Rinaldi, Ciro Roberto (2010) PREFERENTIAL NUCLEAR ACCUMULATION OF JAK2V617F IN CD34+ BUT NOT IN GRANULOCYTIC, MEGAKARYOCYTIC OR ERYTHROID CELLS OF PATIENTS WITH PHILADELPHIA-NEGATIVE MYELOPROLIFERATIVE NEOPLASIA. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||V617FJAK2, NUCLEAR JAK2, MPN|
|Date Deposited:||03 Dec 2010 10:42|
|Last Modified:||30 Apr 2014 19:43|
Recently, Dawson et al. identified a previously unrecognized nuclear role for JAK2 in the phosphorylation of the tyrosine 41 of the histone H3 with the exclusion of HP1a from chromatin and resulting in a disregulation of several JAK2-regulated genes such as LMO2 in haematopoietic cell lines and in one case on peripheral CD34+ cells from a JAK2V617F mutated primary myelofibrosis (PMF) patient. Activation of JAK2 by chromosomal translocations or point mutations is a frequent event in haematological malignancies particularly in Philadelphia negative myeloproliferative disorders (MPNs). To investigate and confirm a possible nuclear localization of JAK2 in presence of V617F mutation, we stably transfected K562 with pMSCV-Puro-JAK2V617F construct and compare with K562 expressing pMSCV-Puro-wild type-(WT)-JAK2 and performed immunofluorescence and western blot analysis. To confirm the in vitro results we searched the possible nuclear localization of JAK2 in total BM of 10 patients affected by all types of JAK2V617F positive MPNs [PMF n=3, polycitemia vera (PV) n=3, essential thrombocythemia (ET) n=4] and 5 patients with WT MPNs (PMF n=2 , ET n=3). To define which cells show nuclear JAK2, we selected by fluorescence activated cell sorting (FACS) 4 cell populations: CD34+, CD15+, CD41+ and CD71+ cells from total BM of 3 JAK2-mutated-MPNs (1 ET, 1 PV, 1 early PMF). Confocal immunofluorescent images on nuclear and cytoplasmic fractions confirmed nuclear JAK2 in K562 although with the strongest nuclear signal in JAK2V617F expressing cells. This latter was also seen by western blot analysis which showed nuclear and cytoplasmic JAK2 only in JAK2V617F expressing K562 comparing with untransfected and WT cells. No differences in JAK2 nuclear signal was observed by the addiction of the nuclear export inhibitor leptomycin B suggesting that export is not involved in nuclear JAK2 shuttling. We found a strong nuclear signal within the nuclei of 3-5% of mononucleated cells in 10 of 10 JAK2 mutated patients but not in un-mutated cases. We found nuclear JAK2 in CD34+ cells but not in other cell populations of the 3 studied patients. Western blot performed on nuclear and cytoplasmic fractions of the JAK2V617FCD34+ cells confirmed the result. No nuclear JAK2 was detected in differentiated erythroid, granulocytic or megakaryocytic colonies obtained from all the studied patients (n=15). We also described how JAK2V617F up-regulates LMO2 in K562 and in JAK2V617F-positive CD34+ cells and that the selective JAK2 inhibitor AG490 normalizes the LMO2 levels in V617F positive K562 and restores the cytoplasmic localization of JAK2. Our data corroborate the recent findings, obtained in hematopoietic cell lines, of a role of JAK2 in direct nuclear signaling. Furthermore we report, for the first time, a nuclear JAK2 in total BM and in sorted CD34+ cells of patients affected by all subtypes of JAK2 mutated MPNs and not in patients with WT diseases. We described also the absence of nuclear JAK2 in sorted mature cells and in differentiated colonies derived from the same patients. Possible chromatin modification due to JAK2 nuclear localization has to be better assessed in patients, where further studies are needed to understand the effects of mutated JAK2 in the nuclei, its target proteins and consequences on gene expression. This intriguing insight reveals a new scenario in the pathogenesis of malignant haematopoiesis and in myeloproliferative phenotype.
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