Non-apoptotic Pathways Research Articles

Abstract 5119: Smac mimetic inhibits tumorigenicity and growth of glioblastoma by promoting differentiation of glioblastoma cancer stem-like cells

Abstract Inhibitors of apoptosis (IAP) proteins are often overexpressed in human cancers including glioblastoma (GBM) and are well characterized for their antiapoptotic properties. Recently, a broader role of IAP proteins in tumor biology has been described, as they also regulate several non-apoptotic signal transduction pathways. Therefore, we investigated the effect of non-toxic concentrations of small-molecule Smac mimetics, which antagonize IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary GBM specimens. Since CSLCs have been implicated in resistance to chemo- and radiotherapy and tumor recurrence, they are considered as critical therapeutic targets in GBM. Here, we identify a novel function of Smac mimetic in regulating differentiation of GBM CSLCs by activating NF-κB. Smac mimetic at non-lethal doses stimulates morphological changes associated with differentiation of GBM CSLCs. Smac mimetic increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that Smac mimetic induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that Smac mimetic triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks the Smac mimetic-stimulated increase in GFAP and differentiation. Interestingly, this Smac mimetic-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, Smac mimetic does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, Smac mimetic treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models of GBM and significantly increases the survival of mice bearing GBM. By identifying a novel role of Smac mimetic in promoting the differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs. Citation Format: Aurelie Tchoghandjian, Claudia Jennewein, Ines Eckhardt, Stefan Momma, Dominique Figarella-Branger, Simone Fulda. Smac mimetic inhibits tumorigenicity and growth of glioblastoma by promoting differentiation of glioblastoma cancer stem-like cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5119. doi:10.1158/1538-7445.AM2014-5119

Mitochondria-derived reactive oxygen species mediate caspase-dependent and -independent neuronal deaths.

Mitochondrial dysfunction and oxidative stress are implicated in many neurodegenerative diseases. Mitochondria-targeted drugs that effectively decrease oxidative stress, protect mitochondrial energetics, and prevent neuronal loss may therefore lend therapeutic benefit to these currently incurable diseases. To investigate the efficacy of such drugs, we examined the effects of mitochondria-targeted antioxidants MitoQ10 and MitoE2 on neuronal death induced by neurotrophin deficiency. Our results indicate that MitoQ10 blocked apoptosis by preventing increased mitochondria-derived reactive oxygen species (ROS) and subsequent cytochrome c release, caspase activation, and mitochondrial damage in nerve growth factor (NGF)-deprived sympathetic neurons, while MitoE2 was largely ineffective. In this paradigm, the most proximal point of divergence was the ability of MitoQ10 to scavenge mitochondrial superoxide (O2(-)). MitoQ10 also prevented caspase-independent neuronal death in these cells demonstrating that the mitochondrial redox state significantly influences both apoptotic and nonapoptotic pathways leading to neuronal death. We suggest that mitochondria-targeted antioxidants may provide tools for delineating the role and significance of mitochondrial ROS in neuronal death and provide a new therapeutic approach for neurodegenerative conditions involving trophic factor deficits and multiple modes of cell death.

Carbamoylating activity associated with the activation of the antitumor agent laromustine inhibits angiogenesis by inducing ASK1-dependent endothelial cell death.

The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis.

The Many Roads to Cell Death: Gaining a Practical Understanding of Apoptosis, Necrosis, and Autophagy

Cell death is, ironically, an essential part of life. In recent years, the study and understanding of cell death pathways has been dramatically transformed by the insights gained into non-apoptotic pathways, including necro-apoptosis and autophagy, together with a deeper understanding of the mechanism of the apoptotic cascade. New discoveries have been enabled by cutting-edge technologies, particularly in the realm of cytometry and cell-death–specific markers. In this webinar, the latest insights into cell death pathways will be discussed, including the molecular markers and cellular changes that characterize each pathway. Viewers will also learn practical cytometry-based strategies for dissecting cell death pathways, and how to use the data to better understand the pathophysiology of diseases such as cancer as well as to uncover new targets for drug discovery and development.

Histiocytoid Sweet Syndrome With Haloed Myeloid Cells Masquerading as a Cryptococcal Infection

: Histiocytoid Sweet syndrome is a histopathologic variant of Sweet syndrome characterized by an infiltrate of mononuclear cells that have a histiocytic appearance and represent immature granulocytes. The primary histopathologic differential diagnosis for histiocytoid Sweet syndrome includes leukemia cutis and true histiocytic dermatoses. However, it does not usually include a deep mycotic infection. Herein, we describe a case of histiocytoid Sweet syndrome in a 75-year-old man in which histopathologic examination showed a dense dermal infiltrate composed of mature neutrophils and numerous yeast-like mononuclear cells with a surrounding halo, suspicious for cryptococcal organisms. Immunohistochemical and histochemical studies demonstrated expression of myeloperoxidase by the yeast-like forms and an absence of periodic acid-Schiff and mucicarmine staining. Microbiologic culture studies were negative for fungal organisms. The case met all the criteria for Sweet syndrome and, given the cytomorphology of the mononuclear cells with vesicular irregularly rounded nuclei and myeloperoxidase expression, the case was most consistent with histiocytoid Sweet syndrome. The perinuclear haloes in this case most likely represent cytoplasmic vacuolization of the immature, histiocytoid appearing neutrophils secondary to a novel nonapoptotic caspase-independent death pathway. This case highlights the histopathologic similarities that may be present between cryptococcal infection and histiocytoid Sweet syndrome, and the utility of immunohistochemical markers and histochemical staining to differentiate between the two entities.

The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation

Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.

Cancer Therapy: Targeting Mitochondria and other Sub-cellular Organelles

Tumour cell death is required for the clearance of malignant cells and is a vital part of the mechanism of natural tumour suppression. Cancer cells, having acquired multiple deregulated pathways involving several cellular oragenelles, are capable of disrupting these normally finely tuned processes thereby evading both physiological and therapeutic intervention. Although current available data indicate the dependence of successful tumour cell clearance on classical apoptotic pathways (intrinsic and/or extrinsic pathways), there is now evidence suggesting that alternative apoptotic and non-apoptotic pathways may effectively contribute to tumour cell death. The mitochondria, proteasomes, endoplasmic reticulum, Golgi apparatus, lysosomes and lysosome-related organelles of tumour cells exhibit a number of deregulations which have been identified as potential druggable targets for successful rational drug design and therapy. In this review, we summarise the roles of these cellular organelles in tumour initiation and establishment as well as current trends in development of agents that target deregulations in these organelles.

Regulated necrosis: the expanding network of non-apoptotic cell death pathways

Cell death research was revitalized by the understanding that necrosis can occur in a highly regulated and genetically controlled manner. Although RIPK1 (receptor-interacting protein kinase 1)- and RIPK3-MLKL (mixed lineage kinase domain-like)-mediated necroptosis is the most understood form of regulated necrosis, other examples of this process are emerging, including cell death mechanisms known as parthanatos, oxytosis, ferroptosis, NETosis, pyronecrosis and pyroptosis. Elucidating how these pathways of regulated necrosis are interconnected at the molecular level should enable this process to be therapeutically targeted.

Methyl jasmonate: putative mechanisms of action on cancer cells cycle, metabolism, and apoptosis.

Methyl jasmonate (MJ), an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1) arrests cell cycle, inhibiting cell growth and proliferation, (2) causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis) pathways, (3) detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4) induces reactive oxygen species mediated responses, (5) stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6) inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7) inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents.

Chapter Nine - Structural Studies of Death Receptors

This chapter describes reports of the structural characterization of death ligands and death receptors (DRs) from the tumor necrosis factor (TNF) and TNF receptor families. The review discusses the interactions of these proteins with agonist ligands, inhibitors, and downstream signaling molecules. Though historically labeled as being implicated in programmed cell death, the function of these proteins extends to nonapoptotic pathways. The review highlights, from a structural biology perspective, the complexity of DR signaling and the ongoing challenge to discern the precise mechanisms that occur at the point of DR activation, including how the degree to which the receptors are induced to cluster may be related to the nature of the impact upon the cell. The potential for posttranslational modification and receptor internalization to play roles in DR signaling is briefly discussed.

Abstract 102: The Detrimental Role Of iPLA2β In Ischemia/reperfusion Injury

Background: Ca 2+ -independent phospholipase A2 (iPLA2) is crucial for a caspase-independent non-apoptotic cell death signaling pathway and involved in nuclear shrinkage induced by hypoxia. PLA2 family hydrolyzes the sn-2 ester bond in phospholipids to release free fatty acids and lysophospholipids. PLA2 inhibitors are known to prevent ischemic-reperfusion (I/R) injury. Here, we attempted to clarify the role of iPLA2β in myocardial I/R injury using cardiac-specific iPLA2β-deficient mice. Methods: Pla2g6 gene-targeting vector was constructed using mouse BAC genomic library. The ES clones with targeted homologous recombination were injected into blastocyst mouse embryos. Floxed iPLA2β mice were crossed with transgenic mice expressing Cre recombinase under the control of the α-myosin heavy chain ( αMHC ) promoter to generate cardiac-specific iPLA2β-deficient mice ( pla2g6 flox/flox αMHC-Cre + : CKO). We used pla2g6 flox/flox αMHC-Cre - littermates as controls (CTRL). We performed transthoracic M-mode echocardiographic analysis to examine the cardiac function of CKO and CTRL. Next, we subjected CKO and CTRL to I/R injury by using Langendorff-perfused mouse heart model. The heart was subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. After the reperfusion, infarct area was determined with 1% triphenyltetrazolium chloride, and apoptotic cell death was detected by in situ TUNEL assay on heart sections. We exposed isolated adult cardiomyocytes from CKO and CTRL to various stimuli, such as Ca 2+ ionophore, H 2 O 2 and staurosporine, to examine the involvement of necrosis and apoptosis. Results: CKO and CTRL mice were born at the expected Mendelian ratio. There were no differences in cardiac function between CKO and CTRL. The functional recovery during reperfusion was significantly greater in CKO than in CTRL. CKO exhibited a significant decrease in the size of myocardial infarct. There were no significant differences in necrotic cell death induced by necrotic stimuli in vitro, while apoptotic cell death tended to decrease in CKO heart. Conclusions: These results suggest that iPLA2β exacerbates apoptotic cell death in I/R.

The dark side of TRAIL signaling

The dark side of TRAIL signaling

Innate immunity in the Grid2 Lc/+ mouse model of cerebellar neurodegeneration: glial CD95/CD95L plays a non-apoptotic role in persistent neuron loss-associated inflammatory reactions in the cerebellum

BackgroundThere is growing evidence that the death receptor CD95 has a wider role in non-apoptotic functions. In the brain, it may contribute to neural death and to the associated inflammatory reaction via a non-apoptotic pathway. Brain injury triggers an inflammatory reaction in which the CD95/CD95L system acts principally through peripheral cells recruited to the lesion. In cases of inflammation within the brain, with no blood–brain barrier leakage, the role of the CD95/CD95L system is thus unclear. We investigated the possible role of CD95 and CD95L in such conditions, by studying the relationships between glial cell activation, neuron death and CD95/CD95L expression in the cerebellum of the Lurcher (Grid2Lc/+) mutant mouse, a model of cerebellar neurodegeneration.MethodsGlial cells in slices of wild-type and Lurcher mouse cerebella were observed by light microscopy at various ages overlapping periods of neuron loss and of pre- and post-neurodegeneration. Subcellular organization was studied by electron microscopy. We assessed CD95 levels by western blotting, RT-PCR and glial cell cultures. The levels of CD95L and IL-6 were studied by ELISA and a biological assay, respectively.ResultsIn the Grid2Lc/+cerebellum, neuron loss triggers a typical, but abnormally persistent, inflammatory reaction. We identified two phases of astrogliosis: an early burst of large glial cell activation, peaking at postnatal days 25 to 26, coinciding with peak cerebellar neuron loss, followed by a long period of slow decline indicating that the strength of the glial reaction is modulated by neuron mortality rates. Comparisons of time-courses of glial cell activation, cytokine production and neuron loss revealed that the number of surviving neurons decreased as CD95 increased. Thus, CD95 cannot be directly involved in neuron death, and its role must be limited to a contribution to the inflammatory reaction. The upregulation of CD95 likely on astrocytes coincides with increases in the levels of IL-6, a cytokine produced principally by astrocytes, and soluble CD95L.ConclusionsThese results suggest that CD95 and soluble CD95L contribute, via non-apoptotic signaling, to the inflammatory reaction initiated early in neuron death within the Grid2Lc/+ cerebellum.

Abstract B05: Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells

Abstract The development of anti-cancer therapeutics that target non-apoptotic pathways has received increasing attention from the scientific community in recent years. The prevalence of chemoresistance and the characterization of defective apoptotic pathways in cancer has promoted interest in the expansion of current therapeutic regimens to include drugs that induce non-apoptotic cell death. From a phenotypic screen we have identified a small molecule named C6 that induces caspase-independent, non-apoptotic cell death in chemoresistant patient-derived breast cancer cells. Additionally, C6 is also selectively cytotoxic against cancer cells compared to normal mammary epithelial cells. In an effort to characterize this small molecule's mechanism of action and identify relevant biological pathways that might be used as therapeutic drug targets, we have utilized a photoaffinity pull-down strategy to identify biological binding partners of C6. Our photoaffinity pull-down studies have revealed Mitsugumin 23 (MG23), an endoplasmic reticulum-bound transmembrane protein capable of ion channel formation, as a binding partner for C6. Additionally, we have identified a metabolic component of C6-induced cell death through the use of mitochondrial respiration measurements, metabolomic analyses, and mitochondrial transmission electron microscopy (TEM) imaging. Metabolic studies have identified mitochondrial respiration defects, excess lactic acid production, and gross changes in mitochondrial morphology as a result of C6 treatment. Collectively, our findings suggest a role for ionic imbalance and subsequent metabolic disruption in this form of caspase-independent cell death. Our ongoing work is focused on further mechanistic characterization of this non-apoptotic pathway as a potential target for breast cancer drug development. Citation Format: Rachel M. Vaden, Keith M. Gligorich, Dawne N. Shelton, Guoying Wang, Cindy B. Matsen, Ryan E. Looper, Matthew S. Sigman, Bryan E. Welm. Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B05.

The death receptor Fas mediates a non-apoptotic signaling cascade in apoptosis resistant CD4+ T cells via the MAPK pathway. (P1304)

Abstract The Fas death receptor mediates peripheral tolerance by apoptotic deletion of autoreactive T cells through Restimulation Induced Cell Death (RICD). Previously, we have shown that within a pool of T cells, effector memory were more sensitive to Fas-induced apoptosis than activated naïve CD4 T cells due to efficient DISC formation. Recently, we found that FasL induces an early MAPK response in the form of pJNK activation, preferentially in activated naïve, but not memory T cells. In apoptosis resistant FADD or Caspase-8 deficient Jurkat T cells, the pJNK response is exaggerated indicating that in the absence of a functional Fas apoptotic response, an alternate non-apoptotic pathway is elicited. Further, in HH cells, a CTCL resistant to Fas apoptosis, pJNK activation was upregulated when compared to HUT78, a highly apoptosis sensitive cell line. In an attempt to identify the upstream activators of Fas-induced pJNK, we studied the role of the kinase RIP1, a known component of the Fas DISC. Inhibition of RIPK1 decreased pJNK activation in Jurkat cells and RIP deficient Jurkat cells were unable to activate pJNK upon FasL stimulation. Immunoprecipitation studies also indicated a preferential recruitment of RIPK1 to the Fas DISC in apoptosis resistant T cells. These preliminary results indicate a role for Fas mediated non-apoptotic MAPK signaling in early activated naïve T cells that precedes its traditional apoptotic role in the elimination of effector memory cells.

Identification of a small molecule that induces ATG5-and-cathepsin-l-dependent cell death and modulates polyglutamine toxicity

Non-apoptotic cell death mechanisms are largely uncharacterized despite their importance in physiology and disease [1]. Here we sought to systematically identify non-apoptotic cell death pathways in mammalian cells. We screened 69,612 compounds for those that induce non-canonical cell death by counter screening in the presence of inhibitors of apoptosis and necrosis. We further selected compounds that require active protein synthesis for inducing cell death. Using this tiered approach, we identified NID-1 (Novel Inducer of Death-1), a small molecule that induces an active, energy-dependent cell death in diverse mammalian cell lines. NID-1-induced death required components of the autophagic machinery, including ATG5, and the lysosomal hydrolase cathepsin L, but was distinct from classical macroautophagy. Since macroautophagy can prevent cell death in several contexts, we tested and found that NID-1 suppressed cell death in a cell-based model of Huntington's disease, suggesting that NID-1 activates a specific pathway. Thus the discovery of NID-1 identifies a previously unexplored cell death pathway, and modulating this pathway may have therapeutic applications. Furthermore, these findings provide a proof-of-principle for using chemical screening to identify novel cell death paradigms.

Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury

Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol-induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol-induced hepatocyte injury or inflammation. Because receptor-interacting protein kinase (RIP) 3-mediated necroptosis, a nonapoptotic cell death pathway, is implicated in a variety of pathological conditions, we tested the hypothesis that ethanol-induced liver injury is RIP3-dependent and RIP1-independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)-deficient mice, indicating CYP2E1-mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol-induced steatosis, hepatocyte injury, and expression of proinflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding, and inhibition of RIP1 kinase by necrostatin-1 did not attenuate ethanol-induced hepatocyte injury. Ethanol-induced apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive nuclei and accumulation of cytokeratin-18 fragments in the liver, was independent of RIP3. CYP2E1-dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol-induced hepatocyte injury is RIP3-dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy.

Efficacy and safety of bendamustine for the treatment of patients with recurring Hodgkin lymphoma

The management of patients with Hodgkin lymphoma (HL) recurring after stem cell transplantation (SCT) and multiply relapsed disease remains challenging. We report on 41 such patients who received bendamustine hydrochloride, a bifunctional mechlorethamine derivative mechanistically unrelated to traditional alkylators, after a median of four prior chemotherapy lines, including SCT in 85% of cases. Bendamustine was given at doses of 90-120 mg/m(2) every 21 or 28 d. At first assessment (2-4 cycles), the overall response rate (ORR) was 78% with 12 (29%) complete (CR) and 20 (49%) partial responses (PR). Upon treatment prolongation to 6-8 courses, 40% of PRs progressed, yielding a final ORR of 58% with 31% of CRs. Eight patients (two CRs, six PRs) were subsequently allotransplanted. Median progression-free and overall survival exceeded 11 and 21 months respectively; complete responders displayed a median disease-free survival above 9 months with a relapse rate of only 30%. Outcomes were independent of disease chemosensitivity, previous transplant and bendamustine dose-intensity. No life-threatening or unexpected adverse events occurred. Within the limits of a retrospective analysis and schedule heterogeneity, these results appear very encouraging and prompt prospective trials to confirm bendamustine as a valuable option in the palliative setting and in cytoreductive strategies before allotransplantation.

Fas activates lipolysis in a Ca2+-CaMKII-dependent manner in 3T3-L1 adipocytes

Fas (CD95) is a member of the tumor necrosis factor (TNF) receptor superfamily and plays a crucial role in the induction of apoptosis. However, like TNF, Fas can induce nonapoptotic signaling pathways. We previously demonstrated that mice lacking Fas specifically in adipocytes are partly protected from diet-induced insulin resistance, potentially via decreased delivery of FAs to the liver, as manifested by lower total liver ceramide content. In the present study, we aimed to delineate the signaling pathway involved in Fas-mediated adipocyte lipid mobilization. Treatment of differentiated 3T3-L1 adipocytes with membrane-bound Fas ligand (FasL) significantly increased lipolysis after 12 h without inducing apoptosis. In parallel, Fas activation increased phosphorylation of ERK1/2, and FasL-induced lipolysis was blunted in the presence of the ERK-inhibitor U0126 or in ERK1/2-depleted adipocytes. Furthermore, Fas activation increased phosphorylation of the Ca(2+)/calmodulin-dependent protein kinases II (CaMKII), and blocking of the CaMKII-pathway (either by the Ca(2+) chelator BAPTA or by the CaMKII inhibitor KN62) blunted FasL-induced ERK1/2 phosphorylation and glycerol release. In conclusion, we propose a novel role for CaMKII in promoting lipolysis in adipocytes.

313 Interaction of Inflammation and Hyperoxia in Neonatal White Matter Damage

Intrauterine infection/inflammation are major causes of preterm birth. The dramatic rise of oxygen tissue tension compared to intrauterine conditions amounts to relative hyperoxia in preterm infants. Both, infection/inflammation and hyperoxia...