Inactivation of wild-type p53 by a dominant negative mutant renders MCF-7 cells resistant to tubulin-binding agent cytotoxicity.

The present study was performed to gain insight into the role of p53 on the cytotoxicity of tubulin-binding agents (TBA) on cancer cells. Drug sensitivity, cell cycle distribution and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the MN-1 cell line containing wild-type p53 (wt-p53) and the MDD2 line, containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to the cytotoxic effects of vinblastine and paclitaxel than the MN1 cell line. MN1 cells, but not MDD2 cells, displayed wt-p53 protein accumulation as well as p21/WAF1 and cyclin G1 induction after exposure to TBA. Both cell lines arrested at G(2)/M after drug treatment. However exposure of MN1 cells to TBA resulted in a stronger variation in mitochondrial membrane potential, associated with cleavage of PARP, and more apoptosis, as measured by annexin V expression. After exposure to vinblastine, Raf 1 kinase activity was reduced in MDD2 cells but not in MN1 cells. Addition of flavopiridol to vinblastine- and paclitaxel-treated cells reversed the MDD2-resistant phenotype by inducing G(1)cell cycle arrest and inhibiting endoreduplication. We conclude that the p53 status of cancer cells influences their sensitivity to TBA cytotoxicity. This effect is likely to involve differences in the apoptotic cascade.

Phospholipids reacylation and palmitoylcoa control tumour necrosis factor-alpha sensitivity.

From the hypothesis that in TNF-alpha-resistant cells the activity of mitochondrial phospholipase A2 could be reversed by a lysophospholipid acyltransferase, we report that the mitochondrial reacylation of phosphatidylcholine as phosphatidylethanolamine was considerably higher in C6 (TNF-alpha-resistant) than in WEHI-164 (TNF-alpha-sensitive) cells. TNF-alpha did not modify the phospholipids' reacylation in C6, while in WEHI-164 it was increased several-fold. These results suggest that TNF-alpha is not sufficient to restore the barrier permeability in sensitive cells, but may be enough to explain the absence of permeability change in resistant cells. AcylCoA esters, depending on whether the acyl group is unsaturated or saturated (palmitic acid), could control membrane permeability either by participating in the reacylation of phospholipids or keeping the pore in a closed state. The analysis of the endogenous acylCoA ester pools of both cell lines show that the amount of palmitoylCoA is higher in resistant than sensitive cell lines. TNF-alpha treatment does not change these results.

A possible involvement of endogenous polyamines in the TNF-alpha cellular sensitivity.

A critical step in the cytotoxic action mechanism of tumor necrosis factor-alpha (TNF-alpha) involves, among mitochondrial dysfunctions, an early change of the inner membrane permeability displaying the characteristics of permeability transition. Cytosolic polyamines, especially spermine, are known to inhibit it. Our results show that spermine is only detectable in the TNF-alpha resistant C6 cells while N1-acetylspermidine is present in the TNF-alpha sensitive WEHI-164 cells, and putrescine and spermidine are found in both. TNF-alpha treatment does not change this distribution but only induces a quantitative alteration in TNF-alpha sensitive cells. Omission of glutamine (energetic substrate) from the culture media alters neither the TNF-alpha responsiveness of both cell lines nor their polyamine distributions, only their quantitative polyamine contents.

Increase of mitochondrial PLA2-released fatty acids is an early event in tumor necrosis factor alpha-treated WEHI-164 cells.

We have previously reported that TNF induced changes in mitochondrial enzymes, one of which, succinate-dehydrogenase, is specifically activated in various TNF-sensitive cell lines. In an attempt to further characterize the mechanism of trans-membrane signalling at the mitochondrial level, we have oriented our investigation to the study of phospholipase A2 activity localized in this organelle isolated from TNF-treated WEHI-164 cells. Under physiological conditions, this enzyme has a very low basal activity near the resting state, while under TNF treatment its activity is dramatically increased. This event is induced by TNF concentrations which also cause cytolysis; however, the activity of this enzyme is increased several hours before maximum cytotoxicity occurs. The activation of the mitochondrial phospholipase A2 is not arachidonoyl or fatty acid selective, as is the case for the cytosolic species. Phospholipase A2 and succinate-dehydrogenase display higher activities simultaneously under TNF treatment. This change in activity is linked to morphologic modifications. Mitochondria in particular display an orthodox state characterized by a large and clear matrix space and few crests.

Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase.

We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164.

[Biosynthesis of pulmonary glycoconjugates. Mechanism of glucosylation of protein and lipid acceptors]. / Biosynthèse des glycoconjugués pulmonaires. IV. Mécanisme de la glucosylation des accepteurs protéinique et lipidique.

In sheep lung microsomes, we have shown that glucosyl-transferases catalize the transfer of glucose from UDP-glucose into four different acceptors. The glucosylated products obtained are as follows: - a glucosyl-phosphoryl-polyprenol (product A) extractable by chloroform/methanol (2:1 by volume). - a product B extractable by chloroform/methanol/water (10:10:3 by volume). The product B was a mixture of five glycolipids, one of them having a chromatographic behaviour similar to the behaviour of a tetrahexosylceramide (asia-lo-GM1). - a product C insoluble in water and organic solvents which has been demonstrated to be a glycoproteinic compound. The molecular weight of this product C was 160 000 as estimated by gel-filtration. The carbohydrate moiety is composed of small oligosaccharides which are found to be attached by O-glycosidic bond to the protein chain. This linkage is not a collagen-like bond.

[Mannosylation of lipid and protein acceptors in sheep lung microsomes]. / Mannosylation des accepteurs lipidiques et protéiniques des microsomes de poumons de mouton.

In Sheep lungs, we have shown that a mannosyl-transferase activity is involved at microsomal level. This enzymatic system is able to catalyze the incorporation of mannose from GDP-mannose into two different endogenous acceptors: lipids and proteins. The mannolipid has the properties of a mannosyl-phosphoryl-polyprenol. The kinetic studies of product biosynthesis demonstrate that no precursor-product relationship exists in this system; this is an original behaviour of the lung in glycosylation phenomenon.