Selective inhibition of phosphatidylinositol phospholipase C by cytotoxic ether lipid analogues.

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) has been shown to be a direct inhibitor of Swiss 3T3 fibroblast and BG1 ovarian adenocarcinoma cell cytosolic phosphoinositide selective phospholipase C (PIPLC) using [3H]-phosphatidylinositol-(4, 5)-bisphosphate ([3H]PIP2) as the substrate. The inhibition occurred when ET-18-OCH3 was incorporated into the [3H]PIP2 substrate micelles, with 50% inhibition (IC50) occurring at a ET-18-OCH3: [3H]PIP2 ratio of 0.04, or an assay concentration of 0.4 microM, and when ET-18-OCH3 was added directly to the incubation, with an IC50 of 9.6 microM. Lipid prepared from cells exposed to cytotoxic concentrations of ET-18-OCH3 for 18 h also inhibited PIPLC with an IC50 less than 1 microM. The noncytotoxic analogue 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine inhibited PIPLC when incorporated into the [3H]PIP2 substrate micelles, but lipid from cells grown with 5 microM 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine did not inhibit PIPLC. BG1 cells, which were more sensitive than Swiss 3T3 fibroblasts to growth inhibition by ET-18-OCH3, had a cytosolic PIPLC activity one-third that of Swiss 3T3 cells. NIH 3T3 cells exhibited the same sensitivity to growth inhibition by ET-18-OCH3 as Swiss 3T3 cells and had a similar level of PIPLC. v-sis NIH 3T3 cells were relatively resistant (greater than 3-fold) to growth inhibition by ET-18-OCH3 and had a cytosolic PIPLC activity more than twice that of the wild type cells. ET-18-OCH3 was a weak inhibitor, IC50 greater than 100 microM, of phospholipase D activity in NIH 3T3 cell membranes. In intact NIH 3T3 cells ET-18-OCH3 at cytotoxic concentrations did not inhibit phospholipase D or phosphatidylcholine-selective phospholipase C activity. The results show that the ether lipid analogues at cytotoxic concentrations are selective inhibitors of PIPLC and that the inhibition of PIPLC may be related to the growth inhibitory activity of the ether lipid analogues.

Increased NAD(P)H:(quinone-acceptor)oxidoreductase activity is associated with density-dependent growth inhibition of normal but not transformed cells.

The activity of DT-diaphorase [NAD(P)H:(quinone-acceptor)oxidoreductase] is increased 7-fold in wild-type BALB/c 3T3T cells as they reach confluence and become density growth arrested. Harvesting and replating the cells at low density resulted in a loss of DT-diaphorase with a half time of 7 h, and removal of serum from high-density growth-arrested cells resulted in a decrease in DT-diaphorase with a half time of 3 days. Platelet-derived growth factor and insulin together, but not singly, maintain elevated DT-diaphorase levels in high-density growth-arrested BALB/c 3T3T cells. The increase in DT-diaphorase at high density diminished proportionately to the extent of transformation in four cell lines, 4NQO-3T3T, UV-3T3T, EJras-3T3T. and CSV3-1-3T3T. The most transformed cell line, CSV3-1-3T3T, showed no increase in DT-diaphorase at high density. Since there was no increase in DT-diaphorase mRNA in high-density growth-arrested wild-type BALB/c 3T3T cells compared to rapidly growing cells, the increase in DT-diaphorase activity at high density is most likely due to posttranslational events. High-density growth-arrested wild-type BALB/c 3T3 cells exhibited a greater sensitivity to growth inhibition by the antitumor quinone diaziquone [1,4-cyclohexadiene-1,4- dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ether], which is metabolically activated by DT-diaphorase, than do low-cell-density, growth-arrested cells. The significance of the increase in DT-diaphorase at high cell density in normal cells and its loss in transformed cells may be related to the phenomenon of density-dependent growth inhibition in nontransformed but not in transformed cells.

The anomaly of pyridine nucleotide synergism in carbon tetrachloride metabolism.

Carbon tetrachloride metabolism was examined in hepatic microsomes isolated from control and phenobarbital-treated Sprague-Dawley rats to determine the mechanism of pyridine nucleotide synergism. An NADPH generator increased metabolism two fold as determined by lipid peroxidation. Addition of NADH to the reaction system did not alter the maximum velocity, but did decrease the Km for NADPH from 61 microM to 7.6 microM in control and from 21 microM to 6.3 mM PB microsomes. Addition of NAD+ produced an increase in metabolism similar to NADH. Substrates and competitive inhibitors of nucleotide pyrophosphatase also enhanced CCl4 metabolism. A high correlation (r = 0.947) was indicated between the percent inhibition of nucleotide pyrophosphatase and the percent synergism of NADPH-catalyzed CCl4 metabolism. Thus, pyridine nucleotide synergism in CCl4 metabolism appears to result from the increased availability of NADPH produced by a decreased degradation of the NADPH by the nucleotide pyrophosphatase.

Microsomal metabolism of carbon tetrachloride: participation of pyridine nucleotide synergism.

The role of pyridine nucleotide synergism in CCl4 metabolism was evaluated for its potential contribution to enhanced lipid peroxidation. Male Sprague-Dawley rats receiving either no treatment (control) or treatment with phenobarbital (PB) were used to prepare hepatic microsomes. Metabolism was evaluated in the presence and absence of an NADPH generator system and in the presence and absence of NADH. The generator system produced a greater extent of metabolism for both control and PB microsomes. NADH-catalyzed CCl4 metabolism occurred to a similar extent in control and PB microsomes, amounting to 9-10% and 5-6% of the NADPH rate in control and PB microsomes, respectively. Synergism by NADH occurred at the lowest concentrations of NADPH, apparently decreasing the Km for NADPH and having little effect on the Vmax. Addition of NAD+ produced synergism, as did the addition of 5' AMP, an inhibitor of nucleotide pyrophosphatase. Thus, the synergistic increase in CCl4 metabolism produced by NADH may occur in part from an increased availability of NADPH, as a result of decreased degradation, rather than by electron donation from NADH.