Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases.
BACKGROUND
Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology.


OBJECTIVE
To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden.


METHODS
Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-ΔΔCt). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers.


RESULTS
In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers.


CONCLUSIONS
The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.
Department of Clinical, Toxicological and Bromatological Analysis, University of São Paulo, Ribeirão Preto School of Pharmaceutical Sciences, Ribeirão Preto, Brazil.
Raquel
R
Tognon
Elainy Patrícia Lino
EP
Gasparotto
Janine Marie Gisele
JM
Leroy
Gislane Lelis Vilela
GL
Oliveira
Renata Peres
RP
Neves
Rita de Cássia Viu
Rde C
Carrara
Simone
S
Kashima
Dimas Tadeu
DT
Covas
Mary
M
Santana
Elizabeth Xisto
EX
Souto
Maria Aparecida
MA
Zanichelli
Carlos Eduardo Engel
CE
Velano
Belinda Pinto
BP
Simões
Fernando Lopes
FL
Alberto
Kozue
K
Miyashiro
Ana Maria
AM
de Souza
Gustavo Pessini
GP
Amarante-Mendes
Fabíola Attié
FA
de Castro
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018952:Antigens, CD34 / Q000032:analysis; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D007962:Leukocytes / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D053218:Receptors, Death Domain / Q000096:biosynthesis* / Q000235:genetics; D055815:Young Adult
D018952:Antigens, CD34; D012333:RNA, Messenger; D053218:Receptors, Death Domain; C507924:JAK2 protein, human; D053614:Janus Kinase 2