Thioredoxin reductase and its inhibitors.

Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis.

DNA damage response in monozygotic twins discordant for smoking habits.

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Retinoic acid targets DNA-methyltransferases and histone deacetylases during APL blast differentiation in vitro and in vivo.

The acute promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion product recruits histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activities on retinoic acid (RA)-target promoters causing their silencing and differentiation block. RA treatment induces epigenetic modifications at its target loci and restores myeloid differentiation of APL blasts. Using RA-sensitive and RA-resistant APL cell lines and primary blasts, we addressed the functional relevance of the aberrant methylation status at the RA-target promoter RARbeta2 and the mechanism by which methylation is reversed by RA. RA decreased DNMT expression and activity, which correlated with demethylation at specific sites on RARbeta2 promoter/exon-1, and the ability of APL blasts to differentiate in vitro and in vivo. None of these events occurred in an RA-resistant APL cell line containing a PML-RARalpha defective for ligand binding. The specific contribution of the HDAC and DNMT pathways to the response of APL cells to RA was also tested by inhibiting these enzymatic activities with TSA and/or 5-azacytidine. In RA-responsive and RA-resistant APL blasts, TSA and 5-azacytidine induced specific changes on the chromatin state at RA-target sites, increased the RA effect on promoter activity, endogenous RA-target gene expression and differentiation. These results extend the rationale for chromatin-targeted treatment in APL and RA-resistant leukemias.