Spontaneous intramedullary apoptosis is present in disorders other than myelodysplasia

Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders characterized by peripheral cytopenias in the presence of normo- or hypercellular dysplastic marrow. It has been suggested that premature intramedullary apoptosis may contribute to this phenomenon. We used terminal dUTP nick-end labeling (TUNEL) of bone marrow biopsy specimens and cytocentrifuge preparations from patients with MDS and a variety of other hematopoietic disorders to determine whether there is increased intramedullary apoptosis in MDS and whether any such effect is specific to MDS. TUNEL labeling of bone marrow from 24 patients with MDS revealed significant positivity in 10 of 11 patients with refractory anemia (RA), five of seven with RA and excess of blasts (RAEB), all three patients with RAEB in transformation (RAEB-t), and all three patients with RA with ring sideroblasts (RARS). The percent of positive cells ranged from 5 to 50% but showed no apparent correlation with morphological subtype. In a series of 29 patients with acute leukemia, 17 showed significant positivity (13 of 13 with myeloid disease: three M1, seven M2, one M3, two M4; four of 16 patients with lymphoid disease: one Burkitt-type lymphoma, two null acute leukemia, and one common acute lymphoid leukemia). Intramedullary apoptosis was associated with myeloid or early committed progenitor cells and was highest in secondary acute myeloid leukemia (AML). Normal bone marrow samples from 12 individuals showed no evidence of apoptosis. Our results suggest that an increased level of intramedullary apoptosis is apparent in both patients with MDS and those with AML; those with secondary AML have the highest levels. The relative absence of such findings in lymphoid malignancy suggests that the apoptotic pathways are different in this lineage.

Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B-cell lymphomas, respectively

Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB targets and their expression distinguished small and aggressive B-cell lymphomas, respectively. Interestingly, in the vast majority of B-cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Addiction to Runx1 is partially attenuated by loss of p53 in the Eµ-Myc lymphoma model

The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the Eµ-Myc model of Burkitt's lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary Eµ-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established Eµ-Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status.

The role of reciprocal regulation between TAL1 and miRNA expression in T-cell leukemia progression

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2015

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Mecanismo de acción de la oncotoxina de Bacillus thuringiensis var.thuringiensis

Tesis (Doctorado en Ciencias con Especialidad en Microbiología) UANL

Caractérisation structurale et fonctionnelle des interactions impliquant TFIIH et les domaines de transactivation viraux

Le facteur de transcription IIH (TFIIH) joue un rôle crucial dans la transcription et dans la réparation de l’ADN. La sous-unité Tfb1/p62 (levure et humain) de TFIIH interagit avec de nombreux facteurs de transcription (p53, NFκB, TFIIEα) et de réparation (Rad2/XPG and Rad4/XPC) (1). La majorité des interactions avec Tfb1/p62 requiert le domaine d’homologie à la Pleckstrin (PH) localisé dans la région N-terminal de la protéine (2, 3). Ce domaine PH forme des complexes avec des domaines de transactivation acide provenant de protéines cibles impliquées dans la transcription et la réparation de l’ADN. De récentes études ont montré que Tfb1/p62 est une cible pour les protéines virales telles que la protéine VP16 du virus de l’herpès simplex (HSV) de type 1, la protéine E1 du virus du papillome humain (VPH) et la protéine EBNA-2 du virus Epstein-Barr (EBV) (4, 5). Ces protéines virales interagissent avec la sous-unité Tfb1/p62 par un domaine de transactivation acide suggérant une interaction similaire à ce qui est observé chez les facteurs de transcription humains comme p53. Ce mémoire présente une caractérisation structurelle et fonctionnelle du complexe formé par la protéine virale EBNA2 et la protéine humaine Tfb1/p62. L’analyse est faite en utilisant le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire (RMN) et une expérience de transactivation chez la levure. Cette étude amène une plus grande compréhension des protéines impliquées dans les maladies comme le lymphome de Burkitt et le lymphome de Hodgkin qui sont souvent associées à l’infection à l’EBV (revue dans (6)) et caractérise une cible potentielle pour un antiviral.

Sobrevida de pacientes pediátricos con enfermedad de Hodgkin en la Fundación Hospital de la Misericordia

Propósito: en Colombia el linfoma es el tercer tipo de cáncer en los niños (3). El objetivo del presente trabajo fue establecer la sobrevida global en pacientes menores de 18 años con enfermedad de Hodgkin en la Fundación Hospital de la Misericordia Bogotá - Colombia en el periodo comprendido entre el 1 de Enero de 2000 hasta el 30 de enero de 2007. Métodos: se realizó un estudio de sobrevida en 49 pacientes tratados con protocolos T1 (2000 – 2004) y T2 (2005 – 2007). Resultados: de 1687 casos de tumores sólidos en 22 años en el servicio de oncología, 440 corresponden a linfoma y 206 a EH (12%). 49 pacientes fueron tratados con los esquemas de quimioterapia sometidos a análisis. 37 (75,5%) pacientes eran niños, el promedio de edad fue 9 años. Más del 90% presentaban enfermedad supradiafragmática, la histología más frecuente fue esclerosis nodular. El estado IIIB se observó en 15 pacientes (30,6%). La sobrevida global de niños sin remisión con tratamiento de primera línea, fue 0%, con estado IV 60%, de acuerdo a tratamiento: T1 fue de 76% y con T2 95%. Discusión: el presente análisis muestra que la sobrevida global con T2 es mayor que con T1, identifica como factores de riesgo de mal pronóstico: la no remisión con tratamiento de primera línea y el estado de la enfermedad. Los síntomas B, el subtipo histológico y la edad no mostraron diferencias significativas pronosticas.

Polimorfismos de los genes ABCB1 y CYP2C8 en pacientes sometidos a TASPE. Análisis farmacogenético del Paclitaxel en la toxicidad y movilización de progenitores hemopoyéticos

Fundamentos. La eficacia de paclitaxel junto con rhG-CSF en la movilización de progenitores hematopoyéticos, se ha probada en pacientes hematológicos. Farmacogenéticamente el paclitaxel presenta una alta variabilidad inter-individual. Los genes CYP2C8 y ABCB1 involucrados en su metabolismo y transporte podrían afectar dicha variabilidad inter-individual. Objetivo. Evaluar en una cohorte retrospectiva de pacientes sometidos a TASPE, el efecto de algunos polimorfismos de nucleótido simple (del gen CYP2C8 y del gen ABCB1) sobre la eficacia en la movilización y toxicidad hematológica inducida por del paclitaxel. Materiales y Métodos. Un grupo de 107 pacientes recibieron paclitaxel y rhG-CSF como esquema movilizador. Los polimorfismos genotipados fueron para los genes ABCB1 rs1045642 A>G, ABCB1 rs2032582 C>A, ABCB1 rs2032582 C>T, CYP2C8 rs10509681 C>T, y CYP2C8 rs11572080 A>G. Resultados. El uso de paclitaxel logró éxito movilizador en más del 80% de los pacientes con linfomas o mieloma (p=0,0021), pero no lo fue en la leucemia aguda. En pacientes con mieloma la variable G>rs1045642 del gen ABCB1 se asoció con mala movilización (p= 0,018) y mayor toxicidad hematológica (p= 0,034). El alelo C>rs10509681 del gen CYP2C8 se relacionó con mayor toxicidad en pacientes con linfoma (p= 0,045) y mieloma múltiple (p=0,042), y portadores del alelo TT en homocigosis presentaron una mayor toxicidad hematológica comparada con los portadores CC o CT (p= 0,027). Conclusión. Este estudio sugiere que los SNPs de las variables alélicas analizadas en los genes CYP2C8 y ABCB1 en algunos grupos de pacientes inciden en la capacidad movilizadora y afectan el grado de toxicidad hematológica.

Ideación suicida y algunos factores biopsicosociales asociados con estas en pacientes adultos con leucemia, linfomas, cáncer gástrico o cáncer colorrectal Informe de investigación

El cáncer es una de las enfermedades con mayor impacto en la población mundial, debido a que genera alteraciones en las áreas de ajuste de los individuos, su núcleo familiar y social circundante. El presente estudio tiene como objetivo establecer la prevalencia de ideación suicida en pacientes con cáncer de tejidos blandos (leucemias y linfomas) y cáncer gástrico (colorectal). Fue un estudio exploratorio descriptivo, de la línea de investigación en Psicooncología y Cuidado Paliativo a la cual pertenece el proyecto: “Conducta suicida (ideación suicida, intención suicida y suicidio frustrado) y factores biopsicosociales asociadas a esta en pacientes con cáncer”. La población que participó en este estudio fueron pacientes adultos del Centro de Investigaciones oncológicas de la clínica San Diego CIOSAD. De esta investigación se puede concluir que la prevalencia de ideación suicida en pacientes con estos tipos de cáncer es de 4.9% (N=10). Los factores asociados a la ideación suicida fueron: número de hijos, dolor y ansiedad en la última semana y estrato socioeconómico

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated With Cu2+ ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu2+ to Cu+ and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu+ oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides. (C) 2007 Elsevier Inc. All rights reserved.

Epstein-Barr virus: clinical and epidemiological revisits and genetic basis of oncogenesis

Epstein-Barr virus (EBV) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. The virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (HHV4). It was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. It was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. Clinically, EBV primary infection is almost silent, persisting as a life-long asymptomatic latent infection in B cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. Following reactivation of the virus from latency due to immunocompromised status, EBV was found to be associated with several tumors. EBV linked to oncogenesis as detected in lymphoid tumors such as Burkitt's lymphoma (BL), Hodgkin's disease (HD), post-transplant lymphoproliferative disorders (PTLD) and T-cell lymphomas (e.g. Peripheral T-cell lymphomas; PTCL and Anaplastic large cell lymphomas; ALCL). It is also linked to epithelial tumors such as nasopharyngeal carcinoma (NPC), gastric carcinomas and oral hairy leukoplakia (OHL). In vitro, EBV many studies have demonstrated its ability to transform B cells into lymphoblastoid cell lines (LCLs). Despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these EBV- associated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (LVPs) upregulated in both tumors and LCLs. In this review, we summarize some clinical and epidemiological features of EBV- associated tumors. We also discuss how EBV latent genes may lead to oncogenesis in the different clinical malignancies

Balanoposthitis caused by Pseudomonas aeruginosa co-producing metallo-beta-lactamase and 16S rRNA methylase in children with hematological malignancies

Balanoposthitis is defined as the inflammation of the glans penis and its foreskin. In the presence of other underlying medical conditions, this localized infection may spread systemically, serving as a source of fever and bacteremia in neutropenic males. Two rare cases of balanoposthitis caused by a clonally related Pseudomonas aeruginosa isolate co-producing the SPM-1 metallo-beta-lactamase and the novel 16S rRNA methylase RmtD are described. Four multidrug-resistant (MDR) P. aeruginosa isolates were successively recovered from glans/foreskin swabs and urine cultures from two uncircumcised pediatric patients, one with Burkitt`s non-Hodgkin`s lymphoma and one with acute lymphoblastic leukemia. Clinically, preputial colonization by MDR P. aeruginosa evolved to severe balanoposthitis with glans/foreskin lesions as a source of fever. Combination therapy of ciprofloxacin and/or aztreonam (systemic) plus polymyxin B (topical) was effective once reversion of the neutropenic condition was achieved. Although P. aeruginosa remains an unusual cause of balanoposthitis, these cases should alert the physician to the potential pathogenicity of this bacterium. Furthermore, co-production of metallo-beta-lactamase and 16S rRNA methylase has a potential impact on the empirical management of complicated infections caused by P. aeruginosa. Crown Copyright (C) 2009 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. All rights reserved.