Search

Scenarios for future climate predict an increase in precipitation amounts and frequency of rain events, resulting in higher air humidity and soil moisture at high latitudes, including in northern Europe. We analysed the effects of artificially elevated environmental humidity (air relative humidity and soil moisture) on leaf gas exchange, water relations, growth and phenology of silver birch (Betula pendula) trees growing at the free air humidity manipulation experimental site situated in the hemiboreal vegetation zone, in eastern Estonia, with no occurring water deficit to the trees. The environmental humidity manipulation did not significantly affect the water relations traits but did affect some leaf gas exchange parameters, growth and phenology of the trees. Elevated air humidity (H) did not influence photosynthetic capacity and stomatal conductance, while the trees exhibited higher stomatal sensitivity to leaf-to-air vapour pressure difference compared with the trees at ambient conditions (C) or at elevated soil moisture (I). H trees demonstrated reduced height growth and foliage biomass, increased allocation to stem radial growth and prolonged leaf retention in autumn compared with the C trees. Increased air humidity supports longer leaf retention and growth period, but this does not translate into increased growth parameters at the tree level. The changes in tree growth in response to increasing atmospheric humidity could plausibly be explained by (i) retardation of foliage development and (ii) changes in resource allocation, causing a shift in the ratio of photosynthetic to non-photosynthetic tissues in favour of the latter. Under high atmospheric evaporative demand, higher stomatal sensitivity in H trees induces faster stomatal closure, which may result in carbon starvation. A future rise in atmospheric humidity at high latitudes may lead to reduced tree growth and forest productivity, in contrast to the predicted future of forests., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Spring wheat ( Triticum aestivum L.) remains an important alternative to winter wheat cultivation at Northern latitudes due to high risk of overwintering or delayed sowing of winter wheat. We studied nine major agronomic traits in a set of 299 spring wheat genotypes in trials across 12-year-site combinations in Lithuania, Latvia, Estonia, and Norway for three consecutive years. The dataset analyzed here consisted of previously published phenotypic data collected in 2021 and 2022, supplemented with additional phenotypic data from the 2023 field season collected in this study. We combined these phenotypic datasets with previously published genotypic data generated using a 25K single nucleotide polymorphism (SNP) array that yielded 18,467 markers with a minor allele frequency above 0.05. Analysis of these datasets via genome-wide association study revealed 18 consistent quantitative trait loci (QTL) replicated in two or more trials that explained more than 5% of phenotypic variance for plant height, grain protein content, thousand kernel weight, or heading date. The most consistent markers across the tested environments were detected for plant height, thousand kernel weight, and days to heading in eight, five, and six trials, respectively. No beneficial effect of the semi-dwarfing alleles Rht-B1b and Rht-D1b on grain yield performance was observed across the 12 tested trials. Moreover, the cultivars carrying these alleles were low yielding in general. Based on principal component analysis, wheat genotypes developed in the Northern European region clustered separately from those developed at the southern latitudes, and markers associated with the clustering were identified. Important phenotypic traits, such as grain yield, days to heading, grain protein content, and thousand kernel weight were associated with this clustering of the genotype sets. Interestingly, despite being adapted to the Nordic environment, genotypes in the Northern set demonstrated lower grain yield performance across all tested environments. The results indicate that spring wheat germplasm harbors valuable QTL/alleles, and the identified trait-marker associations might be useful in improving Nordic-Baltic spring wheat germplasm under global warming conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Aleliūnas, Gorash, Armonienė, Tamm, Ingver, Bleidere, Fetere, Kollist, Mroz, Lillemo and Brazauskas.)

Key Message: QPm.NOBAL-3A is an important QTL providing robust adult plant powdery mildew resistance in Nordic and Baltic spring wheat, aiding sustainable crop protection and breeding. Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici, poses a significant threat to bread wheat (Triticum aestivum L.), one of the world's most crucial cereal crops. Enhancing cultivar resistance against this devastating disease requires a comprehensive understanding of the genetic basis of powdery mildew resistance. In this study, we performed a genome-wide association study (GWAS) using extensive field trial data from multiple environments across Estonia, Latvia, Lithuania, and Norway. The study involved a diverse panel of recent wheat cultivars and breeding lines sourced from the Baltic region and Norway. We identified a major quantitative trait locus (QTL) on chromosome 3A, designated as QPm.NOBAL-3A, which consistently conferred high resistance to powdery mildew across various environments and countries. Furthermore, the consistency of the QTL haplotype effect was validated using an independent Norwegian spring wheat panel. Subsequent greenhouse seedling inoculations with 15 representative powdery mildew isolates on a subset of the GWAS panel indicated that this QTL provides adult plant resistance and is likely of race non-specific nature. Moreover, we developed and validated KASP markers for QPm.NOBAL-3A tailored for use in breeding. These findings provide a critical foundation for marker-assisted selection in breeding programs aimed at pyramiding resistance QTL/genes to achieve durable and broad-spectrum resistance against powdery mildew., (© 2024. The Author(s).)

VEGF inhibition in gastric cancer has a proven benefit in the second line setting. Pazopanib, an oral tyrosine kinase inhibitor, selectively inhibits VEGFR-1, -2 and -3, c-kit and PDGF-R resulting in inhibition of angiogenesis. This open-label randomized phase II trial (2:1) investigated the efficacy of combining pazopanib with FLO (5-fluorouracil, oxaliplatin) vs FLO alone (internal control arm) as first-line treatment in patients with advanced adenocarcinoma of the stomach and gastroesophageal junction (GEJ). Eighty-seven patients were randomized and 78 patients were eligible and evaluable (PaFLO arm 51 patients, FLO arm 27 patients). The PFS rate at 6 months (primary endpoint) was 34% in the PaFLO arm vs 30% in the FLO arm. Comparing PaFLO with FLO median PFS was 4.66 months (95% confidence interval [CI] 2.87-6.46) vs 4.47 months (95% CI 1.79-7.14) (95% CI, hazard ratio [HR] 0.96 (0.60-1.55), P = .882 [exploratory]); median OS was 10.19 months (95% CI 5.46-14.92) vs 7.33 months (95% CI 4.93-9.73), (95% CI HR 1.01 [0.62-1.65], P = .953, exploratory), disease control rate was 72% vs 59%. PaFLO was well tolerable, toxicities were slightly higher in the PaFLO arm. Major adverse events were loss of appetite, nausea, fatigue, diarrhea, neutropenia and thrombocytopenia. Adding pazopanib to chemotherapy shows signs of efficacy but no major improvement in this randomized phase 2 trial. The PFS at 6 months in both arms was lower than expected from the literature. Biomarkers identifying subgroups who benefit and novel combinations are needed. ClinicalTrials.gov: NCT01503372., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: 5-Fluorouracil (FU) is one of the most commonly used cytostatic drugs in the systemic treatment of cancer. Treatment with FU may cause severe or life-threatening side effects and the treatment-related mortality rate is 0.2-1.0%., Summary: Among other risk factors associated with increased toxicity, a genetic deficiency in dihydropyrimidine dehydrogenase (DPD), an enzyme responsible for the metabolism of FU, is well known. This is due to variants in the DPD gene (DPYD). Up to 9% of European patients carry a DPD gene variant that decreases enzyme activity, and DPD is completely lacking in approximately 0.5% of patients. Here we describe the clinical and genetic background and summarize recommendations for the genetic testing and tailoring of treatment with 5-FU derivatives. The statement was developed as a consensus statement organized by the German Society for Hematology and Medical Oncology in cooperation with 13 medical associations from Austria, Germany, and Switzerland. Key Messages: (i) Patients should be tested for the 4 most common genetic DPYD variants before treatment with drugs containing FU. (ii) Testing forms the basis for a differentiated, risk-adapted algorithm with recommendations for treatment with FU-containing drugs. (iii) Testing may optionally be supplemented by therapeutic drug monitoring., (© 2020 S. Karger AG, Basel.)

Suberin fatty acids (SFAs) isolated from outer birch bark were investigated as an antimicrobial agent and biomaterial in nanofibrous mats intended for wound treatment. Electrospinning (ES) was used in preparing the composite nonwoven nanomats containing chloramphenicol (CAM; as a primary antimicrobial drug), SFAs, and polyvinylpyrrolidone (as a carrier polymer for ES). The X-ray powder diffraction, differential scanning calorimetry, scanning electron microscopy, atomic force microscopy, and texture analysis were used for the physicochemical and mechanical characterization of the nanomats. ES produced nanofibrous mats with uniform structure and with an average fiber diameter ranging from 370 to 425 nm. Microcrystalline SFAs and crystalline CAM were found to undergo a solid-state transformation during ES processing. The ES process caused also the loss of CAM in the final nanofibers. In the texture analysis, the SFAs containing nanofibers exhibited significantly higher maximum detachment force to an isolated pig skin (p < 0.05) than that obtained with the reference nanofibers. CAM exists in an amorphous form in the nanofibers which needs to be taken into account in controlling the physical storage stability. In conclusion, homogeneous composite nanofibrous mats for wound healing can be electrospun from the ternary mixture(s) of CAM, SFAs, and polyvinylpyrrolidone., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Electrospinning is an effective method in preparing polymeric nanofibrous drug delivery systems (DDSs) for topical wound healing and skin burn therapy applications. The aim of the present study was to investigate a new synthetic graft copolymer (Soluplus) as a hydrophilic carrier polymer in electrospinning of nanofibrous DDSs. Soluplus (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG)) was applied in the nonwoven nanomats loaded with piroxicam (PRX) as a poorly water-soluble drug. Raman spectroscopy, X-ray powder diffraction, differential scanning calorimetry, and scanning electron microscopy (SEM) were used in the physical characterization of nanofibrous DDSs. According to the SEM results, the drug-loaded PCL-PVAc-PEG nanofibers were circular in cross-section with an average diameter ranging from 500 nm up to 2  µm. Electrospinning stabilized the amorphous state of PRX. In addition, consistent and sustained-release profile was achieved with the present nanofibrous DDSs at the physiologically relevant temperature and pH applicable in wound healing therapy. In conclusion, electrospinning can be used to prepare nanofibrous DDSs of PCL-PVAc-PEG graft copolymer (Soluplus) and to stabilize the amorphous state of a poorly water-soluble PRX. The use of this synthetic graft copolymer can open new options to formulate nanofibrous DDSs for wound healing.

The prediction of transcription factor binding sites in genomic sequences is in principle very useful to identify upstream regulatory factors. However, when applying this concept to genomes of multicellular organisms such as mammals, one has to deal with a large number of false positive predictions since many transcription factor genes are only expressed in specific tissues or cell types. We developed TS-REX, a database/software system that supports the analysis of tissue and cell type-specific transcription factor-gene networks based on expressed sequence tag abundance of transcription factor-encoding genes in UniGene EST libraries. The use of expression levels of transcription factor-encoding genes according to hierarchical anatomical classifications covering different tissues and cell types makes it possible to filter out irrelevant binding site predictions and to identify candidates of potential functional importance for further experimental testing. TS-REX covers ESTs from H. sapiens and M. musculus, and allows the characterization of both presence and specificity of transcription factors in user-specified tissues or cell types. The software allows users to interactively visualize transcription factor-gene networks, as well as to export data for further processing. TS-REX was applied to predict regulators of Polycomb group genes in six human tumor tissues and in human embryonic stem cells.

We present 60 patients with refractory (n=8) or relapsed (n=52) adult ALL who received allogeneic hematopoietic SCT (HSCT) with (n=41) or without (n=19) prior reinduction chemotherapy. In our center, omission of reinduction is recommended if a suitable donor is promptly available, tumor burden is moderate and disease features suggest a highly aggressive course. Overall survival (OS) of the whole cohort at 1, 2 and 5 years was 42, 33 and 28%, respectively. Leukemia-free survival at 1, 2 and 5 years was 37, 33 and 24%. Deaths were due to relapse (n=25), acute or chronic GVHD (n=7), infections (n=8) or toxicity (n=4). Interestingly, patients who did not receive reinduction before HSCT had better outcomes than patients who received reinduction with OS at 1, 2 and 5 years being 58 vs 34%, 47 vs 25% and 47 vs 18%, respectively (P=0.039). Importantly, even achievement of a second CR after reinduction was not associated with improved survival compared to patients directly proceeding to HSCT. We conclude that patients who undergo HSCT for refractory or relapsed ALL can achieve long-term survival. In selected patients, reinduction chemotherapy can be omitted if immediate HSCT is feasible.

Fusarium infections are associated with high mortality after allogeneic stem cell transplantation. We report on successful treatment of a disseminated cutaneous Fusarium proliferatum infection using liposomal amphotericin B and terbinafine. In vitro susceptibility tests of antifungal drugs suggest that terbinafine is a potent additional antifungal drug for disseminated cutaneous fusariosis.

Survivin, an inhibitor of apoptosis (IAP) protein plays a dual role in regulation of mitosis and inhibition of apoptosis. Survivin is expressed in embryonic and fetal organs as well as in most human cancers, but not in normal differentiated adult tissues. In this study we investigated the molecular mechanism involved in overexpression of survivin in acute myeloid leukemia (AML). We used methylation specific PCR (MSP) and bisulfite sequencing to analyze the methylation status of the survivin promoter in primary AML samples and normal peripheral blood mononuclear cells (PBMCs). Both, in patients with de novo AML and normal control samples an unmethylated survivin promoter was present. Mutational analysis of the proximal survivin promoter revealed three single nucleotide polymorphisms (SNPs), where the frequently occurred polymorphism (G/C) at position -31 was detectable in both, AML blasts and healthy PBMCs and showed no significant impact on prognosis in de novo AML patients. These results suggest that the methylation status of the survivin promoter and occurrence of these SNPs within the promoter region of the survivin gene appear to be of minor importance in leukemogenesis.

Aegera, under license from Idera Pharmaceuticals, is developing AEG-35156, a 19-mer phosphorothioate antisense oligonucleotide targeting the caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP) messenger RNA, for the potential treatment of cancer. Several clinical trials are ongoing and include: two phase I monotherapy clinical trials for the potential treatment of cancer and in patients with solid tumors; a phase I combination clinical trial of AEG-35156 with docetaxel in locally advanced, metastatic, or recurrent solid tumors; four phase I/II combination clinical trials for the potential treatment of pancreatic cancer, advanced breast cancer, advanced NSCLC, and acute myeloid leukemia. Mild to moderate adverse effects were observed in early phase clinical trials. Aegera plans to initiate randomized phase III trials if tolerable side effects and evidence of activity are demonstrated in phase I/II clinical trials.

The outcome of patients with acute lymphoblastic leukemia (ALL) receiving therapeutic donor lymphocyte infusions (DLIs) in relapse after stem cell transplantation (SCT) is poor. We analyzed the impact of prophylactic DLIs in ALL on chimerism and sustained complete remission (CR). Eighty-five patients with ALL were allografted between January 1998 and September 2004. Twenty-six of them received prophylactic DLIs and were included in this analysis. A total of 12 of 13 patients, who were treated with mixed chimerism (MC) converted to complete donor chimerism (92%) and 10 of 12 patients had persistent donor chimerism and sustained CR during subsequent follow-up. Overall, 18 of 26 patients developed graft-versus-host disease (GVHD) after DLIs (69%), acute GVHD in 46 and chronic GVHD in 62%. After a median follow-up of 42 months (14-72) after SCT, 18 of 26 patients (70%) are alive, 16 in CR. Probability of event-free survival (EFS) for patients treated with DLIs is 62%, and overall survival is 70% at 3 years. Our preliminary data support a graft-versus-leukemia effect of prophylactic DLIs able to induce stable donor chimerism and ongoing CR after SCT. As the accompanying GVHD rate was considerable, careful selection of patients for prophylactic DLIs is mandatory.

Survivin, a member of the inhibitor of apoptosis protein family, is involved in both, inhibition of apoptosis and regulation of cell division. Because of the tumor-specific expression of survivin, the reduction of its expression is an important therapeutic option in the treatment of malignant diseases. Thus, we analyzed the transcriptional regulation of survivin in order to establish survivin as a target gene for new therapeutic approaches. Here, we describe a novel regulatory region within the survivin promoter. After treatment with phorbol 12-myristate-13-acetate, the early growth response (Egr)-1 transcription factor binds to the sequence 5'GAGGGGGCG 3' within the human survivin promoter in vitro and in entire cells. In reporter-gene assays and overexpression experiments, survivin is downregulated following exogenous expression of wildtype Egr-1. Using p53 wildtype and mutated cell lines, we show that Egr-1 negatively regulates survivin expression and sensitizes cell lines to TRAIL-induced apoptosis., ((c) 2007 Wiley-Liss, Inc.)

Therapeutical hyperthermia has been considered for cancer therapy since William Coley observed tumour remission after induction of fever by bacterial toxins at the end of the 19th century. Because fever is associated with a variety of immunological reactions, it has been suspected, that therapeutical hyperthermia might also activate the immune system in a reproducible manner and thereby positively influence the course of the disease. During the last decade, new insight has been gained regarding the immunological changes taking place during therapeutic hyperthermia. In this chapter, we review the most relevant data known about the effect of hyperthermia on the immune system with special focus on alterations induced by therapeutical whole-body hyperthermia (WBH) in cancer patients.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in most human cancers, but undetectable in normal differentiated adult tissue in vivo. Because of this cancer-related expression, survivin is a promising target for cancer therapy. To determine the expression and prognostic role of survivin in acute myeloid leukemia (AML), we investigated the mRNA expression pattern of survivin and of the splice variants survivin-2B and survivin-DeltaEx3 in adult (n = 74) and children (n = 31) with de novo AML using RT-PCR. Survivin was the predominant transcript variant in AML cells, whereas significantly lower levels of survivin-2B and survivin-DeltaEx3 were observed (p < or = 0.0001). Neither expression of survivin nor of any splice variant correlated with maturation stage (FAB subtypes, immunophenotype) or cytogenetic risk groups. For AML cases treated according to AMLCG92 (adult) and AML-BFM93 (children) protocols, respectively, expression patterns were correlated with clinical data: in adult AML (n = 51), low expression of survivin-2B correlated with a better overall survival (p = 0.05; mean survival time 19 months vs. 9 months) and a better eventfree survival (p < or = 0.01; 27 months vs. 10 months). In childhood AML (n = 31), high survivin-DeltaEx3 expression was associated with a shorter overall survival (p < or = 0.05; 24 months vs. 43 months). We conclude that certain survivin splice variants have potential prognostic impact for long-term therapy outcome in adult as well as childhood de novo AML.

Nucleic acid molecules have emerged as versatile tools with promising utility as therapeutics for human diseases. The specificity of hybridization of an antisense oligonucleotide (AS ODN) to the target mRNA makes the AS strategy attractive to selectively modulate the expression of genes involved in the pathogenesis of malignant or non-malignant diseases. One AS drug has been approved for local therapy of cytomegalovirus retinitis, and a number of AS ODN are currently tested in clinical trials including ODN that target bcl-2, survivin, and DNA methyltransferase. The clinical studies indicate that AS ODN are well tolerated and may have therapeutic activity. In this overview, we summarize therapeutic concepts, clinical studies, and new promising molecular targets to treat human cancer with AS ODN.

Preclinical and clinical studies indicate a role for AS ODNs (antisense oligonucleotides) as therapeutics for malignant diseases. The principle of antisense technology is the sequence-specific binding of an AS ODN to the target mRNA, resulting in a translational arrest. The specificity of hybridization makes antisense strategy attractive to selectively modulate the expression of genes involved in the pathogenesis of malignant diseases. One antisense drug has been approved for local therapy of CMV (cytomegalovirus) retinitis, and a number of AS ODNs are currently being tested in clinical trials, including AS ODN targeting Bcl-2, XIAP (X-linked inhibitor of apoptosis protein) and TGF-beta-2 (transforming growth factor beta-2). AS ODNs are well tolerated and may have therapeutic activity. In particular, an AS ODN to Bcl-2 has been tested in phase III clinical trials in chronic lymphocytic leukaemia, multiple myeloma and malignant melanoma. In this review, therapeutic concepts, clinical studies and new promising molecular targets to treat malignancies with AS ODNs are summarized.

We investigated human rights concerns related to migration, living and working conditions, and access to HIV/AIDS services and reproductive health services for Burmese women in Thailand. Vulnerability to HIV/AIDS for Burmese women stemmed from abuses they experienced: gender and ethnic discrimination, including violence; unsafe migration and trafficking; labor and sexual exploitation; and denial of health care. Despite having bound itself to human rights laws, the Thai government is failing to fulfill its obligations to Burmese women, with particularly devastating impacts for their well-being, including the risk of HIV/AIDS. Moreover, as our documentation shows, this failure to incorporate human rights concerns into its national response to the epidemic virtually guarantees that HIV/AIDS will continue to be a problem in Thailand.

The demethylating effect of 5-aza-2' deoxycytidine (decitabine, DAC) has been well characterized. The molecular events downstream of methylation inhibition are less well known. Here, DAC was shown to induce apoptosis in acute myeloid leukemia (AML) cells (p53 mutant and wild type) but not in epithelial or normal peripheral blood mononuclear cells. Apoptosis was characterized by activation of the mitochondrial but not the receptor death pathway, as demonstrated by the release of cytochrome c and loss of mitochondrial membrane potential. Western blotting and enzyme assays showed that caspase-3, but not caspase-6 or caspase-8, were activated. Decitabine induced expression of the cell cycle inhibitor p21, arresting AML cell lines in G1 of the cell cycle. Expression of p21 was induced irrespective of the methylation status of its promoter, mediated instead via reexpression of the tumor suppressor p73, an upstream regulator of p21. The promoter of p73 was hypermethylated in AML cell lines in vitro and in primary AML cells ex vivo but not in DAC-resistant epithelial cells. In conclusion, DAC acts on leukemic myeloid cells via caspase activation, which may be dependent on demethylation of the hypermethylated p73 promoter and consequent reexpression of p73.

To assess the role of allogeneic stem cell transplantation (SCT) after reduced-intensity conditioning (RIC) in acute leukaemias, we retrospectively compared 25 patients with acute lymphoblastic leukaemia or acute myelogenous leukaemia after RIC to a historical group of 50 matched controls after high-dose conditioning. Engraftment, acute GvHD and severe infections were comparable in both groups. During the observation period, 1/25 patients (4%) after RIC and 14/50 (28%) after standard SCT died due to transplant-related causes; cumulative nonrelapse mortality (NRM) was 4% after RIC and 24% after standard SCT (P=0.029). In total, 15/25 patients (60%) relapsed after RIC and 20/50 (40%) after standard SCT; probability of disease-free survival (DFS) at 3 years was 43% after RIC and 49% after standard SCT (NS). Overall survival (OS) was 40% after RIC and 37% after standard SCT (NS). Stage of disease, cytogenetic risk profile, acute and chronic GvHD, chimerism status at day 90 and severe infections after transplantation were risk factors with significant impact on DFS and/or OS. In retrospective analysis, patients with acute leukaemias who receive RIC because of contraindications against standard SCT have a comparable outcome to standard SCT, but the higher relapse rate warrants further studies.

The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (DeltaPsim). Activation of caspase-3 (but not -6 and -8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdR-mediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells., (2004 Wiley-Liss, Inc.)

The methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR, decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after 5-Aza-CdR treatment in AML. In the AML cell line OCI-AML2, Aza-CdR induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (> or =2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with 5-Aza-CdR ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with 5-Aza-CdR. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5' region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected 5-Aza-CdR-inducible genes. We detected hypermethylation exclusively in the 5' region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P< or =0.004). In contrast, all other analyzed 5-Aza-CdR-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of 5-Aza-CdR.

The Hodgkin cell line HD-MyZ is resistant to apoptosis induced by tumor necrosis factor alpha (TNFalpha). In the present work, we show that pretreatment with TNFalpha sensitized the cells to apoptosis induced by antineoplastic agents and ceramide. TNFalpha pretreatment resulted in enhanced cleavage and activity of caspase-3 upon addition of etoposide, epirubicin or ceramide. No caspase-8 activation was detectable, although caspase-8 could be activated in cell-free extracts. Inhibition of caspase-8 by z-IETD-fmk did not block the sensitizing effect of TNFalpha. Furthermore, exogenous ceramide, a mediator of TNFalpha signaling, could not substitute for TNFalpha in sensitization to drug-induced apoptosis. In contrast, we observed mitochondrial changes following cotreatment of cells with TNFalpha and drugs. Mitochondrial permeability transition, cytochrome c release and subsequent processing of caspase-9 preceded the onset of apoptosis, and were enhanced by TNFalpha pretreatment. Interestingly, although transcription factor NF-kappaB protected HD-MyZ cells from drug-induced apoptosis, TNFalpha-mediated sensitization was independent of NF-kappaB, since overexpressing a dominant-negative IkappaB mutant did not alter the TNFalpha effect. Sensitization for drug-induced apoptosis by TNFalpha was abrogated by Bcl-x(L). Thus, the sensitizing effect of TNFalpha is mediated by the mitochondrial pathway and involves processing of caspase-2, -3 and -9, but appears to be independent of caspase-8 processing, Bid cleavage and NF-kappaB signaling. Therefore, sensitization by TNFalpha is mediated at least in part through different pathways, as reported for TRAIL. There, sensitization occurs through a FADD/caspase-8-dependent mechanism. Regarding TNFalpha, the sensitizing effect was also observed in myeloid leukemia cells. Therefore, TNFalpha or alternate molecules activating its pathways might be useful as sensitizers for chemotherapy in hematological malignancies.