Hypermethylation of CXCR4 promoter in CD34+ cells from patients with primary myelofibrosis.
Constitutive mobilization of CD34(+) cells in patients with primary myelofibrosis (PMF) has been attributed to proteolytic disruption of the CXCR4/SDF-1 axis and reduced CXCR4 expression. We document here that the number of circulating CD34(+)/CXCR4(+) cells in PMF patients, as well as the cellular CXCR4 expression, was directly related to CXCR4 mRNA level and that reduced CXCR4 mRNA level was not due to SDF-1-induced downregulation. To address whether epigenetic regulation contributes to defective CXCR4 expression, we studied the methylation status of the CXCR4 promoter using methylation-specific polymerase chain reaction and methylation-specific sequencing in the JAK2V617F-positive HEL cell line and in CD34(+) cells. We found that CD34(+) cells from PMF patients, unlike those from normal subjects, presented hypermethylation of CXCR4 promoter CpG island 1. Following incubation with the demethylating agent 5-Aza-2'-deoxycytidine (5-AzaD), the percentage of PMF CD34(+) cells expressing CXCR4 increased 3-10 times, whereas CXCR4 mRNA level increased approximately 4 times. 5-AzaD-treated PMF CD34(+) cells displayed almost complete reversal of CpG1 island 1 hypermethylation and showed enhanced migration in vitro in response to SDF-1. These data point to abnormal methylation of the CXCR4 promoter as a mechanism contributing to constitutive migration of CD34(+) cells in PMF. Disclosure of potential conflicts of interest is found at the end of this article.
Department of Hematology, University of Florence, 50134 Florence, Italy.
Costanza
C
Bogani
Vanessa
V
Ponziani
Paola
P
Guglielmelli
Cristophe
C
Desterke
Vittorio
V
Rosti
Alberto
A
Bosi
Marie-Caroline
MC
Le Bousse-Kerdilès
Giovanni
G
Barosi
Alessandro M
AM
Vannucchi
D018952:Antigens, CD34 / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D005434:Flow Cytometry / Q000379:methods; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D009154:Mutation; D016133:Polymerase Chain Reaction; D055728:Primary Myelofibrosis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000235:genetics* / Q000378:metabolism
D018952:Antigens, CD34; C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D012333:RNA, Messenger; D019718:Receptors, CXCR4