Lucille Lacoste

Comparative biosynthetic pathway of androstenol and androgens.

It has been shown recently that androstenol and androstanol could modulate gene expression through the nuclear orphan receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor). Although, in the pig, androstenol is produced in high amounts and is active as a pheromone, its role in the human is ill defined. Androstenol possesses a structure similar to that of androgens, with the exception that it does not possess an oxygen at position 17 that is crucial for androgenic and estrogenic activity. It has been shown that human and boar testis homogenates could produce androstenol, but details of the biosynthetic pathway had not yet been elucidated. It has also been shown recently that androstenol could modulate the activity of CAR and PXR and the expression of some cytochrome P450 drug-metabolizing enzymes. We wanted to determine the precise biosynthetic pathway of androstenol and other closely related steroids. Using transformed human embryonic kidney (HEK-293) cells that stably express 3 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, we have shown that these enzymes are able to efficiently transform the precursor 5,16-androstadien-3 beta-ol into androstenol. We thus provided evidence that androstenol, the ligand for CAR and PXR, is produced by the biosynthetic pathway of sex steroids.

Derepression of the Glutamine Synthetase in Neuroblastoma Cells at Low Concentrations of Glutamine

Abstract: Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro‐2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10‐fold increase in glutamine synthetase specific activity (15‐fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 mM glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 mM). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C‐6 glioma cells and glial cells in primary cultures.

High metabolization of catecholestrogens by type 1 estrogen sulfotransferase (hEST1).

Recently, two types of estrogen sulfotransferase, chronologically named types 1 and 2 estrogen sulfotransferase (hEST1 and hEST2), have been described. Since hEST2 selectively catalyzes the sulfonation of ethinyl estradiol as well as that of estrone (E1) and estradiol (E2), but poorly the sulfonation of catecholestrogens, we wanted to assess the ability of hEST1 to metabolize these compounds. We overexpressed hEST1 in Escherichia coli in fusion with GST, then purified the enzyme using a glutathione affinity column, and obtained GST-free enzyme by digestion with thrombin. Using [35S]-phosphosadenosine phosphosulfate (PAPS) as cofactor, we showed that hEST1 efficiently metabolizes the transformation of 2-OH-E2 and 2-OH-E1. However, the transformation of 4-OH-E1 and 4-OH-E2 is much less efficient. Our results also show that hEST1 metabolizes more efficiently E2 than E1. Since hEST1 mRNA is produced from the same gene as MPST using different alternative promoters and since it is expressed in most breast cancer cells (MCF-7, ZR-75-1, T47-D, MDA-231, and MDA-418), studies of the expression and activity of hEST1 will be most important to have a better knowledge about its involvement in the control of the genotoxicity of estrogens and catecholestrogens.

Synthesis of an inhibitor of glutamyl-tRNA synthetase

Abstract A specific inhibitor of glutamyl-tRNA synthetase, analog of glutamyl-adenylate, the N 6 -benzoyl-L-glutamol AMP, has been synthesized. This compound does not inhibit glutaminyl-tRNA synthetase.

Human type 1 estrogen sulfotransferase: catecholestrogen metabolism and potential involvement in cancer promotion.

Abstract: Using purified human type 1 estrogen sulfotransferase (hEST1), we show that the best substrate for this enzyme is 2‐hydroxy‐catecholestrogen. The enzyme also catalyzes the transformation of 4‐hydroxy‐estrogens and 16‐hydroxy‐estrogens, but with a lower affinity. We also present evidence to indicate that estrogen sulfotransferase may play a role in processes other than the detoxification and elimination of steroids. Indeed, hEST1 may also be involved in the production of stable precursors for local steroid biosynthesis or in the activation of promutagenic estrogen metabolites into carcinogens.

Non-linear kinetics of glutamyl-tRNA synthesis catalyzed by high molecular weight complexes from rat brain neuronal cells but not from glial cells

High molecular weight complexes of aminoacyl-tRNA synthetases isolated from rat brain catalyze the formation of glutamyl-tRNA with an initial lag time of the order of 1 min, as previously reported for the formation of glutamyl-tRNA and glutaminyl-tRNA catalyzed by similar complexes from bovine brain (Vadeboncoeur and Lapointe, Eur. J. Biochem., 109 (1980) 581-587). To determine the type(s) of brain cell(s) where this phenomenon occurs, we have studied the kinetics of glutamyl-tRNA formation catalyzed by high molecular weight complexes of aminoacyl-tRNA synthetases isolated from neuronal and from glial cells, either transformed (Neuro-2A and C6), or from primary cultures, or isolated from rat brain. The delay in the formation of glutamyl-tRNA was observed only in the case of neuronal cells isolated from rat brain, whereas a delay in the formation of glutaminyl-tRNA was also seen in these cells, as well as in neuronal cells in primary culture and in synaptosomes. The kinetics of formation of aspartyl-tRNA and valyl-tRNA catalyzed by high molecular weight complexes from all these cells was linear.