Molecular basis of human ATM kinase inhibition.

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.

Endothelin-1 stimulates proliferation of first-trimester trophoblasts via the A- and B-type receptor and invasion via the B-type receptor.

CONTEXT: Endothelin-1 (ET-1) stimulates proliferation and invasion of first-trimester human trophoblast cells. OBJECTIVE: To test the hypothesis that ET-1 effects are mediated by different receptor subtypes [ET receptor (ETR)-A and ETR-B]. DESIGN: The location of ETR in trophoblast cell columns (wk 6-12) was investigated by immunohistochemistry and autoradiography. Trophoblasts were isolated from first-trimester human placentas and proliferative and invasive subpopulations separated using an integrin α6 antibody. Cells were incubated for 24 h with 10 µm ET-1 and different ETR antagonists: PD142893 (unselective), BQ-610 (ETR-A), and RES-701-1 (ETR-B). After ETR down-regulation by antisense oligonucleotides, proliferation (thymidine incorporation, protein synthesis) and invasion (Matrigel invasion) were measured. ETR expression in isolated cells was analyzed by Western blotting and semiquantitative RT-PCR. RESULTS: Both ETR are expressed in both subpopulations in the cell column with predominance of ETR-A in the proximal part and proliferative subpopulation, whereas ETR-B is present at similar levels in both subpopulations. These results were confirmed at the mRNA level. ET-1 increased proliferation (maximum 267% of control) and invasion (maximum 288% of control) of first-trimester trophoblasts. The mitogenic ET-1 effect was inhibited (P < 0.05) by 40-80% with each receptor antagonist and by 44 and 40%, respectively, by ETR-A and ETR-B antisense oligonucleotides. The invasion-promoting effect was almost completely blocked in the presence of the ETR-B antagonists. CONCLUSION: The effect of ET-1 on cell proliferation in first-trimester trophoblasts is mediated by both ETR, whereas its effect on invasion is mediated predominantly by ETR-B. These effects are in line with the receptor subtype location.

Inhibition of LDL oxidation by antioxidants.

Low density lipoprotein (LDL) consists of about 3000 fatty acids (50% polyunsaturated) and a single molecule apolipoprotein B (500 kDa). The endogenous antioxidants of LDL consist mainly of tocopherols and few carotenoids, which protect the PUFAS against oxidation. That native LDL contains traces of oxidation products has not been proved yet. Oxidatively modified LDL (oLDL) exhibits cytotoxic and chemotactic activities, furthermore it leads to foam cell formation, a critical step in atherogenesis. The oxidation of LDL is a free radical process and leads to various aldehydic products. The oxidation of LDL is initiated by cells as well as by transition metals like Cu2+. In both cases the oxidation goes through three consecutive phases. The lag-phase is characterized by minimal degradation of PUFAs but a loss of the antioxidants. Thereafter the PUFAs are oxidized to lipid hydroperoxides, which are only intermediates (propagation-phase). These intermediates will decompose to aldehydic products, accompanied by several additional changes in the LDL particle (decomposition-phase). For increased macrophage uptake oLDL must reach the late decomposition-phase; the presence of lipid hydroperoxides in LDL is not sufficient. It is suggested that binding of aldehydes to free amino groups of Apo B is the reason for macrophage uptake. This is supported by the finding that antibodies against aldehyde-modified LDL are able to recognize oxidized LDL in atherosclerotic lesions. Antioxidants like alpha-tocopherol are able to protect LDL against oxidation. The duration of the lag-phase shows a linear relationship with the content of alpha-tocopherol in LDL. Yet the efficiency of alpha-tocopherol to protect LDL shows strong individual variation.

Effect of oral supplementation with D-alpha-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation.

Twelve clinically healthy subjects participated in a vitamin E supplementation study. Eight were given daily dosages of 150, 225, 800, or 1200 IU RRR-alpha-tocopherol for 21 days (two persons per dose) and four received placebo. Prior, during, and after the supplementation period, alpha-tocopherol, gamma-tocopherol, and carotenoids were determined in plasma and low density lipoprotein (LDL). The maximum levels of alpha-tocopherol were 1.7- to 2.5-times the baseline values in plasma and 1.7- to 3.1-times in LDL. A high correlation existed between alpha-tocopherol in plasma and LDL. gamma-Tocopherol significantly decreased in plasma and LDL during vitamin E supplementation. No significant influence on the lipoprotein and lipid status and carotenoid levels of the participants occurred throughout the supplementation. The resistance of LDL against copper-mediated oxidation was also measured. The oxidation resistance of LDL was significantly higher during vitamin E supplementation. However, the efficacy of vitamin E in protecting LDL varied from person to person. The statistical evaluation of all data gave a correlation of r2 = 0.51 between alpha-tocopherol in LDL and the oxidation resistance as measured by the length of the lag-phase preceding the oxidation of LDL. No association was seen between levels of carotenoids and vitamin E in plasma and LDL. The present study clearly shows that in humans the oxidation resistance of LDL can be increased by vitamin E supplementation.

Time course study of lipid peroxidation induced by N2-methyl-9-hydroxyellipticinium acetate or celiptium in rat renal cortex.

Celiptium is an ellipticine derivative with renal toxic side effects. It has recently been characterized as a lipid overload in proximal tubular cells where loss of total phospholipids (in particular phosphatidylethanolamine) and of polyunsaturated fatty acids are linked to the accumulation of unsaturated free fatty acids and aldehydes. A time course study of celiptium-induced peroxidative damage showed that a single dose of 40 mg/kg of celiptium induced no change in total or individual phospholipids of rat renal cortex. On the other hand, free fatty acids and thiobarbituric acid reactive substances increased as early as 1 hr after celiptium injection. 4-Hydroxynonenal (4-HNE) also increased whereas polyunsaturated fatty acids levels decreased at 6 and 24 hr. After 24 hr no change was detected in microsomal phospholipids. In contrast, the brush-border membranes showed alterations such as decrease in total phospholipids and polyunsaturated fatty acids levels accompanied by increase in aldehydes. It appears that peroxidative damage occurs in brush-border membranes of celiptium-treated rat kidneys with preferential losses of phosphatidylethanolamine (PE, 30%) and phosphatidylcholine (PC, 14%).

Effect of antioxidants on oxidative modification of LDL.

Human low density lipoprotein (LDL) with a molecular mass of 2.5 million contains on average 1300 molecules of polyunsaturated fatty acids (PUFAs) bound in the different lipid classes. The predominant antioxidant in LDL is alpha-tocopherol, with an average of 6 molecules in each LDL particle. The other substances with potential antioxidant activity are: gamma-tocopherol, beta-carotene, alpha-carotene, lycopene, cryptoxanthin, cantaxanthin, phytofluene and ubiquinol-10. Each is present in amounts of only 1/20th to 1/300th of that of alpha-tocopherol. If LDL is exposed to oxidative conditions (Cu++ ions, macrophages) a lag phase precedes the oxidation of PUFAs. During the lag phase the antioxidants disappear with alpha-tocopherol the first to go and beta-carotene the last. The lag phase, which can readily be determined, is an index of the oxidation resistance of LDL. If LDL is loaded with vitamin E in vitro its oxidation resistance increases linearly with its alpha-tocopherol content according to the equation, y = kx+a. The same relationship is applicable if the alpha-tocopherol content of LDL is increased by taking oral vitamin E. Daily doses of 150, 225, 800 and 1200 IU RRR-alpha-tocopherol increased the LDL alpha-tocopherol on average to 138%, 158%, 144% and 215% of the initial value, the oxidation resistance being increased to 118%, 156%, 135% and 175%, respectively. The efficiency of vitamin E-dependent (= k) and the vitamin independent (= a) oxidation resistance seem to be subject specific with strong individual variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of vitamin E in preventing the oxidation of low-density lipoprotein.

The fatty acid composition, antioxidants, and the oxidation resistance of the low-density lipoproteins (LDL) from a number of different donors were determined. The oxidation resistance of LDL, as determined in vitro by the duration of the lag-phase in copper ion-induced oxidation, did not correlate with the alpha-tocopherol content of the LDL. By supplementating plasma with vitamin E, the alpha-tocopherol content of LDL could be increased from approximately 9 to 30 mol/mol LDL and also the oxidative resistance increased nearly linearly with increasing alpha-tocopherol content. The results indicate that alpha-tocopherol is an important, yet not the only parameter that determines the oxidation resistance of LDL.

Antioxidants and resistance against oxidation of porcine LDL subfractions.

The objective of this study was to determine the level of antioxidants, the content of fatty acids and peroxidation products, and the resistance against oxidation of native porcine LDL1 and LDL2. There were no significant differences in the fatty acid distribution of both native low density lipoprotein (LDL) subfractions, which was similar to that of human LDL. The total amount of alpha- and gamma-tocopherol of pig LDL was significantly lower than in human LDL, and beta-carotene, lycopene, and retinyl esters were totally absent. Levels of thiobarbituric acid-reacting substances (TBARS) and lipid peroxides in freshly isolated pig LDL subfractions were below or only slightly above the detection limit. The susceptibility to oxidation of both LDL subfractions was investigated by addition of Cu2+ as prooxidant. The results show that pig LDL subfractions are much more susceptible to oxidation as measured by the duration of the lag phase preceding the onset of rapid lipid peroxidation. From the low content of vitamin E one would expect even much shorter lag phases. The possibility therefore exists that pig LDL contains additional, and as yet unidentified, antioxidants.

Evidence for 4-hydroxyalkenals in rat renal cortex peroxidized by N2-methyl-9-hydroxyellipticinium acetate or Celiptium.

The antitumor drug celiptium is an ellipticine derivative whose nephrotoxic pathogenesis implicates a lipid peroxidation process. It has been shown that hydrophobic lipid deposits overload the proximal tubular cells. Histochemistry with Holczinger's technique has demonstrated that these deposits are free fatty acids. In this study, the fatty acid analysis of phospholipids and neutral lipids was performed in rat renal cortex 4 and 8 days following a single i.v. dose of 20 mg/kg celiptium and showed: (1) a loss of polyunsaturated fatty acids within total phospholipids and a loss of phosphatidylethanolamine with a preferential decrease of arachidonic (20:4) and docosahexaenoic (22:6) acids; (2) an increase of free fatty acid levels with an increase in oleic (18:1) and linoleic (18:2) acids; (3) an increase of thiobarbituric acid-reactive substances or aldehydes. The analysis of these aldehydes showed significant amounts of 4-hydroxyalkenals, mainly the presence of 4-hydroxynonenal on day 4 and of a hydroxyaldehyde with a chromatographic behavior very similar to 4-HNE on day 8. We conclude that celiptium induced a preferential decrease of phosphatidylethanolamine linked to the formation of unsaturated free fatty acids and of 4-hydroxyalkenals. The toxic side-effects of these breakdown products produced in the proximal tubular cell are discussed in light of the lipid peroxidation process involved in the renal toxicity of celiptium.

Effects of low- and high-density lipoproteins on the proliferation of human breast cancer cells in vitro: differences between hormone-dependent and hormone-independent cell lines.

The influence of low- and high-density lipoproteins on the proliferation of human breast cancer cells in culture was studied. We compared total cell number after incubation for 48 hr in culture medium containing or lacking plasma lipoproteins. Marked differences were found between hormone-dependent (MCF-7, T-47-D, ZR-75) and hormone-independent (MDA-MB-231, HBL-100) mammary tumor cell lines. The cells also reacted differently on the different lipoproteins offered in the medium. Human low-density lipoproteins (LDL) exhibited a marked stimulation of the growth of hormone-independent cell lines but no or only toxic effects upon the hormone-sensitive lines. Human high-density lipoproteins (HDL) stimulated the proliferation of all cell lines in a dose-dependent manner but hormone-independent cells showed a higher response. These findings point towards different utilizations of nutrients in hormone-dependent and hormone-independent cells.

Influence of morphology and malignancy of three new human melanoma cell lines on the cyclooxygenase pathway.

Three newly established human melanoma cell lines (WU-BI, PN-JC, MJ-ZJ) of different morphology and different stage of malignancy were incubated with ionophore A23187 (2.5 to 40 microM) or arachidonic acid (AA, 6.25 to 100 microM). PGF2 alpha, 6-keto-PGF1 alpha, PGE2, TXB2 and 2,3-dinor-TXB2 from isolated cells and supernatants were measured by negative ion chemical ionization gas chromatography/mass spectrometry (GC/MS). PGE2 decreased in the fibroblastoid MJ-ZJ cells from 36.7 ng/mg cell protein about 70% (A23187) and about 20% (AA), respectively. However, in the cell supernatant PGE2 increased up to 295.4 +/- 66.5 ng/mg cell protein. Production of PGF2 alpha and PGE2 increased up to 5.7 +/- 1.2 ng/mg cell protein for polydendritic WU-BI cells and spindle shaped PN-JC cells. Up to 9.3 +/- 4.3 ng PGF2 alpha and 13.4 +/- 4.7 ng PGE2 was measured for WU-BI and PN-JC in the cell supernatants. All three melanoma cell lines completely lacked formation of 6-keto-PGF1 alpha, TXB2, and 2,3-dinor-TXB2.

Continuous monitoring of in vitro oxidation of human low density lipoprotein.

The kinetics of the oxidation of human low density lipoprotein (LDL) can be measured continuously by monitoring the change of the 234 nm diene absorption. The time-course shows three consecutive phases, a lag-phase during which the diene absorption increases only weakly, a propagation phase with a rapid increase of the diene absorption and finally a decomposition phase. The increase of the dienes is highly correlated with the increase of MDA or lipid hydroperoxides. The duration of the lag-phase is determined by the endogenous antioxidants contained in LDL (vitamin E, carotenoids, retinylstearate). Water-soluble antioxidants (ascorbic acid, urate) added in micromolar concentrations prolong the lag-phase in a concentration-dependent manner. The determination of the lag-phase is a convenient and objective procedure for determining the susceptibility of LDL from different donors towards oxidation as well as effects of pro- and antioxidants.

Interaction of lipoprotein Lp(a) and low density lipoprotein with glycosaminoglycans from human aorta.

The lipoprotein complexing activity of glycosaminoglycans (GAG) prepared from human aortas with lipoprotein Lp(a) in comparison to low density lipoproteins (LDL) was determined tubidimetrically in the presence of Ca++. In control experiments, purified chondroitin-6 sulfate and proteoglycans (PG) were used. Lp(a) exhibited approximately a threefold higher reactivity. Analyzing the chemical composition of the complexes, we found that Lp(a) had greater than fourfold higher binding capacity for GAG. The binding capacity of Lp(a) to PG was 3.4-fold higher as compared to LDL. The binding capacity of both lipoproteins for chondroitin-6 sulfate was only 50% in comparison to GAG, but again Lp(a) was four times more reactive. Neuraminidase treatment of LDL or Lp(a) did not interfere with GAG or chondroitin-6 sulfate binding. If, on the other hand, Lp(a) was treated with dithiothreitol and the Lp(a)-specific protein (apoprotein [apo] a) was removed, the GAG binding was reduced by about 45%. Apo a by itself gave no insoluble complexes with GAG. LDL and Lp(a)-s GAG and -LP(a)-PG complexes were incubated with mouse peritoneal macrophages (MPM), and the stimulation of cholesteryl ester formation was studied. At identical lipoprotein cholesterol concentrations, Lp(a)-GAG complexes exhibited a 1.3-fold higher stimulation of cholesterol esterification as compared to LDL-GAG. This difference was even more striking if lipoproteins were compared at a molar basis. PG-lipoprotein complexes were much more active with respect to interactions with MPM. The highest amount of cholesterol ester formation upon incubation with MPM was found with PG-Lp(a) complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of various lipoproteins from normal and dyslipoproteinemic plasma with mouse peritoneal macrophages.

In this study we tried to elucidate the atherogenicity of various plasma lipoproteins with respect to their capability of foam cell formation. Mouse peritoneal macrophages (MPM) were incubated with increasing amounts of lipoproteins and the incorporation of 14C oleate into the cholesteryl ester fraction was followed. The results may be summarized as follows: freshly isolated Lp(a) behaves very similar to normal LDL causing no or little increase in CE formation in MPM. Lp(a) treated with dextran sulfate as well as with antibodies to Apo-a, strongly interact with scavenger receptors causing massive accumulation of CE in MPM. The abnormal lipoproteins from patients suffering from liver disease, LP-X, HDL-E cause no increase in CE formation of MPM. They behave very similar to artificial PL/FC liposomes. If on the other hand these abnormal lipoproteins are mixed with Ac-LDL, a synergistic effect was observed causing an approx. 30 per cent increase in CE-formation as compared to Ac-LDL alone. This was caused by a net transfer of FC from abnormal lipoproteins to Ac-LDL alone. This was caused by a net transfer of FC from abnormal lipoproteins to Ac-LDL. It is concluded that the lipoproteins studied in this report by itself exert no atherogenic function in MPM. They may, however, aggravate the atherogenicity of other processes known to be involved in the development of vascular diseases.

Cholesterol esterification in mouse peritoneal macrophages in the presence of pathological human plasma lipoproteins.

Mouse peritoneal macrophages were incubated with abnormal lipoproteins (LP-X, HDL-E, VLDL-p, IDl-p and LDL-p) from a patient with secondary deficiency in phosphatidylcholine-sterol acyltransferase, or with phosphatidylcholine/cholesterol liposomes, and the stimulation of cholesteryl ester formation was studied. Acetylated low density lipoproteins served as a control. It was found that macrophages incubated with LP-X, the other pathological lipoproteins or with liposomes did not show an enhanced cholesterol esterification. Also HDL-E had no effect despite of its high apoE content and the fact that apoE has been postulated to be the agonist in beta-VLDL binding to macrophages.