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Epigenetic Down-Regulation of Death-associated Protein Kinase in Lung Cancers¹

Hamon Center for Therapeutic Oncology Research [S. T., K. O. T., K. M., J. L. R., U. G. S., A. P., N. S., A. F. G.] and Department of Pathology [U. G. S., N. S., A. F. G.], University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593; First Department of Internal Medicine, Sapporo Medical University, Sapporo 060-8543, Japan [M. T.]; Molecular Screening Laboratory, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612-9497 [M. S. T.]; and British Columbia Cancer Agency, Vancouver, V5Z 355 Canada [S. L.]

Purpose: Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine kinase involved in apoptosis. Aberrant methylation of DAPK was reported in lung cancers by methylation-specific PCR. However, we were unable to relate methylation with gene silencing with the same methodology. Our goals were to develop a methodology that related methylation with gene silencing and use it to study the state of the gene in lung cancers.

Experimental Design and Results: Using a semiquantitative real-time reverse transcription-PCR, DAPK expression was lower in lung cancers than in corresponding nonmalignant bronchial epithelial cells in five of six primary short-term cultures. In continuous cell lines, mRNA expression was down-regulated, as well as compared with nonmalignant bronchial epithelial cells, and its protein was not detected by Western blotting in 17 of 23 (74%) cell lines. We investigated methylation status of 5' flanking region of DAPK by combined bisulfite restriction analysis and bisulfited DNA sequencing. Aberrant methylation was detected in 21 of 48 (44%) cell lines, 2 of 6 primary cultured tumors, and 14 of 38 (37%) primary lung cancers, although varying degrees of methylation were noticed. Furthermore, bisufite sequence data suggested that aberrant methylation might occur selectively at some CpG dinucleotides in cell lines which had absent expression. Treatment with 5-aza-2'-deoxycytidine restored DAPK expression in heavily methylated cell lines tested, and histone deacetylase inhibitor trichostatin A alone restored DAPK expression in some methylated cell lines as well.

Conclusions: Our major findings are: (a) DAPK expression is frequently down-regulated in lung cancers; (b) aberrant methylation of DAPK is frequent in lung cancers, although considerable heterogeneity of methylation is present, and some specific CpG dinucleotides are often methylated in expression negative lung cancers; and (c) besides methylation and histone deacetylation, there may be other mechanisms for down-regulation of DAPK expression.

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