Laura I. Kornblihtt

JAK2V617F mutation in platelets from essential thrombocythemia patients: correlation with clinical features and analysis of STAT5 phosphorylation status.

Objective:  JAK2V617F mutation rate in granulocytes from essential thrombocythemia (ET) patients ranges from 12% to 57%. Our aim was to evaluate the frequency of this mutation in the megakaryocyte/platelet lineage, and to analyze its clinical associations in ET. In addition, we determined whether this mutation leads to constitutive phosphorylation of STAT5 in platelets.

State-of-the-Art Review : Effectiveness of Anagrelide in the Treatment of Symptomatic Patients with Essential Thrombocythemia

We prospectively evaluated the effect of anagrel ide on platelet counts and the clinical manifestations of micro vascular circulation disturbances in 17 newly diagnosed pa tients with essential thrombocythemia. Ten patients had symp toms related to thrombocythemia, eight at the time of starting anagrelide treatment. The platelet counts before anagrelide treatment and during maintained remission of essential throm bocythemia by anagrelide were 980 (range, 610-2030) and 378 (range, 212-546) x 109/L, respectively. Spontaneous platelet aggregation was found in 6 patients (35%), which disappeared on remission of essential thrombocythemia in five cases (P = 0.02). Essential thrombocythemia-related microvascular thrombotic and hemorrhagic symptoms disappeared with the normalization of platelet count in all cases during maintained remission of essential thrombocythemia by long term continu ous anagrelide treatment with a follow-up period of between 2 and 6 years. However, ET-related symptoms reappeared in three patients, coinciding with increased platelet count up to 600 x 109/L caused by anagrelide dose reduction. We conclude that reduction of increased platelet to normal (< 400 x 109/L) in symptomatic patients with essential thrombocythemia through use of maintained anagrelide treatment is associated with the disappearance of spontaneous platelet aggregation and the complete relief of thrombotic and hemorrhagic manifestations.

Moyamoya Syndrome in an Adolescent With Essential Thrombocythemia. Successful Intracranial Carotid Stent Placement

Background— Essential thrombocythemia (ET) is a chronic myeloproliferative disorder with increased frequency of thrombotic events, including transient ischemic attacks (TIAs) and stroke. Moyamoya syndrome is a rare cerebrovascular disease characterized by progressive occlusion of intracerebral arteries with a typical “puff of smoke” angiographic pattern. We report the development of moyamoya syndrome in a patient with ET. Case Description— The patient is an 18-year-old female who presented at age 13 with recurrent TIAs. Persistent thrombocytosis was found, a diagnosis of ET was made, and treatment with anagrelide was started. Despite normal platelet counts, she experienced recurrent TIAs and stroke. Severe stenosis of the supraclinoid segment of the left internal carotid artery (LICA) and abnormal collateral vessels were found, and moyamoya syndrome was diagnosed on the basis of the characteristic angiographic appearance. An intracranial stent was placed in the LICA, and since then, she has had an uneventful outcome after a 46-month follow-up. Conclusion— To our knowledge, the development of moyamoya phenomenon has not been reported in ET, and the relationship between these 2 disorders remains unclear. Besides, this is the first intracranial carotid stent implanted successfully in a patient with moyamoya.

Inhibitory effect of melatonin on platelet activation induced by collagen and arachidonic acid

Abstract: Melatonin, an indolamine synthesized in the pineal gland, is known to have antiprostanoid activity. The inhibition of platelet aggregation induced by melatonin has been proposed to take place through the cyclooxygenase pathway. In the present study, we found that melatonin has a marked inhibitory effect on collagen, arachidonic acid (AA), adenosine diphosphate (ADP), epinephrine, and A23187‐induced aggregation in platelet‐rich plasma. On the other hand, using metrizamide‐filtered platelets resuspended in Tyrodes buffer, melatonin fails to suppress AA‐induced platelet aggregation and 14C‐5‐HT release. Under the same conditions, melatonin inhibits collagen‐induced platelet activation; however, the addition of threshold doses of AA (0.3 mM) abrogates this effect. These studies suggest that melatonin also inhibits platelet function at a stage preceding the cyclooxygenase‐dependent pathway.

Synergism of arsenic trioxide and MG132 in Raji cells attained by targeting BNIP3, autophagy, and mitochondria with low doses of valproic acid and vincristine.

We previously demonstrated that arsenic trioxide (ATO) and proteasome inhibitor MG132 synergistically induced cell death in promonocytic leukaemia cell line U937 but were antagonistic in Burkitts lymphoma cell line Raji. Here we explore the role of autophagy, expression of BNIP3, and mitochondrial mass, in determining whether ATO and MG132 interaction can be shifted from antagonism to synergism in Raji cells. Treatment with ATO+MG132 increased the percentage of cells with collapsed mitochondrial membrane potential (MMP) in U937 cells, but had no effect in Raji cells. Mitochondria were found in cytoplasmic marginal location in U937 cells but at perinuclear location in Raji cells. ATO+MG132 increased mitochondrial mass in U937 cells but decreased it in Raji cells, while autophagy was increased in both cell lines. BNIP3 was expressed in U937 cells at cytoplasmic marginal locations and was hardly detected in Raji cells. Histone deacetylase (HDAC) inhibitor valproic acid (VPA) increased expression of BNIP3 in Raji cells at perinuclear locations. However antagonism between ATO and MG132 was increased in the presence of low doses of VPA. Addition of vincristine (VCR) blocked autophagy, while VPA+VCR treatment of Raji cells at sub-cytotoxic doses caused BNIP3 and mitochondria to redistribute to cytoplasmic peripheral location and increased mitochondrial mass. ATO+MG132 in the presence of subcytotoxic doses of VPA+VCR caused collapse of MMP in Raji cells, while interaction between ATO and MG132 shifted from antagonism to synergism. We conclude that synergism between ATO and MG132 was attained in Raji cells by disruption of the perinuclear mitochondrial cluster, blockage of selective autophagy of mitochondria (mitophagy) by VCR, increased mitochondrial mass, and upregulation of BNIP3 by VPA.

Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion.

Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitts lymphoma-derived Raji cells, by the Chou-Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O₂⁻) levels. Our results showed that combined arsenite+MG132 produced low levels of O₂⁻ at 6h and 24h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80μM hydrogen peroxide together with arsenite+MG132 changed synergism on cell death to antagonism at all effect levels while increasing O₂⁻ levels. Arsenite+hydrogen peroxide also resulted in antagonism with increased O₂⁻ levels in U937 cells. In Raji cells, arsenite+MG132 also produced low levels of O₂⁻ at 6h and 24h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite+CAPE showed high levels of O₂⁻ production at 6h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O₂⁻ levels at early time points after exposure.

Monocyte IL-2Rα expression is associated with thrombosis and the JAK2V617F mutation in myeloproliferative neoplasms

The development of bone marrow fibrosis and thrombosis are main causes of morbidity in essential thrombocythemia (ET). Monocyte activation has been associated to the production of fibrosis-related cytokines and pro-thrombotic factors. The aim of this study was to identify new markers of monocyte activation in Phi-negative myeloproliferative neoplasms and to search for their relationship with clinical features. Forty-five patients comprising 30 ET, eight myelofibrosis and seven polycythemia vera were included. We evaluated the alpha subunit of IL-2 receptor (CD25) on monocytes, basal and LPS-induced IL-1beta release from mononuclear cells, and monocyte TGF-beta mRNA content. Patients who had thrombotic events displayed higher monocyte CD25 levels (6.2%) than those without symptoms (1.3%) and controls (2.6%), p=0.0006. JAK2V617F-positive patients had higher monocyte CD25 expression levels (4.7%), than JAK2V617F-negative (2.6%), p=0.0213. Patients with myeloproliferative neoplasms had similar monocyte CD25 expression than controls, both, in basal conditions and after cell adhesion. IL-1beta release and TGF-beta mRNA levels were normal. In conclusion, increased monocyte CD25 expression is associated with history of thrombosis and is also up-regulated in patients harboring JAK2V617F mutation. The finding of increased CD25 levels together with normal IL-1beta and TGF-beta production reveals a selective monocyte activation profile in myeloproliferative neoplasms.

Median Effect Dose and Combination Index Analysis of Cytotoxic Drugs Using Flow Cytometry

Targeted therapy is a strategy of anticancer treatment that aims to interfere with processes of tumorigenesis, cancer progression and metastasis by selectively affecting key molecules of tumor cells (Armand et al., 2007; Favoni & Florio, 2011; Gross-Goupil & Escudier, 2010). Targeted therapies are directed to small molecules participating in different mechanisms that control cell survival through cellular proteins or signalling pathways (Mueller et al., 2009; Zahorowska et al., 2009). Targeted therapies may offer enhanced efficacy, enhanced selectivity, and less toxicity. However, targeting selective molecules and pathways often induces the activation of redundant mechanisms and enhances the emergence of resistant cells due to selective pressure (Woodcock et al., 2011). This is one of the reasons why the effects of targeted agents are not durable when used alone, and often result in drug resistance and clinical relapse.

Prognostic Implications of Cytogenetic Features in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) are a heterogeneous group of hematologic diseases characterized by refractory cytopenia(s) and variable risk of leukemic progression. Cytogenetic analysis is important in day-to-day clinical practice helping to define subgroups of MDS patients who share similarities in the course of the disease. There are recurring aberrations affecting chromosomes 5, 7, 8, and 20. While all of them do not suggest a therapeutic approach, their presence has been considered as a risk indicator since the original international prognostic scoring system (IPSS) was published. The most recent cytogenetic stratifications tried to find the prognostic significance of less frequent alterations which have been longer included in the intermediate group. Moreover, monitoring of karyotype changes is suggested to evaluate cytogenetic response to treatments and the acquisition of new aberrations associated to an unfavorable outcome. This review focuses on different cytogenetic risk stratifications that have been published during the past 20 years and the molecular background of the most relevant chromosomal findings.

Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses

Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitts lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5–15 μM low‐dose range (LDR) of CCCP. Within the 20–50 μM high‐dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3‐(2,4‐dichloro‐5‐methoxyphenyl)‐2,3‐dihydro‐2‐thioxo‐4(1H)‐quinazolinone (Mdivi‐1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage.