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Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL

Oncogene volume 27, pages 490–498 (2008)Cite this article

Abstract

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-α2b or IFN-β). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2′-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a >3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-α2b and IFN-β; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5′CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-β or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-β and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-α2b, IFN-β and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.

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Acknowledgements

We thank Alex Rodriguez for technical assistance of FACS analyses, MethylGene for providing the DNMT1 antibody, Thomas S Griffith, University of Iowa for pCMV-FLAG-DR4 plasmid and Dustin Thomas for critical reading of the manuscript. This work was supported by NIH R01 CA 90914 and GCRC M01 RR-018390.

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  1. S I Bae and V Cheriyath: These authors contributed equally to the work.

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  1. Center for Hematology and Oncology Molecular Therapeutics, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH, USA

    S I Bae, V Cheriyath, B S Jacobs, F J Reu & E C Borden

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  1. S I Bae
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Correspondence to E C Borden.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Bae, S., Cheriyath, V., Jacobs, B. et al. Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL. Oncogene 27, 490–498 (2008). https://doi.org/10.1038/sj.onc.1210655

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