MALT1-dependent cleavage of CYLD promotes NF-κB signaling and growth of aggressive B-cell receptor-dependent lymphomas.

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies.

Clinical Utility of Plasma-Based Comprehensive Molecular Profiling in Advanced Non-Small-Cell Lung Cancer.

Comprehensive molecular profiling (CMP) plays an essential role in clinical decision making in metastatic non-small-cell lung cancer (mNSCLC). Circulating tumor DNA (ctDNA) analysis provides possibilities for molecular tumor profiling. In this study, we aim to explore the additional value of centralized ctDNA profiling next to current standard-of-care protocolled tissue-based molecular profiling (SoC-TMP) in the primary diagnostic setting of mNSCLC in the Netherlands. METHODS: Pretreatment plasma samples from 209 patients with confirmed mNSCLC were analyzed retrospectively using the NGS AVENIO ctDNA Targeted Kit (Roche Diagnostics, Basel, Switzerland) and compared with paired prospective pretreatment tissue-based molecular profiling from patient records. The AVENIO panel is designed to detect single-nucleotide variants, copy-number variations, insertions or deletions, and tyrosine kinase fusion in 17 genes. RESULTS: Potentially targetable drivers were detected with SoC-TMP alone in 34.4% of patients. Addition of clonal hematopoiesis of indeterminate potential-corrected, plasma-based CMP increased this to 39.7% (P < .001). Concordance between SoC-TMP and plasma-CMP was 86.6% for potentially targetable drivers. Clinical sensitivity of plasma-CMP was 75.2% for any oncogenic driver. Specificity and positive predictive value were more than 90% for all oncogenic drivers. CONCLUSION: Plasma-CMP is a reliable tool in the primary diagnostic setting, although it cannot fully replace SoC-TMP. Complementary profiling by combined SoC-TMP and plasma-CMP increased the proportion of patients who are eligible for targeted treatment.

Schedule-Dependent Synergy Between the Histone Deacetylase Inhibitor Belinostat and the Dihydrofolate Reductase Inhibitor Pralatrexate in T-and B-cell Lymphoma Cells in vitro.

Pralatrexate (Folotyn; PLX) and belinostat (Beleodaq; BLS) are registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and are being considered for other lymphomas. In this study we investigated whether BLS had the ability to potentiate the cytotoxicity of PLX. A panel of lymphoma cell lines was used for the combination studies: the B-cell SUDHL-4, SUDHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. Uptake of PLX was mediated by the reduced folate carrier (RFC). PLX showed a 6-fold better RFC substrate affinity compared to methotrexate, and 2-fold better than levoleucovorin (l-LV). Sensitivity expressed as the concentration that resulted in 50% growth inhibition (IC50) after 72 hr exposure to PLX varied from 2.8 to 20 nM and for BLS from 72 to 233 nM, independent of the background of the cell lines. The interaction between BLS and PLX was studied using the median-drug effect analysis. At a fixed molar ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: around 1.0). In three selected cell lines (SUDHL-4, SUDHL-5, and HT) sequential exposure (24 h pretreatment with BLS, followed by 48 h to PLX + BLS), did not improve interaction (CI: 0.9-1.4). As an alternative approach a non-fixed ratio was used by exposing SUDHL-4, SUDHL-5, and HT cells to IC25 concentrations of either BLS or PLX in combination with the other drug. Exposure to IC25 of PLX did not decrease the IC50 for BLS (CI from 0.6-1.2), but exposure to IC25 of BLS markedly increased PLX sensitivity (low CIs from 0.40 to 0.66). Mechanistic studies focused on induction of apoptosis, and showed cleavage of predominantly caspase-9 in HT and SUDHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of PLX and BLS showed additivity in various lymphoma cell lines, with a schedule-dependent synergism in B-cell lymphoma. Based on these data, proficient inhibition of HDAC activity by BLS holds promise in sensitization of tumor cells to PLX.

A New and Validated Clinical Prognostic Model (EPI) for Enteropathy-Associated T-cell Lymphoma.

PURPOSE: Enteropathy-associated T-cell lymphoma (EATL) is a rare intestinal non-Hodgkin lymphoma with a poor, though variable prognosis. The International Prognostic Index (IPI) and the prognostic index for peripheral T-cell lymphoma (PIT) have limited predictive value for outcome of EATL. The purpose of this study was to develop and validate a prognostic model for EATL, which can identify high-risk patients who need more aggressive therapy. EXPERIMENTAL DESIGN: This retrospective multicenter study was based on 92 patients and included 45 patients diagnosed with EATL between 1999 and 2009 from the Netherlands and 47 patients from England and Scotland, diagnosed with EATL between 1994 and 1998. A new EATL prognostic index (EPI) was constructed using the RPART (recursive partitioning and regression trees) procedure. Validation was performed applying the bootstrap method. RESULTS: Three risk groups were distinguished (P < 0.0001): a high-risk group, characterized by the presence of B-symptoms [median overall survival (OS) of 2 months]; an intermediate-risk group, comprising patients without B-symptoms and an IPI score ≥ 2 (7 months); and a low-risk group, representing patients without B-symptoms and an IPI score of 0 to 1 (34 months). Internal validation showed stability of statistical significance and prognostic discrimination. In contrast with the IPI and PIT, the EPI better classified patients in risk groups according to their clinical outcome. CONCLUSIONS: Our new, validated, prognostic model EPI accurately predicts survival outcome in EATL and may be used for patient selection for new therapeutic strategies and evaluation of clinical trials.

Treatment response in enteropathy associated T-cell lymphoma; survival in a large multicenter cohort.

Enteropathy-associated T-cell lymphoma (EATL) is a T-cell Non-Hodgkin Lymphoma which is highly associated with celiac disease. The prognosis of EATL has been considered poor and there are no standardized treatment protocols. Here, we evaluate treatment response and survival of EATL patients in a large multicenter cohort. A total of 61 patients diagnosed with EATL were analyzed. Various treatment regimens were applied in EATL during the past fifteen years including either monotherapy consisting of chemotherapy or resection, or combination therapy with both aforementioned regimens whether or not combined with stem-cell transplantation (SCT). Overall, 50/61 patients (82%) died after a median of 7.4 months. One- and five-year overall survival was 40 and 11%, respectively. Median follow-up in the survivors was 26 months. Patients treated with the most aggressive treatment, that is, resection, chemotherapy and autologous SCT, showed the most favourable outcome with complete remission in all patients, the lowest relapse rate and one- and five-year overall survival of 100 and 33%, respectively, although overall survival in this group was not significantly better as compared to patients treated with surgery and chemotherapy. This study indicates that combination treatment is superior compared to monotherapy. Whether or not consolidation therapy with autologous SCT may improve survival needs to be substantiated in a larger randomized international trial.

18F-FDG or 3'-deoxy-3'-18F-fluorothymidine to detect transformation of follicular lymphoma.

UNLABELLED: Considering the different treatment strategy for transformed follicular lymphoma (TF) as opposed to follicular lymphoma (FL), diagnosing transformation early in the disease course is important. There is evidence that (18)F-FDG has utility as a biomarker of transformation. However, quantitative thresholds may require inclusion of homogeneous non-Hodgkin lymphoma subtypes to account for differences in tracer uptake per subtype. Moreover, because proliferation is a hallmark of transformation, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) might be superior to (18)F-FDG in this setting. To define the best tracer for detection of TF, we performed a prospective a head-to-head comparison of (18)F-FDG and (18)F-FLT in patients with FL and TF. METHODS: (18)F-FDG and (18)F-FLT PET scans were obtained in 17 patients with FL and 9 patients with TF. We measured the highest maximum standardized uptake value (SUVmax), defined as the lymph node with the highest uptake per patient, and SUVrange, defined as the difference between the SUVmax of the lymph node with the highest and lowest uptake per patient. To reduce partial-volume effects, only lymph nodes larger than 3 cm(3) (A50 isocontour) were analyzed. Scans were acquired 1 h after injection of 185 MBq of (18)F-FDG or (18)F-FLT. To determine the discriminative ability of SUVmax and SUVrange of both tracers for TF, receiver-operating-characteristic curve analysis was performed. RESULTS: The highest SUVmax was significantly higher for TF than FL for both (18)F-FDG and (18)F-FLT (P < 0.001). SUVrange was significantly higher for TF than FL for (18)F-FDG (P = 0.029) but not for (18)F-FLT (P = 0.075). The ability of (18)F-FDG to discriminate between FL and TF was superior to that of (18)F-FLT for both the highest SUVmax (P = 0.039) and the SUVrange (P = 0.012). The cutoff value for the highest SUVmax of (18)F-FDG aiming at 100% sensitivity with a maximum specificity was found to be 14.5 (corresponding specificity, 82%). For (18)F-FLT, these values were 5.1 and 18%, respectively. When the same method was applied to SUVrange, the cutoff values were 5.8 for (18)F-FDG (specificity, 71%) and 1.5 for (18)F-FLT (specificity, 36%). CONCLUSION: Our data suggest that (18)F-FDG PET is a better biomarker for TF than (18)F-FLT PET. The proposed thresholds of highest SUVmax and SUVrange should be prospectively validated.

Methylation-mediated repression of PRDM14 contributes to apoptosis evasion in HPV-positive cancers.

Promoter methylation of the transcription factor PRDM14 (PRDI-BF1 and RIZ domain containing 14) represents a highly frequent event in human papillomavirus (HPV)-induced cervical cancers and cancer precursor lesions. Here, we aimed to assess the functional consequences of PRDM14 promoter methylation in HPV-induced carcinogenesis. PRDM14 promoter methylation, expression and consequences of ectopic PRDM14 expression were studied in HPV16-positive cervical and oral cancer cell lines (SiHa, CaSki and 93VU147T), human embryonic kidney 293 (HEK293T) cells and primary human foreskin keratinocytes (HFK). PRDM14 mRNA expression was restricted to HEK293T and HFK cells, and could be upregulated in SiHa cells upon DNA methylation inhibition. Ectopic expression of PRDM14 in SiHa, CaSki and 93VU147T cells resulted in significantly more apoptotic cells, as measured by annexin V labelling, compared to HEK293T and HFK cells. MRNA profiling of 41 apoptosis regulators identified NOXA and PUMA as candidate target genes involved in PRDM14-mediated apoptosis induction. Full-length PRDM14 transactivated both NOXA and PUMA promoters. Transactivation was abolished upon deletion of the PRDM14 DNA binding domain. This suggests that NOXA and PUMA expression is directly regulated by PRDM14, which in case of NOXA was linked to a consensus PRDM14 binding motif in the promoter region. Taken together, these results suggest that PRDM14 acts as a regulator of NOXA and PUMA-mediated apoptosis induction, thereby providing evidence for a tumour suppressive role in HPV-induced carcinogenesis. The contribution of methylation-mediated gene silencing of PRDM14 to apoptosis evasion in HPV-positive cancer cells offers novel therapeutic options for HPV-induced cancers.

Expression of apoptosis regulating proteins identifies stage II and III colon cancer patients with high risk of recurrence.

BACKGROUND AND OBJECTIVES: Deregulation of apoptosis related genes may be associated with poor outcome in cancer. Aim of the present study was to investigate the prognostic role of expression levels of apoptosis related proteins in stage II and III colon cancer. METHODS: From tumor samples of 386 stage II and III colon cancer patients, DNA was isolated and tissue microarrays were constructed. Expression of Bcl-2, Bcl-X, BAX, XIAP, Fas, FasL and c-FLIP was evaluated and PCR-based microsatellite instability analysis was performed. RESULTS: High FasL expressing tumors were associated with high disease recurrence rates in stage II colon cancer patients overall, as was low Bcl-X expression in microsatellite stable stage II patients. In stage II patients, a multivariable model based on FasL and Bcl-XL expression revealed a significant association with disease free survival (DFS). In stage III colon cancer patients, low Bcl-2, low BAX and low Fas expression levels were associated with worse outcome. In these patients a multivariable model based on angioinvasion and Bcl-2, Fas and FasL expression was significantly associated with DFS. CONCLUSIONS: Stage II patients with low Bcl-X and high FasL protein expression levels and stage III patients with low Fas, high FasL and low Bcl-2 expression could be considered as high risk for disease recurrence.

Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients.

BACKGROUND: New treatment modalities are needed for the treatment of cancers of the head and neck region (HNSCC). Survivin is important for the survival and proliferation of tumor cells and may therefore provide a target for immunotherapy. Here we focused on the ex vivo presence and in vitro induction of survivin specific T cells. METHODS: Tetramer staining and ELIspot assays were used to document the presence of survivin specific T cells in patient derived material, and to monitor the presence and persistence of survivin specific T cells after repeated in vitro stimulation with autologous dendritic cells. RESULTS: Ex vivo analysis showed the presence of survivin-specific T cells in the peripheral blood (by tetramer analysis) and in the draining lymph node (by ELIspot analysis) in a HNSCC and a locally advanced breast cancer patient respectively. However, we were unable to maintain isolated survivin specific T cells for prolonged periods of time. For the in vitro generation of survivin specific T cells, monocyte derived DC were electroporated with mRNA encoding full length survivin or a survivin mini-gene together with either IL21 or IL12 mRNA. Western blotting and immunohistochemical staining of dendritic cell cytospin preparations confirmed translation of the full length survivin protein. After repeated stimulation we observed an increase, followed by a decrease, of the number of survivin specific T cells. FACS sorted or limiting dilution cloned survivin specific T cells could not be maintained on feeder mix for prolonged periods of time. Protein expression analysis subsequently showed that activated, but not resting T cells contain survivin protein. CONCLUSIONS: Here we have shown that survivin specific T cells can be detected ex vivo in patient derived material. Furthermore, survivin specific T cells can be induced in vitro using autologous dendritic cells with enforced expression of survivin and cytokines. However, we were unable to maintain enriched or cloned survivin specific T cells for prolonged periods of time. Endogenous expression of survivin in activated T cells and subsequent fratricide killing might explain our in vitro observations. We therefore conclude that survivin, although it is a universal tumor antigen, might not be the ideal target for immunotherapeutic strategies for the treatment of cancer of the head and neck.

Differential IL-13 production by small intestinal leukocytes in active coeliac disease versus refractory coeliac disease.

A small fraction of coeliac disease (CD) patients have persistent villous atrophy despite strict adherence to a gluten-free diet. Some of these refractory CD (RCD) patients develop a clonal expansion of lymphocytes with an aberrant phenotype, referred to as RCD type II (RCDII). Pathogenesis of active CD (ACD) has been shown to be related to gluten-specific immunity whereas the disease is no longer gluten driven in RCD. We therefore hypothesized that the immune response is differentially regulated by cytokines in ACD versus RCDII and investigated mucosal cytokine release after polyclonal stimulation of isolated mucosal lymphocytes. Secretion of the T(H)2 cytokine IL-13 was significantly higher in lamina propria leukocytes (LPLs) isolated from RCDII patients as compared to LPL from ACD patients (P = 0.05). In patients successfully treated with a gluten-free diet LPL-derived IL-13 production was also higher as compared to ACD patients (P = 0.02). IL-13 secretion correlated with other T(H)2 as well as T(H)1 cytokines but not with IL-10 secretion. Overall, the cytokine production pattern of LPL in RCDII showed more similarities with LPL isolated from GFD patients than from ACD patients. Our data suggest that different immunological processes are involved in RCDII and ACD with a potential role for IL-13.

Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

Enteropathy associated T-cell lymphoma and its precursor lesions.

Enteropathy Associated T-cell Lymphoma (EATL) is an intestinal tumour of intra-epithelial lymphocytes. Based on morphology, immunohistochemistry and genetic profile EATL can be divided into two groups. EATL type I is a large cell lymphoma which is highly associated with Coeliac Disease (CD) and mostly presents with malabsorption, weight loss and CD-related symptoms. EATL type II consists of small to medium-sized cells and presents often with obstruction or perforation of the small bowel. This type of EATL has no known association with CD. When EATL has been diagnosed a thorough diagnostic work-up is needed. This work-up preferably includes video capsule enteroscopy (VCE), double-balloon enteroscopy (DBE), computed tomography (CT) combined with 18F-fluorodeoxyglucose positron emission tomography scan (18F-FDG-PET scan) if possible and magnetic resonance enteroclysis (MRE). Nowadays, most EATL patients are treated with chemotherapy mostly preceded by resection of the tumour and followed by stem cell transplantation. Despite these therapies outcome of EATL remains very poor with a 5-year survival of 8-20%. In order to improve survival prospective multicentre trials, studying new therapies are needed. The combination of chemotherapy, monoclonal antibodies and/or apoptosis inducing small molecules might be a potential treatment for EATL in the (nearby) future.

Molecular targeted therapies for diffuse large B-cell lymphoma based on apoptosis profiles.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of adult non-Hodgkin lymphoma and is treated with chemotherapy in combination with rituximab. Despite this aggressive therapy, the disease is fatal in 30-40% of patients. Inhibition of the apoptosis signalling pathways is strongly related to response to chemotherapy and eventual clinical outcome. In order to survive, lymphoma cells depend on disruption of the apoptosis pathway by mutations in apoptosis inducing genes or by continuous expression of anti-apoptotic proteins. The development of molecules targeting these apoptosis inhibitors provides a very promising opportunity to specifically target tumour cells without toxicity to non-malignant cells in DLBCL patients. Sensitivity for most of these antagonists can be predicted based on biological markers, suggesting the possibility of pre-defining patients who will most likely benefit from these targeted therapies. Experimental therapies aimed at restoring the upstream apoptosis pathway or targeting apoptosis inhibitors are currently being tested in clinical trials and are expected to be effective particularly in chemotherapy-refractory DLBCL, providing hope for patients who are refractory to current therapies.

Small-molecule XIAP antagonist restores caspase-9 mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells.

Clinical outcome in patients with primary nodal diffuse large B-cell lymphomas (DLBCLs) is correlated with expression of inhibitors of the intrinsic apoptosis pathway, including X-linked inhibitor of apoptosis protein (XIAP). XIAP suppresses apoptosis through inhibiting active caspase-3, caspase-7, and caspase-9. In this study, we investigated to see if the small-molecule XIAP antagonist 1396-12 induces cell death in cultured lymphoma cells of patients with DLBCL. Treatment with this XIAP antagonist resulted in relief of caspase-3 inhibition and in induction of apoptosis in 16 of 20 tested DLBCL samples. Sensitivity to the XIAP antagonist was observed in both chemotherapy-refractory and -responsive DLBCL, but did not affect peripheral blood mononuclear cells and tonsil germinal-center B cells from healthy donors. XIAP antagonist-sensitive samples were characterized by high expression levels of XIAP, relatively low expression levels of Bcl-2, and by constitutive caspase-9 activation. These data indicate that the small-molecule XIAP antagonist can induce apoptosis in cultured DLBCL cells and therefore should be considered for possible development as a therapy for these patients. In vitro sensitivity to the XIAP antagonist can be predicted based on biological markers, suggesting the possibility of predefining patients most likely to benefit from XIAP antagonist therapy.

Inhibition of the intrinsic apoptosis pathway downstream of caspase-9 activation causes chemotherapy resistance in diffuse large B-cell lymphoma.

PURPOSE: Inhibition of the apoptosis cascade is an important cause of therapy resistance in diffuse large B-cell lymphomas (DLBCL). In this study, we investigated possible mechanisms and expression levels of apoptosis-related genes in the apoptosis pathway that may be responsible for differences in chemotherapy sensitivity between DLBCL patients. EXPERIMENTAL DESIGN: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9-mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry. RESULTS: Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist. CONCLUSIONS: We conclude that the intrinsic, caspase-9-mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.

Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis.

BACKGROUND: Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes. METHODS: H9c2 cells (rat cardiomyoblast cells) and adult rat cardiomyocytes were incubated with Hcy and were analyzed for cell viability. Furthermore, we determined the effects of Hcy on intracellular mediators related to cell viability in cardiomyocytes, namely NOX2, reactive oxygen species (ROS), mitochondrial membrane potential (DeltaPsi (m)) and ATP concentrations. RESULTS: We found that incubation of H9c2 cells with 0.1 mM D,L-Hcy (= 60 microM L-Hcy) resulted in an increase of DeltaPsi (m) as well as ATP concentrations. 1.1 mM D,L-Hcy (= 460 microM L-Hcy) induced reversible flip-flop of the plasma membrane phospholipids, but not apoptosis. Incubation with 2.73 mM D,L-Hcy (= 1.18 mM L-Hcy) induced apoptosis and necrosis. This loss of cell viability was accompanied by a thread-to-grain transition of the mitochondrial reticulum, ATP depletion and nuclear NOX2 expression coinciding with ROS production as evident from the presence of nitrotyrosin residues. Notably, only at this concentration we found a significant increase in S-adenosylhomocysteine which is considered the primary culprit in HHC. CONCLUSION: We found concentration-dependent effects of Hcy in cardiomyocytes, varying from induction of reversible flip-flop of the plasma membrane phospholipids, to apoptosis and necrosis.