Expression of p15ink4b gene during megakaryocytic differentiation of normal and myelodysplastic hematopoietic progenitors

Abstract

In myelodysplastic syndrome (MDS), the expression of the cyclin-dependent kinase inhibitor p15 ink4B (p15) is frequently decreased because of the aberrant methylation of the gene promoter; p15 is normally up-regulated during megakaryocytic differentiation. It was hypothesized that p15 methylation and deregulation of gene expression contribute to defective megakaryocytopoiesis in patients with MDS. Here it is shown that the increasing autocrine production of TGF-␤1 stimulates megakaryocytic differentiation in normal CD34 ؉ cells and that p15 mediates, at least in part, this effect. This TGF-␤1-dependent pathway is altered in MDS CD34 ؉ progenitors because of p15 methylation. The demethylating agent 2-deoxyAZAcytidin can restore the normal demethylated state of the p15 gene and increase its expression. Nevertheless, MDS CD34 ؉ cells only poorly differentiate to the megakaryocytic lineage. These findings suggest that p15 methylation occurs in a neoplastic clone with a profound defect of cell proliferation, survival, and differentiation that cannot be overcome by using a demethylating drug. IntroductionWe have recently shown that expression of the cyclin-dependent kinase inhibitor (CDKI) p15 ink4B (p15) is up-regulated during in vitro granulocytic and megakaryocytic differentiation of normal CD34 ϩ hematopoietic progenitors. 1,2 An aberrant methylation of CpG islands in the p15 promoter region commonly occurs in myelodysplastic syndromes (MDS), such as refractory anemia with excess of blasts (RAEB) or RAEB in transformation (RAEB-t), and is associated with the loss of p15 expression. 3,4 An important function of p15 is to mediate extracellular antimitotic signals, and p15 has been identified as the effector of the G1-arrest induced by transforming growth factor ␤1 (TGF-␤1) in a keratinocyte cell line. 5 TGF-␤1 is a multifunctional hematopoietic regulator. 6 With respect to megakaryocytopoiesis, in vitro studies suggest that TGF-␤1 exerts an inhibitory effect on early and late stages of megakaryocytopoiesis, affecting both megakaryocytic differentiation and endomitosis. [7][8][9] Megakaryocytic dysplasia and thrombocytopenia are frequent findings in patients with RAEB and RAEB-t. We hypothesize that the loss of p15 expression contributes to the impaired megakaryocytic differentiation in these patients.The aims of the present study were to clarify the association between TGF-␤1 and p15 in normal megakaryocytic differentiation and to address the role of p15 methylation in the altered megakaryocytopoiesis of MDS. Study designPatients CD34 ϩ cells were isolated from bone marrow samples of 5 RAEB-free donors undergoing marrow harvest for allogeneic transplantation and from 11 patients affected by RAEB. Diagnosis was established according to the French-American-British classification. 10 The proportion of blast in the bone marrow ranged between 15% and 20%, and all patients had platelet counts less than 60 ϫ 10 9 /L. Cytogenetic analysis was performed in 7 of 11 patients: 6 patients had normal karyotype, and one patient s...

Cyclin D–Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization

“…45 In contrast, less is known about the role of INK4 family members in endomitosis. One study revealed that p15 ink4b expression was up-regulated in CD41 ϩ cells generated ex vivo from human CD34 ϩ cells, 46 while another reported that p16 decreased upon megakaryocytic differentiation. 47 Our observation that GATA-1-deficient megakaryocytes express nearly 10-fold less p16 suggests that GATA-1 may directly repress p16 transcription in the course of megakaryocyte maturation.…”

Hypermethylation of GpG islands in the promoter region of p15INK4b in acute promyelocytic leukemia represses p15INK4b expression and correlates with poor prognosis

“…b-Actin was used in each PCR reaction as internal control according to the method previously described. 7,18 BM mononuclear cells and water were used, respectively, as positive and negative controls. PCR products were separated in 2% agarose gel and transferred onto nylon membranes (Amersham, Arlington Heights, IL, USA), and standard hybridization was performed using a p15-specific DNA probe.…”

Differentiation induction as a treatment for hematologic malignancies

“…However, methylated genes can be activated by the combination of HDACi and 5 Aza-2' deoxycytidine (Cameron et al, 1999). Genes known to be methylated in MDS and myeloid leukemia include the cell cycle inhibitor p15 INK4b , p16 and E-cadherin (Teo®li et al, 2001;Toyota et al, 2001). Hypermethylation of p15 and other key genes may contribute to the di erentiation block seen in MDS.…”