DNA methylation analysis of the HIF-1α prolyl hydroxylase domain genes PHD1, PHD2, PHD3 and the factor inhibiting HIF gene FIH in invasive breast carcinomas.
OBJECTIVE
Hypoxia-inducible factor-1 (HIF-1) activity is regulated by prolyl hydroxylase (PHD1, PHD2, PHD3) and factor inhibiting HIF-1 (FIH) that target the α subunit of HIF-1 (HIF-1α) for proteosomal degradation. We hypothesised that the elevated HIF-1α level is due in some tumours to epigenetic silencing by DNA hypermethylation of the promoter region of one or more of the PHDs and FIH genes. The aims were to define the presence or absence of promoter methylation of PHDs and FIH in cell lines of various sources and breast carcinomas and, if present, determine its effect on mRNA and protein expression.


RESULTS
Tumour cell lines (n = 20) and primary invasive breast carcinomas (n = 168) were examined for promoter region DNA methylation using methylation-sensitive high-resolution melting. There was evidence of PHD3 but not of PHD1, PHD2 or FIH DNA methylation in breast cancer (SkBr3) and leukaemic (HL60 and CCRF-CEM) cell lines, but there was no evidence of methylation in any of 168 breast cancers. Only the high-level PHD3 methylation seen in leukaemic cell lines correlated with absent mRNA and protein expression.


CONCLUSIONS
Methylation-induced epigenetic silencing of PHD1, PHD2, PHD3 and FIH is unlikely to underlie up-regulated HIF-1α expression in human breast cancer but may play a role in other tumour types.
Huang
Katie T
KT
Department of Pathology, Molecular Pathology Research and Development Laboratory, Peter MacCallum Cancer Centre, Melbourne, Vic., Australia.
Mikeska
Thomas
T
Dobrovic
Alexander
A
Fox
Stephen B
SB
D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics*; D002277:Carcinoma / Q000201:enzymology / Q000235:genetics*; D019175:DNA Methylation; D049308:Dioxygenases / Q000235:genetics*; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D006899:Mixed Function Oxygenases; D009687:Nuclear Proteins / Q000235:genetics*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C439790:HIF1AN protein, human; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C447780:EGLN2 protein, human; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases