Promoter Hypermethylation and Quantitative mRNA Expression of the Retinoic Acid Receptor β2 Gene in Patients with Acute Myeloid Leukemia.

Abstract

Introduction: Treatment with Retinoic Acid (RA) has proved to be successful in acute myeloid leukemia (AML) patients harboring the t(15;17) aberration (Dubois et al. Blood 83;3264,1994). Promoter hypermethylation of the RA receptor Retinoic Acid Receptor β2 (RARβ2), a putative tumor-suppressor-gene, has been associated with gene silencing in several cancer types. Moreover, lack of RA response has been correlated to RARβ2 promoter hypermethylation (Sirchia et al. Cancer Research62;2455, 2002). Other studies have shown promoter hypermethylation of the this gene in t(15;17) patients and induction of demethylation, re-expression of the gene, and cell differentiation following treatment with RA (Croce et al. Science295;1079, 2002). Based on these findings we found it of interest to analyze the promoter methylation and the transcriptional status of the RARβ2 gene in a cohort of AML patients. Materials and Methods: Mononuclear cells from either peripheral blood (PB) and/or bone marrow (BM) from 229 AML patients were cryopreserved in DMSO and fetal calf serum. DNA and/or RNA were purified on a MagNa-Pure LC robot (Roche Diagnostic). Bisulfite treatment and Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE) for promoter hypermethylation analysis were performed as previously described (Aggerholm et al. Cancer Research59;436, 1999). Transcription levels were determined by real-time quantitative RT-PCR (RQ-PCR), which was performed in a 7700 Sequence Detector System (Applied Biosystems). Results: We first determined that promoter methylation of the gene was absent in BM from healthy individuals. In contrast, bisulfite-DGGE analysis showed that 38% (87/229) of AML patients were intra- and interindividually heterogeneously methylated. While methylation was found to be present in all AML subtypes, (Table), patients harboring the inv(16) fusion transcript were significantly more often hypermethylated when compared to the other AML subgroups (χ2, p = 0.0003). Somewhat puzzling, RQ-PCR analysis of BM cells from 77 AML patients showed an increase in the transcription level of RARβ2 in 42% of the patients when these were compared to the transcription levels in BM from healthy individuals. Still, the group of inv(16)+ patients differed significantly from the other subgroups by being more often transcriptional inactive (χ2, p = 0.00004). Comparisons of the RARβ2 transcription levels in the methylated and unmethylated groups of patients revealed a repressive effect of methylation in spite of the general transcriptional up-regulation of the gene (student’s T-test, p = 0.006). Conclusion: Promoter hypermethylation of RARβ2 in AML patients is present in all subtypes. Moreover, a correlation between methylation and transcriptional expression could be demonstrated even though the overall transcription level of the gene was up-regulated. Thus, it seems that successful RA treatment in t(15,17)+ patients cannot be correlated to the promoter hypermethylation status of the RARβ2 gene. On the other hand, the demonstration of a higher frequency of hypermethylation and gene silencing in inv(16)+ patients could indicate that the fusion protein created from this lesion might be involved in the mechanisms behind such epigenetically changes. Distribution of methylated and unmethylated patients with different fusion transcripts Fusions Transcript Methylated Unmethylated t(8;21) 4 9 t(15;17) 3 4 inv (16) 14 4 dupMLL 5 7 Others 1 4 No fusion transcript 60 114 Total 87 142 Table 1