DAP Kinase Research Articles

Identification of Target Antigens of Antifibroblast Antibodies in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) may be classified as idiopathic (IPAH) or familial (FPAH) or associated with various conditions and exposures such as dexfenfluramine intake (Dex-PAH) or systemic sclerosis (SSc-PAH). Because fibroblast dysfunction has been identified in SSc and IPAH and antifibroblast antibodies (AFAs) with a pathogenic role have been detected in the serum of SSc patients, we used a proteomic approach combining two-dimensional electrophoresis and immunoblotting to identify the target antigens of AFAs in such patients. To identify target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Sera from 24 patients with IPAH, 6 with FPAH, 6 with Dex-PAH, and 12 with SSc-PAH were collected. We pooled sera from sets of three patients with PAH classification and SSc-PAH based on autoantibody profile. Sera from 14 healthy blood donors were also pooled and used as a control. Serum IgG antibodies in the pools of patients with IPAH (n = 8), FPAH (n = 2), Dex-PAH (n = 2), and SSc-PAH (n = 4) recognized 103 +/- 31, 63 +/- 20, 78 +/- 11, and 81 +/- 12 protein spots, respectively, whereas serum IgG antibodies from healthy control subjects recognized 43 +/- 22 protein spots. Twenty-one protein spots were specifically recognized by the serum IgG antibodies from patients with PAH. We identified 16 of the protein spots as vimentin, calumenin, tropomyosin 1, heat shock proteins 27 and 70, glucose-6-phosphate-dehydrogenase, phosphatidylinositol 3-kinase, DAP kinase, and others. These proteins are involved in regulation of cytoskeletal function, cell contraction, oxidative stress, cell energy metabolism, and other key cellular pathways. AFAs detected in patients with PAH recognize cellular targets playing key roles in cell biology and maintenance of homeostasis.

DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress

Endothelial cells are actively involved in regulating the exchanges between blood and tissues. This function is tightly dependent on actin cytoskeleton dynamics and is challenged by a wide variety of stimuli, including oxidative stress. In endothelial cells, oxidative stress quickly activates the extracellular-signal-regulated kinase (ERK) MAP kinase, which results in the phosphorylation of tropomyosin. Here, we investigated further the mechanisms of tropomyosin phosphorylation and its function in actin remodeling. We identified, for the first time, death-associated protein kinase 1 (DAP kinase 1) as the kinase that phosphorylates tropomyosin-1 in response to ERK activation by hydrogen peroxide (H(2)O(2)). We also report that the phosphorylation of tropomyosin-1 mediated by DAP kinase occurs on Ser283. Moreover, the expression of the pseudophosphorylated tropomyosin mutant Ser283Glu triggers by itself the formation of stress fibers in untreated cells, and the effect is maintained in H(2)O(2)-treated cells in which DAP kinase expression is knocked-down by siRNA. By contrast, the expression of the nonphosphorylatable tropomyosin mutant Ser283Ala is not associated with stress fibers and leads to membrane blebbing in response to H(2)O(2). Our finding that tropomyosin-1 is phosphorylated downstream of ERK and DAP kinase and that it helps regulate the formation of stress fibers will aid understanding the role of this protein in regulating the endothelial functions associated with cytoskeletal remodeling.

DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress

The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP kinase (DAPk), a cell death promoting Ser/Thr protein kinase, plays a main role in oxidative stress-induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knockdown of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process that displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress.

Promoter Hypermethylation for Molecular Nodal Staging in Non–Small Cell Lung Cancer

Even among cases of non-small cell lung cancer (NSCLC) in the most favorable stage (IA), the disease-specific mortality is 25% or greater. One plausible explanation implicates the simplistic standard pathologic procedures used to designate lymph node involvement. A more sensitive assessment of the nodal status may improve staging. To determine the prognostic impact of detecting an abnormal molecular event (promoter hypermethylation in a set of relevant genes) in histologically uninvolved lymph nodes in resected NSCLC. In this retrospective analysis of archived material, we examined DNA extracted from lymph nodes of stage I NSCLC (n = 180). Patients underwent surgery between 1991 and 1995 in a single institution. Methylation-specific polymerase chain reaction was used to detect promoter hypermethylation in a panel of 8 genes. Survival data were extracted from the computerized database at the Tumor Registry. Evidence of promoter hypermethylation in at least 1 gene was detected in 67% of these N0 nodes. The most commonly hypermethylated gene was E-cadherin (53%). The hypermethylation frequency for the remaining genes were as follows: APC, 5%; p16, 9%; MGMT, 11%; hMLH1, 15%; RASSF1A, 4%; DAP kinase, 9%; and ATM, 19%. The presence of promoter hypermethylation in 2 or more genes did not influence the overall, median, or 5-year survival rates. Identifying promoter hypermethylation (in our panel) in N0 lymph nodes in stage I NSCLC cannot be recommended for clinical decision making. Molecular abnormalities, including those found in cancer by qualitative methylation-specific polymerase chain reaction, are not synonymous with established, histologically detectable metastasis.

Dlk/ZIP kinase, a novel Ser/Thr‐specific protein kinase with multiple functions

AbstractDlk/ZIP kinase is a member of the DAP kinase family which has been implicated in apoptosis. However, Dlk/ZIP kinase does not induce apoptosis per se. Rather, downregulation of Dlk/ZIP kinase by siRNA results in apoptosis and multinucleated cells suggesting that Dlk/ZIP kinase fulfills a vital function in processes other than apoptosis. Indeed, Dlk/ZIP kinase interacts with several transcription and splicing factors pointing to a role in transcription, presumably via chromatin modification. Moreover, it appears to participate in regulation of cell polarity and contractile processes in non‐muscle or smooth muscle cells. Finally, Dlk/ZIP kinase seems to play a role in mitosis, particularly cytokinesis. Thus, rather than being a proapoptotic kinase, Dlk/ZIP kinase participates in diverse and vital processes.

Frequent DAP kinase but not p14 or Apaf-1 hypermethylation in B-cell chronic lymphocytic leukemia

Dysregulation of apoptosis, and thus the p14/DAP kinase/HDM2/p53/Apaf-1 pathway, is potentially important in carcinogenesis. Chronic lymphocytic leukemia (CLL), uncommon in the Chinese, is a disease characterized by impaired apoptosis, of the neoplastic lymphocytes. Hypermethylation of p14, DAP kinase and Apaf-1 was studied by methylation-specific polymerase chain reaction (MSP) with primers for methylated (M-MSP) and unmethylated (U-MSP) alleles in 50 diagnostic marrow samples from patients with CLL. Chinese CLL patients had an indolent course similar to Caucasians with median overall survival (OS) of 96 months, which was adversely affected by advanced Rai stage (projected 5-year OS = 72% and 39% for Rai < or = 2 and Rai > 2; P = 0.01). DAP kinase was methylated in 18 (36%) patients while p14 and Apaf-1 were completely unmethylated in all the primary CLL samples. There was no correlation between DAP kinase hypermethylation and age, sex, poor-risk karyotype, lymphocyte count and Rai stage at diagnosis. Projected OS for patients with and without DAP kinase hypermethylation were 59 and 57% (P = 0.91). DAP kinase, but not p14 and Apaf-1, of the DAP kinase/p14/HDM2/p53/Apaf-1 pathway is frequently hypermethylated in CLL, but not of prognostic significance. Moreover Chinese patients with CLL share a similarly indolent clinical course, and this is the first comprehensive study on p14, DAP kinase and Apaf-1 hypermethylation in CLL.

E-cadherin and DAP kinase in pancreatic adenocarcinoma and corresponding lymph node metastases

E-cadherin and DAP kinase have been implicated as 'invasion suppressor' genes in human cancer. The aim of this study was to analyze the methylation status of E-cadherin and DAP kinase and the expression of the protein in the metastatic lesions and to compare it with the expression in the primary tumor. Methylation-specific PCR of the DAP kinase and E-cadherin promoter was performed in 28 primary adenocarcinomas of the pancreas and in 13 corresponding regional lymph node metastases. The presence of E-cadherin and DAP kinase protein was assessed by immunohistochemistry. Metastatic lymph nodes showed a significant different expression profile from the primary tumor. E-cadherin methylation was observed in 8/28 (29%) and loss of protein expression was observed in 16/28 (57%) of pancreatic carcinomas. E-cadherin methylation was observed in 7/13 (54%) and loss of protein expression was observed in 11/13 (85%) lymph node metastases (p=0.047). DAP kinase methylation occurred in 11/28 (39%) pancreatic carcinomas and loss of protein expression was observed in 13/28 (46%). DAP kinase was methylated in 6/13 (46%) lymph node meta-stases and loss of protein expression was observed in 10/13 (77%) (p=0.039). Comparing primary tumor and corresponding lymph node metastases in 13 cases, the status of E-cadherin methylation was discordant in 2 cases. The protein expression pattern of E-cadherin and DAP kinase was discordant in 4 and 3 cases respectively. Unmethylated tumor samples did not express E-cadherin in 12 and DAP kinase protein in 6 cases. Our results demonstrate that reduction of E-cadherin and DAP kinase expression is more frequent in lymph node metastases than in the primary tumor and methylation of the promoter region contributes to this reduction; however, an alternative mechanism of inactivation seems to exist.

DNA hypermethylation status of multiple genes in soft tissue sarcomas

The aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor-related genes. To determine the clinicopathological significance of gene promoter methylation in soft tissue sarcomas, we examined the promoter methylation status of 10 tumor-related genes in 65 soft tissue sarcomas and 19 adjacent non-neoplastic tissues by methylation-specific PCR. The methylation frequencies of tumor-related genes tested in soft tissue sarcomas were 17 (26%) for RASSF1A, 11 (17%) for DAP kinase, 10 (15%) for MGMT, nine (14%) for GSTP1, eight (12%) for PTEN, six (9%) for p16 and hMLH1, five (8%) for hMSH2, two (3%) for p14, and one (2%) for RB. Promoter methylation of these genes was not recognized in non-neoplastic tissues. All those cases of soft tissue sarcoma that had MGMT methylation, with the exception of one case of malignant peripheral nerve sheath tumor, showed large tumor size (≥10 cm) or recurrence. Moreover, eight of 10 cases with MGMT methylation revealed high American Joint Committee on Cancer stage. Seven of 10 cases (70%) with MGMT methylation showed a loss of MGMT expression by immunohistochemistry. In addition, MGMT methylation status had a statistically significant correlation with a loss of MGMT expression (P=0.014). In conclusion, although methylation of tumor-related genes was a relatively rare event in soft tissue sarcomas, methylation was tumor-specific. Of 10 tumor-related genes, cases with MGMT methylation had a tendency to be aggressive behavior. Moreover, MGMT methylation was closely associated with a loss of MGMT expression. Although our findings need to be extending to a large series, promoter methylation of tumor-related genes is likely to have an association with the pathogenesis of soft tissue sarcomas. Furthermore, MGMT methylation may be associated with tumor aggressiveness and the inactivation of MGMT gene.

Nuclear Localization of the Serine/Threonine Kinase DRAK2 Is Involved in UV-Induced Apoptosis

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2), a member of the DAP kinase family, is a serine/threonine kinase capable of inducing apoptosis. Here we studied the relationship between DRAK2 intracellular localization and apoptosis, and found that UV light acts as a stimulus for apoptosis induced by DRAK2. The intracellular location of DRAK2 depended on the cell line: DRAK2 was found primarily in the nuclei of NRK, NIH3T3, and Caco-2 cells while it was present primarily in the cytoplasm of ACL-15, HeLa, and WI-38 cells. Overexpression of Myc-tagged DRAK2 led to apoptosis-like cell death in NRK cells, but not in ACL-15 cells. A GFP fusion protein of DRAK2 was spontaneously localized to the nucleus of ACL-15 cells and resulted in cell death. Nuclear localization and cell death were also observed with DRAK2(1-293) fused to the NLS of SV40 but not with DRAK2(1-293) alone. These results suggested that nuclear accumulation of DRAK2 and the resulting increase in the endogenous level of its kinase activity are required for cell death. UV irradiation caused nuclear accumulation of endogenous DRAK2 in ACL-15 cells, which was followed by apoptosis-like cell death. Knockdown of DRAK2 expression by siRNA partially suppressed UV-induced apoptosis. These results suggest that DRAK2 plays a role in UV induced apoptosis.

The Tumor Suppressor DAP Kinase Is a Target of RSK-Mediated Survival Signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.

DRAK2, a lymphoid-enriched DAP kinase, regulates the TCR activation threshold during thymocyte selection

DAP kinases are a family of serine/threonine kinases known to regulate intrinsic apoptotic processes. DAP-related apoptotic kinase-2 (DRAK2) is highly expressed in lymphoid organs, with differential expression during thymocyte development. Low levels of transcript were observed in CD4/CD8 double-positive (DP) and double-negative populations, whereas single-positive thymocytes possessed elevated levels. Ex vivo stimulation of DP thymocytes with phorbol myristate acetate or antibodies that activate the TCR complex led to the accumulation of DRAK2 in a protein kinase C- and MAP Kinase-dependent fashion. Although DAP kinase family members are thought to potentiate apoptosis, ectopic expression of DRAK2 using retroviral transduction of primary T cells and NIH3T3 fibroblasts failed to decrease rates of survival, suggesting that DRAK2 expression is not sufficient to promote apoptosis. Rather, our results demonstrate that DRAK2 is a primary response gene activated by TCR stimulation in DP thymocytes. Further, we observed that DRAK2 controlled the threshold for calcium signaling in the thymus since positively selected Drak2-deficient thymocytes displayed a reduced requirement for TCR cross-linking. These findings are consistent with a role for DRAK2 in thymocyte selection and lymphoid maturation, and demonstrate that DRAK2 transduces non-apoptotic signals during thymocyte differentiation.

Hypermethylation of cyclin D2 and DAP kinase is associated with the lobular subtype of breast cancer

Hypermethylation of cyclin D2 and DAP kinase is associated with the lobular subtype of breast cancer

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

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

Biologie des cancers bronchiques

The bronchial multistep carcinogenesis is a cascade of molecular genetic alterations responsible for the histological continuum from squamous metaplasia to invasive bronchial carcinoma. These genetic alterations are responsible for the acquired properties of the transformed bronchial cell that lead to an invasive cancer. Many genes involved in the control of G1-S transition are mutated in lung cancer, such as the tumor suppressor genes Rb, p16, p14 ARF and p53. The later is an actual cellular gatekeeper at the crossroads of DNA repair control and apoptosis signaling, this process being activated when carcinogene–induced DNA alterations are irreversible. Other genes regulating apoptosis pathways are often altered in lung cancer such as RasSF1, the main tumor suppressor gene on 3p chromosome and the DAP kinase gene. Genes that are responsible for growth factors self sufficiency of bronchial cancer cells code for tyrosine kinase receptors to growth factors (EGF-R, HGF-R, erbB2 etc.) and can be amplified and/or mutated in lung carcinomas. Other genes promoting growth factors self sufficiency code for intra-cellular proliferative signal transduction proteins such as the GTPase Ras or the serine threonine kinase Raf, both mutated in a subset of adenocarcinomas. Many of these genes are also involved in angiogenesis signaling that is critical for tumor growth. At last, genes coding for cell adhesion and cell motility proteins are frequently altered in lung cancer. These are genes coding for receptors to extra-cellular matrix, for components of this matrix, for matrix proteases or their inhibitors, for cytoplasmic regulators of actin cytoskeleton, or secreted proteins controlling cell migration and therefore cancer cell metastasis. Such a molecular dissection of cell signalization involved in bronchial carcinogenesis will lead in a near future to the design of “biological modifiers” interfering with those signalizations. These “ targeted” inhibitors could be the bona fide candidates to be used in human therapeutics, as pro-apoptotic, anti-metastatic, or antigiogenic agents. They probably will take place in multi-modal therapeutic strategies, associated to classical cytotoxic chemotherapy, radiotherapy or as adjuvant treatments after surgery, in order to avoid tumoural relapses.

Aberrant DNA Methylation Patterns in Chronic Idiopathic Myelofibrosis.

Chronic idiopathic myelofibrosis (CIMF) is a clonal myeloproliferative disorder characterized by bone marrow fibrosis, angiogenesis and extramedullary hematopoiesis. No specific genetic defect underlying the disease has been identified so far. The spectrum of cytogenetic abnormalities in CIMF includes del (13q), del (20q) and partial trisomy 1q as well as abnormalities of chromosomes 1, 7 and 9. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function in cancerogenesis. In order to investigate the role of DNA methylation changes in the pathogenesis of CIMF, we have analyzed the methylation status of the promoter-associated CpG islands of 13 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in peripheral blood cells from 20 adult patients with CIMF. The frequency of aberrant methylation among the patient samples was 25.0 % (5/20) for SOCS-1 and 5.0 % (1/20) for E-cadherin, MGMT, p73 as well as TIMP-2. There was no hypermethylation of p15, p16, RARbeta, DAP kinase 1, SOCS-3, hMLH1, TIMP-3 and RASSF1A. We detected at least one hypermethylated gene promoter region in 35.0 % (7/20) of the primary patient samples. Our data indicate that hypermethylation of tumor suppressor genes is a common event in CIMF. Epigenetic modification of genes regulating growth factor signaling, cell adhesion and DNA repair may, in addition to genetic aberrations, contribute to the malignant phenotype in CIMF. The exploration of our growing knowledge about epigenetic aberrations in tumorigenesis may help develop novel strategies in diagnosis and treatment of CIMF for the future.

Death-associated protein kinase (DAP kinase) alteration in soft tissue leiomyosarcoma: Promoter methylation or homozygous deletion is associated with a loss of DAP kinase expression

The death-associated protein kinase ( DAP kinase) was initially identified as a positive mediator of programmed cell death induced by interferon-γ. To investigate the potential role and the alteration of the DAP kinase gene in soft tissue leiomyosarcoma (LMS), we first searched for homozygous deletion and promoter hypermethylation in 45 LMSs for which genomic DNA was available, using differential PCR and methylation-specific PCR, respectively. Promoter methylation was recognized in 10 of 45 cases (22%), and homozygous deletion was detected in 3 of 45 cases (7%). p53 mutation was detected in 11 of 45 LMS cases (24%). Cases with DAP kinase alteration or p53 mutation showed a close correlation with high French Federation of Cancer Centers grade or with poor prognosis ( P = 0.0244, P = 0.0491, respectively). Next, to determine that DAP kinase promoter methylation or homozygous deletion is involved in the down-regulation of DAP kinase expression, we examined the expression of DAP kinase protein by immunohistochemistry. Decreased expression of DAP kinase protein was recognized in 13 of 45 LMS cases (29%). Seven of 13 cases (54%) with decreased expression of DAP kinase protein revealed promoter methylation or homozygous deletion of DAP kinase, and the methylation status or homozygous deletion of its gene showed a close correlation with decreased DAP kinase expression ( P = 0.0300). In conclusion, although DAP kinase alteration was relatively rare, DAP kinase alteration and/or p53 mutation may associate with tumor progression in soft-tissue LMSs. Furthermore, although further detailed analyses are necessary, promoter methylation or homozygous deletion status of DAP kinase may present a major alternative mechanism of a loss of or decrease in DAP kinase expression.

DNA methylation changes in multiple myeloma.

Using a candidate gene approach, we analyzed the methylation status of the promoter-associated CpG islands of 11 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in five multiple myeloma (MM) cell lines and 56 patients with malignant plasma cell disorders. The frequency of aberrant methylation among the patient samples was 46.4% for SOCS-1, 35.7% for p16, 21.4% for E-cadherin, 12.5% for DAP kinase and p73, 1.8% for p15, MGMT as well as RARbeta, and 0% for TIMP-3, RASSF1A and hMLH1. We found at least one hypermethylated gene in 80.4% of the primary patient samples, while 33.9% harbored two or more hypermethylated genes. For the first time, we show that p73 may be hypermethylated in MM and thus be involved in the pathogenesis of plasma cell disorders. Hypermethylation of p16 at diagnosis was associated with a poorer prognosis. In patients with plasma cell leukemia, we found frequent simultaneous hypermethylation of p16, E-cadherin and DAP kinase. We conclude that aberrant methylation of tumor suppressor genes is a common event in malignant plasma cell disorders and that there is a correlation between methylation patterns and clinical characteristics in MM patients.

Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells.

Type V collagen is a "minor" component of normal human breast stroma, which is subjected to over-deposition in cases of ductal infiltrating carcinoma (DIC). We reported that, if used as a culture substrate for the DIC cell line 8701-BC, it exhibited poorly-adhesive properties and restrained the proliferative and motile behavior of the cell subpopulation able to attach onto it. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of 8701-BC cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by type V collagen was of the apoptotic type by (i) microscopic detection and quantitation of Apoptag-labeled cells, (ii) analysis of the expression levels of selected genes coding for apoptosis-linked factors, caspases, and stress-response proteins by conventional and semi-quantitative multiplex PCR, and (iii) evaluation of the extent of caspase activation by chromogenic assay. We report here that type V collagen is able to determine an increase in the percentage of Apoptag-positive cells, to up-regulate Bcl-xS, Bad, Dap kinase, hsf-1, mthsp75, caspase-1, -5, -8, -9, and -14, whilst down-regulating Bcl-2, Bcl-xbeta, and hsp60. Treatment of cell lysates with chromogenic tetrapeptide substrates specific for caspase-1, -5, -8, and -9 demonstrated a marked increase of enzymatic activity in the presence of type V collagen. Our data validate 8701-BC cell line as a suitable "in vitro" model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on primary DIC cells.

DAP-kinase as a target for drug design in cancer and diseases associated with accelerated cell death

Misregulated cell death, which can result in either the excessive, inappropriate elimination of cells, or in the insufficient removal of damaged or malignant cells, has been associated with numerous diseases. Here we discuss an important molecular regulator of cell death, DAP-kinase (DAPk), which presents a promising target for therapeutic intervention. A structure-functional analysis of this calcium-regulated Ser/Thr kinase which promotes cell death will be presented, and emphasis will be placed on particular disease models in which its modulation might affect clinically-relevant cell death processes.

DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation.

DAP-kinase (DAPk) is a Ser/Thr kinase that regulates cytoplasmic changes associated with programmed cell death. It is shown here that a GFP-DAPk fusion, which partially localized to actin stress fibers, induced extensive membrane protrusions. This phenotype correlated with changes in myosin-II distribution and with increased phosphorylation of the myosin-II regulatory light chain (RLC). A mutant lacking the cytoskeletal-interacting region (GFP-DAPkDeltaCyto) displayed diffuse cytoplasmic localization, and induced peripheral membrane blebbing, instead of the extensive protrusions. In contrast, deletion of the ankyrin repeats led to mislocalization of the kinase to focal contacts, where it failed to elicit any changes in cell morphology. While both wild-type DAPk and DAPkDeltaCyto induced RLC phosphorylation independently of the Rho-activated kinase ROCK, only the wild type led to increases in stress-fiber associated phospho-RLC. Thus, the precise intracellular localization of DAPk is critical for exposure to its substrates, including the RLC, which mediate varying morphologic changes.