The Anti-Leukemia Effect of Ascorbic Acid: From the Pro-Oxidant Potential to the Epigenetic Role in Acute Myeloid Leukemia.

Data derived from high-throughput sequencing technologies have allowed a deeper understanding of the molecular landscape of Acute Myeloid Leukemia (AML), paving the way for the development of novel therapeutic options, with a higher efficacy and a lower toxicity than conventional chemotherapy. In the antileukemia drug development scenario, ascorbic acid, a natural compound also known as Vitamin C, has emerged for its potential anti-proliferative and pro-apoptotic activities on leukemic cells. However, the role of ascorbic acid (vitamin C) in the treatment of AML has been debated for decades. Mechanistic insight into its role in many biological processes and, especially, in epigenetic regulation has provided the rationale for the use of this agent as a novel anti-leukemia therapy in AML. Acting as a co-factor for 2-oxoglutarate-dependent dioxygenases (2-OGDDs), ascorbic acid is involved in the epigenetic regulations through the control of TET (ten-eleven translocation) enzymes, epigenetic master regulators with a critical role in aberrant hematopoiesis and leukemogenesis. In line with this discovery, great interest has been emerging for the clinical testing of this drug targeting leukemia epigenome. Besides its role in epigenetics, ascorbic acid is also a pivotal regulator of many physiological processes in human, particularly in the antioxidant cellular response, being able to scavenge reactive oxygen species (ROS) to prevent DNA damage and other effects involved in cancer transformation. Thus, for this wide spectrum of biological activities, ascorbic acid possesses some pharmacologic properties attractive for anti-leukemia therapy. The present review outlines the evidence and mechanism of ascorbic acid in leukemogenesis and its therapeutic potential in AML. With the growing evidence derived from the literature on situations in which the use of ascorbate may be beneficial in vitro and in vivo, we will finally discuss how these insights could be included into the rational design of future clinical trials.

Absence of FGFR3-TACC3 rearrangement in hematological malignancies with numerical chromosomal alteration.

FGFR-TACC, found in different tumor types, is characterized by the fusion of a member of fibroblast grown factor receptor (FGFR) tyrosine kinase (TK) family to a member of the transforming acidic coiled-coil (TACC) proteins. Because chromosome numerical alterations, hallmarks of FGFR-TACC fusions are present in many hematological disorders and there are no data on the prevalence, we studied a series of patients with acute myeloid leukemia and myelodysplastic syndrome who presented numerical alterations using cytogenetic traditional analysis. None of the analyzed samples showed FGFR3-TACC3 gene fusion, so screening for this mutation at diagnosis is not recommended.

Acute Promyelocytic Leukemia: Update on the Mechanisms of Leukemogenesis, Resistance and on Innovative Treatment Strategies.

This review highlights new findings that have deepened our understanding of the mechanisms of leukemogenesis, therapy and resistance in acute promyelocytic leukemia (APL). Promyelocytic leukemia-retinoic acid receptor α (PML-RARa) sets the cellular landscape of acute promyelocytic leukemia (APL) by repressing the transcription of RARa target genes and disrupting PML-NBs. The RAR receptors control the homeostasis of tissue growth, modeling and regeneration, and PML-NBs are involved in self-renewal of normal and cancer stem cells, DNA damage response, senescence and stress response. The additional somatic mutations in APL mainly involve FLT3, WT1, NRAS, KRAS, ARID1B and ARID1A genes. The treatment outcomes in patients with newly diagnosed APL improved dramatically since the advent of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). ATRA activates the transcription of blocked genes and degrades PML-RARα, while ATO degrades PML-RARa by promoting apoptosis and has a pro-oxidant effect. The resistance to ATRA and ATO may derive from the mutations in the RARa ligand binding domain (LBD) and in the PML-B2 domain of PML-RARa, but such mutations cannot explain the majority of resistances experienced in the clinic, globally accounting for 5-10% of cases. Several studies are ongoing to unravel clonal evolution and resistance, suggesting the therapeutic potential of new retinoid molecules and combinatorial treatments of ATRA or ATO with different drugs acting through alternative mechanisms of action, which may lead to synergistic effects on growth control or the induction of apoptosis in APL cells.

Retinoic acid and arsenic trioxide sensitize acute promyelocytic leukemia cells to ER stress.

Retinoic acid (RA) in association with chemotherapy or with arsenic trioxide (ATO) results in high cure rates of acute promyelocytic leukemia (APL). We show that RA-induced differentiation of human leukemic cell lines and primary blasts dramatically increases their sensitivity to endoplasmic reticulum (ER) stress-inducing drugs at doses that are not toxic in the absence of RA. In addition, we demonstrate that the PERK pathway, triggered in response to ER stress, has a major protective role. Moreover, low amounts of pharmacologically induced ER stress are sufficient to strongly increase ATO toxicity. Indeed, in the presence of ER stress, ATO efficiently induced apoptosis in RA-sensitive and RA-resistant APL cell lines, at doses ineffective in the absence of ER stress. Our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.

PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL.

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Nucleophosmin/B26 regulates PTEN through interaction with HAUSP in acute myeloid leukemia.

PTEN (phosphatase and tensin homolog deleted in chromosome 10) is a bona fide dual lipid and protein phosphatase with cytoplasmic (Cy) and nuclear localization. PTEN nuclear exclusion has been associated with tumorigenesis. Nucleophosmin (NPM1) is frequently mutated in acute myeloid leukemia (AML) and displays Cy localization in mutated nucleophosmin (NPMc+) AML. Here we show that NPM1 directly interacts with herpes virus-associated ubiquitin specific protease (HAUSP), which is known as a PTEN deubiquitinating enzyme. Strikingly, PTEN is aberrantly localized in AML carrying NPMc+. Mechanistically, NPM1 in the nucleus opposes HAUSP-mediated deubiquitination and this promotes the shuttle of PTEN to the cytoplasm. In the cytoplasm, NPMc+ prevents HAUSP from deubiquitinating PTEN, causing the latter to stay in the cytoplasm where it is polyubiquitinated and degraded. Our findings delineate a new NPM1-HAUSP molecular interaction controlling PTEN deubiquitination and trafficking.

[The clinical relevance of FDG-PET imaging in severe traumatic brain injuries]. / Relevancia clínica de la FDG-PET en los traumatismos craneoencefálicos graves.

AIM: To evaluate the usefulness of positron emission tomography (PET) as a predictor of long-term disability after a severe traumatic brain injury (TBI). PATIENTS AND METHODS: Fifty-six patients who had sustained a severe TBI were assessed with a broad battery of cognitive and functional scales at baseline and 6-months after inclusion in a multidisciplinary rehabilitation program. All patients underwent a FDG-PET at baseline. A physician blind to clinical data performed a semiquantitative analysis (normal vs altered) of functional neuroimaging (PET), including four cortical and three subcortical areas. The total number of lesions (cortical, subcortical and total) was correlated to the intensity of the TBI and to clinical data at admission and at follow-up. RESULTS: All patients showed changes in cerebral metabolism, being the thalamus the area most frequently affected. The degree of cerebral hypometabolism showed a significant correlation with TBI severity, functional disability, global outcome and cognitive impairment not only at baseline but also at follow-up. CONCLUSIONS: According to our results, FDG-PET may be a useful tool when studying brain dysfunction after severe TBI. FDG-PET findings correlate with the TBI severity, and with the level of patients' disability, as well as with the degree of memory and intelligence impairment. However, clinical variables related to the severity of the TBI, still are the best predictors of functional outcome after TBI.

Relevancia clínica de la FDG-PET en los traumatismos craneoencefálicos graves / The clinical relevance of FDG-PET imaging in severe traumatic brain injuries

Resumen. Objetivo. Demostrar la utilidad de la tomografía por emisión de positrones (PET) como predictor de la discapacidad a largo plazo tras un traumatismo craneoencefálico (TCE). Pacientes y métodos. Se evaluó neuropsicológica y funcionalmente a 56 pacientes que habían sufrido un TCE grave al inicio y aproximadamente seis meses después de su inclusión en un programa de rehabilitación multidisciplinar. A todos los pacientes se les realizó una tomografía por emisión de positrones con fluordeoxiglucosa al inicio del tratamiento. De forma ciega, se determinó la presencia o ausencia de alteraciones en cuatro áreas corticales y tres subcorticales, y se determinaron tres índices cualitativos de metabolismo cerebral (cortical, subcortical y total). Los índices de metabolismo se correlacionaron con las variables relacionadas con la gravedad del traumatismo, y con la situación cognitiva y funcional de los pacientes en el momento de realizar la PET y al finalizar el programa de rehabilitación. Resultados. Todos los pacientes mostraron alteraciones en el metabolismo cerebral, y el tálamo fue el área más frecuentemente afectada. La intensidad del hipometabolismo cerebral se correlacionó significativamente con la gravedad del TCE y con la alteración cognitiva y funcional tanto al inicio como al final del tratamiento. Conclusiones. Las técnicas de neuroimagen funcional presentan una excelente sensibilidad para detectar alteraciones tras un TCE, además de ofrecer una buena correlación anatomoclínica. No obstante, las variables relacionadas con la gravedad del TCE, siguen siendo las mejores predictoras de la discapacidad resultante tras un TCE (AU)

Summary. Aim. To evaluate the usefulness of positron emission tomography (PET) as a predictor of long-term disability after a severe traumatic brain injury (TBI). Patients and methods. Fifty-six patients who had sustained a severe TBI were assessed with a broad battery of cognitive and functional scales at baseline and 6-months after inclusion in a multidisciplinary rehabilitation program. All patients underwent a FDG-PET at baseline. A physician blind to clinical data performed a semiquantitative analysis (normal vs altered) of functional neuroimaging (PET), including four cortical and three subcortical areas. The total number of lesions (cortical, subcortical and total) was correlated to the intensity of the TBI and to clinical data at admission and at follow-up. Results. All patients showed changes in cerebral metabolism, being the thalamus the area most frequently affected. The degree of cerebral hypometabolism showed a significant correlation with TBI severity, functional disability, global outcome and cognitive impairment not only at baseline but also at follow-up. Conclusions. According to our results, FDG-PET may be a useful tool when studying brain dysfunction after severe TBI. FDG-PET findings correlate with the TBI severity, and with the level of patients’ disability, as well as with the degree of memory and intelligence impairment. However, clinical variables related to the severity of the TBI, still are the best predictors of functional outcome after TBI (AU)

Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia.

Mutations in the Nucleophosmin (NPM1) gene have been recently described to occur in about one-third of acute myeloid leukemias (AML) and represent the most frequent genetic alteration currently known in this subset. These mutations generate an elongated NPM1 protein that localizes aberrantly in the cytoplasm. In analogy with Flt3 alterations, NPM1 mutations are mostly detectable in AML with normal karyotype and their recognition may be relevant to identify distinct response to treatment. Hence, in addition to conventional karyotyping and RT-PCR of fusion genes, combined analysis of both Flt3 and NPM1 mutations will be increasingly relevant in the genetic diagnosis work-up of AML. We developed a multiplex RT-PCR assay followed by capillary electrophoresis to simultaneously analyze NPM1 and Flt3 gene alterations (NFmPCR assay). The assay was validated in leukemic cell RNAs extracted from 38 AML patients, which had been previously characterized for Flt3 status by conventional RT-PCR. Direct sequencing of NPM1 RT-PCR products was carried out in 15 cases to verify results obtained by capillary electrophoresis. Both NPM1 sequencing and conventional RT-PCR Flt3 results showed 100% concordance with the results of the NFmPCR assay. We suggest that this assay may be introduced in routine analysis of genetic alterations in AML.

Minimally invasive silicon probe for electrical impedance measurements in small animals.

It is commonly accepted that electrical impedance provides relevant information about the physiological condition of living tissues. Currently, impedance measurements are performed with relatively large electrodes not suitable for studies in small animals due to their poor spatial resolution and to the damage that they cause to the tissue. A minimally invasive needle shaped probe for electrical impedance measurements of living tissues is presented in this paper. This micro-probe consists of four square platinum electrodes (300 microm x 300 microm) on a silicon substrate (9 mm x 0.6 mm x 0.5 mm) and has been fabricated by using standard Si microelectronic techniques. The electrodes are not equally spaced in order to optimise the signal strength and the spatial resolution. Characterisation data obtained indicate that these probes provide high spatial resolution (measurement radius <4 mm) with a useful wide frequency band going from 100 Hz to 100 kHz. A series of in vivo experiments in rat kidneys subjected to ischemia was performed to demonstrate the feasibility of the probes and the measurement system. The impedance modulus and phase were measured at 1 kHz since this frequency is sufficiently low to permit the study of the extracellular medium. The extracellular pH and K+ were also simultaneously measured by using commercial miniaturised Ion Selective Electrodes. The induced ischemia period (45 min) resulted in significant changes of all measured parameters (Delta/Z/ approximately 65%; DeltapH approximately 0.8; DeltaK+ approximately 30 mM).

Triplication (/alphaalphaalpha anti3.7) or deletion (-alpha3.7/) association in Argentinian beta-thalassemic carriers.

Prevalence of alpha gene triplication or deletion in beta-thalassemia carriers was studied in 109 unrelated individuals in Rosario, Argentina. In different populations -alpha(3.7) allele presents a higher prevalence than alphaalphaalpha(anti3.7); thus, alpha-thalassemia associated with beta-thalassemia is more frequently observed. Nevertheless, this event was detected in only one case (0.9%), while the association with alpha triplication was present in two subjects (1.8%).

Multiparametric monitoring of ischemia-reperfusion in rat kidney: effect of ischemic preconditioning.

BACKGROUND: Microelectrode technology is a promising tool for monitoring kidney ischemia and the changes induced by its therapeutic management. Ischemic preconditioning, that is, brief ischemic periods before sustained ischemia, has been shown to protect several organs, including the kidney, from ischemia-reperfusion injury. We tested whether the effect of preconditioning could be appraised by real-time measurement of parameters representative of tissue hypoxia. METHODS: In a sample of pentobarbital-anesthetized and mechanically ventilated rats, we studied the effect of renal ischemic preconditioning (10-min ischemia and 10-min reflow interval) on subsequent ischemia-reperfusion (45 min and 60 min). Renal tissue electrical impedance, extracellular pH, and potassium concentration [K+] were measured continuously by implanted microelectrodes. RESULTS: Ischemia induced an early, rapid rise in extracellular potassium and impedance module, followed by a phase of slower increase, whereas pH decreased rapidly, reaching a plateau. Preconditioning treatment did not cause significant changes in interstitial pH and [K+] but increased ischemic tissue impedance. During reperfusion, the three variables recovered progressively; however, after a decline, electrical impedance showed a clear postischemic increase. This rise was suppressed by preconditioning. CONCLUSIONS: Real-time measurement of any of the three parameters showed capability for early detection of ischemia. In contrast with findings in myocardial tissue, preconditioning in the kidney did not increase potassium cell loss during ischemia or improve ischemic acidosis or tissue impedance. Electrical impedance increased for a second time during reperfusion, indicating the presence of a postischemic cellular edema; concealing this episode was the most noticeable effect of the preconditioning treatment.

Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol.

Alterations in the FLT3 gene, including internal tandem duplications (ITDs) and D835 mutations occur frequently in acute myelogenous leukemia. We investigated the prevalence and clinico-biological correlations of FLT3 ITDs and D835 mutations in 90 patients with acute promyelocytic leukemia (APL) receiving the AIDA protocol. Twenty patients in which both presentation and relapse material was available were analyzed sequentially. Thirty-three patients (37%) harbored the ITD, and seven (7.7%) the D835 mutation in blasts obtained at diagnosis. Presence of ITDs was strongly associated with high WBC count (P = 0.0001), M3 variant (P = 0.0004), and the short (BCR3) PML/RARalpha isoform (P = 0.003). There was no difference in response to induction in the two ITD+ve and ITD-ve groups, while a trend towards inferior outcome was observed for ITD+ve cases when analyzing disease-free survival (DFS) and relapse risk (RR). These differences, however, did not reach statistical significance. Sequential studies showed variable patterns in diagnostic and relapse material, ie ITD (-ve/-ve, +ve/+ve, +ve/-ve, -ve/+ve) and D835 (-ve/-ve, +ve/-ve, -ve/+ve). Our results indicate that FLT3 alterations are associated in APL with more aggressive clinical features and suggest that these lesions may not play a major role in leukemia progression.

A novel splice acceptor site mutation of the alpha2-globin gene causing alpha-thalassemia.

A novel nondeletional alpha-thalassemia mutation that affects RNA processing, changing the alpha2 IVS-II-142 splice acceptor consensus sequence from AG to AA, has been detected in an Argentinian patient with Hb H disease and her daughter.

A new polymorphim (G->A) in the psizeta1 globin gene.

BACKGROUND AND OBJECTIVES: a-globin cluster polymorphisms are obtained with specific restriction enzymes (Xba I, Eco RI, Sac I, Apa I, Bgl II, etc) that can also have implications for genetic analysis. DESIGN AND AND METHODS: We studied three unrelated patients; one from Argentina, one from Spain and one from Australia but of Polish origin. Genomic DNA was digested with several different restriction enzymes and probes, amplified and sequenced with an ABI Prism 310 sequencer. RESULTS: In the three patients an abnormal 26 kb band appeared when they were studied with restriction enzyme Bgl II and z probe. A fragment of 944 bp was amplified with primers that cover from -280 to +714 bp of the recognition sequence of Bgl II enzyme (AGATCT) localized 5' from pseudogene z1. After digestion of this PCR product with Bgl II, two fragments of 714 and 280 bp were produced in normal controls, whereas in patient #1 the PCR fragment was undigested and in patients 2 and 3 both undigested and digested fragments were observed. Sequencing of the PCR fragment showed that in all three patients it was the same polymorphism (G->A) at nucleotide 153171 of the 16 p sequence found in the Bgl II recognition site that changed to AAATCT. INTERPRETATION AND CONCLUSIONS: We describe a new polymorphism in the yz1 first exon Bgl II restriction site (G->A). The polymorphism is associated in cis with haplotype -a3.7. The fragment obtained by PCR enabled us to corroborate the presence of the polymorphism quickly without having to use complicated sequencing techniques.

Modified salting-out method for DNA isolation from newborn cord blood nucleated cells.

The present work describes modification of a widely used salting-out procedure to rapidly extract DNA suitable for PCR, using the ARMS method to amplify a target sequence in the beta-globin gene. The salting-out DNA extraction procedure did not completely remove or decrease the presence of inhibitors to PCR in a considerable number of cord blood samples. By introducing a simple phenol/chloroform step, before ethanol precipitation of the nucleic acid, to certain samples, we were able to eliminate or substantially reduce the presence of inhibitors to PCR without having to re-extract the samples.