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Gastric cancer is one of the most common malignant cancers globally. Chemotherapy resistance remains a major obstacle in the treatment of gastric cancer, and the molecular mechanisms underlying drug resistance are still not well understood. We previously reported that Zipper interacting protein kinase (ZIPK), also known as death‐associated protein kinase3, exerts an oncogenic effect on gastric cancer via activation of Akt/NF‐κB signaling and promotion of stemness. Here, we explored the roles of ZIPK in cisplatin resistance. We report that ZIPK enhances cell proliferation and invasion and reduces the antitumor activity of cisplatin in gastric cancer. In addition, our western blot data suggest that ZIPK activated the IL‐6/STAT3 signaling pathway. Furthermore, ZIPK increased the expression of IL‐6 and multidrug‐resistance genes. Using the STAT3 inhibitor stattic to block the IL‐6/STAT3 signaling pathway strongly increased the sensitivity of ZIPK‐expressed cells to cisplatin. In conclusion, ZIPK may play a role in cisplatin resistance through activation of the IL‐6/ STAT3 signaling pathway. Inhibition of STAT3 in gastric cancer overexpressing ZIPK might have potential to improve the efficacy of cisplatin., ZIPK activates the IL‐6/STAT3 signaling pathway via Ser727 phosphorylation in gastric cancer cells and increases IL‐6‐induced STAT3‐dependent transcription. Effective blockade of STAT3 activity by the STAT3 inhibitor stattic sensitized gastric cancer cells to cisplatin and reduced expression of the cisplatin resistance‐related genes G3BP2, THOC1, ATP7A, and OTUD1.

OBJECTIVE DNA Methylation of the tumour suppressor gene leading to gene silencing plays an important role in thyroid tumour development. The purpose was to determine the DNA methylation status of phosphatase and tensin homolog (PTEN) and death-associated protein kinase (DAPK) in patients with thyroid nodules and to explore whether they can be used as molecular diagnostic tools to differentiate benign and malignant thyroid nodules. DESIGN, PATIENTS AND MEASUREMENTS Thyroid tissue and blood samples were obtained from normal healthy individuals (controls) and patients suffering from clinically diagnosed thyroid nodular disease [papillary thyroid carcinoma (PTC), adenoma and nodular goitre]. DNA methylation level, mRNA expression and protein expression of PTEN and DAPK in the thyroid tissues and peripheral blood were detected using methylation-specific PCR, semi-quantitative reverse transcription PCR and Western blot, respectively. Diagnostic sensitivity, specific and accuracy of detection were evaluated between blood and thyroid tissue. RESULTS There was a significant increase in the level of DNA methylation of PTEN and DAPK in PTC (P

DAPK, a transcriptional target of the p53 protein, has long been characterized as a tumor suppressor that acts as a negative regulator in multiple cellular processes. However, increasing studies have suggested that the role of DAPK may vary depending on cell type and cellular context. Thus far, the expression and function of DAPK in clear cell renal cell carcinoma (ccRCC) remain ambiguous. Since ccRCC behaves in an atypical way with respect to p53, whether the p53-DAPK axis functions normally in ccRCC is also an intriguing question. Here, tissue specimens from 61 ccRCC patients were examined for DAPK expression. Functional studies regarding apoptosis, growth, and migration were used to determine the role of DAPK in renal cancer cells. The validity of the p53-DAPK axis in ccRCC was also determined. Our study identified DAPK as a negative regulator of ccRCC, and its expression was reduced in certain subgroups. However, the p53-DAPK axis was disrupted due to upregulation of miR-34a-5p under stressed conditions. miR-34a-5p was identified as a novel repressor of DAPK acting downstream of p53. Inhibition of miR-34a-5p can correct the p53-DAPK axis disruption by upregulating DAPK protein and may have potential to be used as a therapeutic target to improve outcomes for ccRCC patients.

The paracellular gap formed by endothelial cell (EC) contraction is fundamental for endothelium permeability, but the mechanism underlying EC contraction has yet to be determined. Here, we identified the zipper-interacting protein kinase (ZIPK) as the kinase for EC contraction and myosin light chain (MLC) phosphorylation. Inhibition of ZIPK activity by pharmacological inhibitors and small interfering RNAs led to a significant decrease in the mono- and diphosphorylation of MLCs along with a contractile response to thrombin, suggesting an essential role of ZIPK in EC paracellular permeability. To assess the role of ZIPK in vivo, we established mouse lines with conditional deletion of Zipk gene. The endothelium-specific deletion of Zipk led to embryonic lethality, whereas the UBC-Cre(ERT2)–mediated deletion of Zipk by tamoxifen induction at adulthood caused no apparent phenotype. The induced deletion of Zipk significantly inhibited ischemia-reperfusion–induced blood-brain barrier dysfunction and neuronal injuries from middle cerebral artery occlusion and reperfusion, as evidenced by reduced infarct and edema volume, attenuated Evans blue dye leakage, and improved neuronal behavior. We thus concluded that ZIPK and its phosphorylation of MLC were required for EC contraction and ischemic neuronal injuries. ZIPK may be a prospective therapeutic target for stroke.—Zhang, Y., Zhang, C., Zhang, H., Zeng, W., Li, S., Chen, C., Song, X., Sun, J., Sun, Z., Cui, C., Cao, X., Zheng, L., Wang, P., Zhao, W., Zhang, Z., Xu, Y., Zhu, M., Chen, H. ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.

Cross‐linking of the B‐cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post‐translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death‐associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti‐IgM‐dependent manner. DAPK inhibition mimics ibrutinib‐induced repression of both IEG mRNA and histone H3 phosphorylation and has anti‐proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti‐tumour effect than ibrutinib, by repressing both anti‐IgM‐ and CD40L‐dependent activation., Transcription involves alteration of histones, the proteins surrounding DNA. Here, we show that phosphorylation of histone H3 mediated by the protein DAPK3 does not impact on transcription itself, but is required for co‐transcriptional mRNA processing of key pro‐proliferative genes. Inactivation of DAPK3 not only prevents mRNA processing of these genes but correlates with accumulation of their unprocessed pre‐mRNA transcripts.

For many decades, kinases have predominantly been characterized as oncogenes and drivers of tumorigenesis, because activating mutations in kinases occur in cancer with high frequency. The oncogenic functions of kinases relate to their roles as growth factor receptors and as critical mediators of mitogen‐activated pathways. Indeed, some of the most promising cancer therapeutic agents are kinase inhibitors. However, cancer genomics studies, especially screens that utilize high‐throughput identification of loss‐of‐function somatic mutations, are beginning to shed light on a widespread role for kinases as tumor suppressors. The initial characterization of tumor‐suppressing kinases— in particular members of the protein kinase C (PKC) family, MKK4 of the mitogen‐activated protein kinase kinase family, and DAPK3 of the death‐associated protein kinase family— laid the foundation for bioinformatic approaches that enable the identification of other tumor‐suppressing kinases. In this review, we discuss the important role that kinases play as tumor suppressors, using several examples to illustrate the history of their discovery and highlight the modern approaches that presently aid in the identification of tumor‐suppressing kinases. © 2018 IUBMB Life, 71(6):738–748, 2019

Zipper-interacting protein kinase (ZIPK) is a Ser/Thr kinase that mediates a variety of cellular functions. Analogue-sensitive kinase technology was applied to the study of ZIPK signaling in coronary artery smooth muscle cells. ZIPK was engineered in the ATP-binding pocket by substitution of a bulky gatekeeper amino acid (Leu93) with glycine. Cell-permeable derivatives of pyrazolo[3,4-d]pyrimidine provided effective inhibition of L93G-ZIPK (1NM-PP1, IC50 , 1.0 μM; 3MB-PP1, IC50 , 2.0 μM; and 1NA-PP1, IC50 , 8.6 μM) but only 3MB-PP1 had inhibitory potential (IC50 > 10 μM) toward wild-type ZIPK. Each of the compounds also attenuated Rho-associated coiled-coil containing protein kinase (ROCK) activity under experimental conditions found to be optimal for inhibition of L93G-ZIPK. In silico molecular simulations showed effective docking of 1NM-PP1 into ZIPK following mutational enlargement of the ATP-binding pocket. Molecular simulation of 1NM-PP1 docking in the ATP-binding pocket of ROCK was also completed. The 1NM-PP1 inhibitor was selected as the optimal compound for selective chemical genetics in smooth muscle cells since it displayed the highest potency for L93G-ZIPK relative to WT-ZIPK and the weakest off-target effects against other relevant kinases. Finally, the 1NM-PP1 and L93G-ZIPK pairing was effectively applied in vascular smooth muscle cells to manipulate the phosphorylation level of LC20, a previously defined target of ZIPK.

Death-associated protein kinase (DAPK) is a serine/threonine kinase and a tumour suppressor gene. Diffuse large B-cell lymphomas with inactivated DAPK through hypermethylation of a CpG island is known to result in a biologically aggressive phenotype in humans. This retrospective study was carried out to analyse the prognostic significance of DAPK CpG island hypermethylation in canine lymphoma. We hypothesized that DAPK CpG island hypermethylation can be a negative prognostic indicator in dogs with nodal high-grade B-cell lymphoma. Forty-seven dogs with high-grade B-cell lymphoma, according to the updated Kiel classification, were evaluated after being treated with a CHOP (vincristine, cyclophosphamide, doxorubicin and prednisolone)-based chemotherapy protocol. The methylation status of the DAPK CpG island was examined by methylation-specific PCR. Progression-free survival (PFS) and overall survival (OS) were compared using the Kaplan-Meier analysis and log-rank test. The cox proportional hazard regression model was used to evaluate the effect of multiple variables. Hypermethylation of the DAPK CpG island was detected in 21 of the 47 dogs. The PFS and OS in dogs with the hypermethylation (median: 220 and 266 days, respectively) were significantly shorter than those of dogs without hypermethylation (median: 301 and 412 days, respectively) (PFS, P = .036; OS, P = .007). In the multivariate analysis, hypermethylation of the DAPK CpG island remained an independent prognostic factor in predicting shortened PFS (P = .047) and OS (P = .021) as well as clinical substage b. Overall, hypermethylation of the DAPK CpG island was a negative prognostic factor in canine high-grade B-cell lymphoma.

Herein we report a CRISPR‐Cas9‐mediated loss‐of‐function kinase screen for cancer cell deformability and invasive potential in a high‐throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High‐ranking candidates identified included well‐reported tumor suppressor kinases, such as chk2, IKK‐α, p38 MAPKs, and DAPK2. A high‐ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR‐based on‐chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.

Protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In this study, we sought to determine whether PIAS3 inhibits cell growth in non-small cell lung cancer cell lines by inducing apoptosis. Our results demonstrate that overexpression of PIAS3 promotes mitochondrial depolarization, leading to cytochrome c release, caspase 9 and 3 activation and poly (ADP-ribose) polymerase cleavage. This intrinsic pathway activation was associated with decreased Bcl-xL expression and increased Noxa expression and was independent of p53 status. Furthermore, PIAS3 inhibition of STAT3 activity was also p53 independent. Microarray experiments were performed to discover STAT3-independent mediators of PIAS3-induced apoptosis by comparing the apoptotic gene expression signature induced by PIAS3 overexpression with that induced by STAT3 siRNA. The results showed that a subset of apoptotic genes was uniquely expressed only after PIAS3 expression. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy and its potential to synergize with Bcl-2 targeted inhibitors.

Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field.

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Aberrant DNA methylation and concomitant transcriptional silencing of death-associated protein kinase 1 (DAPK1) have been demonstrated to be key pathogenic events in chronic lymphocytic leukemia (CLL). In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), however, the presence of elevated DNA methylation levels has been a matter of continued controversy. Several studies demonstrated highly variable frequencies of DAPK1 promoter methylation by the use of methylation-specific PCR (MSP). By quantitative high-resolution assessment, we demonstrate that aberrant DNA methylation is an extremely rare event in this region. We observed elevated levels just in one out of 246 (0.4%) AML patients, all 42 MDS patients were unmethylated. In conclusion, we present a refined DAPK1 methylation analysis in a large representative patient cohort of AML and MDS patients proofing almost complete absence of elevated DNA methylation. Our results highlight the importance of quantitative measurements for translational research questions on primary patient specimens, particularly.

Alzheimer's disease (AD) is an aging-related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP-ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up-regulated the lysoso-mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3-methylade-nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ-induced apoptosis.

Death-associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)-induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB-Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch-repeat domain and BTB domain, respectively. The KLHL20-Cul3-ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20-mediated DAPK ubiquitination is suppressed in cells receiving IFN-alpha or IFN-gamma, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN-induced autophagy. This study identifies KLHL20-Cul3-ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN-induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.

Summary Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16, O6-methyl guanine DNA methyl transferase (MGMT), death-associated protein kinase (DAPK) and E-cadherin (ECAD); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16, MGMT, DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

A. H. Hainsworth, R. C. Allsopp, A. Jim, J. F. Potter, J. Lowe, C. J. Talbot and R. J. Prettyman (2010) Neuropathology and Applied Neurobiology36, 17–24 Death-associated protein kinase (DAPK1) in cerebral cortex of late-onset Alzheimer's disease patients and aged controls Aims: Here our objective was to detect the pro-apoptotic serine/threonine kinase death-associated protein kinase (DAPK1) in aged human cerebral cortex and to test the hypothesis that DAPK1 abundance is associated with late-onset Alzheimer's disease (AD). Methods: Using Western analysis and immunohistochemistry we evaluated post mortem frontal cerebral cortex from patients with severe AD (mean age 76 years, range 66–91, n = 11, all male), and from control cases without serious central nervous system illness (mean age 77 years, range 61–95, n = 12, all male). We also examined brains of Tg2576 transgenic mice (males, aged 16–21 months), a model for chronic amyloid-induced brain injury. Results: Immunohistochemical labelling showed DAPK1 expression in cortical neurones of human cortex and axonal tracts within subcortical white matter, both in AD and in control brains. Western analysis confirmed DAPK1 expression in all samples, although expression was very low in some control cases. DAPK1 abundance in the AD group was not significantly different from that in controls (P = 0.07, Mann–Whitney test). In brains of Tg2576 mice DAPK1 abundance was very similar to that in wild-type littermates (P = 0.96, Mann–Whitney test). Conclusion: We found that DAPK1 was expressed in neurones of aged human frontal cortex, both in AD and in control cases.

Summary Promoter hypermethylation plays an important role in the inactivation of cancer-related genes. This abnormality occurs early in leukemogenesis and seems to be associated with poor prognosis in myelodsplastic syndrome (MDS). The identification of more inactivated tumor suppressor genes contributing to the development of MDS may lead to further elucidation of the biology of this disease and help to identify novel targets for therapy. In this study, the methylation status of death-associated protein kinase 1 (DAPK1) gene promoter was analyzed by using methylation-specific polymerase chain reaction in bone marrow (BM) samples from 59 patients with different stages of MDS. The abnormal methylation of the DAPK1 gene was found in 37 of 59 (62.7%) MDS cases. The correlation was significant between the sex and the methylation status of DAPK1 promoter in MDS patients (R = 0.332, P = 0.010). Furthermore, methylation status of DAPK1 promoter was associated with the percentage of BM blasts (R = 0.346, P = 0.010) and International Prognosis Scoring System (IPSS) groups (R = 0.278, P = 0.034). The estimated 50% survival time of the methylated DAPK1 group and unmethylated group was 20 and 33 months, respectively. There was no significant difference between these two groups (χ2 = 0.652, P = 0.419). Multivariate analysis identified the age older than 50 years, the Int-2/high-risk categories of IPSS and the advanced stage MDS (RAEB-1/RAEB-2) in WHO classification as negative prognostic factors (P

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO(LRRK2) self-interaction. Interestingly, ROCO(LRRK2) fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO(LRRK2) reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO(LRRK2) fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.

Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.

Penile carcinoma is a rare disease, accordingly there are few studies on molecular changes, and these results also vary greatly. A total of 26 penile squamous cell carcinomas in Japanese men were studied with respect to HPV, p53 alterations, and methylation of gene promoter region. HPV-DNA was detected in three of 26 patients (11.5%). Overexpression of p53 was observed in 13 of 26 patients (50%), and p53 gene mutations were detected in four of 26 patients (15.4%). The frequency of methylation was as follows: DAPK, 26.9% (7/26); FHIT, 88.4% (23/26); MGMT, 19.2% (5/26); p14, 3.8% (1/26); p16, 23.1% (6/26); RAR-beta, 23.1% (6/26); RASSF1A, 11.5% (3/26); and RUNX3, 42.3% (11/26). As for correlation between HPV and p53 alterations, and methylation status, mutations of the p53 gene were detected only in HPV-negative patients, and methylation was more frequently found in HPV-negative than in HPV-positive patients. The present results suggest that the majority of penile squamous cell carcinomas in Japanese men are unrelated to HPV, and gene alterations accumulate more frequently in HPV-unrelated penile carcinomas.

BACKGROUND Aberrant DNA methylation of the CpG islands for cancer-related genes is among the earliest and most frequent alterations in cancer and may be useful for diagnosing cancer or evaluating recurrent disease. METHODS In this study, a 3-dimensional (3-D), polyacrylamide gel-based DNA microarray coupled with linker-polymerase chain reaction (PCR) was developed to detect hypermethylation of CpG islands in multiple genes from a large group of different samples. The authors determined the frequency of aberrant promoter methylation of 15 genes in 28 resected primary nonsmall cell lung cancers (NSCLCs) and in 12 corresponding nonmalignant lung tissues. RESULTS Methylation frequencies in the tumor samples were detected in 18% of samples for the breast cancer 1 gene BRCA1, in 43% of samples for the tissue inhibitor of metalloproteinase 3 gene TIMP-3, in 38% of samples for the cyclin-dependent kinase inhibitor 4A gene p16INK4a, in 54% of samples for the cadherin 13 gene CDH13, in 50% of samples for the death-associated protein kinase gene DAPK, in 11% of samples for the E-cadherin gene ECAD, in 25% of samples for the insulin-like growth factor binding protein 7 gene IGFBP7, in 18% of samples for the Ras association domain family 1 gene RASSF1, in 68% of samples for the adenomatous polyposis coli gene APC, in 7% of samples for the cyclin-dependent kinase inhibitor gene p15, in 18% of samples for the CD44 cell adhesion molecule gene, in 29% of samples for the human Mut-L homolog gene hMLH, in 32% of samples for the human telomerase reverse transcriptase gene hTERT, in 64% of samples for the calcitonin gene-related polypeptide α gene CALCA, and in 54% of samples for the estrogen receptor gene ER; however, methylation was not observed in the majority of corresponding nonmalignant tissues. Six samples in from 28 tumors had >6 genes methylated, and 1 sample had 13 genes methylated. Methylation of these genes was correlated with some clinicopathologic patient characteristics. CONCLUSIONS This study demonstrated that a 3-D microarray could be used to detect DNA hypermethylation and provided a high-throughput platform for DNA hypermethylation analysis. Cancer 2008. © 2008 American Cancer Society.

Objectives/Hypothesis: Promoter hypermethylation of tumor suppressor genes is common in head and neck cancer as well as other primary cancers resulting in epigenetic gene silencing. Tissue inhibitor of metalloproteinase-3 (TIMP-3) has been shown to have promoter hypermethylation in several solid tumors, but has not been identified in head and neck squamous cell carcinoma (HNSCC). Our objective was to determine if TIMP-3 promoter was hypermethylated in HNSCC, if there was any correlation with death associated protein kinase (DAPK), a tumor suppressor whose promoter has been hypermethylated at high levels in HNSCC, and if any clinical factors influence hypermethylation of either of these genes. Study Design: Prospective study. Methods: Tumor samples from 124 patients with HNSCC were evaluated for promoter hypermethylation for TIMP-3 and DAPK using quantitative methylation specific polymerase chain reaction (qMSP). We compared both TIMP-3 and DAPK hypermethylation in HNSCC with each other as well as with other clinical variables. Results: We found that TIMP-3 was hypermethylated in approximately 71.8% of the tumor samples and DAPK was hypermethylated in 74.2%. The presence of TIMP-3 and DAPK promoter hypermethylation was significantly higher than in control specimens. More importantly, TIMP-3 and DAPK hypermethylations in these samples were highly correlated with a concordance of 78% (P < .001). DAPK was also correlated with current alcohol consumption (P < .028), but neither TIMP-3 nor DAPK hypermethylation was significantly correlated with other clinical variables or with survival. Conclusion: TIMP-3 promoter hypermethylation is elevated in HNSCC and is highly correlated with DAPK hypermethylation, implying a functional relationship between these genes.

Accumulating evidence implicates epigenetic changes such as hypermethylation in carcinogenesis. We investigated whether DNA methylation of 5 tumor suppressor genes in pleural fluid samples could aid in diagnosis of malignant effusion. In samples from 47 patients with malignant pleural effusions and 34 with nonmalignant effusions, we used a methylation-specific polymerase chain reaction to detect aberrant hypermethylation of the promoters of the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a), ras association domain family 1A (RASSF1A), apoptosis-related genes, death-associated protein kinase (DAPK), and retinoic acid receptor beta (RARbeta). Promoter hypermethylation was associated with malignant effusion for MGMT (Odds ratio (OR) = infinity), p16(INK4a) (OR = infinity), RASSF1A (OR = 13.8; CI, 1.71-112), and RARbeta (OR = 3.17; CI, 1.10-9.11), but not for DAPK. Instead, DAPK methylation was associated with the length of smoking (p < 0.05). Patients with hypermethylation of MGMT, p16(INK4a), RASSF1A or RARbeta were 5.68 times more likely to have malignant effusions than patients without methylation (p = 0.008). Methylations per patient were more numerous for lung cancer than nonmalignant pulmonary disease (0.915 vs. 0.206, p < 0.001). Sensitivity, specificity, and positive predictive value of methylation in one or more genes for diagnosis of malignant effusion were 59.6%, 79.4%, and 80.0% respectively. In conclusion, aberrant promoter methylation of tumor suppressor genes in pleural fluid DNA could be a valuable diagnostic marker for malignant pleural effusion.

Gastric cancer is the second most common cause of cancer deaths worldwide. The identification of molecular genetic parameters that are associated with response to chemotherapy and prognosis is of utmost interest. We examined methylation of the apoptosis-related genes, TMS1 and DAPK, in 81 primary gastric cancers using methylation-specific PCR and compared their methylation status with clinicopathological findings. Aberrant methylation of TMS1 and DAPK genes was detected in 26 (32.1%) tumors and in 18 (22.2%) tumors, respectively. The overall survival of patients with both methylated genes was significantly shorter compared with those with only one methylated gene or no methylated genes (p = 0.0003). Neither gene methylation had any relation to other clinicopathological findings. Next, we examined 43 patients treated by 5-fluorouracil-based chemotherapy, who had distant metastasis or recurrence after radical resection, to determine the relation between chemosensitivity and methylation. The response rate was lower in patients with either methylation than without (TMS1: 22.2% vs. 48.0%; DAPK: 21.4% vs. 44.8%). Overall survival tended to be shorter in the patients with both methylations compared with either or no methylations (p = 0.0806). The time to progression of patients with methylation of TMS1 or DAPK was significantly shorter than patients without methylation (TMS1: p = 0.0123; DAPK: p = 0.0464). Furthermore, the time to progression of patients with both methylated genes was significantly shorter than patients with one methylation or no methylation (p = 0.0082). In conclusion, TMS1 and DAPK methylation might predict the prognosis and response to chemotherapy in gastric cancer.

Epigenetic silencing of tumor suppressor genes by promoter hypermethylation has been shown for a variety of genes in bladder cancer. Various p53 target genes have been investigated, but only few demonstrated promoter hypermethylation when semiquantitative detection methods were applied. To address to the question whether promoter methylation of novel p53 effector genes is a common event in transitional cell carcinoma of the bladder, we selected the p53 target genes apoptotic protein-activating factor (APAF-1), Caspase 8 (CASP-8), death-associated protein kinase, (DAPK-1) and insulin-like growth-factor-binding protein-3 (IGFBP-3), performing quantitative methylation-specific real-time PCR. The individual level of methylation (normalized index of methylation) was correlated with clinicopathological features as well as the biological behavior of the superficial and muscleinvasive tumors. Tissue was obtained from 110 tumor patients and 20 patients without urological malignancy. The median follow-up of the tumor patients was 52 months. Hypermethylation of the promoter region in tumor specimens was common for APAF-1 (100%), DAPK-1 (74%) and IGFBP-3 (66%), but not for CASP-8 (3.6%). It was seen less frequently and with undetectable or low methylation levels in the normal urothelium group. The APAF-1 methylation levels significantly correlated with tumor stage and tumor grade. The APAF-1 and IGFBP-3 methylation levels were able to separate tumors with higher recurrence risk from low-risk tumors in nonmuscleinvasive and muscleinvasive tumors. In multivariate analysis, APAF-1 and IGFBP-3 methylation levels were independent prognostic markers for recurrence in superficial bladder tumors. This study provides new insights into the role of promoter methylation of selected p53 target genes. The extent of promoter methylation of specific genes offers additional prognostical information and is associated with the outcome in patients with nonmuscleinvasive and muscleinvasive bladder cancer.

Netrin-1 receptors UNC5H (UNC5H1-4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase.

The prevalence of methylation of the p16, DAPK and RASSF1A genes was investigated in lung adenocarcinoma from smokers, former uranium miners and never smokers. The association between a common genetic alteration in adenocarcinoma, mutation of the K-ras gene and methylation of these genes, as well as survival was examined. Adenocarcinomas from 157 smokers, 46 never smokers and 34 former uranium miners were evaluated for methylation of the p16, DAPK and RASSF1A genes using the methylation-specific PCR assay. Comparisons were also made to prevalences of methylation of the MGMT gene and mutation of the K-ras gene previously examined in these tumors. The prevalence of methylation for all genes was similar between adenocarcinomas from smokers and never smokers, although the prevalence for methylation of the p16 gene tended to be higher in smokers compared to never smokers. A significantly higher prevalence for p16 methylation was seen in central vs. peripheral lung tumors. At least 1 gene was methylated in 35% of stage I tumors, whereas 2 and ≥3 genes were methylated in 40% and 16% of tumors, respectively. Methylation of all genes was independent of K-ras mutation, whereas methylation of the DAPK and RASSF1A genes was positively associated. Environmental tobacco smoke, the strongest lung cancer risk factor among never smokers, induces adenocarcinoma in part through inactivation of the p16, DAPK and RASSF1A genes. Adenocarcinomas may develop through 2 distinct processes: multiple gene inactivations through promoter hypermethylation and activation of the K-ras gene. © 2004 Wiley-Liss, Inc.

Background Hypermethylation of CpG islands in the promoter region of death-associated protein kinase (DAP-kinase) coupled with the loss of γ-interferon-induced apoptosis have been reported in B-cell malignancies suggesting a role in pathogenesis or prognosis. Along with B-cell malignancies, pediatric lymphomas also include T-cell non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma, and Hodgkin disease, each with unique prognoses. The purpose of this study was to elucidate epigenetic changes in the DAP-kinase promoter region of pediatric cases to determine associations with aberrant hypermethylation. Procedures Thirty-nine cases of different lymphoid pathology [10 Burkitt lymphoma, 1 B-cell NHL; 7 T-cell lymphoblastic lymphoma; 4 anaplastic large cell lymphoma (LCL); 2 B-cell LCL; 14 nodular sclerosing Hodgkin disease (NSHD); and 1 B-cell acute lymphoblastic leukemia (ALL)] had methylation-specific polymerase chain reaction performed on bisulfite-treated DNA, which distinguishes the methylation status of the promoter region, and DAP-kinase mRNA expression assays performed on available specimens. Results In normal lymphocytes, the CpG islands in the promoter region were unmethylated, as were the T-cell lymphoblastic lymphoma and anaplastic LCL. In contrast, 100% of the Burkitt lymphoma (10/10) and B-cell ALL (1/1) were hypermethylated. Of the specimens with mRNA available, 7/8 Burkitt lymphoma had no DAP-kinase mRNA expression compared to normal expression in 3/3 and 4/4 T-cell lymphoblastic lymphoma and NSHD, respectively. Conclusions In these pediatric lymphoid tumors, hypermethylation of the DAP-kinase promoter region with associated loss of DAP-kinase gene expression was associated with B-cell malignancies and thus may be important in the development and/or provide a prognostic tool in B- cell lymphomas. Med Pediatr Oncol 2003;41:527–531. © 2003 Wiley-Liss, Inc.

Aberrant promoter hypermethylation is a mechanism of tumour suppressor gene inactivation. We explored aberrant promoter hypermethylation of multiple genes in 88 human immunodeficiency virus (HIV)-non Hodgkin lymphomas (NHL), 25 post-transplant lymphoproliferative disorders (PTLD) and five common variable immunodeficiency (CVI)-related NHL. Twenty-six of 79 (32.9%) HIV-NHL, eight of 14 (57.1%) PTLD and two of five (40.0%) CVI-NHL showed aberrant hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT). Aberrant hypermethylation of death-associated protein-kinase (DAP-K) occurred in 70 of 84 (83.3%) HIV-NHL, 19 of 25 (72.0%) PTLD and three of five (60.0%) CVI-NHL. These data implicate MGMT and DAP-K hypermethylation in lymphomagenesis of immunodeficient hosts. In particular, promoter hypermethylation of DAP-K represents the most frequent molecular alteration yet identified in immunodeficiency-related lymphomas.

It is important to understand the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death. Defects in apoptosis are implicated in both tumorigenesis and drug resistance, and these defects are cause of chemotherapy failures. These studies should explain the relationship between cancer genetics and treatment sensitivity, and should enable a more rational approach to anticancer drug design and therapy. Lung cancer is a major cause of cancer deaths throughout the world. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) represent the two major categories of lung cancer that differ in their sensitivity to undergo apoptosis. The role of apoptosis regulation in lung cancer with major focus on the differential sensitivities of the major subtypes is reviewed.

Promoter hypermethylation is an alternative mechanism of gene silencing in human cancers including gastric cancer. While intestinal metaplasia (IM) is generally regarded as a precancerous lesion of the stomach, our study examines the presence of gene promoter hypermethylation in IM of patients with and without gastric cancer. We examined 31 samples of gastric cancer, 36 gastric IM (21 associated with gastric cancer and 15 from noncancer patients) and 10 normal gastric biopsies. Tissues containing foci of IM were carefully microdissected from paraffin-embedded section. Bisulfite-modified DNA was examined for gene promoter hypermethylation in DAP-kinase, E-cadherin, GSTP1, p14, p15, p16, RASSF1A and hMLH1 by methylation-specific-PCR. None of the control gastric tissues had hypermethylation detected, but gene promoter hypermethylation was frequently detected in gastric cancer and IM. The mean number of methylated genes in cancer and IM was 3.0 and 1.4, respectively (p < 0.0001). Methylation in IM from cancer patients was all associated with concurrent methylation in the corresponding tumor samples. The numbers of methylated genes were similar in IM obtained from cancer and noncancer patients. By examining the methylation patterns of these genes, 3 differential methylation patterns were recognized: hypermethylation was more frequent in cancer than in IM (DAP-kinase, p14, p15 and p16); comparable frequencies of methylation in cancer and IM (E-cadherin and hMLH1); and no methylation (GSTP1). Aberrant methylation in tumor-related genes is frequently detected in gastric IM of both cancer and noncancer patients, suggesting their early involvement in the multistep progression of gastric carcinogenesis.

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with 32P release and a point mutant of Thr 38.

Death-associated protein kinase (DAP-kinase) is Ca2+/calmodulin-dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon-γ-induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP-kinase, the tissue distribution and developmental changes in mRNA expression of DAP-kinase were investigated by Northern blot and in situ hybridization analyses. DAP-kinase mRNA was predominantly expressed in brain and lung. In brain, DAP-kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP-kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP-kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP-kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP-kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP-kinase and neuronal cell death. J. Neurosci. Res. 58:674–683, 1999. © 1999 Wiley-Liss, Inc.

The primary objective of this study is to identify prognostic site-specific epigenetic changes in surgically treated Stage I and II nonsmall cell lung cancer (NSCLC) patients by quantifying methylation levels at multiple CpG sites within each gene promoter. Paraffin-embedded tumors from stage Ib, IIa and IIb in training and validation groups of 75 and 57 surgically treated NSCLC patients, respectively, were analyzed for p16, MGMT, RASSF1, RASSF5, CDH1, LET7, DAPK and PTEN promoter hypermethylation. Hypermethylation status was quantified individually at multiple CpG sites within each promoter by pyrosequencing. Molecular and clinical characteristics with time to recurrence (TTR) and overall survival (OS) were evaluated. Overall average promoter methylation levels of MGMT and RASSF1 were significantly higher in smokers than in nonsmokers (p = 0.006 and p = 0.029, respectively). Methylation levels of the p16 promoter were significantly higher in squamous cell carcinoma than in adenocarcinoma (p = 0.020). In univariate analysis, hypermethylation of RASSF1 at CpG sites −53 and −48 and PTEN at CpG site −1310 were the significantly associated with shorter TTR (p = 0.002 and p < 0.000, respectively). Hypermethylation of PTEN at −1310 and DAPK at −1482 were most significantly associated with outcome in multivariate analysis. These results show that methylation of specific promoter CpG sites in PTEN, RASSF1 and DAPK is associated with outcome in early stage surgically treated NSCLC.