Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

A randomised, phase II trial of the DNA-hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer.

Background: Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2'-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer.

Methods: Patients progressing 6-12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma.

Results: After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%).

Conclusions: With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies.

Aza-annulation of enaminones with itaconic anhydride:kinetic preference for exocyclic enamide products

Enaminones react with itaconic anhydride in methylene chloride at room temperature to give exocyclic enamides sis the major products. These can be readily equilibrated to the thermodynamically more stable endocyclic enamides. In substrates where the exocyclic isomer could not be formed only intractable materials were produced from this reaction. An intermediate in this two step process was detected and identified by proton and C-13 NMR spectroscopy. In two cases chiral enaminones were employed and the relative stereochemistry at the new chiral centre in the product was established by a crystal structure of compound 27.

Aza-annulation of enaminones with crotonyl chloride - Formal reversal of regioselectivity.

The regiochemistry of aza-annulation of enaminones with alpha,beta-unsaturated acid chlorides bearing hydrogen atoms on the gamma-carbon is reversed when triethylamine is used as mediator. When the reaction was carried out at lower temperatures as 3-acyl beta,gamma-unsaturated compound could be isolated which cyclised to the desired product under thermal or basic conditions. The nature of this intermediate strongly suggests that a vinyl ketene is the active acylating agent. (C) 1997 Elsevier Science Ltd.

Smac-derived Aza-peptide As an Aminopeptidase-resistant XIAP BIR3 Antagonist

The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smac-derived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla-Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics.

Azaspiracid: First evidence of protein binding in shellfish

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC–MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures.

AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.

Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine.

AIMS
To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the in?uence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or in?ammatory bowel disease (IBD).
METHODS
Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94?A and IVS2+21A?C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined.
RESULTS
Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients,P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A?C variants among ALL and IBD patients had signi?cantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients,P = 0.047 in IBD patients). The study con?rmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a signi?cant association between inosine triphosphatase IVS2+21A?C variants with thrombocytopenia (P = 0.012).
CONCLUSIONS
Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose
individualization.

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer

We characterized Fas immunoreactivity, functionality and its role in the response to mitomycin-C (MMC) chemotherapy in vitro in cell lines and in vivo in bladder washings from 23 transitional cell carcinoma of the bladder (TCCB) patients, harvested prior to and during MMC intravesical treatment. Having established the importance of functional Fas, we investigated the methylation and exon 9 mutation as mechanisms of Fas silencing in TCCB. For the first time, we report p53 up-regulation in 9/14 and Fas up-regulation in 7/9 TCCB patients during intravesical MMC treatment. Fas immunoreactivity was strong in the TCCB cell line T24 and in 17/20 (85%) tumor samples from patients with advanced TCCB. T24 and HT1376 cells were resistant to MMC and recombinant Fas ligand, whilst RT4 cells were responsive to Fas ligand and MMC. Using RT4 cells as a model, siRNA targeting p53 significantly reduced MMC-induced p53 and Fas up-regulation and stable DN-FADD transfection decreased MMC-induced apoptosis, suggesting that functional Fas enhances chemotherapy responses in a p53-dependent manner. In HT1376 cells, 5-aza-2-deoxycytidine (12 µM) induced Fas immunoreactivity and reversed methylation at CpG site -548 within the Fas promoter. This site was methylated in 13/24 (54%) TCCB patient samples assessed using Methylation-Specific Polymerase Chain Reaction. There was no methylation at either the p53 enhancer region within the first intron or at the SP-1 binding region in the promoter and no mutation within exon 9 in tumor DNA extracted from 38 patients. Methylation at CpG site -548 is a potential target for demethylating drugs.

Biphenyl dioxygenase-catalysed cis-dihydroxylation of tricyclic azaarenes: chemoenzymatic synthesis of arene oxide metabolites and furoquinoline alkaloids

Biotransformation of acridine, dictamnine and 4-chlorofuro[2,3-b]quinolone, using whole cells of Sphingomonas yanoikuyae B8/36, yielded five enantiopure cyclic cis-dihydrodiols, from biphenyl dioxygenase-catalysed dihydroxylation of the carbocyclic rings. cis-Dihydroxylation of the furan ring in dictamnine and 4-chlorofuro[2,3-b] quinoline, followed by ring opening and reduction, yielded two exocyclic diols. The structures and absolute configurations of metabolites have been determined by spectroscopy and stereochemical correlation methods. Enantiopure arene oxide metabolites of acridine and dictamnine have been synthesised, from the corresponding cis-dihydrodiols. The achiral furoquinoline alkaloids robustine, gamma-fagarine, haplopine, isohaplopine-3,3'-dimethylallylether and pteleine have been obtained, from either cis-dihydrodiol, catechol or arene oxide metabolites of dictamnine.

THE SYNTHESIS OF BRIDGED-RING CARBOCYCLES AND HETEROCYCLES VIA PALLADIUM CATALYZED REGIOSPECIFIC CYCLIZATION REACTIONS

A catalyst system comprising 10 mol % (Pd(OAc) and 20 mol % PPh3 effects the cyclisation of aryl halides onto proximate alkenes via 5-, 6-, and 7-exo-trig, and 7-endo-trig processes giving a variety of bridged-ring carbo- and hetero-cycles in excellent yield. Double bond isomerisation in the product is rarely encountered and may be suppressed by the addition of Tl(1) salts. One example of diastereospecific bis-cyclisation is given and the crystal structure of 1-aza-2-sulphonyl-3,4-benzobicyclo[3.2.1]nona-6-ene is reported.

New insights into the causes of human illness due to consumption of azaspiracid contaminated shellfish

Azaspiracid (AZA) poisoning was unknown until 1995 when shellfish harvested in Ireland caused illness manifesting by vomiting and diarrhoea. Further in vivo/vitro studies showed neurotoxicity linked with AZA exposure. However, the biological target of the toxin which will help explain such potent neurological activity is still unknown. A region of Irish coastline was selected and shellfish were sampled and tested for AZA using mass spectrometry. An outbreak was identified in 2010 and samples collected before and after the contamination episode were compared for their metabolite profile using high resolution mass spectrometry. Twenty eight ions were identified at higher concentration in the contaminated samples. Stringent bioinformatic analysis revealed putative identifications for seven compounds including, glutarylcarnitine, a glutaric acid metabolite. Glutaric acid, the parent compound linked with human neurological manifestations was subjected to toxicological investigations but was found to have no specific effect on the sodium channel (as was the case with AZA). However in combination, glutaric acid (1mM) and azaspiracid (50nM) inhibited the activity of the sodium channel by over 50%. Glutaric acid was subsequently detected in all shellfish employed in the study. For the first time a viable mechanism for how AZA manifests itself as a toxin is presented.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.