KG-1 and KG-1a model the p15 CpG island methylation observed in acute myeloid leukemia patients.
p15 and p16 are tumor suppressor genes that have 5' CpG islands and both are subject to hypermethylation associated with their transcriptional inactivation in hematological malignancies. In this study, we used sodium bisulfite sequencing to obtain a complete map of the 5-methylcytosine status of 80 CpGs covering approximately 900 bp in the 5' p15 CpG island, and 53 CpGs covering approximately 700 bp in the 5' p16 CpG island in the hematopoietic cell lines HL60, KG-1, and KG-1a, two normal human bone marrow samples (NBM), and eight cytosine arabinoside (ara-C)-resistant adult acute myeloid leukemia (AML) patients. We found methylation of the p15 CpG island in 75% of the AML cases studied spread throughout the 5' region analyzed but only minimal methylation of p15 in NBM. Further, the p16 CpG island was not aberrantly methylated in NBM or the eight AML patients studied. Two distinct modes of p15 methylation in AML were identified, variegated and complete. Interestingly, KG-1 and KG-1a model the methylation of p15 observed in AML, where KG-1 methylation is variegated and KG-1a methylation is complete. Both KG-1 and KG-1a had no detectable p15 mRNA or protein. These results demonstrate that rather than continuous increases in p15 methylation, surprisingly two punctuated modes of aberrant p15 methylation, variegated and complete, were observed in vitro and in vivo. Thus aberrant methylation of tumor suppressor genes is not a binary switch but in the case of p15 occurs in two independent and stable states.
Dodge
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JE
Department of Pharmacology and Toxicology, University of Arizona, Arizona Cancer Center, Tucson, AZ 85724-5024, USA. dodge@cvrc.mgh.harvard.edu
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D001853:Bone Marrow; D018797:Cell Cycle Proteins; D018899:CpG Islands; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D018922:HL-60 Cells; D006657:Histones / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D015470:Leukemia, Myeloid, Acute / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013447:Sulfites; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D006657:Histones; D011994:Recombinant Proteins; D013447:Sulfites; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D013755:Tetradecanoylphorbol Acetate; C009279:sodium bisulfite