Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes

Abstract

We previously reported that the hypermethylation of the p15 INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15 INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15 INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15 INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15 INK4B -methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15 INK4B methylation were detected in both patients.

Methylation of p15INK4B is common, is associated with deletion of genes on chromosome arm 7q and predicts a poor prognosis in therapy-related myelodysplasia and acute myeloid leukemia

“…Second, the pronounced p15 methylation observed in the myeloid disorders has been located strictly to the malignant clone of cells, unrelated to their different stage of differentiation. 35 Third, demethylating therapy of patients with MDS with decitabine has been shown to reduce p15 methylation and restore expression of the p15 protein in clinically responding patients. 36 Fourth, p15 methylation seems to be a specific event, since the p14 and p16 genes, located within the same 25 kb fragment of chromosome band 9p21 as the p15 gene, were rarely or never methylated in t-MDS and t-AML.…”

Expression levels of DNA methyltransferase genes do not correlate with p15INK4B gene methylation in myelodysplastic syndromes

“…1 We also reported that the p15 INK4B gene is methylated not only in the blast population, but also in differentiated cells among MDS patients, suggesting that p15 INK4B methylation is an early event in MDS. 2,3 However, the molecular mechanism(s) responsible for aberrant methylation of the p15 INK4B gene in MDS remains obscure.…”

“…We extracted DNA and total RNA from cell lines and BM-MNCs using standard procedures and methylation-specific PCR (MSP) detected p15 INK4B gene methylation as described. 2 Real-time quantitative RT-PCR evaluated expression levels of DNMT1, 3a, 3b mRNA. We prepared first-strand cDNA from 5 mg of total cellular RNA using a random hexadeoxynucleotide primer and M-MLV reverse transcriptase To standardize the integrity of RNA, the efficiency of the RT-PCR and the amount of applied RNA, we amplified glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA using the GAPDH control Reagent kit (PE Applied Biosystems, Foster City, CA, USA) as described by the manufacturer.…”

Gene silencing of the p15/INK4B cell-cycle inhibitor by hypermethylation: an early or later epigenetic alteration in myelodysplastic syndromes?

“…This is supported by work analyzing granulocyte and monocyte peripheral blood fractions (Aoki et al 1 ; Daskalakis M, Jones PA, Lü bbert M, unpublished data), and results derived from erythroid and nonerythroid colonies from MDS bone marrow generated in soft agar. 18 Evidence for p15 hypermethylation as an early event particularly in secondary MDS stems from a study examining mononuclear bone marrow cells from 10 patients having received curative treatment for malignant lymphoma. In each patient, increased p15 methylation was detected despite normal or only mildly abnormal marrow morphology, corroborating its high incidence in secondary MDS in the absence of blast expansion.…”