Epigenetic down-regulation of death-associated protein kinase in lung cancers

Shinichi Toyooka, Kiyomi O. Toyooka, Kuniharu Miyajima, Jyotsna L. Reddy, Minoru Toyota, Ubradka G. Sathyanarayana, Asha Padar, Melvyn S. Tockman, Stephen Lam, Narayan Shivapurkar, Adi F. Gazdar

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)3034-3041
Number of pages8
JournalClinical Cancer Research
Volume9
Issue number8
Publication statusPublished - Aug 1 2003
Externally publishedYes

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Death-Associated Protein Kinases
Epigenomics
Methylation
Lung Neoplasms
Down-Regulation
Cell Line
decitabine
Gene Silencing
trichostatin A
Epithelial Cells
Polymerase Chain Reaction
Histone Deacetylase Inhibitors
5' Flanking Region
Protein-Serine-Threonine Kinases
DNA Sequence Analysis
Histones
Reverse Transcription

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Toyooka, S., Toyooka, K. O., Miyajima, K., Reddy, J. L., Toyota, M., Sathyanarayana, U. G., ... Gazdar, A. F. (2003). Epigenetic down-regulation of death-associated protein kinase in lung cancers. Clinical Cancer Research, 9(8), 3034-3041.

Epigenetic down-regulation of death-associated protein kinase in lung cancers. / Toyooka, Shinichi; Toyooka, Kiyomi O.; Miyajima, Kuniharu; Reddy, Jyotsna L.; Toyota, Minoru; Sathyanarayana, Ubradka G.; Padar, Asha; Tockman, Melvyn S.; Lam, Stephen; Shivapurkar, Narayan; Gazdar, Adi F.

In: Clinical Cancer Research, Vol. 9, No. 8, 01.08.2003, p. 3034-3041.

Research output: Contribution to journalArticle

Toyooka, S, Toyooka, KO, Miyajima, K, Reddy, JL, Toyota, M, Sathyanarayana, UG, Padar, A, Tockman, MS, Lam, S, Shivapurkar, N & Gazdar, AF 2003, 'Epigenetic down-regulation of death-associated protein kinase in lung cancers', Clinical Cancer Research, vol. 9, no. 8, pp. 3034-3041.
Toyooka S, Toyooka KO, Miyajima K, Reddy JL, Toyota M, Sathyanarayana UG et al. Epigenetic down-regulation of death-associated protein kinase in lung cancers. Clinical Cancer Research. 2003 Aug 1;9(8):3034-3041.
Toyooka, Shinichi ; Toyooka, Kiyomi O. ; Miyajima, Kuniharu ; Reddy, Jyotsna L. ; Toyota, Minoru ; Sathyanarayana, Ubradka G. ; Padar, Asha ; Tockman, Melvyn S. ; Lam, Stephen ; Shivapurkar, Narayan ; Gazdar, Adi F. / Epigenetic down-regulation of death-associated protein kinase in lung cancers. In: Clinical Cancer Research. 2003 ; Vol. 9, No. 8. pp. 3034-3041.
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abstract = "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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AU - Toyooka, Kiyomi O.

AU - Miyajima, Kuniharu

AU - Reddy, Jyotsna L.

AU - Toyota, Minoru

AU - Sathyanarayana, Ubradka G.

AU - Padar, Asha

AU - Tockman, Melvyn S.

AU - Lam, Stephen

AU - Shivapurkar, Narayan

AU - Gazdar, Adi F.

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N2 - 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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