Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.
Resistance of human renal cell carcinoma (RCC) and melanoma to the apoptosis-inducing effects of IFNs was postulated to result from epigenetic silencing of genes by DNA methylation, a common feature of human cancers. To reverse silencing, 5-AZA-deoxycytidine (5-AZA-dC) or selective depletion of DNA methyltransferase 1 (DNMT1) by phosphorothioate oligonucleotide antisense (DNMT1 AS) were employed in cells resistant (<5% terminal deoxynucleotidyl transferase-mediated nick-end labeling positive) to apoptosis induction by IFN-alpha2 and IFN-beta (ACHN, SK-RC-45, and A375). 5-AZA-dC and DNMT1 AS similarly depleted available DNMT1 protein and, at doses that did not cause apoptosis alone, resulted in apoptotic response to IFNs. The proapoptotic tumor suppressor RASSF1A was reactivated by DNMT1 inhibitors in all three cell lines. This was associated with demethylation of its promoter region. IFNs augmented RASSF1A protein expression after reactivation by DNMT1 inhibition. In IFN-sensitive WM9 melanoma cells, expression of RASSF1A was constitutive but also augmented by IFNs. RASSF1A small interfering RNA reduced IFN-induced apoptosis in WM9 cells and in DNMT1-depleted ACHN cells. Conversely, lentiviral expression of RASSF1A but not transduction with empty virus enabled IFN-induced apoptosis. IFN induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL-neutralizing antibody inhibited apoptotic response to IFN in RASSF1A-expressing ACHN cells. Accordingly, RASSF1A markedly sensitized to recombinant TRAIL. Normal kidney epithelial cells, although expressing RASSF1A, did not undergo apoptosis in response to IFN or TRAIL but had >400-fold higher TRAIL decoy receptor 1 expression than transduced ACHN cells (real-time reverse transcription-PCR). Results identified RASSF1A as regulated by IFNs and participating in IFN-induced apoptosis at least in part by sensitization to TRAIL.
Reu
Frederic J
FJ
The Cleveland Clinic Foundation Taussig Cancer Center, Cleveland, Ohio 44195, USA.
Leaman
Douglas W
DW
Maitra
Ratan R
RR
Bae
Soo In
SI
Cherkassky
Leonid
L
Fox
Mark W
MW
Rempinski
Donald R
DR
Beaulieu
Normand
N
MacLeod
A Robert
AR
Borden
Ernest C
EC
D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000172:deficiency; D019008:Drug Resistance, Neoplasm; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D016899:Interferon-beta / Q000494:pharmacology*; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; C411762:RASSF1 protein, human; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D025521:Tumor Suppressor Proteins; D016899:Interferon-beta; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human