The mouse Gm853 gene encodes a novel enzyme: Leucine decarboxylase.

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. ODC-antizyme inhibitors (AZINs) are homologous proteins of ODC, devoid of enzymatic activity but acting as regulators of polyamine levels. The last paralogue gene recently incorporated into the ODC/AZINs family is the murine Gm853, which is located in the same chromosome as AZIN2, and whose biochemical function is still unknown. By means of transfection assays of HEK293T cells with a plasmid containing the coding region of Gm853, we show here that unlike ODC, GM853 was a stable protein that was not able to decarboxylate l-ornithine or l-lysine and that did not act as an antizyme inhibitor. However, GM853 showed leucine decarboxylase activity, an enzymatic activity never described in animal cells, and by acting on l-leucine (Km=7.03×10-3M) it produced isopentylamine, an aliphatic monoamine with unknown function. The other physiological branched-chain amino acids, l-valine and l-isoleucine were poor substrates of the enzyme. Gm853 expression was mainly detected in the kidney, and as Odc, it was stimulated by testosterone. The conservation of Gm853 orthologues in different mammalian species, including primates, underlines the possible biological significance of this new enzyme. In this study, we describe for the first time a mammalian enzyme with leucine decarboxylase activity, therefore proposing that the gene Gm853 and its protein product should be named as leucine decarboxylase (Ldc, LDC).

Attenuated JNK signaling in multidrug-resistant leukemic cells. Dual role of MAPK in cell survival.

Having found previously that leukemic cells with multidrug resistant (MDR) phenotype, but not their sensitive counterparts, exhibit collateral sensitivity to cold stress in a P-gp-dependent manner, our aim was to study the signaling pathways involved in this phenomenon in sensitive (L1210) and resistant cells (L1210R and CBMC-6). It was observed that the acquisition of MDR phenotype by leukemic cells or their transfection with the extrussion pump, P-gp, modifies the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. More specifically, cold stress provoked the activation of c-Jun N-terminal kinase (JNK) in sensitive cells, while attenuated JNK signaling was observed in MDR cells. This effect was also observed, although with less intensity, in P-gp-transfected cells. Using pharmacological inhibitors to determine the role of MAPK in leukemic cell survival in physiological conditions or under cold stress, a dual temperature-dependent role was observed for JNK in MDR cell survival. At 37°C JNK is necessary for the survival of parental, resistant and P-gp-transfected cells; however, the use of inhibitors of either extracellular signal-regulated protein kinase (ERK) or JNK significantly counteracts cold-induced death of resistant and P-gp-transfected cells, supporting a role for ERK and JNK in cold-stress induced cell death. Finally, a connectivity model concerning MAPK is proposed, summarizing how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. These findings on MDR cells may assist in the design of specific therapeutic strategies to complement current chemotherapy.

Collateral sensitivity to cold stress and differential BCL-2 family expression in new daunomycin-resistant lymphoblastoid cell lines.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells is one of the main causes of chemotherapy failure in cancer, and, usually, is due to the increased expression of P-glycoprotein (MDR-1, P-gp, ABCB1), a pump that expels chemotherapeutics from the cell and/or regulates apoptosis. Thus, it is fundamental to find drugs or stress stimuli with a capacity to induce apoptosis in such cells and to identify the mechanisms involved. We address this matter in human cells and establish new daunomycin (DNM)-resistant cell lines (IM-9R) by exposing the parental lymphoblastic cells (IM-9) to increasing doses of the anti-neoplastic drug, daunomycin. The resistance level of IM-9R cell lines, MDR-1 expression and functionality, collateral sensitivity and Bcl-2 and caspases protein expression are analyzed. As a result, we show for the first time that, unlike the parental cells, human lymphoblastic resistant cells exhibit collateral sensitivity to cold stress, confirming that this phenomenon is not exclusive to murine leukemic cells, but a broader one associated with the acquisition of drug resistance. Furthermore, the new resistant cell lines undergo a significant increase in active caspase-3 and -9 levels and drastic changes in Bcl-2 family protein expression during the process of MDR phenotype acquisition.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

Dehydroepiandrosterone-sulfate modifies human fatty acid composition of different adipose tissue depots.

BACKGROUND: Dehydroepiandrosterone-sulfate (DHEA-S) has been described as a protector agent against obesity-related pathologies, although the mechanism of action is still unknown. We have shown that DHEA-S acts on adipose tissue (AT), altering the fatty acid (FA) profile in rodents. Thus, we could hypothesize that some of the beneficial effects shown by DHEA-S in humans are related to a modification of the human AT-FA profile. The present study examines this question and whether this effect is tissue-dependent. METHODS: Paired visceral and subcutaneous AT biopsies were obtained from 20 patients who had undergone bariatric surgery. These samples were subjected to primary adipose culture and incubated for 24 h with 1 µM DHEA-S. The FA profile of both control and treated samples were analyzed by gas chromatography. RESULTS: A reduction in total saturated fatty acids (SFA), the n-6 family of polyunsaturated fatty acids (PUFA) and the n-6/n-3 PUFA ratio was observed after DHEA-S treatment, whereas monounsaturated fatty acids (MUFA) increased. In addition, DHEA-S altered the percentage of several individual FA, decreasing palmitic acid and increasing vaccenic acid in both AT. All estimated desaturase activity ratios slightly increased after DHEA-S treatment, although only the increase of delta-6-desaturase index in both depots reached statistical significance. No depot-specific action of DHEA-S was found between subcutaneous and visceral AT. CONCLUSIONS: In vitro, DHEA-S modifies the AT-FA composition towards a better metabolic profile to a similar extent in the subcutaneous and visceral adipose depots, in both of which a decrease in SFA and increased MUFA are observed after treatment. This effect could help to explain the beneficial effects attributed to DHEA-S. Further studies, however, are required to determine whether the effect of DHEA-S on adipose tissue in vitro is conserved in vivo.

Effect of dehydroepiandrosterone on protein and fat digestibility, body protein and muscular composition in high-fat-diet-fed old rats.

The main objective of the present study was to examine the effects of dehydroepiandrosterone (DHEA) on the digestive efficiency of dietary protein and fat. Second, we analysed the specific changes in muscle composition induced by the hormone. DHEA was given in the diet (0 x 5 %, w/w) to 75-week-old, high-fat-fed Sprague-Dawley rats (n 11) for 13 weeks; age- and weight-matched rats fed on the same diet without DHEA supplementation were used as controls (n 10). To determine dietary protein and fat apparent digestibility coefficients, 1-week 24 h faecal depositions were collected. In parallel, urine N was assessed. These assays were performed twice, in the short term (2-week treatment) and in the long term (13-week treatment). Body and gastrocnemius muscle compositions were also analysed. The present results show that DHEA decreased energy intake, body weight, body fat, adipocyte size and number (P<0 x 001). The feed efficiency ratio indicates that DHEA-treated rats were less efficient in transforming nutrients fed into their own biomass. Also, a short-term reduction in protein digestibility (P<0 x 05) and in body-protein degradation (P<0 x 01) was found in DHEA-treated rats, resulting in an increased content of body protein (P<0 x 05). Gastrocnemius muscles were smaller, as a result of fat (P<0 x 05) but not protein reduction. In conclusion, we confirm the slimming effect of DHEA and, for the first time, we demonstrate that DHEA has an effect at the digestive level. The anti-obesity properties of DHEA could be related to a reduction in protein digestibility in the short term and a protective effect on body protein with a selective mass loss from body fat.