Constitutive cytoplasmic localization of p21Waf1/Cip1 affects the apoptotic process in monocytic leukaemia

In the present study, we analysed the expression and localization of p21Waf1/Cip1 in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-κB (NF-κB) independent, results in predominantly cytoplasmic localized p21 protein. In acute monocytic leukaemia (M4, M5), monocytic blasts (N=12) show constitutive cytoplasmic p21 expression in 75% of the cases, while in myeloid leukaemic blasts (N=10), low nuclear and cytoplasmic localization of p21 could be detected, which is also PKC dependent. Constitutive p21 expression in monocytic leukaemia might have important antiapoptotic functions. This is supported by the finding that in U937 cells overexpressing p21, VP16-induced apoptosis is significantly reduced (20.0±0.9 vs 55.8±3.8%, P<0.01, N=5), reflected by a reduced phosphorylation of p38 and JNK. Similarly, AML blasts with high cytoplasmic p21 were less sensitive to VP16-induced apoptosis as compared to AML cases with low or undetectable p21 expression (42.25 vs 12.3%, P<0.01). Moreover, complex formation between p21 and ASK1 could be demonstrated in AML cells, by means of coimmunoprecipitation. In summary, these results indicate that p21 has an antiapoptotic role in monocytic leukaemia, and that p21 expression is regulated in a PKC-dependent and NF-κB independent manner.

DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

Background: Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. Methodology/Principal Findings: To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). Conclusions/Significance: We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.Tina Bianco-Miotto, Damian J. Hussey, Tanya K. Day, Denise S. O'Keefe and Alexander Dobrovi

Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung

Phagocytes not only coordinate acute inflammation and host defense at mucosal sites, but also contribute to tissue damage. Respiratory infection causes a globally significant disease burden and frequently progresses to acute respiratory distress syndrome, a devastating inflammatory condition characterized by neutrophil recruitment and accumulation of protein-rich edema fluid causing impaired lung function. We hypothesized that targeting the intracellular protein myeloid cell leukemia 1 (Mcl-1) by a cyclin-dependent kinase inhibitor (AT7519) or a flavone (wogonin) would accelerate neutrophil apoptosis and resolution of established inflammation, but without detriment to bacterial clearance. Mcl-1 loss induced human neutrophil apoptosis, but did not induce macrophage apoptosis nor impair phagocytosis of apoptotic neutrophils. Neutrophil-dominant inflammation was modelled in mice by either endotoxin or bacteria (Escherichia coli). Downregulating inflammatory cell Mcl-1 had anti-inflammatory, pro-resolution effects, shortening the resolution interval (R(i)) from 19 to 7 h and improved organ dysfunction with enhanced alveolar–capillary barrier integrity. Conversely, attenuating drug-induced Mcl-1 downregulation inhibited neutrophil apoptosis and delayed resolution of endotoxin-mediated lung inflammation. Importantly, manipulating lung inflammatory cell Mcl-1 also accelerated resolution of bacterial infection (R(i); 50 to 16 h) concurrent with enhanced bacterial clearance. Therefore, manipulating inflammatory cell Mcl-1 accelerates inflammation resolution without detriment to host defense against bacteria, and represents a target for treating infection-associated inflammation

LSK Derived LSK– Cells Have a High Apoptotic Rate Related to Survival Regulation of Hematopoietic and Leukemic Stem Cells

A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin−Sca-1+c-Kit- (LSK−) cell population derived from HSC-containing Lin−Sca-1+c-Kit+ (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK− population. In contrast, the LSK− population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK− population. The transition of LSK to LSK− cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK− cells in the regulation of LSK cells and LSCs

Mutations with epigenetic effects in myeloproliferative neoplasms and recent progress in treatment: Proceedings from the 5th International Post-ASH Symposium

Immediately following the 2010 annual American Society of Hematology (ASH) meeting, the 5th International Post-ASH Symposium on Chronic Myelogenous Leukemia and BCR-ABL1-Negative Myeloproliferative Neoplasms (MPNs) took place on 7–8 December 2010 in Orlando, Florida, USA. During this meeting, the most recent advances in laboratory research and clinical practice, including those that were presented at the 2010 ASH meeting, were discussed among recognized authorities in the field. The current paper summarizes the proceedings of this meeting in BCR-ABL1-negative MPN. We provide a detailed overview of new mutations with putative epigenetic effects (TET oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), isocitrate dehydrogenase (IDH) and enhancer of zeste homolog 2 (EZH2)) and an update on treatment with Janus kinase (JAK) inhibitors, pomalidomide, everolimus, interferon-α, midostaurin and cladribine. In addition, the new ‘Dynamic International Prognostic Scoring System (DIPSS)-plus' prognostic model for primary myelofibrosis (PMF) and the clinical relevance of distinguishing essential thrombocythemia from prefibrotic PMF are discussed

Recasting the cancer stem cell hypothesis: Unification using a continuum model of microenvironmental forces

Purpose of review Here, we identify shortcomings of standard compartment-based mathematical models of cancer stem-cells, and propose a continuous formalism which includes the tumor microenvironment. Recent findings Stem-cell models of tumor growth have provided explanations for various phenomena in oncology including, metastasis, drug- and radio-resistance, and functional heterogeneity in the face of genetic homogeneity. While some of the newer models allow for plasticity, or de-differentiation, there is no consensus on the mechanisms driving this. Recent experimental evidence suggests that tumor microenvironment factors like hypoxia, acidosis, and nutrient deprivation have causative roles. Summary To settle the dissonance between the mounting experimental evidence surrounding the effects of the microenvironment on tumor stemness, we propose a continuous mathematical model where we model microenvironmental perturbations like forces, which then shape the distribution of stemness within the tumor. We propose methods by which to systematically measure and characterize these forces, and show results of a simple experiment which support our claims

Macrophage Glucose-6-Phosphate Dehydrogenase Stimulates Proinflammatory Responses with Oxidative Stress

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that regulates cellular redox potential. In this study, we demonstrate that macrophage G6PD plays an important role in the modulation of proinflammatory responses and oxidative stress. The G6PD levels in macrophages in the adipose tissue of obese animals were elevated, and G6PD mRNA levels positively correlated with those of proinflammatory genes. Lipopolysaccharide (LPS) and free fatty acids, which initiate proinflammatory signals, stimulated macrophage G6PD. Overexpression of macrophage G6PD potentiated the expression of proinflammatory and prooxidative genes responsible for the aggravation of insulin sensitivity in adipocytes. In contrast, when macrophage G6PD was inhibited or suppressed via chemical inhibitors or small interfering RNA (siRNA), respectively, basal and LPS-induced proinflammatory gene expression was attenuated. Furthermore, macrophage G6PD increased activation of the p38 mitogen-activated protein kinase (MAPK) and NF-??B pathways, which may lead to a vicious cycle of oxidative stress and proinflammatory cascade. Together, these data suggest that an abnormal increase of G6PD in macrophages promotes oxidative stress and inflammatory responses in the adipose tissue of obese animals.open5

Involvement of Lgl and Mahjong/VprBP in Cell Competition

Mahjong is a novel Lethal giant larvae-binding protein that plays a vital role in cell competition in both flies and mammals

Chronic Myeloid Leukemia Stem Cell Biology

Leukemia progression and relapse is fueled by leukemia stem cells (LSC) that are resistant to current treatments. In the progression of chronic myeloid leukemia (CML), blast crisis progenitors are capable of adopting more primitive but deregulated stem cell features with acquired resistance to targeted therapies. This in turn promotes LSC behavior characterized by aberrant self-renewal, differentiation, and survival capacity. Multiple reports suggest that cell cycle alterations, activation of critical signaling pathways, aberrant microenvironmental cues from the hematopoietic niche, and aberrant epigenetic events and deregulation of RNA processing may facilitate the enhanced survival and malignant transformation of CML progenitors. Here we review the molecular evolution of CML LSC that promotes CML progression and relapse. Recent advances in these areas have identified novel targets that represent important avenues for future therapeutic approaches aimed at selectively eradicating the LSC population while sparing normal hematopoietic progenitors in patients suffering from chronic myeloid malignancies

Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins