The Fine LINE: Methylation Drawing the Cancer Landscape

LINE-1 (L1) is the most abundant mammalian transposable element that comprises nearly 20% of the genome, and nearly half of the mammalian genome has stemmed from L1-mediated mobilization. Expression and retrotransposition of L1 are suppressed by complex mechanisms, where the key role belongs to DNA methylation. Alterations in L1 methylation may lead to aberrant expression of L1 and have been described in numerous diseases. Accumulating evidence clearly indicates that loss of global DNA methylation observed in cancer development and progression is tightly associated with hypomethylation of L1 elements. Significant progress achieved in the last several years suggests that such parameters as L1 methylation status can be potentially utilized as clinical biomarkers for determination of the disease stage and in predicting the disease-free survival in cancer patients. In this paper, we summarize the current knowledge on L1 methylation, with specific emphasis given to success and challenges on the way of introduction of L1 into clinical practice.

Copper(II) and Palladium(II) Complexes with Cytotoxic and Antibacterial Activity

The synthesis of eight square pyramidal copper complexes with general structure [Cu(IL)(AL)H2O]2+, where IL represents various methylated 1,10-phenanthrolines, and AL represents either 1S,2S- or 1R,2R-diaminocyclohexane, is reported, with the complexes synthesised as both the perchlorate and chloride salts. The crystal structures of [Cu(1,10-phenanthroline)(1S,2S-diaminocyclohexane](ClO4)2·H2O and [Cu(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane](ClO4)2·1.5H2O are reported. Four square planar palladium complexes with general structure [Pd(IL)(AL)]Cl2 have also been synthesised. These complexes were synthesised in order to investigate the structure–activity relationship against both cancer cell lines and bacterial cultures. The copper complexes display anticancer activity similar to cisplatin and 1,10-phenanthroline (phen) in the L1210 murine leukaemia cell line. Methylation of the phen increased the copper complex cytotoxicity by approximately four-fold, compared with the non-methylated complex. No significant difference in activity was observed by altering the chirality of the diaminocyclohexane ligand. The copper complexes demonstrated antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli; however, high levels of toxicity (30–60 % of death) were observed in the nematode Caenorhabditis elegans. The copper complexes have also been shown to act as DNA nucleases, with the ability to cleave plasmid DNA in the presence of hydrogen peroxide. The palladium complexes all have half maximal inhibitory concentration (IC50) values of ~10 μM in the L1210 cell line, with no significant difference in the cytotoxicity of any of the compounds tested. Minimal antibacterial activity of the palladium complexes was observed.

Genomic Imprinting of Dopa decarboxylase in Heart and Reciprocal Allelic Expression with Neighboring Grb10

ABSTRACT By combining a tissue-specific microarray screen with mouse uniparental duplications, we have identified a novel imprinted gene, Dopa decarboxylase (Ddc), on chromosome 11. Ddc_exon1a is a 2-kb transcript variant that initiates from an alternative first exon in intron 1 of the canonical Ddc transcript and is paternally expressed in trabecular cardiomyocytes of the embryonic and neonatal heart. Ddc displays tight conserved linkage with the maternally expressed and methylated Grb10 gene, suggesting that these reciprocally imprinted genes may be coordinately regulated. In Dnmt3L mutant embryos that lack maternal germ line methylation imprints, we show that Ddc is overexpressed and Grb10 is silenced. Their imprinting is therefore dependent on maternal germ line methylation, but the mechanism at Ddc does not appear to involve differential methylation of the Ddc_exon1a promoter region and may instead be provided by the oocyte mark at Grb10. Our analysis of Ddc redefines the imprinted Grb10 domain on mouse proximal chromosome 11 and identifies Ddc_exon1a as the first example of a heart-specific imprinted gene.

Hypomethylation Induction and Molecular Response after Decitabine Therapy in Chronic Myelomonocytic Leukemia (CMML).

Decitabine has shown therapeutic activity in patients with MDS and CMML. The mechanisms of response to therapy remain incompletely understood. In particular, the relative contribution of this drug’s ability to induce hypomethylation and cytotoxicity remains unclear. To address this issue, we studied the dynamics of neoplastic cell clearance during decitabine treatment determined by quantitative monitoring of the mutant allele using pyrosequencing. DNA extracted from peripheral blood mononuclear cells from consented patients with CMML in a decitabine phase II study were first screened for JAK2 and NPM1 mutations as previously reported. We identified three patients with mutations (two with JAK2 mutation, one with NPM1 mutation) and samples at multiple points during therapy were available. All three carried normal karyotype. LINE repetitive element methylation and several other gene specific methylations were also assessed. In the three patients, LINE methylation decreased after each cycle of therapy, and recovered to near baseline after the drug was stopped (e.g. during the first cycle, average relative hypomethylation from baseline was 13.9% at day 12 and 6.5% at day 28). At the same time, the proportion of circulating neoplastic cells decreased slowly after the first cycle (decrease by 19.3% at day 12 and 13.5% at day 28). A substantial decrease in mutant allele percentage was observed after cycles 2, 3, and 2 in patients 1, 2, and 3, respectively. Clinical complete responses were achieved along with molecular responses at cycles 5, 4 and 2, respectively. Patients 1 and 2 showed complete disappearance of detectable neoplastic clones, and had sustained remissions (duration 1.5 and 2.5 years). In patient 3, the proportion of neoplastic cells was lower than baseline but still detectable at clinical remission, and the remission only lasted 8 months. We conclude that neoplastic cell clearance after decitabine therapy in CMML is observed after several courses of therapy, and is initially seen concurrently with hypomethylation. While LINE methylation returns to its steady state values after completion of decitabine infusion, the tumor elimination process slowly continues. Our data suggest a non-cytotoxic mechanism of action for the drug, whereby the biology of the neoplastic clone is altered by hypomethylation, leading to delayed clearances of unknown mechanism. Possibilities include an immune response and effects on the neoplastic (or normal) stem cells. Figure Figure

Improvement of formaldehyde-scavenging ability of condensed tannins by ammonia treatment

Condensed tannins have formaldehyde gas-scavenging ability. Tannins consisting of phloroglucinol-type A-rings, such as prodelphinidin and procyanidin, had higher formaldehyde-scavenging ability than those consisting of resorcinol-type A-rings, such as prorobietinidin and profisetinidin. This ability was markedly improved by ammonia treatment. The increase in formaldehyde-scavenging ability was especially notable in the case of condensed tannins with a high ratio of pyrogallol-type B-rings, such as prorobietinidin and prodelphinidin. To clarify the factors affecting improvement of the formaldehyde-scavenging ability by ammonia treatment, the reaction behavior of condensed tannins during ammonia treatment was studied. Analyses using 13C-NMR and on-line methylation pyrolysis/gas chromatography/mass spectrometry revealed that on treatment of condensed tannins with ammonia, amino-substitution at C-4′ of the pyrogallol-type B-ring occurred to form a 4′-amino-3′,5′-dihydroxybenzene-type B-ring. It can be assumed that the introduction of a 4′-amino-3′,5′-dihydroxybenzene-type B-ring into the tannin molecule results in improved formaldehyde-scavenging ability.