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Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases
  1. Raquel Tognon1,
  2. Elainy Patrícia Lino Gasparotto1,
  3. Janine Marie Gisele Leroy2,
  4. Gislane Lelis Vilela Oliveira1,
  5. Renata Peres Neves1,
  6. Rita de Cássia Viu Carrara3,
  7. Simone Kashima3,
  8. Dimas Tadeu Covas3,
  9. Mary Santana4,
  10. Elizabeth Xisto Souto4,
  11. Maria Aparecida Zanichelli4,7,
  12. Carlos Eduardo Engel Velano5,
  13. Belinda Pinto Simões5,
  14. Fernando Lopes Alberto6,
  15. Kozue Miyashiro6,
  16. Ana Maria de Souza1,
  17. Gustavo Pessini Amarante-Mendes2,
  18. Fabíola Attié de Castro1
  1. 1Department of Clinical, Toxicological and Bromatological Analysis, University of São Paulo, Ribeirão Preto School of Pharmaceutical Sciences, Ribeirão Preto, Brazil. INCT-IF
  2. 2Department of Immunology, University of São Paulo, Institute of Biomedical Sciences, São Paulo, Brazil
  3. 3Ribeirão Preto Center for Transfusion Medicine, Hospital das Clínicas, University of São Paulo, Ribeirão Preto School of Medicine, Ribeirão Preto, Brazil
  4. 4Brigadeiro Hospital of São Paulo, São Paulo, Brazil
  5. 5Department of Clinical Medicine, University of São Paulo at Ribeirão Preto School of Medicine, Ribeirão Preto, Brazil
  6. 6Fleury Medicine and Health, São Paulo, Brazil
  7. 7Institute for cancer treatment in children-ITACI
  1. Correspondence to Professor Fabiola Attié de Castro, Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP, Laboratório de Hematologia, Av do Café, s/n Ribeirão Preto, SP, Brazil CEP: 14040-903; castrofa{at}fcfrp.usp.br

Abstract

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.

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

Subjects and 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.

  • Apoptosis
  • myelofibrosis
  • myeloproliferative disease
  • polycythaemia
  • thrombocythaemia

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Footnotes

  • Funding RT is the recipient of a fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (No 08/54387-5), EPLG and RPN are recipients of fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and GLVO is the recipient of a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This work was supported by a FAPESP grant (No 06/50094-8).

  • Ethics approval This study was conducted with the approval of the Brigadeiro Hospital and Hospital das Clínicas of the University of São Paulo at Ribeirão Preto School of Medicine ethics committees (No 014/06 and No 10529/2006, respectively).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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