TNF-alpha can stimulate a variety of kinases with the ability to activate non-muscle myosin II. As a result, increases in actin filament formation and actomyosin contractility (AMC) have been reported in response to TNF-alpha. These events are thought to play an important role in mediating TNF-alpha induced apoptosis but how they do so is unclear. In this study we prevented non-muscle myosin II activation in response to TNF-alpha by treating cells with the myosin light chain kinase (MLCK) inhibitor ML-7 or through isoform specific siRNA knockdown of myosin IIA and IIB. We found that treatment with ML-7 or knockdown of myosin IIB, but not IIA, impaired the cleavage of caspase 3 and caspase 8 as well as nuclear condensation in response to TNF-alpha. During this cell death process myosin II seemed to function independent of AMC since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF-alpha induced caspase cleavage. Immunoprecipitation studies revealed associations of myosin IIB with clathrin and FADD in response to TNF-alpha suggesting a role for myosin IIB in TNFR1 endocytosis and DISC formation. Taken together these findings suggest that myosin IIB activation promotes TNF-alpha cell death signaling in a manner independent of its force generating property.

DAPK-1 is calcium-calmodulin regulated protein kinase involved in multiple cellular pathways including apoptosis, autophagy, cell survival and motility. The cytokine TNF-α has been reported to induce the degradation of DAPK-1. Here I identified the protease cathepsin B as a novel binding partner of DAPK-1 that protects DAPK-1 from TNF-α induced degradation. Using deletion mutants of DAPK-1, I mapped the cathepsin B binding domain on DAPK-1 to amino acids 836-947. Overexpression of this mini-protein DAPK-1(836-947) facilitated degradation of full-length DAPK-1 and apoptosis induced by TNFR-1. Moreover, siRNA mediated knock-down of DAPK-1 enhanced TNF-α induced apoptosis, confirming the role of DAPK-1 as a survival factor in the TNF-α signalling pathway. In addition, a splice variant of DAPK-1, which I have called s-DAPK-1, was discovered. s-DAPK-1 shares part of DAPK-1’s ankyrin repeats region and cytoskeletal binding domain, and possesses an unique tail region, which contains a cleavage site at its first two amino acids. Unlike DAPK-1, s-DAPK-1 does not contribute to apoptosis induced by high level of MEK/ERK signalling, but it does mimic DAPK-1’s function to induce membrane blebbing. The proteolytically processed form of s-DAPK-1 is more active in the induction of membrane blebbing, which may be due to its higher stability compared to that of full-length s-DAPK-1, suggesting that the tail region can control s-DAPK-1 stability and activity. Co-transfection of s-DAPK-1 and DAPK-1 leads to reduction in DAPK-1 expression level, suggesting a role for s-DAPK-1 to regulate DAPK-1 stability. The kinase domain of DAPK-1 is the region required for s-DAPK-1 to promote DAPK-1 degradation. Surprisingly, s-DAPK-1 does not bind directly to DAPK-1, suggesting that the interaction is indirect and mediated by as yet unidentified accessory proteins. Finally, the experiments with proteasome and lysosome inhibitors indicated that s-DAPK-1 induces DAPK-1 degradation via both lysosome and proteasome pathways.

DNR pažaidų atitaisymas ir apoptozė - dvi pagrindinės grandys, palaikančios žmogaus genomo vientisumą. Sutrikus šiems procesams, ląstelė išgyvena, nepaisant susikaupusių DNR pažaidų ir sudaromas pagrindas tolesnei transformacijai. Tyrimu įvertinome DNR pažaidų atitaisymo funkcijoje dalyvaujančių (hMLH1, MGMT) ir su apoptoze susijusių (DAPK-1, CASP8) genų epigenetinio reguliavimo - metilinimo aspektus pakopiniame skrandžio onkogenezės procese. Šio mokslinio tyrimo metu pirmą kartą buvo nustatytas skirtingas hMLH1 geno metilinimo dažnis atskirose skrandžio anatominėse dalyse atrofiniu pangastritu sergančiųjų audinyje. Įvertinta, kad hMLH1 geno metilinimas sergančiųjų skrandžio vėžiu aplinkiniame nenavikiniame audinyje sietinas su pacientų amžiumi. Išgyvenamumo analizės rezultatai parodė, kad MGMT geno metilinimas agresyvios skrandžio vėžio histologinės formos atveju yra geresnės prognozės rodiklis. Tyrimo metu nustatėme mokslinėje periodikoje neaprašytų tirtųjų genų metilinimo derinių sąsajų su klinikiniais, morfologiniais ir prognoziniais onkologinės ligos ypatumais. / DNA repair and apoptosis are two main pathways supporting the integrity of human genome. After the disturbance of these processes the cell survives, despite the accumulation of DNA lesions, and in this way a basis for a subsequent transformation is formed. In our research we evaluated the epigenetic regulation - methylation - aspects of genes participating in DNA repair function (hMLH1 and MGMT) and also of apoptosis-related genes (DAPK-1, CASP8) in relation to a stepwise gastric oncogenesis process. During this investigation a different hMLH1 gene methylation observation frequency in tissues obtained from separate anatomical parts of the stomach in atrophic pangastritis patients was determined for the first time. It was estimated, that hMLH1 gene methylation in tumor-surrounding non-cancerous tissue in gastric cancer patients could be associated with patient age. Results of survival analysis indicated that MGMT gene methylation is an indicator of better prognosis in case of diffuse form of gastric cancer. During the study we determined some additional associations (not described in previous publications) between methylation combinations of analyzed genes and clinical, morphological and prognostic features of oncological illness.

Indiana University-Purdue University Indianapolis (IUPUI) / Modulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.

Indiana University-Purdue University Indianapolis (IUPUI) / Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.