Apoptotic Signal Transduction Research Articles

Inhibition of cytochrome c release by 10-N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat

The release of cytochrome c from the mitochondria to the cytosol is a critical step for downstream caspase-mediated apoptotic signal transduction in ischemia-reperfusion (I/R)-induced myocardial tissue injury. 10-N-nonyl acridine orange (NAO), a cardiolipin-specific dye, has been shown to inhibit Bid-mediated cytochrome c release from isolated mitochondria in vitro; however, the possible protective effects of NAO and the mechanisms underlying the protection from myocardial I/R-induced tissue injury in a rat model are unknown. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion. All rats received either vehicle or NAO (100 microg/kg iv) 10 min before the occlusion. The infarct size in the heart at 24 h after reperfusion was significantly reduced in NAO-treated rats compared with vehicle-treated rats. NAO treatment significantly reduced the cytosolic cytochrome c contents and caspase-9 activity in the ischemic region but did not affect caspase-8 activity. Furthermore, NAO treatment markedly suppressed the translocation of truncated Bid, a proapoptotic Bcl-2 family member, to the mitochondrial fraction. NAO also suppressed the mitochondrial swelling and oxygen uptake stimulated by calcium overload. The results suggest that NAO possesses protective effects against myocardial I/R injury, which may be due to the suppression of cytochrome c release through blockade of truncated Bid translocation to mitochondria and inhibition of the opening of mitochondrial permeability transition pores.

A Phase 1b Clinical Trial of Dacetuzumab in Combination with Rituximab and Gemcitabine: Multiple Responses Observed in Patients with Relapsed Diffuse Large B-Cell Lymphoma.

Abstract 586 Background:Dacetuzumab (SGN-40), a humanized monoclonal antibody that targets CD40, mediates antitumor activity through multiple mechanisms of action, including effector cell functions (ADCC/ADCP) and direct apoptotic signal transduction. Ongoing evaluation of the molecule has led to 2 additional key findings: a gene signature that predicts sensitivity to single-agent dacetuzumab, and upregulation of the pro-apoptotic and chemosensitizing transcription factor TAp63αa when dacetuzumab is combined with rituximab (R) and gemcitabine (GEM). In prior single-agent dacetuzumab trials, several patients who were immediately treated with GEM after progressing achieved unexpected responses. These observations provided rationale for further analysis of the combination of dacetuzumab, R and GEM. Methods:A phase 1b dose-escalation study to evaluate the safety and adverse event profile of dacetuzumab, R and GEM was conducted in patients with relapsed diffuse large B-cell lymphoma (DLBCL). Patients received dacetuzumab at cohort-specific doses of 8 or 12 mg/kg IV weekly in combination with R (375 mg/m2 IV for 1 cycle, then once/28-day cycle) and GEM (1000 mg/m2 IV weekly or biweekly) in a 3 + 3 dose-escalation scheme. A maximum number of 8 cycles was permitted. Eligible patients included those with de novo DLBCL, including transformed lymphoma and follicular grade 3, who failed potentially curative therapy. Results:Thirty-one patients with a median age of 68 years (range 29–80) were enrolled and treated in the study (3 patients in each of 3 cohorts + 22 in an expansion cohort). The median number of prior regimens was 3 (range 1–18); 10 patients (32%) had transformed histology and 4 (13%) had prior ASCT. The median number of cycles received was 3 (range 1–8), although 11 patients are still on treatment. Three patients in the first cohort required dose reduction of GEM due to transient, non-dose-limiting thrombocytopenia. The protocol was thus amended to administer GEM on a biweekly schedule. No dose-limiting toxicity was observed, and MTD was not defined. Based on pharmacokinetic modeling suggesting no significant difference in exposure between dose levels, 8 mg/kg of dacetuzumab was chosen as the dose for the expansion cohort. The combination of dacetuzumab, R and GEM was generally well tolerated. Across all cohorts, the most common adverse events (> 15%) were nausea (42%), thrombocytopenia (35%), fatigue (32%), headache (29%), anorexia (19%), dyspnea (19%), pyrexia (19%), diarrhea (16%), and neutropenia (16%), the majority of which were Grade 1 or 2. Grade 3/4 adverse events related to dacetuzumab occurred infrequently. One patient experienced Grade 3 stress cardiomyopathy that resolved; this patient had a history of congestive heart failure. One patient developed pneumonitis in the left lung and acute respiratory distress syndrome, and subsequently died due to pulmonary toxicity; this patient had received prior radiation therapy to the left chest. There was preliminary evidence of antitumor activity. Of the 28 patients with response assessments, 7 patients achieved CR, 8 PR, 7 SD, and 6 PD per investigator assessment of response, yielding an objective response rate of 54%. Of interest, 1 patient who previously failed ASCT achieved a CR and was able to undergo allogeneic transplant. To date, this patient remains in CR. Conclusion:The combination of dacetuzumab, R and biweekly GEM was generally well tolerated. Evidence of antitumor activity with this combination is encouraging as multiple responses were observed in a population with heavily pre-treated patients. Further investigation with a randomized trial is warranted to more fully characterize safety and efficacy and to determine if sensitivity to this combination correlates with the previously identified single-agent predictive gene signature. Disclosures:Forero-Torres:Seattle Genetics, Inc.: Research Funding. Off Label Use: Gemcitabine has not been FDA approved for treatment of lymphoma. Dacetuzumab is an investigational agent.. Bartlett:Seattle Genetics, Inc.: Research Funding. Nasta:Seattle Genetics, Inc.: Research Funding; Genentech: Speakers Bureau. Northfelt:Seattle Genetics, Inc.: Research Funding. Beaven:Seattle Genetics, Inc.: Research Funding. Myint:Seattle Genetics, Inc.: Research Funding. Whiting:Seattle Genetics, Inc.: Employment, Equity Ownership. Drachman:Seattle Genetics, Inc.: Employment, Equity Ownership. Moskowitz:Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

FoxO3a Governs Early Microglial Proliferation and Employs Mitochondrial Depolarization with Caspase 3, 8, and 9 Cleavage During Oxidant Induced Apoptosis

Microglia of the central nervous system have a dual role in the ability to influence the survival of neighboring cells. During inflammatory cell activation, microglia can lead to the disposal of toxic cellular products and permit tissue regeneration, but microglia also may lead to cellular destruction with phagocytic removal. For these reasons, it is essential to elucidate not only the underlying pathways that control microglial activation and proliferation, but also the factors that determine microglial survival. In this regard, we investigated in the EOC 2 microglial cell line with an oxygen-glucose deprivation (OGD) injury model of oxidative stress the role of the "O" class forkhead transcription factor FoxO3a that in some scenarios is closely linked to immune system function. We demonstrate that FoxO3a is a necessary element in the control of early and late apoptotic injury programs that involve membrane phosphatidylserine externalization and nuclear DNA degradation, since transient knockdown of FoxO3a in microglia preserves cellular survival 24 hours following OGD exposure. However, prior to the onset of apoptotic injury, FoxO3a facilitates the activation and proliferation of microglia as early as 3 hours following OGD exposure that occurs in conjunction with the trafficking of the unphosphorylated and active post-translational form of FoxO3a from the cytoplasm to the cell nucleus. FoxO3a also can modulate apoptotic mitochondrial signal transduction pathways in microglia, since transient knockdown of FoxO3a prevents mitochondrial membrane depolarization as well as the release of cytochrome c during OGD. Control of this apoptotic cascade also extends to progressive caspase activation as early as 1 hour following OGD exposure. The presence of FoxO3a is necessary for the expression of cleaved (active) caspase 3, 8, and 9, since loss of FoxO3a abrogates the induction of caspase activity. Interestingly, elimination of FoxO3a reduced caspase 9 activity to a lesser extent than that noted with caspase 3 and 8 activities, suggesting that FoxO3a in relation to caspase 9 may be more reliant upon other signal transduction pathways potentially independent from caspase 3 and 8.

Protein Kinase Cε Inhibits UVR-Induced Expression of FADD, an Adaptor Protein, Linked to both Fas- and TNFR1-Mediated Apoptosis

Protein kinase C (PKC)epsilon overexpression in FVB/N transgenic mice sensitized skin to UVR-induced development of squamous cell carcinomas and suppressed formation of sunburn cells, which are DNA-damaged keratinocytes undergoing apoptosis. Here, we elucidated the mechanisms associated with the inhibition of UVR-induced appearance of sunburn cells in PKCepsilon transgenic mice. We found that the inhibition of UVR-induced sunburn cell formation in PKCepsilon transgenic mice may be the result of the inhibition of the expression of Fas, Fas ligand, and the mammalian death adaptor protein termed Fas-associated with death domain (FADD). The adaptor protein FADD is the key component of the death-inducing signaling complex of both Fas and tumor necrosis factor receptor 1. A decreased expression of epidermal FADD was observed after a single UVR exposure. However, a complete loss of FADD expression was found after four (Monday, Wednesday, Friday, and Monday) repeated UVR exposures. FADD transmits apoptotic signals from death receptors to the downstream initiator caspase-8 and connects to the mitochondrial intrinsic apoptotic signal transduction pathway by the cleavage of Bid, a Bcl-2 family member. PKCepsilon-mediated loss of FADD expression inhibited UVR signals to the activation of both extrinsic and intrinsic apoptotic pathways.

CDK11p58 Phosphorylation of PAK1 Ser174 Promotes DLC2 Binding and Roles on Cell Cycle Progression

CDK11(p58), a CDK11 family Ser/Thr kinase, is a G2/M specific protein and contributed to regulation of cell cycle, transcription and apoptotic signal transduction. Recently, CDK11(p58) has been reported to exert important functions in mitotic process, such as the regulation of bipolar spindle formation and sister chromatid cohesion. Here, we identified p21 activated kinase 1 (PAK1) as a new CDK11(p58) substrate and we mapped a new phosphorylation site of Ser174 on PAK1. By mutagenesis, we created PAK1(174A) and PAK1(174E), which mimic the dephosphorylated and phosphorylated form of PAK1; further analysis showed PAK1(174E) could be recruited to myosin V motor complex through binding to dynein light chain 2 (DLC2). PAK1(174E) could accelerate the mitosis progression in a nocodazole blocked cell model, while PAK1(174A) exhibited an opposite role. Our results indicated PAK1 may serve as a downstream effector of CDK11(p58) during mitosis progression.

Sulindac Sulfide Differentially Induces Apoptosis in Smac-Proficient and -Deficient Human Colon Cancer Cells

Sulindac, the non-steroidal anti-inflammatory drug has shown promise in the prevention of colon cancer but the molecular mechanisms by which it mediates such effects remain to be elucidated. Sulindac sulfide is the major active metabolite of sulindac and believed to be responsible for mediating the effects of sulindac. Previously, our group and others have shown that sulindac sulfide induces apoptosis by engaging death receptor and mitochondrial pathways and that a cross-talk exists between these two pathways during sulindac sulfide-induced apoptosis. Second mitochondrial-derived activator (Smac) is an important pro-apoptotic molecule that activates caspases by antagonizing the inhibitors of apoptosis (IAPs). In this study, we have utilized Smac-proficient and -deficient human colon cancer cells to investigate the role of Smac during sulindac sulfide-induced apoptosis and found that Smac deficiency affects sulindac sulfide-induced apoptosis in human colon cancer cells. Sulindac sulfide-induced apoptosis is coupled with upregulation of death receptor 5 (DR5), and activation of caspases 3, 9 and 8 in Smac-proficient cells. In Smac-deficient cells, although sulindac sulfide-induced DR5 upregulation is not altered, activation of caspases 3, 9 and 8 is affected. Smac deficiency also abrogates sulindac sulfide-induced cytochrome c release from mitochondria into cytosol. Our results, therefore, demonstrate that Smac is involved in sulindac sulfide-induced apoptotic signal transduction in human colon cancer cells and highlight the existence of a potential cross-talk between Smac and cytochrome c.

Interactions between apoptotic signal transduction and capacitation in human spermatozoa

Capacitation of sperm is a prerequisite for successful fertilization, determined by hyperactivated motility, increased tyrosine phosphorylation (TyrP) and membrane changes. However, the exact molecular mechanism is not fully clarified. The calpain-calmodulin-system is essential for membrane fusion during capacitation. Recently, interactions with caspase (CP) activation, a main feature of apoptotic cells, were postulated. The objective of our study was to examine interactions between apoptosis signalling and the calpain-calmodulin-system during capacitation. Semen samples from 20 healthy donors were incubated in human tubal fluid at 37 degrees C, 5% CO(2) for 3 h without additives (control), with 3% BSA (capacitation), 10 microM calpain-inhibitor III, 20 microM CP-1 inhibitor or 20 microM calmodulin-antagonist. Capacitation was monitored by computer assisted sperm motion analyzer, chlortetracycline (CTC)-assay and western blot (TyrP). Activation of caspases and integrity of transmembrane mitochondrial potential (TMP) were evaluated by flow cytometry. Capacitation, as measured by CTC assay, increased TyrP levels and hyperactivation, resulted in inactivation of CP-9, CP-3 and improved integrity of the TMP. Inhibition of calpain and CP-1 during capacitation reduced the capacitation-related parameters, but did not lead to apoptosis. Inhibition of calmodulin resulted in blocking of capacitation and stimulation of apoptosis. Interaction of the capacitation and apoptosis signalling systems seems to enable the capacitation process by prevention of apoptosis.

Smac deficiency affects endoplasmic reticulum stress-induced apoptosis in human colon cancer cells

Thapsigargin (TG) is a sesquiterpen lactone that inhibits the endoplasmic reticulum (ER) calcium ATPases to disrupt calcium homeostasis and consequently induces ER stress. We have previously reported that TG induces apoptosis by engaging the death receptor 5 (DR5) and the intrinsic pathways. Second mitochondrial-derived activator (Smac) is an important modulator of apoptosis that induces activation of caspases by antagonizing inhibitors of apoptosis (IAPs). In this study, we have utilized Smac-proficient and -deficient human colon cancer cells to investigate the effects of Smac deficiency during ER-stress-induced apoptosis. Our results indicate that Smac deficiency considerably affects ER stress-induced apoptosis in human colon cancer cells. For example, ER stress inducing agent TG upregulates DR5, and activates caspases 3, 9 and 8 in Smac-proficient cells. In Smac-deficient cells, although TG-induced DR5 upregulation is not affected, activation of caspases 3, 9 and 8 is affected. Smac deficiency also affects TG-induced cytochrome c release from mitochondria into cytosol suggesting the existence of a potential cross-talk between Smac and cytochrome c. Thus, our results indicate that ER stress-induced apoptosis also engages Smac for transduction of apoptotic signals in human colon cancer cells and that a potential feedback signaling between Smac and cytochrome c appears to modulate the intrinsic pathway of apoptosis.

MCF-7 breast carcinoma cells do not express caspase-3

Apoptosis, a fundamental process essential for normal tissue homeostasis and development, is closely associated with the activation of a class of aspartate-specific cysteinedependent proteases, called caspases, that lead to the demise of the cell via limited proteolysis of a multitude of cellular substrates [1, 2]. Caspases are expressed as inactive zymogens that become activated upon cleavage by other caspases in a so-called caspase activation cascade, or by mere oligomerization instigated by the formation of large multi-protein complexes such as the death-inducing signaling complex (DISC) or the apoptosome [3, 4]. Whereas DISC formation occurs via the so-called extrinsic death pathway that is instigated by activation of members of the death receptor family such as CD95, tumor necrosis factor receptor (TNF-R) or the receptors of the TNF-Rrelated apoptosis-inducing ligand (TRAIL), the apoptosome is formed following activation of mitochondria and is hence termed the intrinsic or mitochondrial death pathway. Based on their order in cell death pathways, caspases can be divided into initiator (caspase-2, -8, -9, and -10) and effector (caspase-3, -6, and -7) caspases. Among them, caspase-3, a member of the latter group, is absolutely crucial for apoptosis induction, as this enzyme is not only activated downstream of both the extrinsic and intrinsic death pathway, it is also responsible for the cleavage of the majority of substrates known so far [1, 5]. More importantly, with the proteolysis of discrete substrates, caspase-3 evokes some of the typical morphological and biochemical alterations associated with apoptosis. For instance, whereas the caspase-3-mediated cleavages of a-fodrin, gelsolin, rho-associated kinase-1 (ROCK-1) and p21-activated kinase 2 (PAK2) contribute to membrane blebbing, cleavage of the inhibitor of the caspase-activated DNase (ICAD) leads to the typical DNA fragmentation pattern observed in apoptosis [1]. Furthermore, with the cleavagemediated activation of the calcium-independent phospholipase A2 and subsequent production of the chemotactic phospholipid lysophosphatidylcholine, caspase-3 appears to be also responsible for the generation of so-called ‘‘eatme’’ signals that induce migration of phagocytes to the site of apoptotic cell death [6]. Thus, caspase-3 not only instigates and pursues the demise of a cell, but, in addition, makes sure that the corpse is properly disposed, a function crucial for avoiding inflammatory processes. Hence, determination of the processing and activation of caspase-3 are common means to assess apoptotic signal transduction pathways in numerous cell lines of varying origin. Curiously, several reports still claim the presence of caspase-3 in the breast carcinoma cell line MCF-7 in which this enzyme is supposed to contribute to apoptosis signaling [7–15]. This was not only demonstrated indirectly via fluorometric assay systems measuring caspase-3-like activities—that might be also elicited by the closely related caspase-7—but by Western blotting analyses demonstrating directly the presence of this protease in MCF-7 cells. However, in addition to the lack of caspase-10 [16], an initiator caspase in the extrinsic death pathway, MCF-7 cells do also not express caspase-3 [17]. Ten years ago, we demonstrated unambiguously that the lack of caspase-3 in these cells is caused by a 47-base pair deletion within exon 3 of the CASP3 gene resulting in the skipping of this exon during premRNA splicing and introduction of a premature stop codon at position 42 that completely abrogates translation of the CASP-3 mRNA [17]. Although caspase-3-deficient MCF-7 R. U. Janicke (&) Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, University of Dusseldorf, Universitatsstrasse 1, 40225 Dusseldorf, Germany e-mail: Janicke@uni-duesseldorf.de

Tacrolimus-Induced Apoptotic Signal Transduction Pathway

Tacrolimus (FK506) has been widely used as an immunosuppressant. We examined the effects of FK506 on expression of apoptotic signal transduction pathway proteins of Jurkat human T lymphocytes. We investigated the effects of FK506 on apoptosis, cell viability, caspase family protein activity, Western blotts of Bcl-2, Bak, Fas, Fas-L, CDK4, and cyclin D1, as well as reactive oxygen species (ROS) generation and mitochondrial membrane potential transition. Cells were cultured in the presence or absence of FK506. Flow cytometric analysis was performed after staining with propidium iodide. Viability of Jurkat cells was decreased by the addition of FK506 in dose- and time- dependent manner. FK506-induced cytotoxicity was characterized by G0/G1 phase cell cycle arrest. FK506-induced cell death was confirmed by apoptosis characterized by nuclear fragmentation and caspase-3 protease activation. FK506 induced no change in catalytic activity of caspase-6, -8, and -9 proteases. No change in expression of Bcl-2 protein was noted but we confirmed increased expression of Bak protein. No changes of expressions of Fas and Fas-L were seen. Increased expressions of CDK4 and cyclin D1 were identified. In addition, pharmacological scavenging study of ROS, including H 2O 2, revealed that cytotoxicity was achieved by generation of ROS, which might modulate Bak protein expression and mitochondrial dysfunction. In conclusion, FK506-induced cell death was apoptotic, characterized by nuclear fragmentation and caspase-3 activation. FK506 induced G0/G1 phase cell cycle arrest via expression of CDK4 and cyclin D1. Apoptosis was also achieved by generation of H 2O 2, which modulated Bak protein expression and mitochondrial dysfunction.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Promotes Migration of Human Bone Marrow Multipotent Stromal Cells

Adult multipotent stromal cells (MSCs), also known as mesenchymal stem cells, represent an important source of cells for the repair of a number of damaged tissues. Both bone marrow (BM)-derived and amniotic MSCs expressed detectable surface levels of two (tumor necrosis factor-related apoptosis-inducing ligand receptor 2 [TRAIL-R2] and TRAIL-R4) of four transmembrane TRAIL receptors. Although the best-characterized activity of TRAIL-R2 is the transduction of apoptotic signals, neither recombinant TRAIL (rTRAIL) nor infection with an adenovirus-expressing TRAIL induced cytotoxic effects on MSCs. Moreover, whereas rTRAIL did not affect proliferation or differentiation of MSCs along the osteogenic and adipogenic lineages, it significantly promoted the migration of human MSCs in range of concentrations comparable to that of soluble TRAIL in human plasma (100 pg/ml). Since rTRAIL induced the rapid phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in MSC cultures and pretreatment with pharmacological inhibitors of the ERK1/2 pathway efficiently counteracted the rTRAIL-induced human MSC migration, these data indicate that ERK1/2 is involved in mediating the ability of rTRAIL to stimulate MSC migration. Taking into consideration that the soluble factors able to induce MSC migration have not been extensively characterized, our current data indicate that the TRAIL/TRAIL-R system might play an important role in the biology of MSCs. Disclosure of potential conflicts of interest is found at the end of this article.

Blockade of processing/activation of caspase-3 by hypoxia

Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells. However, it is largely unknown how hypoxia-inhibited Bax translocation attenuates TRAIL-induced apoptosis. Here, we demonstrate that despite its inhibitory activity in TRAIL-induced apoptosis, hypoxia does not affect TRAIL-triggered proximal apoptotic signaling events, including caspase-8 activation and Bid cleavage. Instead, hypoxia inhibited processing of caspase-3, leading to incomplete activation of the caspase. Importantly, hypoxia-blocked translocation of Bax to the mitochondria significantly inhibited releasing the mitochondrial factors, such as cytochrome c and Smac/DIABLO, to the cytosol in response to TRAIL. It is well-known that complete processing/activation of caspase-3 requires Smac/DIABLO released from mitochondria. Therefore, our data indicate that an engagement of the apoptotic mitochondrial events leading to caspase-3 activation is blocked by hypoxia. Our data shed new light on understanding of the apoptotic signal transduction and targets regulated by tumor hypoxia.

MPYS, a Novel Membrane Tetraspanner, Is Associated with Major Histocompatibility Complex Class II and Mediates Transduction of Apoptotic Signals

Although the best-defined function of type II major histocompatibility complex (MHC-II) is presentation of antigenic peptides to T lymphocytes, these molecules can also transduce signals leading alternatively to cell activation or apoptotic death. MHC-II is a heterodimer of two transmembrane proteins, each containing a short cytoplasmic tail that is dispensable for transduction of death signals. This suggests the function of an undefined MHC-II-associated transducer in signaling the death response. Here we describe a novel plasma membrane tetraspanner (MPYS) that is associated with MHC-II and mediates its transduction of death signals. MPYS is unusual among tetraspanners in containing an extended C-terminal cytoplasmic tail (approximately 140 amino acids) with multiple embedded signaling motifs. MPYS is tyrosine phosphorylated upon MHC-II aggregation and associates with inositol lipid and tyrosine phosphatases. Finally, MHC class II-mediated cell death signaling requires MPYS-dependent activation of the extracellular signal-regulated kinase signaling pathway.

Doxorubicin induced apoptosis in oocytes—Mechanism and possible executers

9611 Background: Premature ovarian failure and infertility are well-known side-effects observed in young girls and reproductive-age women treated for cancer. Long-term consequences of chemotherapy observed on non-target tissues, such as ovaries are substantial. Oocytes are among the cells most sensitive to collateral damage from chemotherapy, raising concern and potential health risks for women surviving diseases such as breast cancer, Hodgkin’s disease and leukemia. Our study aims at exploring the lethal effect doxorubicin exerts on oocytes. Methods: Retrieved ovulated or ovarian mouse oocytes, served as our biological model. Oocytes were exposed to doxorubicin and studied at several time-intervals. Using state of the art confocal microscopy, we performed fluorescent immunohistochemistry to follow doxorubicin localization within the oocytes as well as possible cellular targets at various post-treatment intervals. To evaluate doxorubicin induced apoptosis, we also used specific activity assays to trace potential effectors such as caspases and calpain. Results: Doxorubicin appears to be localized simultaneously at two cellular compartments - the nucleus (being the known original target site, as expected), where it induces acute obliteration of the DNA, and the mitochondria. We demonstrated activation of the mitochondria by doxorubicin as reflected by changes in its membrane potential. The effect on the mitochondria appears to be direct and is not inhibited by caspase2-inhibitor, an effector that mediates signals of the nuclear pathway. Exploring the apoptotic cascade within the oocyte, we showed that doxorubicin induces caspase-12 activation, probably via calpain activation. Conclusions: Our results imply that doxorubicin elicits a unique apoptotic signal transduction pathway within the oocyte via direct co-localization within the mitochondria and its activation. Possible cellular effectors appeared to be caspase12 and calpain, as formerly described in cardiomyocytes. Revealing the cellular mechanisms by which doxorubicin executes oocyte destruction might shed light on chemotherapy- induced infertility and could serve in discovering biological keys needed for protecting the germ cells against chemotherapy-induced damage. No significant financial relationships to disclose.

Cytotoxic action mode of a novel porphyrin derivative isolated from harmful red tide dinoflagellate Heterocapsa circularisquama

Heterocapsa circularisquama is known to cause lethal effect on bivalves, but toxic effect on fish has not been reported yet. Recently, we have found that H. circularisquama has potent light-dependent hemolytic toxins. Based on the chemical structural analysis, one of the hemolytic toxins named H2-a was found to be a novel porphyrin derivative with similar structure to pyropheophorbide a methyl ester (PME), a well-known photoactive hemolytic agent (Miyazaki et al., Aquatic Toxicol. 2005;73:382--393). To clarify the cytotoxic action mode of H2-a, we examined the effects of H2-a on HeLa cells in comparison with PME. The cytotoxicities of both reagents were strictly light dependent, and no significant cytotoxic effects including cellular morphological changes were induced without light illumination. The dose response curves revealed that H2-a showed stronger cytotoxicity to HeLa cells than PME. Fluorescence microscopic observation suggested that H2-a tends to accumulate in the plasma membrane, whereas PME seems to distribute entire cytoplasm. Although PME induced typical apoptotic nuclear morphological changes and DNA fragmentation in HeLa cells, no such apoptosis-inducing ability of H2-a was observed. Among the radical scavengers, histidine significantly inhibited the cytotoxic activity of H2-a, suggesting the involvement of singlet oxygen in the cytotoxicity. These results suggest that the cytotoxic mechanism of H2-a is necrotic rather than apoptosis differing from PME, even though these are structurally quite similar to each other. The relatively high affinity of H2-a to the plasma membrane might result in the potent and quick cytotoxicity without induction of apoptotic signal transduction.

Original article: TRAIL mediated signaling in human mast cells: the influence of IgE‐dependent activation

Background: Mast cells activation through FcɛRI cross‐linking has a pivotal role in the initiation of allergic reactions. The influence of this activation on programmed cell death of human mast cells has not yet been clarified. This study evaluates the influence of IgE‐dependent activation alone and in synergy with TRAIL on the expression of molecules involved in the apoptotic signal transduction.Methods: Human cord blood derived mast cells (CBMC) were cultured with myeloma IgE followed by activation with anti‐human IgE. The expression of proteins involved in apoptotic signal transduction was assessed by immunoblot analysis. To test the effect of activation on a pro‐apoptotic stimulus, activated, IgE‐treated and resting CBMC were incubated with TRAIL, or in a medium with suboptimal concentrations of stem cell factor (SCF).Results: In accordance with a previous study of ours, it was found that IgE‐dependent activation increased TRAIL‐induced caspase‐8 and caspase‐3 cleavage. However, it did not have a significant influence on CBMC death induced by SCF withdrawal. IgE‐dependent activation increased the expression of FLIP and myeloid cell leukemia 1 (MCL‐1) anti‐apoptotic molecules as well as the pro‐apoptotic one, BIM. In addition, a decrease in BID expression was observed. TRAIL could reverse the increase in FLIP but did not influence the upregulation of MCL‐1 and of BIM.Conclusions: These findings suggest that IgE‐dependent activation of human mast cells induces an increase in both pro‐survival and pro‐apoptotic molecules. We therefore hypothesized that IgE‐dependent activation may regulate human mast cell apoptosis by fine‐tuning anti‐apoptotic and pro‐apoptotic factors.

Alteration of apoptotic signaling molecules as a function of time after radiation in human neuroblastoma cells

Ascertaining the time-dependent regulation of induced apoptosis and radioresistance is important to understand the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity. Accordingly, we investigated the time-dependent expression of apoptosis related genes and radioresistance in neuroblastoma cells. Serum-starved human SK-N-MC cells were exposed to low linear energy transfer (LET) radiation (2 Gy) and incubated for 15, 30, 45 min, and 48 h. Radioresistance was investigated by examining the NF kappa B DNA-binding activity, cellular toxicity, DNA fragmentation, and expression of apoptotic signal transduction molecules. NF kappa B DNA binding activity was analyzed using electrophoretic mobility shift assay (EMSA). Cellular toxicity was measured using MTT assay. DNA fragmentation was quantified by labeling with fluorescein-conjugated deoxynucleotides. Microarray analysis was performed using cDNA microarray and relative gene expression was measured as % GAPDH and, subsequently validated using Q-PCR. Induction of NF kappa B analyzed using EMSA showed an increased DNA-binding activity at all time points investigated. Induced DNA fragmentation was observed after 15, 30, and 45 min post-radiation. Relatively, induced fragmentation was reduced after 48 h. Compared to the untreated controls cellular toxicity was induced with low LET radiation after 15, 30, and 45 min. Conversely, cytotoxicity was relatively less at 48 h after low LET radiation. Microarray analysis after low LET radiation revealed time-dependent modulation of apoptosis-related genes that are involved in radio-adaptation, spontaneous apoptosis-related early-responsive genes and late response genes. These results suggest that the time-dependent regulation of apoptotic response may determine the relationship between the level of spontaneous apoptosis in cells and their radiosensitivity.

Supportive Role of TNF-Family Death Receptors in Hematopoietic Cell Engraftment.

The activity of death receptors of the tumor necrosis factor (TNF) superfamily as negative regulators of distal differentiation in the immuno-hematopoietic system is well characterized. The role of these receptors, classically associated with transduction of apoptotic signals, is unknown. A series of studies have attributed these receptors also negative regulatory functions in ex vivo expanded hematopoietic progenitors, showing variable supporting and suppressive interactions with growth factors. We observed robust upregulation of Fas, TNF-R1, TNF-R2 and TRAIL-R2 in bone marrow homed donor cells, reaching levels of 60–75% expression after 6 days. All putative SCA-1+c-kit+lin− progenitors ubiquitously expressed all four receptors. These receptor-positive cells were largely resistant to apoptotic signals delivered by the corresponding soluble and membranous ligands. Furthermore, there was no evidence of receptor cross-talk in sensitization of the cells to apoptotic death. The engraftment potential of cells was markedly diminished by ex vivo incubation, without any relationship to the viability of the putative progenitors. Taken together these data suggest a supportive role of the “death receptors” in hematopoietic cell engraftment and dissociate the deficient engraftment of cultured cells from apoptosis of progenitors.

Genome-Scale MicroRNA and Small Interfering RNA Screens Identify Small RNA Modulators of TRAIL-Induced Apoptosis Pathway

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4/5 and selectively induces caspase-dependent apoptosis. The RNA interference screening approach has led to the discovery and characterization of several TRAIL pathway components in human cells. Here, libraries of synthetic small interfering RNA (siRNA) and microRNAs (miRNA) were used to probe the TRAIL pathway. In addition to known genes, siRNAs targeting CDK4, PTGS1, ALG2, CLCN3, IRAK4, and MAP3K8 altered TRAIL-induced caspase-3 activation responses. Introduction of the miRNAs let-7c, mir-10a, mir-144, mir-150, mir-155, and mir-193 also affected the activation of the caspase cascade. Putative targets of these endogenous miRNAs included genes encoding death receptors, caspases, and other apoptosis-related genes. Among the novel genes revealed in the screen, CDK4 was selected for further characterization. CDK4 was the only member of the cyclin-dependent kinase gene family that bore a unique function in apoptotic signal transduction.

Pp32/PHAPI determines the apoptosis response of non-small-cell lung cancer

During malignant transformation, cancer cells have to evade cell-intrinsic tumor suppressor mechanisms including apoptosis, thus acquiring a phenotype that is relatively resistant to clinically applied anticancer therapies. Molecular characterization of apoptotic signal transduction defects may help to identify prognostic markers and to develop novel therapeutic strategies. To this end we have undertaken functional analyses of drug-induced apoptosis in human non-small cell-lung cancer (NSCLC) cells. We found that primary drug resistance correlated with defects in apoptosome-dependent caspase activation in vitro. While cytochrome c-induced apoptosome formation was maintained, the subsequent activation of caspase-9 and -3 was abolished in resistant NSCLC. The addition of recombinant pp32/putative human HLA class II-associated protein (pp32/PHAPI), described as a putative tumor suppressor in prostate cancer, successfully restored defective cytochrome c-induced caspase activation in vitro. Conditional expression of pp32/PHAPI sensitized NSCLC cells to apoptosis in vitro and in a murine tumor model in vivo. Immunohistochemical analyses of tumor samples from NSCLC patients revealed that the expression of pp32/PHAPI correlated with an improved outcome following chemotherapy. These results identify pp32/PHAPI as regulator of the apoptosis response of cancer cells in vitro and in vivo, and as a predictor of survival following chemotherapy for advanced NSCLC.