The promoters of the p16INK4aand p15INK4b genes located at chromosome band 9p21 are frequently silenced by CpG island methylation in hematologic malignancies and solid tumors.1 In chronic myelogenous leukemia (CML) the situation is confused because 2 reports have produced contradictory results on p15INK4bmethylation, possibly because of the sensitivity of the techniques used for these studies. By use of restriction endonuclease–based Southern blot, methylation of the 5′ region of p15INK4bwas not detected on 21 samples in contrast to other leukemia types.2 In another study with the more sensitive methylation-specific polymerase chain reaction (MSP) technique, methylation of p15INK4b was detected in 24% (8/34) of CML cases and was associated with progression.3
In the present report, we used MSP4 to study p15INK4b- andp16INK4a-promoter hypermethylation status in 76 cases of CML at various phases of the disease: chronic (50), accelerated (11), myeloblastic transformation (5), and complete cytogenetic remission (10) upon interferon-base regimen5 or imatinib treatment.6 In addition, we tested p14ARF promoter methylation, as this study has not been described yet.
Partial p15INK4b hypermethylation was found for 4 patients (5%), 2 in chronic phase and 2 in blast crisis (patients 11, 12, 15, and 19 in Figure 1), consistent with previous data on a smaller series.2 In all cases, the intensity of the unmethylated bands greatly exceeded that of methylated ones. While the technique is only semiquantitative, this suggests that only a minor part of the leukemic cells was implicated in the hypermethylation process. Patient 19 in myeloid transformation showed slight hypermethylation of thep14ARF promoter (Figure 1). For all the other patients, the p14ARF promoter was unmethylated. The p16INK4a promoter was found unmethylated for all patients (not shown), again in agreement with Herman et al's data.2
Methylation status of
p15INK4b andp14ARF analyzed by MSP. DNA was phenol/chloroform extracted from blood. Promoter methylation was determined by the method of MSP.4 The modified DNA was used as a template for PCR amplification using primers specific for either methylated or unmethylated DNA forp14ARF,7,p15INK4b, andp16INK4a.4 The specificity of the PCR reaction was checked by sequencing the amplified fragments. Control without DNA was performed for each set of PCRs. Placental DNA treated or not in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) was used as positive control for the methylated or the unmethylated form, respectively. Examples are given for patients 9 to 19. M indicates methylated form; U, unmethylated form; P, unmethylated placental DNA; and SssI, methylated placental DNA. The DNA standards (methylated or unmethylated) gave the expected results.
Methylation status of
p15INK4b andp14ARF analyzed by MSP. DNA was phenol/chloroform extracted from blood. Promoter methylation was determined by the method of MSP.4 The modified DNA was used as a template for PCR amplification using primers specific for either methylated or unmethylated DNA forp14ARF,7,p15INK4b, andp16INK4a.4 The specificity of the PCR reaction was checked by sequencing the amplified fragments. Control without DNA was performed for each set of PCRs. Placental DNA treated or not in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) was used as positive control for the methylated or the unmethylated form, respectively. Examples are given for patients 9 to 19. M indicates methylated form; U, unmethylated form; P, unmethylated placental DNA; and SssI, methylated placental DNA. The DNA standards (methylated or unmethylated) gave the expected results.
On the basis of these data, negative regulation by hypermethylation of p14ARF,p15INK4b, and p16INK4a,which contributes to the cell cycle, does not appear to be a frequent inactivating event in CML. This is in contrast with other hematopoietic malignancies, particularly acute myelogenous leukemia (AML), in which p15INK4bhypermethylation is frequent.8 Perhaps more interestingly, in myelodysplastic syndromes hypermethylation ofp15INK4b has been reported to increase with the phase of the disease.9
Supported by Association pour la Recherche Contre le Cancer et Ligue Nationale Contre le Cancer.
