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.

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation.In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy [5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-ight) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into lightlight duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3Hladenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.In a previous study, while analyzing changes in DNA methylation during terminal differentiation of Friend erythroleukemia cells (FELC), some of us have observed that genome-wide DNA hypomethylation is induced by a variety of agents that are known to cause differentiation in these cells (1). Genome-wide hypomethylation has also been observed in developing mouse (2) and rabbit (3) early embryos and in teratocarcinoma mouse cells in response to inducers of differentiation (2,4,5). It is noteworthy that the hypomethylation observed in FELC was found to be transient: demethylation takes place relatively rapidly (within several hours) and is followed by de novo methylation (1). The kinetics of this demethylation process is not consistent with a passive mechanism of hypomethylation-namely, loss of methyl groups by replication of the DNA due to the absence or inhibition of maintenance methylation. In addition to this observation, two other instances have been reported recently that suggest an active mechanism for DNA demethylation.One Hpa II site upstream to the chicken vitelogenin II gene undergoes demethylation in response to estrogen treatment in the absence of replication (6), and a region in the EpsteinBarr virus DNA undergoes demethylation in response to induction by phorbol 12-myristate 13-acetate and butyrate before viral DNA amplification takes place (7). In an attempt to elucidate the mechanism by which active demethylation takes place, we have considered three possible mechanisms: (i) removal of the methyl group from 5-methylcytosine by direct demethylation; (ii) removal of5-methylcytosine and its replacement with cytosine through an enzymatic mechanism not previously described; (iii) removal of a stretch of DNA that would include the 5-methylcytosine moiety by the conventional ...

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The effects of 3‐deazaaristeromycin and 3‐deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S‐Adenosylhomocysteine accumulated in cells incubated with 3‐deazaaristeromycin while S‐3‐deazaadenosylhomocysteine was the major product in cells incubated with 3‐deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S‐adenosylhomocysteine or S‐3‐deazaadenosylhomocysteine, with S‐adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6‐methyladenosine and N6‐methyl‐2′‐O‐methyladenosine were inhibited to the greatest extent, while the synthesis of 7‐methylguanosine and 2′‐O‐methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 μM 3‐deazaaristeromycin or with 10 μM 3‐deazaadenosine and 50 μM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 μM 3‐deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.

CpG islands are distinguishable from the bulk of vertebrate DNA for being unmethylated and CpG-rich. Since CpG doublets are the specific target of eukaryotic DNA methyltransferases, CpG-rich sequences might be expected to be good methyl-accepting substrates in vitro, despite their unmethylated in vivo condition. This was tested using a partially purified DNA-methyltransferase from human placenta and several cloned CpG-rich or CpG-depleted sequences. The efficiency of methylation was found to be proportional to the CpG content for CpG-depleted regions, which are representative of the bulk genome. However, methylation was much less efficient for CpG frequencies higher than 1 in 12 nucleotides, reaching only 60% of the expected level. That suggests that the close CpG spacing typical of CpG-islands somehow inhibits mammalian DNA methyltransferase. The implications of these findings on the in vivo pattern of DNA methylation are discussed.