This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tang, X. Articles by Mao, L. Search for Related Content PubMed PubMed Citation Articles by Tang, X. Articles by Mao, L.

---

Cell Cycle, Cell Death, and Senescence

Hypermethylation of the Death-Associated Protein Kinase Promoter Attenuates the Sensitivity to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Non–Small Cell Lung Cancer Cells ¹

Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Li Mao, Molecular Biology Laboratory, Department of Thoracic/Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Unit 432, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-796-8655. E-mail: lmao{at}mdanderson.org

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Death-associated protein (DAP) kinase plays an important role in IFN-, tumor necrosis factor (TNF)-, 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.

Key Words: DAP kinase • methylation • TRAIL • apoptosis • NSCLC

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Death-associated protein (DAP) kinase is a Ca ²⁺/calmodulin-regulated, 160-kDa serine/threonine, microfilament-bound kinase shown recently to be involved in IFN-, tumor necrosis factor-, or Fas ligand–induced apoptosis (1-3). Aggressiveness of malignant tumors has been associated with methylation of the promoter region of the DAP kinase gene (4-8) and loss of DAP kinase expression (9).

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL, also known as Apo-2L), a member of the tumor necrosis factor ligand family, is a cytokine that can induce a rapid caspase-dependent and tumor-specific apoptosis ( 10-16) through its specific death receptors DR4 and DR5 (17). The activation of DR4 and DR5, like that of Fas/Apo, leads to the activation of the initiator caspase, caspase-8, and its downstream targets (18). TRAIL seems to exert selective toxicity toward neoplastic cells, whereas most normal cells are resistant to TRAIL (19). Multiple factors have been proposed for such resistance, including the presence of the decoy receptors DcR1 and DcR2, the downexpression of DR4 and DR5, the silencing of caspase-8, the inactivity of Akt, and the overexpression of cFLIP and cyclooxygenase-2 ( 20-27).

In this study, we investigated whether DAP kinase plays a role in determining the sensitivity of cells to TRAIL using non-small cell lung cancer (NSCLC) as a model. Our data suggest that the DAP kinase may be involved in TRAIL-induced apoptosis, and restoration of DAP kinase expression may overcome TRAIL resistance in certain NSCLCs.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Establishment of a Multiplex Methylation-Specific PCR
Methylation-specific PCR (MSP; ref. 28) is the most extensively used method for detecting the methylation status of CpG islands in the promoter regions of genes. Combined bisulfite restriction analysis is more quantitative (29) but requires more strict experimental condition. In this study, we combined multiplex PCR and MSP to create multiplex MSP (MMSP). In this method, unmethylated and methylated DNA are amplified simultaneously with two primer sets specific for methylated and unmethylated CpG islands in the DAP kinase promoter. The intensities of PCR products between methylated and unmethylated DNA were used to determine their relative ratios. Our results indicate that MMSP is robust in quantifying methylated DAP kinase promoter (Fig. 1)).

View larger version (38K): [in this window] [in a new window]   FIGURE 1. MMSP consistency for DAP kinase promoter methylation. A. MMSP was done using bisulfite-modified DNA from A549 cells (with unmethylated DAP kinase promoter) and Calu-1 cells (with methylated DAP kinase promoter), with ratios on top of each lane. B. MMSP using bisulfite-modified DNA isolated from NSCLC cell lines (H460, H157, and Calu-1). U, 279-bp unmethylated fragments; M, 218-bp methylated fragments. C. Scanning densitometry was used to measure and analyze the intensity of the fragment signal of B. Relative quantity of the methylation promoter was then calculated.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. Correlation of DAP kinase promoter methylation status and expression of the gene in NSCLC cells. A. Methylation status of the DAP kinase promoter of the 11 NSCLC cell lines. U, 279-bp unmethylated PCR fragment; M, 218-bp methylated PCR fragment. B. DAP kinase mRNA expression in the 11 NSCLC cell lines. Lane 1, DNA size marker. C. Correlation of DAP kinase promoter methylation and expression of the gene based on data from A and B. Points, mean proportions of methylated DAP kinase promoter or relative gene expression level of three independent replicates; bars, unbiased SD.

Expression of DAP Kinase in the NSCLC Cells Negatively Correlates with Its Promoter Methylation Status
Using multiplex reverse transcription-PCR, we examined DAP kinase expression in the 11 lung cancer cell lines. DAP kinase expression was completely or partially silenced in Calu-1, H1792, H157, H460, and SK-MES-1 cells (Fig. 2B), which have methylated CpG islands in their promoters, but not in A549, H226, H292, H522, H596, and H1944 cells, which have an unmethylated promoter. The extent of methylation detected in the DAP kinase promoter was negatively correlated with the DAP kinase mRNA level in the 11 cell lines (r = –0.77; P = 0.003 by linear correlation and regression analysis; Fig. 2C). Of the H460 subclones tested, H460-12 and H460-126, which were completely methylated, had no detectable DAP kinase gene and protein expression (example in Fig. 3C and D)) but H460-14,H460-120, and H460-124, which were unmethylated, had detectable DAP kinase expression (example in Fig. 3C and D).

View larger version (57K): [in this window] [in a new window]   FIGURE 3. Correlation of DAP kinase expression with TRAIL-induced apoptosis in H460 cells. A. Double immunohistochemistry staining of H460 cells. Black nuclear staining, apoptosis; red cytoplasmic staining, expression of DAP kinase. Top right box, control panel without TRAIL treatment. B. Methylation status of two H460 subclones measured by MMSP. U, unmethylated fragment; M, methylated fragment. C. DAP kinase gene expression in H460-12 and H460-124. D. DAP kinase protein expression in H460-12 and H460-124. ß-actin level was used as a loading control. E. TRAIL-induced cell death measured by MTT assay. Top, scanned image of the MTT results; bottom, measured rates of cell death. Points, mean cell death rates of three independent replicates; bars, SD. F. TRAIL-induced DNA fragmentation in the two subclones after TRAIL treatment (80 ng/mL for 12 hours). M, DNA size marker.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Restoration of DAP kinase expression and sensitivity to TRAIL treatment by promoter demethylation. A. DAP kinase expression was restored after treatment with 5ADC at the concentration of 1 or 2 µmol/L. B. MTT assay measuring cell death induced by TRAIL with or without 5ADC in H460-12 cells. Bars, unbiased SD of the triplicates. C. TRAIL-induced DNA fragmentation in H460-12 cells treated with TRAIL with (+) or without (–) 5ADC.

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods References

Of the 11 NSCLC cell lines studied, 5 contained a methylated DAP kinase promoter, including 4 that contained both methylated and unmethylated DAP kinase promoter, indicating that they are heterogeneous either due to differential methylation in one of the two DAP kinase alleles or the presence of subclones.

A highly negative correlation between DAP kinase methylation and gene expression was verified in the 11 NSCLC cell lines (r = –0.77; P = 0.003) and in the H460 subclones ( r= –0.97; P = 0.001; data not shown). The fact that the promoter methylation status was consistent with DAP kinase gene expression indicates that the promoter methylation is the major mechanism to inactivate DAP kinase. Measurement of DAP kinase expression either by immunohistochemistry or in situ MSP would be helpful for predicting the functional status of the gene.

Previous studies have shown that DAP kinase is an important death messenger in IFN-, tumor necrosis factor-, and Fas ligand–mediated apoptosis (3, 9), but DAP kinase involvement with TRAIL-induced cell apoptosis has not been reported. Consistent with this involvement is the observation that cells lacking DAP kinase expression were less sensitive to TRAIL-induced apoptosis than cells expressing DAP kinase. The fact that only cells expressing DAP kinase protein underwent apoptosis (Fig. 3) further supports the involvement of DAP kinase in TRAIL-induced apoptosis.

The expression of TRAIL decoy receptors DcR1 and DcR2 and death receptors DR4 and DR5 as messenger receivers directly influences TRAIL-induced apoptosis. In our previous study, we have shown DR5 was expressed, whereas decor receptors were not expressed in H157 cells (34), yet these cells were resistant to TRAIL (Table 1), suggesting the involvement of other mechanisms for this resistance. We have not observed a strong correlation between the expression of TRAIL receptors and their sensitivity to TRAIL in the 11 cell lines we studied. ² It has been shown that the binding of death ligands to receptors might result in recruitment of the cofactor Fas-associated death domain–containing protein with formation of a death-inducing signaling complex, which results in activation of caspase-8 (18, 35). Recent studies reported that the gene for caspase-8 is silenced preferentially by aberrant promoter methylation in neuroblastomas, bronchial carcinoids, and small cell lung cancer but not in NSCLC (36, 37). Nevertheless, a potential relationship between TRAIL receptors and DAP kinase warrants further investigation.

When NSCLC cells containing a methylated DAP kinase promoter were treated with 5ADC (a demethylation agent), the cells restored expression of DAP kinase and TRAIL sensitivity (Fig. 4). These results support the involvement of DAP kinase in TRAIL-induced apoptosis and provide justification for combination of demethylation agent(s) with death ligand(s) to improve the therapeutic effects.

Although different cancer cell lines carry distinct patterns of methylated promoters and 5ADC treatment sensitized TRAIL response in multiple cell lines ( Fig. 4), ³ it is still possible that activation of other genes by 5ADC treatment contributes to the sensitization. Additional experiments to specifically activate DAP kinase or specifically inhibit DAP kinase may provide more direct evidence to support the role of DAP kinase in rescuing TRAIL-induced apoptosis in NSCLC.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
Cell Lines, Cell Culture, and Cell Subcloning
Human NSCLC cell lines A549, Calu-1, H157, H226, H292, H460, H522, H596, H1792, H1944, and SK-MES-1 were obtained from the American Type Cell Culture (Rockville, MD) and grown in DMEM (Life Technologies, Rockville, MD) containing 10% fetal bovine serum and antibiotics. The cells were maintained at 37°C in a humidified atmosphere consisting of 5% CO ₂ and 95% air in monolayers.

For each subcloning experiment, 30 to 50 H460 parental cells were seeded into a 100-mm dish and incubated for 4 hours; attached single cells were then identified by marking the back of the dish. After being cultured for 7 days, the individual clones originating from the marked single cells were transferred to 24-well plates for further culture before being transferred to 100-mm dishes for further expansion. All the subclones were harvested once on the same day to avoid biological variation in different generations.

cDNA Preparation and Multiplex Reverse Transcription-PCR
Total RNA was extracted and purified from cultured cells using RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Total RNA (1 µg) was used to synthesize cDNA with SuperScript II reverse transcriptase (Life Technologies) in 20 µL, and the final product was diluted to 100 µL. The cDNA was then used for the quantitative assay of DAP kinase expression by multiplex PCR. The multiplex reverse transcription-PCR was carried out under optimal conditions after which a linear correlation between cycle numbers and product amount of PCR was obtained (data not shown). PCR reaction mixture (10 µL) contained 1 µL diluted cDNA sample, 1 unit Hotstart polymerase (Qiagen), 0.1 mmol/L deoxynucleotide triphosphates (dATP + dTTP + dCTP +dGTP), 1 µL DMSO, and 50 ng DAP kinase primers (forward 5'-GACACCGGCGAGGAACTTGGC-3' and reverse 5'-AAAGTCAATGATCTTGATCCGA-3'). The reaction was done according to the following program: at 94°C for 15 minutes for activating the polymerase; for 34 cycles at 94°C for 30 seconds, at 60°C for 1 minute, and at 72°C for 1 minute; and at 72°C for 7 minutes. At the 6th cycle of the 2nd step, ß-actin primers (forward 5'-GTTGCTATCCAGG CTGTGC-3' and reverse 5'-GCATCCTGTCGGCAATGC-3') were added into the reaction mixture as an internal control. The PCR products were electrophoresed on 2.5% agarose gel. Then, the gel was stained with ethidium bromide and photographed under UV light. The absorbance of the DAP kinase specific fragments was measured by scanning densitometry using the absorbance of the internal control (ß-actin) as the standard. The relative amount of the DAP kinase mRNA was calculated as absorbance of DAP kinase/actin.

DNA Isolation and MMSP
DNA was extracted and purified from cultured cells. The cells were collected and digested in 200 µL of digestion buffer containing 50 mmol/L Tris-HCl (pH 8.0), 1% SDS, and 0.5 mg/mL proteinase K and incubated at 42°C for 36 hours. The digested products were purified with phenol-chloroform twice. DNA was then precipitated using the ethanol precipitation method and recovered in distilled DNase-free water. Bisulfite modification of DNA was done as described by Herman et al. (28). Briefly, 1 µg of the DNA from each sample was denatured using NaOH and then treated with sodium bisulfite (Sigma, St. Louis, MO) for 16 hours. After purification using the Wizard DNA purification kit (Promega, Madison, WI), the purified DNA was treated again with NaOH, precipitated with ethanol, and recovered in distilled water. The relative quantity of methylated and unmethylated DAP kinase promoters was determined by the MMSP. The primers specific for unmethylated CpG islands were (forward) 5'-TTGTGAGTTGTTGATTTTTTTTTGT-3' and (reverse) 5'-ATACACAATAAAACACACCAACAAA-3', which cover a 279-bp fragment; the primers specific for methylated CpG islands were (forward) 5'-CGAGTTGTCGAGTTTTTTTCGC-3' and (reverse) 5'-CCGCGCAAAACCCGCAACG-3', which cover a 218-bp fragment. The PCR products were separated in 2.5% of agafrose gel, stained with ethidium bromide, and then photographed under UV light. The absorbances of the unmethylated fragments (U) at 279 bp and methylated fragments (M) at 218 bp were determined by scanning densitometry of the photographs. The proportion of methylation alleles was calculated as M / U + M.

Protein Extraction and Western Blot Analysis
Cellular proteins were collected in lysis buffer containing 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 µg/mL phenylmethylsulfonyl fluoride, 30 µg/mL aprotinin, and 50 mmol/L Tris-HCl (pH 8.0). The samples were then placed on ice for 60 minutes and then centrifuged at 14,000 x g at 4°C for 30 minutes. The protein concentration was measured using a protein assay kit (Bio-Rad, Hercules, CA). Each protein sample (30 µg) was subjected to SDS-PAGE using 8% denaturing polyacrylamide gel. The proteins were then transferred to Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). The nitrocellulose membranes were incubated in a blocking solution containing 5% bovine skim milk in 10 mmol/L PBS containing 0.1% Tween 20 for 1 hour followed by incubation for 3 hours with monoclonal anti-DAP kinase antiserum (DAPK-55, Sigma) at a concentration of 1:1,000 or monoclonal anti-ß-actin (AC15, Sigma) antibody at a concentration of 1:5,000. The membranes were washed with PBS and then incubated with the secondary anti-mouse antibody supplied in the enhanced chemiluminescence kit (Amersham Pharmacia Biotech) for 1 hour. After this incubation, the membranes were washed thrice in PBS, developed in enhanced chemiluminescence solution for 1 to 2 minutes, and exposed to X-ray film for chemiluminescence detection of the positive protein bands.

TUNEL and DAP Kinase Protein Detection by Immunohistochemistry Double Staining
H460 cells were seeded on two glass slides, incubated for 24 hours, and then treated with or without TRAIL (10 ng/mL, Calbiochem, San Diego, CA) for 12 hours. The slides were then fixed in 10% paraformalin [prepared with PBS (pH 7.4)] for 20 minutes and washed in PBS for 5 minutes. The TUNEL assay was done with TUNEL kit (Intergen, Burlington, MA) according to instructions provided by the manufacturer. 3,3'-Diaminobenzidine-conjugated nickel was used as a chromogen to stain the apoptotic cells. The slides were then subjected to double staining with immunohistochemistry to detect the expression of DAP kinase protein by the following procedures: the slides were treated thrice for 5 minutes in a microwave oven with 10 mmol/L citrate buffer (pH 6.0) to retrieve the antigenicity, immersed in methanol containing 0.3% hydrogen peroxidase for 20 minutes to block the endogenous peroxidase, and incubated in 2.5% blocking serum to reduce nonspecific binding. The slides were incubated overnight at 4°C with primary anti-DAP kinase monoclonal antiserum (DAPK-55, Sigma) at a dilution 1:160 and then processed using standard avidin-biotin immunohistochemistry according to the manufacturer's recommendations (Vector Laboratories, Burlingame, CA). Vector NevaRed (Vector Laboratories) was used as a chromogen to detect DAP kinase expression as red in the cytoplasm. Methyl green was used for nuclear counterstaining.

DNA Fragmentation Assay
After treatment with TRAIL, both floating and attached cells were collected, pelleted, and resuspended in Tris-EDTA buffer (pH 8.0). The plasma membrane of the cell was lysed on ice in a mixture of 10 mmol/L Tris-HCl (pH 8.0), 10 mmol/L EDTA, and 0.5% Triton X-100 for 15 minutes. The lysate was centrifuged at 12,000 x g for 15 minutes to separate the soluble (fragmented) from pellet (intact genomic) DNA. The soluble DNA was treated with RNase A (50 µg/mL) at 37°C for 1 hour and proteinase K (100 µg/mL) in 0.5% SDS at 50°C for 2 hours, extracted with phenol-chloroform, precipitated in ethanol, electrophoresed on a 1.8% agarose gel, and stained with ethidium bromide. The gels were then photographed under UV illumination.

Demethylation
Cells were treated with 5ADC (Sigma) at 1 or 2 µmol/L concentrations for 48 hours before further treatment with TRAIL.

MTT Assay
About 30,000 cells of each line were seeded in 96-well plates in 0.1 mL DMEM in triplicate and incubated for 24 hours. The medium was then replaced with medium containing a designated concentration of TRAIL and incubated for another 12 hours. At the end of treatment, 10 µL (5 mg/mL) MTT (Sigma) were added and incubated for 3 hours. The medium containing MTT was absorbed off and washed with PBS carefully, and 0.1 mL DMSO was added to each well. Absorbances of controls (A_c) and experimental samples ( A_t) at a wavelength of 540 nm with background subtraction at 620 nm were measured using the E_max precision microplate reader (Molecular Devices, Sunnyvale CA). Cell death (%) is calculated as 100 x (1 – A_t / A_c).

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 
¹ American Cancer Society grant RPG-98-054; Tobacco Research Fund from the State of Texas; National Cancer Institute grants CA 16620, CA 68437, CA86390, and CA91844 (L. Mao); and Department of Defense grant DAMD17-01-1-01689-1.

W.K. Hong is an American Cancer Society clinical research professor.

² Unpublished data.

³ Unpublished data.

Received March 2, 2004; revised October 21, 2004; accepted November 22, 2004.

	References

Top Notes Abstract Introduction Results Discussion Materials and Methods References

 

1. Deiss LP, Feinstein E, Berissi H, Cohen O, Kimchi A. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the interferon-induced cell death. Genes Dev 1995;9:15–30[Abstract/Free Full Text]
2. Cohen O, Feinstein E, Kimchi A. DAP-kinase is a Ca ²⁺/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J 1997;16:998–1008[CrossRef][Medline]
3. Cohen O, Inbal B, Kissil JL, et al. DAP-kinase participates in TNF-- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol 1999;146:141–8[Abstract/Free Full Text]
4. Shiramizu B, Mick P. Epigenetic changes in the DAP-kinase CpG island in pediatric lymphoma. Med Pediatr Oncol 2003;41:527–31[Medline]
5. Ueki T, Toyota M, Sohn T, et al. Hypermethylation of multiple genes in pancreatic adenocarcinoma. Cancer Res 2000;60:1835–9[Abstract/Free Full Text]
6. Katzenellenbogen RA, Baylin SB, Herman JG. Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. Blood 1999;93:4347–53[Abstract/Free Full Text]
7. Sanchez-Cespedes M, Esteller M, Wu L. Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 2000;60:892–5[Abstract/Free Full Text]
8. Tang X, Khuri FR, Lee JJ, et al. Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small-cell lung cancer. J Natl Cancer Inst 2000;92:1511–6[Abstract/Free Full Text]
9. Inbal B, Cohen O, Polak-Charcon S, et al. DAP kinase links the control of apoptosis to metastasis. Nature 1997;390:180–4[CrossRef][Medline]
10. Pan G, O'Rourke K, Chinnaiyan AM, et al. The receptor for the cytotoxic ligand TRAIL. Science 1998;276:111
11. Sedger LM, Shows DM, Blamton RA, et al. IFN- mediates a novel antiviral activity through dynamic modulation of TRAIL and TRAIL receptor expression. J Immunol 1999;163:920[Abstract/Free Full Text]
12. Yu R, Mandlekar S, Ruben S, Ni J, Kong AN. Tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in androgen-independent prostate cancer cells. Cancer Res 2000;60:2384–9[Abstract/Free Full Text]
13. Thomas WD, Hersy P. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates in CD4 T cell killing of target cells. J Immunol 1998;161:2195–200[Abstract/Free Full Text]
14. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999;5:157–63[CrossRef][Medline]
15. Mitsiades N, Poulaki V, Tseleni-Balafouta S, Koutras DA, Stamenkovic I. Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2000;60:4122–9[Abstract/Free Full Text]
16. Wu M, Das A, Tan Y, Zhu C, Cui T, Wong MC. Induction of apoptosis in glioma cell lines by TRAIL/Apo-2L. J Neurosci Res 2000;61:464–70[CrossRef][Medline]
17. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2002;2:420–30[CrossRef][Medline]
18. Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, Ashkenazi A. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 2000;12:611–20[CrossRef][Medline]
19. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815–8[Abstract/Free Full Text]
20. Kim K, Fisher MJ, Xu S-Q, El-Deiry WS. Molecular determinants of response to TRAIL in killing of normal and cancer cells. Clin Cancer Res 2000;6:335–46[Abstract/Free Full Text]
21. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 1996;271:12687–90[Abstract/Free Full Text]
22. Fanger NA, Maliszewski CR, Schooley K, Griffith TS. Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Exp Med 1999;190:1155–64[Abstract/Free Full Text]
23. Zhang XD, Nguyen T, Thomas WD, Sanders JE, Hersey P. Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types. FEBS Lett 2000;482:193–9[CrossRef][Medline]
24. Hopkins-Donaldson S, Ziegler A, Kurtz S, et al. Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation. Cell Death Differ 2003;10:356–64[CrossRef][Medline]
25. Chen X, Thakkar H, Tyan F, et al. Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer. Oncogene 2001;20:6073–83[CrossRef][Medline]
26. Jonsson G, Paulie S, Grandien A. High level of cFLIP correlates with resistance to death receptor-induced apoptosis in bladder carcinoma cells. Anticancer Res 2003;23:1213–8[Medline]
27. Tang X, Sun YJ, Half E, Kuo MT, Sinicrope F. 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. Cancer Res 2002;62:4903–8[Abstract/Free Full Text]
28. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996;93:9821–6[Abstract/Free Full Text]
29. Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 1997;25:2532–4[Abstract/Free Full Text]
30. Matsumoto H, Nagao M, Ogawa S, et al. Prognostic significance of death-associated protein-kinase expression in hepatocellular carcinomas. Anticancer Res 2003;23:1333–41[Medline]
31. Gonzalez-Gomez P, Bello MJ, Alonso ME, et al. Frequent death-associated protein-kinase promoter hypermethylation in brain metastases of solid tumors. Oncol Rep 2003;10:1031–3[Medline]
32. Nakatsuka S, Takakuwa T, Tomita Y, 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. Cancer Sci 2003;94:87–91[CrossRef][Medline]
33. Yamaguchi S, Asao T, Nakamura J, Ide M, Kuwano H. High frequency of DAP-kinase gene promoter methylation in colorectal cancer specimens and its identification in serum. Cancer Lett 2003;194:99–105[CrossRef][Medline]
34. Wu W, Soria J, Wang L, Kemp BL, Mao L. TRAIL-R2 is not correlated with p53 status and is rarely mutated in non-small cell lung cancer. Anticancer Res 2000;20:4525–30[Medline]
35. Bodmer JL, Holler N, Reynard S, et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol 2000;2:241–3[CrossRef][Medline]
36. Shivapurkar N, Toyooka S, Eby MT, et al. Differential inactivation of caspase-8 in lung cancers. Cancer Biol Ther 2002;1:65–9[Medline]
37. Teitz T, Wei T, Valentine MB, et al. Caspase-8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nat Med 2000;6:529–35[CrossRef][Medline]

This article has been cited by other articles:

				S. Luxen, S. A. Belinsky, and U. G. Knaus Silencing of DUOX NADPH Oxidases by Promoter Hypermethylation in Lung Cancer Cancer Res., February 15, 2008; 68(4): 1037 - 1045. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tang, X. Articles by Mao, L. Search for Related Content PubMed PubMed Citation Articles by Tang, X. Articles by Mao, L.