Specific inhibition of DNMT1 by antisense oligonucleotides induces re-expression of estrogen receptor-alpha (ER) in ER-negative human breast cancer cell lines.
Recent studies have shown that changes in epigenetic regulation, such as DNA methylation and histone acetylation, are associated with silencing of the estrogen receptor a (ER) gene in ER-negative human breast cancer cells. Treatment of these cells with the general DNMT inhibitor, 5-aza-2'deoxycytidine, led to reactivation of functional ER protein. This study addresses the hypothesis that specific inhibition of the maintenance DNA methyltransferase, DNMT1, by antisense oligonucleotides (DNMT1 ASO) is sufficient to re-express the ER gene in ER-negative human breast cancer cell lines. MDA-MB-231 and Hs578t cells were transfected with 100 nM and 150 nM DNMT1 ASO respectively for three consecutive days and evidence of DNMT1 downregulation and functional ER re-expression was sought. Significant growth reduction was observed within 48 hr and persisted after 96 hr. DNMT1 expression was blocked after exposure to DNMT1 ASO as detected by RT-PCR, Western blot and enzymatic assay whereas a mutant DNMT1 ASO had little effect. This was associated with enhanced ER mRNA and protein expression and restoration of estrogen responsiveness in MDA-MB-231 cells as demonstrated by the ability of the induced ER protein to elicit ERE-regulated reporter activity from a luciferase reporter construct. Methylation specific PCR showed that the ER CpG island was minimally demethylated, suggesting that other epigenetic events, introduced by specific DNMT1 inhibition, might also be involved in ER re-expression. Our results suggest that specific inhibition of DNMT1 expression alone is sufficient to re-express ERa in human breast cancer cell lines.
Yan
Lan
L
Sidney Kimmel Comprehensive Cancer at Johns Hopkins University Medical School, Baltimore, Maryland USA.
Nass
Sharyl J
SJ
Smith
Dawn
D
Nelson
William G
WG
Herman
James G
JG
Davidson
Nancy E
NE
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D047628:Estrogen Receptor alpha; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D047628:Estrogen Receptor alpha; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D000077209:Decitabine; D008156:Luciferases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D001374:Azacitidine