Dominique Lautier

Molecular and biochemical features of poly (ADP-ribose) metabolism

In the past five years, poly(ADP-ribosyl)ation has developed greatly with the help of molecular biology and the improvement of biochemical techniques. In this article, we describe the physico-chemical properties of the enzymes responsible for the synthesis and degradation of poly(ADP-ribose), respectively poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase. We then discuss the possible roles of this polymer in DNA repair and replication as well as in cellular differentiation and transformation. Finally, we put forward various hypotheses in order to better define the function of this polymer found only in eucaryotes. (Mol Cell Biochem122: 171–193, 1993)

Kit signaling and negative regulation of daunorubicin-induced apoptosis : role of phospholipase Cγ

Previous studies have demonstrated that activation of Kit by stem cell factor (SCF), its natural ligand, or by gain-of-function point mutation in its intracellular domain, confers significant protection against apoptosis induced by growth factor deprivation or radiation. However, the effects of Kit activation on the cellular response to anti-tumor agents have not been so extensively documented. This study shows that daunorubicin-induced apoptosis and cytotoxicity were reduced in the murine Ba/F3 cells transfected with Kit (Ba/F3-Kit) in the presence of SCF and in Ba/F3 cells transfected with a constitutively active Kit variant (Ba/F3-KitΔ27), compared to either parental Ba/F3 (Ba/F3-wt) or unstimulated Ba/F3-Kit cells. In Ba/F3-wt and in Ba/F3-Kit cells, daunorubicin stimulated within 8–15 min neutral sphingomyelinase and ceramide production but not in SCF-stimulated Ba/F3-Kit or in Ba/F3-KitΔ27 whereas all Ba/F3 cells were equally sensitive to exogenous cell-permeant C6-ceramide. In Ba/F3-Kit, SCF-induced Kit activation resulted in a rapid phospholipase Cγ (PLCγ) tyrosine phosphorylation followed by diacylglycerol release and protein kinase C (PKC) stimulation. U-73122, a PLCγ inhibitor, not only blocked diacylglycerol production and PKC stimulation but also restored daunorubicin-induced sphingomyelinase stimulation, ceramide production, and apoptosis. These results suggest that Kit activation results in PLCγ-mediated PKC-dependent sphingomyelinase inhibition which contributes to drug resistance in Kit-related malignancies.

The role of poly(ADP-ribose) metabolism in response to active oxygen cytotoxicity

These experiments are a continuation of our work describing the effect of H2O2 and O2- on DNA strand breaks, NAD pools and poly(ADP-ribose) synthesis in C3H10T1/2 cells (Lautier et al. (1990) Biochem. Cell Biol. 68, 602-608). The current experiments were carried out firstly to evaluate the polymer synthesis in C3H10T1/2 cells exposed to benzamide, oxygen radicals and hyperthermia. Secondly, using four different protocols for the time of addition and removal of benzamide, the lowest benzamide levels shown to inhibit polymer synthesis were used to study the effect on plating efficiency and colony-forming ability of cells exposed to H2O2 and O2(-). Plating efficiency and colony-forming ability were affected by the active oxygen-species-generating system xanthine-xanthine oxidase and 100 microM benzamide. With higher levels of benzamide, this effect disappeared, and 0.5 to 1 mM benzamide were actually protective against the effects of xanthine-xanthine oxidase, suggesting the involvement of other processes in addition to poly(ADP-ribosyl)ation in response to oxygen radical damage.

Modulation of rhodamine 123 uptake by nigericin in sensitive and multidrug resistant leukemic cells

We have investigated the effect of the ionophore nigericin (NIG) in multidrug resistant (MDR) cells, using intracellular accumulation of the fluorescent dye rhodamine 123 (R123). NIG increased the accumulation of R123 in half of the murine MDR RFLC3 population but not in the human MDR CEM/VLB 100 cells. Co-treatment of RFLC3 with NIG plus verapamil showed additive effect on the accumulation of R123. The increase in R123 accumulation observed in RFLC3 was not the consequence of a direct effect of NIG on P-glycoprotein and was accompanied by a redistribution of the dye throughout the cell and a high cytotoxicity, which prevents the use of NIG as a resistance modulating agent.

Influence of Controlled Glucose Deprivation on Kinetics of Detoxification Mechanisms for Two Intermediate Metabolites, 9-Hydroxybenzo[a]pyrene and 3-Hydroxybenzo[a]pyrene, in 3T3 and RTG2 Cells

Abstract PAH metabolism is known to proceed in two successive steps the first one resulting in the production of activated metabolites which are subsequently transformed by the different pathways involved in the second step. Microspectrofluorometry enables the study of the kinetics of these steps on living intact cells in which no imbalance has been introduced artefactually. We have used this technique in order to check the influence of glucose deprivation on these kinetics. 9 and 3-OH-B(a)P have been selected as fluorescent substrates for there are potential substrates for the different pathways of the second step. The physiological cell status was controlled through the level of the intrinsic cellular fluorescence. Glucose deprivation results in a strong decrease of the experimental rate constants characteristic of the metabolism of 9 and 3-OH-B (a)P both in RTG2 and 3T3 cells. Nevertheless the metabolism of 3-OH-B(a)P in 3T3 cells appears to be less sensitive to glucose starvation, a result which remai...