskey

We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.


2004 ◽  
Vol 321 (1) ◽  
pp. 116-123 ◽  
Author(s):  
Yong-Fu Xiao ◽  
Qingen Ke ◽  
Yu Chen ◽  
James P. Morgan ◽  
Alexander Leaf

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