Altered expression of retinoblastoma (RB) protein in acute myelogenous leukemia does not result from methylation of the Rb promotor

Prior studies demonstrated that expression of the retinoblastoma (RB) protein in acute myelogenous leukemia (AML) is heterogeneous with low expression conferring a poor prognosis, The molecular change(s) responsible for low RE expression in AML are unknown. Since methylation of the RE promoter has been shown to result in decreased expression we hypothesized that this might explain some cases of low RE expression in AML. To investigate this hypothesis Southern blotting and FCR sequencing after bisulfite conversion were used to study the methylation status of the RE gene promoter. DNA and protein lysates were prepared from the mononuclear cell fraction from peripheral blood or bone marrow samples from 46 patients with newly diagnosed AML. By Western blot 16, 22 and 8 patients had low, elevated and hyperphosphorylated patterns of RE expression respectively using previously defined criteria. The SacI endonuclease cuts a 5.7-kb or 6.8-kb fragment, depending on polymorphism, containing the RE promoter, detected by the probe p123M1.8 that covers the RE promoter region and exon I. The methylation sensitive endonuclease SacII cuts twice within a key hairpin loop structure in the RE promoter that contains binding sites for AP1, Spl and RBF1, Others have demonstrated that methylation within this hairpin loop can decrease RE mRNA transcription by up to 92%. Comparison of the SacI and SacI + SacII digestion fragments showed no evidence of methylation in the promoter region of RE in any of the patients studied. DNA from the promoter region of 11 patients with no/low RE expression was subjected to bisulfite conversion and PCR sequencing. No evidence of methylation was seen by this method either. These results suggests that hypermethylation of the RE promoter region is at best an infrequent event in AML and that RE promoter hypermethylation is not the predominant cause of the low levels of RE expression observed in 20% of AML patients.