Hypermethylation of the death-associated protein kinase promoter attenuates the sensitivity to TRAIL-induced apoptosis in human non-small cell lung cancer cells.

Death-associated protein (DAP) kinase plays an important role in IFN-gamma, tumor necrosis factor (TNF)-alpha, or Fas-ligand induced apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand family and can induce caspase-dependent apoptosis in cancer cells while sparing most of the normal cells. However, some of the cancer cell lines are insensitive to TRAIL, and such resistance cannot be explained by the dysfunction of TRAIL receptors or their known downstream targets. We reported previously that DAP kinase promoter is frequently methylated in non-small cell lung cancer (NSCLC), and such methylation is associated with a poor clinical outcome. To determine whether DAP kinase promoter methylation contributes to TRAIL resistance in NSCLC cells, we measured DAP kinase promoter methylation and its gene expression status in 11 NSCLC cell lines and correlated the methylation/expression status with the sensitivity of cells to TRAIL. Of the 11 cell lines, 1 had a completely methylated DAP kinase promoter and no detectable DAP kinase expression, 4 exhibited partial promoter methylation and substantially decreased gene expression, and the other 6 cell lines showed no methylation in the promoter and normal DAP kinase expression. Therefore, the amount of DAP kinase expression amount was negatively correlated to its promoter methylation (r = -0.77; P = 0.003). Interestingly, the cell lines without the DAP kinase promoter methylation underwent substantial apoptosis even in the low doses of TRAIL, whereas those with DAP kinase promoter methylation were resistant to the treatment. The resistance to TRAIL was reciprocally correlated to DAP kinase expression in 10 of the 11 cell lines at 10 ng/mL concentration (r = 0.91; P = 0.001). We treated cells resistant to TRAIL with 5-aza-2'-deoxycytidine, a demethylating reagent, and found that these cells expressed DAP kinase and became sensitive to TRAIL. These results suggest that DAP kinase is involved in TRAIL-mediated cell apoptosis and that a demethylating agent may have a role in enhancing TRAIL-mediated apoptosis in some NSCLC cells by reactivation of DAP kinase.

[1]  A. Ashkenazi,et al.  Targeting death and decoy receptors of the tumour-necrosis factor superfamily , 2002, Nature Reviews Cancer.

[2]  A. Kimchi,et al.  Dap-Kinase Participates in TNF-α–And FAS-Induced Apoptosis and Its Function Requires the Death Domain , 1999, The Journal of cell biology.

[3]  H. Matsumoto,et al.  Prognostic significance of death-associated protein-kinase expression in hepatocellular carcinomas. , 2003, Anticancer research.

[4]  R. Stahel,et al.  Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation , 2003, Cell Death and Differentiation.

[5]  Mian Wu,et al.  Induction of apoptosis in glioma cell lines by TRAIL/Apo‐2l , 2000, Journal of neuroscience research.

[6]  P. Laird,et al.  COBRA: a sensitive and quantitative DNA methylation assay. , 1997, Nucleic acids research.

[7]  F. Sinicrope,et al.  Cyclooxygenase-2 overexpression inhibits death receptor 5 expression and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human colon cancer cells. , 2002, Cancer research.

[8]  F. Khuri,et al.  Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small-cell lung cancer. , 2000, Journal of the National Cancer Institute.

[9]  P. Hersey,et al.  Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types , 2000, FEBS letters.

[10]  K. Schooley,et al.  Human Dendritic Cells Mediate Cellular Apoptosis via Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (Trail) , 1999, The Journal of experimental medicine.

[11]  Y. Tomita,et al.  Hypermethylation of death‐associated protein (DAP) kinase CpG island is frequent not only in B‐cell but also in T‐ and natural killer (NK)/T‐cell malignancies , 2003, Cancer science.

[12]  R. K Srivastava,et al.  Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer , 2001, Oncogene.

[13]  A. Kimchi,et al.  DAP‐kinase is a Ca2+/calmodulin‐dependent, cytoskeletal‐associated protein kinase, with cell death‐inducing functions that depend on its catalytic activity , 1997, The EMBO journal.

[14]  A. Grandien,et al.  High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells. , 2003, Anticancer research.

[15]  양우익.  Hypermethylation of death-associated protein (DAP) kinase CpG island is frequent not only in B-cell but also in T- and natural killer (NK)/T-cell malignancies , 2003 .

[16]  F. Behm,et al.  Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN , 2000, Nature Medicine.

[17]  Arul M. Chinnaiyan,et al.  The Receptor for the Cytotoxic Ligand TRAIL , 1997, Science.

[18]  John D Minna,et al.  Differential Inactivation of Caspase-8 in Lung Cancers , 2002, Cancer biology & therapy.

[19]  M. Alonso,et al.  Frequent death-associated protein-kinase promoter hypermethylation in brain metastases of solid tumors. , 2003, Oncology reports.

[20]  R. Gentz,et al.  An antagonist decoy receptor and a death domain-containing receptor for TRAIL. , 1997, Science.

[21]  I. Stamenkovic,et al.  Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand. , 2000, Cancer research.

[22]  J. Herman,et al.  Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. , 2000, Cancer research.

[23]  B. Shiramizu,et al.  Epigenetic changes in the DAP-kinase CpG island in pediatric lymphoma. , 2003, Medical and pediatric oncology.

[24]  S. Polak‐Charcon,et al.  DAP kinase links the control of apoptosis to metastasis , 1997, Nature.

[25]  J. Herman,et al.  Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. , 1999, Blood.

[26]  S. Marsters,et al.  Induction of Apoptosis by Apo-2 Ligand, a New Member of the Tumor Necrosis Factor Cytokine Family* , 1996, The Journal of Biological Chemistry.

[27]  Michael T. Fisher,et al.  Molecular determinants of response to TRAIL in killing of normal and cancer cells. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[28]  A. Kimchi,et al.  Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. , 1995, Genes & development.

[29]  H. Kuwano,et al.  High frequency of DAP-kinase gene promoter methylation in colorectal cancer specimens and its identification in serum. , 2003, Cancer letters.

[30]  J. Peschon,et al.  IFN-gamma mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression. , 1999, Journal of immunology.

[31]  J. Soria,et al.  TRAIL-R2 is not correlated with p53 status and is rarely mutated in non-small cell lung cancer. , 2000, Anticancer research.

[32]  C. Rauch,et al.  Tumoricidal activity of tumor necrosis factor–related apoptosis–inducing ligand in vivo , 1999, Nature Medicine.

[33]  R. Yu,et al.  Tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in androgen-independent prostate cancer cells. , 2000, Cancer research.

[34]  D. Lawrence,et al.  Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. , 2000, Immunity.

[35]  J. Herman,et al.  Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[36]  松原 弘樹,et al.  腎癌に対するTNF-related Apoptosis-inducing Ligand(TRAIL)を用いたアポトーシス誘導による分子標的治療の検討 , 2003 .

[37]  J. Tschopp,et al.  TRAIL receptor-2 signals apoptosis through FADD and caspase-8 , 2000, Nature Cell Biology.

[38]  R H Hruban,et al.  Hypermethylation of multiple genes in pancreatic adenocarcinoma. , 2000, Cancer research.