Biomedical subjects

S Fulda

Publications and source records attributed to S Fulda.

At least 19 recordsLinked to original sources

PubMed2006

Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance.

Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. Although it usually promotes tumor cell survival under hypoxia, it has also been implied to trigger apoptosis. Although the impact of hypoxia has been extensively studied in many adult solid tumors, its role in most childhood tumors, for example, in rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), has not yet been addressed. Here, we report that hypoxia protects A204 RMS and A673 ES cells against anticancer drug- or tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and that Hif-1alpha plays a key role in conferring apoptosis resistance under hypoxia. Although a functional HIF-1 pathway and proapoptotic proteins such as p53 and Bcl-2/E1B 19 kDa interacting protein 3 were activated under hypoxia in both A204 RMS and A673 ES cells, these cells remained refractory to apoptosis. Concomitant analysis of antiapoptotic proteins revealed that hypoxia induced expression of Bcl-2 and inhibitor of apoptosis proteins (IAP)-2 as well as proteins associated with anaerobic metabolism such as the glucose transporter protein GLUT-1 and the glycolytic enzyme Aldolase A. Specific downregulation of Hif-1alpha by RNA interference significantly enhanced apoptosis under hypoxia by preventing the hypoxia-mediated increase in GLUT-1 expression without altering expression levels of the antiapoptotic proteins Bcl-2 or cIAP-2. Moreover, glucose deprivation-induced apoptosis of A204 RMS and A673 ES cells was inhibited under hypoxic conditions in a Hif-1alpha-dependent manner. As GLUT-1 was induced via Hif-1alpha under hypoxia in A204 RMS and A673 ES, these findings suggest that the Hif-1alpha-mediated increase in glucose uptake plays an important role in conferring apoptosis resistance. Thus, hypoxia-inducible genes may represent novel targets for therapeutic intervention in some pediatric tumors, which warrants further investigation.

Apoptosis↗

PubMed2006

Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy.

Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. One of the most important advances in cancer research in recent years is the recognition that cell death mostly by apoptosis is crucially involved in the regulation of tumor formation and also critically determines treatment response. Killing of tumor cells by most anticancer strategies currently used in clinical oncology, for example, chemotherapy, gamma-irradiation, suicide gene therapy or immunotherapy, has been linked to activation of apoptosis signal transduction pathways in cancer cells such as the intrinsic and/or extrinsic pathway. Thus, failure to undergo apoptosis may result in treatment resistance. Understanding the molecular events that regulate apoptosis in response to anticancer chemotherapy, and how cancer cells evade apoptotic death, provides novel opportunities for a more rational approach to develop molecular-targeted therapies for combating cancer.

Animals↗

PubMed2006

NF-kappaB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

Active Transport, Cell Nucleus↗

PubMed2006

Regulation of TRAIL-induced apoptosis by XIAP in pancreatic carcinoma cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapy because of its relative tumor selectivity. However, many cancers including pancreatic cancer remain resistant towards TRAIL. To develop TRAIL for cancer therapy of pancreatic carcinoma, it will therefore be pivotal to elucidate the molecular mechanisms of TRAIL resistance. Here, we identify X-linked inhibitor of apoptosis (XIAP) as a regulator of TRAIL sensitivity in pancreatic carcinoma cells. Full activation of effector caspases, loss of mitochondrial membrane potential and cytochrome c release following TRAIL treatment were markedly impaired in pancreatic carcinoma cell lines, which poorly responded to TRAIL (PaTuII, PancTu1, ASPC1, DanG), compared to TRAIL-sensitive Colo357 pancreatic carcinoma cells. Stable downregulation of XIAP by RNA interference significantly reduced survival and enhanced TRAIL-induced apoptosis in pancreatic carcinoma cells. Also, downregulation of XIAP significantly increased CD95-induced cell death. Importantly, knockdown of XIAP strongly inhibited clonogenicity of pancreatic cancer cells treated with TRAIL indicating that XIAP promotes clonogenic survival of pancreatic carcinoma cells. Thus, our findings for the first time indicate that targeting XIAP represents a promising strategy to enhance the antitumor activity of TRAIL in pancreatic cancer, which has important clinical implications.

Apoptosis↗

PubMed2006

5-Aza-2'-deoxycytidine and IFN-gamma cooperate to sensitize for TRAIL-induced apoptosis by upregulating caspase-8.

Resistance of tumors to cytotoxic therapy remains a major obstacle in cancer treatment and is often caused by defects in apoptosis programs. Caspase-8, a key mediator of death receptor-induced apoptosis, has previously been reported to be frequently inactivated by epigenetic silencing in many tumors, for example in neuroblastoma or medulloblastoma. Here, we provide for the first time evidence that combined treatment with suboptimal concentrations of the demethylating agent 5-Aza-2'-deoxycytidine (5-dAzaC) and interferon-gamma (IFN-gamma) cooperated to upregulate caspase-8 expression in neuroblastoma and medulloblastoma cells lacking caspase-8. Consequently, activation of caspase-8 and downstream caspases upon addition of TNF-related apoptosis-inducing ligand (TRAIL) was restored by pretreatment with 5-dAzaC and IFN-gamma. Importantly, pretreatment with 5-dAzaC and IFN-gamma acted in concert to significantly enhance TRAIL-induced apoptosis in neuroblastoma and medulloblastoma cells. Inhibition of caspase-8 by dominant-negative caspase-8 or by the relatively specific caspase-8 inhibitior zIETD.fmk inhibited the increase in apoptosis provided by 5-dAzaC and IFN-gamma, indicating that caspase-8 is a key mediator of this sensitization effect. Thus, by demonstrating that 5-dAzaC and IFN-gamma at relatively low individual concentrations cooperate to restore caspase-8 expression and sensitize resistant neuroblastoma and medulloblastoma cells to TRAIL-induced apoptosis, our findings have important implications for novel strategies targeting defective apoptosis pathways in neuroectodermal tumors.

Apoptosis↗

PubMed2005

Pleiotropic effects of sonic hedgehog on muscle satellite cells.

Muscle satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. A regulatory disruption of growth and differentiation of these cells is assumed to result in tumor formation. Here we provide for the first time evidence that sonic hedgehog (Shh) regulates the cell fate of adult muscle satellite cells in mammals. Shh promotes cell division of satellite cells (and of the related model C2C12 cells) and prevents their differentiation into multinucleated myotubes. In addition, Shh inhibits caspase-3 activation and apoptosis induced by serum deprivation. These effects of Shh are reversed by simultaneous administration of cyclopamine, a specific inhibitor of the Shh pathway. Taken together, Shh acts as a proliferation and survival factor of satellite cells in the adult muscle. Our results support the hypothesis of the rhabdomyosarcoma origin from satellite cells and suggest a role for Shh in this process.

Animals↗

PubMed2004

Betulinic acid-induced apoptosis in leukemia cells.

Betulinic acid (BA), a natural component isolated from Birch trees, effectively induces apoptosis in neuroectodermal and epithelial tumor cells and exerts little toxicity in animal trials. Here, we show that BA-induced marked apoptosis in 65% of primary pediatric acute leukemia cells and all leukemia cell lines tested. When compared for in vitro efficiency with conventionally used cytotoxic drugs, BA was more potent than nine out of 10 standard therapeutics and especially efficient in tumor relapse. No crossresistances were found between BA and any cytotoxic drug. Intracellular apoptosis signaling in leukemia tumor cells paralleled the pathway found in neuroectodermal cells involving caspases, but not death receptors. In isolated mitochondria, BA induced release of both cytochrome c and Smac. Taken together, BA potently induces apoptosis in leukemia cells and should be further evaluated as a future drug to treat leukemia.

Adolescent↗

PubMed2004

Daytime variation in performance and tiredness/sleepiness ratings in patients with insomnia, narcolepsy, sleep apnea and normal controls.

Daytime tiredness or sleepiness and deficits in cognitive performance are common complaints in sleep disordered patients. Till now there are few studies comparing patients from different diagnostic groups of sleep disorders in the same experimental protocol. We studied the time course of cognitive functions and subjective alertness in a parallel group design with four groups of patients [narcolepsy, untreated or treated obstructive sleep apnea (OSA), or psychophysiological insomnia] and a control group of subjects without sleep complaints. Each group consisted of 10 subjects, matched for age and gender. After a night with polysomnography, subjects were studied for 10 h from 08:00 hours to 18:00 hours at 20 min intervals under standardized environmental conditions. Four psychological tests were applied, (1) a critical flicker fusion (CFF) test to measure optical fusion threshold (alertness); (2) a paper-and-pencil visual line tracking test (selective attention); (3) a visual analog scale (VAS) for tiredness/sleepiness; and (4) the Tiredness Symptoms Scale (TSS), a 14 items check list. Each test session lasted for 8 min, followed by a 12 min pause. The level and time course of cognitive performance and self-rating data were analysed with hierarchical linear mixed effects models. Cognitive tests showed decrements in alertness and selective attention in untreated patients with insomnia, narcolepsy, and sleep apnea. Narcoleptic patients and untreated OSA had a lower CFF threshold than controls, and for narcoleptic patients the time course differed from that of all other groups. In the visual tracking test the performance of all groups of patients was worse compared with normal controls. Self-rated tiredness/sleepiness was significantly more pronounced in the three groups of untreated patients than in control subjects.

Attention↗

PubMed2001

Activation of apoptosis pathways in peripheral blood lymphocytes by in vivo chemotherapy.

In addition to myelosuppression, anticancer drugs cause rapid and persistent depletion of lymphocytes, possibly by direct apoptosis induction in mature T and B cells. Induction of apoptosis regulators was analyzed in peripheral blood lymphocytes from pediatric patients undergoing first-cycle chemotherapy for solid tumors. In vivo chemotherapy induced a significant increase in lymphocyte apoptosis ex vivo. The activation of initiator caspase-8 and effector caspase-3 and the cleavage of caspase substrates was detected 12 to 48 hours after the onset of therapy. Caspase inhibition by Z-VAD-fmk did not reduce ex vivo lymphocyte apoptosis in all patients, indicating the additional involvement of caspase-independent cell death. No evidence for the involvement of activation-induced cell death was found in the acute phase of lymphocyte depletion as analyzed by activation marker expression and sensitivity for CD95 signaling. Lymphocyte apoptosis in vivo appeared to be predominantly mediated by the mitochondrial pathway because a marked decrease of mitochondrial membrane potential (DeltaPsi(M)) was detected after 24 to 72 hours of treatment, preceded by the increased expression of Bax. Interestingly, despite the use of DNA-damaging agents, p53 remained completely undetectable throughout treatment. In contrast, in vitro treatment with cytarabine and etoposide induced p53 protein, CD95 receptor expression, CD95 sensitivity, and CD95 receptor-ligand interaction in stimulated cycling lymphocytes, but no such induction was seen in resting cells. These data suggest that chemotherapy-induced lymphocyte depletion involves distinct mechanisms of apoptosis induction, such as direct mitochondrial and caspase-dependent pathways in resting cells and p53-dependent pathways in cycling lymphocytes.

Adolescent↗

PubMed2001

Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer.

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.

Apoptosis↗

PubMed2001

Cell type specific involvement of death receptor and mitochondrial pathways in drug-induced apoptosis.

Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-X(L) only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Preclinical and clinical aspects on the use of amifostine as chemoprotector in neuroblastoma patients.

BACKGROUND: In several studies in adults, amifostine (WR-2721) and its active metabolite WR-1065 have shown protection against myelo- and nephrotoxicity of chemotherapeutic agents without compromising cytotoxic efficacy to the tumor. PROCEDURE: In the present study, the effect of amifostine and WR-1065 on neuroblastoma tumor growth and its protective potential for hematotoxicity were investigated. Neither amifostine nor WR-1065 reduced the cytotoxic effect of six drugs commonly used for this tumor when tested on neuroblastoma cells in vitro. RESULTS: In mice carrying human xeno-transplanted neuroblastoma, tumor growth and antitumor activity of chemotherapy were unaffected by amifostine. In addition, hematotoxicity of alkylators was relieved in some cases. In patients with neuroblastoma, amifostine only slightly reduced bone marrow toxicity and was highly emetogenic. CONCLUSIONS: Therefore, amifostine warrants further investigation before its widespread clinical use in the treatment of children with neuroblastoma.

Adolescent↗

PubMed2001

Cognitive dysfunction in sleep disorders.

Fifty-six studies were reviewed that explored cognitive dysfunctions in people with sleep-related breathing disorders (SRBD, 24 studies), insomnia (18 studies), or narcolepsy (14 studies). Individual study outcomes were grouped according to neuropsychological functions. Available evidence was reviewed separately for SRBD, insomnia and narcolepsy. Consistent evidence was found for impaired driving simulation performance in SRBD patients (92.9% of comparisons with control subjects). Other neuropsychological functions with less pronounced impairment included (i) attention span, divided attention and sustained attention for SRBD patients; (ii) attention span, verbal immediate memory and vigilance for insomniac patients, and (iii) sustained attention, vigilance and driving simulation performance for narcoleptic patients. Reduced performance in tasks measuring attention was found to be higher for SRBD and narcoleptic patients (35.9% and 44.2% of all comparisons, respectively) while this rate was lower for insomniac patients (22.8%). Impairment of memory performance in comparison with control subjects was less pronounced for all three groups, with 20.0% for insomnia, 17.1% for SRBD and 15.6% for narcolepsy. In other areas of cognitive functioning, the data did not allow definite conclusions for any of the patient groups.

Journal Article↗

PubMed2000

Apoptotic responsiveness of the Ewing's sarcoma family of tumours to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL).

We investigated the cytotoxic responsiveness of 40 cell lines derived from representatives of the Ewing's sarcoma family of tumours (ESFT), i.e., Ewing's sarcoma (ES), peripheral primitive neuroectodermal tumour (pPNET) and Askin tumour (AT), to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Incubation with TRAIL at 100 ng/ml induced cell death at 24 hr in 19 of 26 ES, 11 of 12 pPNET and 2 of 2 AT cell lines. Half-maximal cell death concentrations (IC(50) values) varied from 0.1 to 20 ng/ml. TRAIL displayed potent cytotoxic activity against freshly derived ESFT cell isolates. Cytotoxicity was associated with phosphatidylserine expression and internucleosomal DNA fragmentation, features characteristic of apoptosis. The apoptotic programme in the sensitive ESFT VH-64 cell line revealed TRAIL-induced activation of FLICE/MACH1 (caspase-8) and CPP32/Yama/apopain (caspase-3) and processing of the prototype caspase substrate poly(ADP-ribose) polymerase. In addition, TRAIL provoked a collapse of the mitochondrial transmembrane potential (DeltaPsi(m)), parallelled by a reduction in ATP levels and release of cytochrome c from mitochondria into the cytosol. Inhibition of caspase-8 and caspase-3 by zIETDfmk and zDEVDfmk, respectively, substantially prevented TRAIL-induced apoptosis. However, zIETDfmk, but not zDEVDfmk, reduced TRAIL-mediated DeltaPsi(m) dissipation, indicating that TRAIL causes mitochondrial dysfunction through caspase-8 acting upstream of mitochondria. While macromolecule synthesis inhibitors (actinomycin D, cycloheximide) augmented susceptibility to TRAIL in TRAIL-responsive cell lines, these agents did not render TRAIL-resistant cell lines susceptible to TRAIL. However, the proteasome inhibitor MG132 sensitised to TRAIL in resistant cell lines. Collectively, these results show that TRAIL initiates effective death in the vast majority (80%) of cell lines derived from ESFT. Since TRAIL provoked cell death in ESFT ex vivo, this cytokine may be a promising drug for the treatment of ESFT in vivo.

Adenosine Triphosphate↗

PubMed2000

Metabolic inhibitors sensitize for CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fas-associated death domain-like interleukin 1-converting enzyme inhibitory protein expression.

Protein or RNA synthesis inhibitors are known to sensitize some resistant cells for death receptor-induced apoptosis. However, the molecular mechanism(s) involved in sensitization have not yet been defined exactly. Here, we report that metabolic inhibitors such as cycloheximide (CHX) or actinomycin D (ActD) sensitize for CD95-induced apoptosis by strongly down-regulating FLIP and RIP expression. Metabolic labeling studies revealed that CHX or ActD inhibited protein or RNA synthesis at concentrations required for sensitization. In contrast to Fas-associated death domain (FADD) or caspase-8, FADD-like interleukin 1-converting enzyme-inhibitory protein (FLIP) and RIP protein levels rapidly decreased upon treatment with CHX or ActD, indicating that both molecules have a high turnover rate. Selective down-regulation of FLIP expression by FLIP antisense oligonucleotides sensitized for CD95-induced apoptosis. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing the recruitment of caspase-8 to the DISC and caspase-8 activation. CHX- or ActD-mediated sensitization to CD95-induced apoptosis was predominantly found in type I cells in which FADD and caspase-8 are recruited to CD95 upon stimulation but not in type II cells in which no DISC formation is detected. Pretreatment with CHX or ActD sensitized for subsequent CD95 stimulation compared with cells without pretreatment. CHX or ActD also reduced XIAP expression and similarly sensitized for tumor necrosis factor-related apoptosis-inducing ligand- or tumor necrosis factor-alpha-induced apoptosis. Because blockade of death receptor triggering by FLIP overexpression has recently been implicated in tumorigenesis and treatment resistance in vivo, strategies to inhibit FLIP expression, e.g., by metabolic inhibitors, may prove to be a useful complementary tool for the treatment of cancer.

Apoptosis↗

PubMed2000

Mitochondrial amplification of death signals determines thymidine kinase/ganciclovir-triggered activation of apoptosis.

Previous clinical experience shows that the efficacy of suicide gene transfer in tumor therapy is limited, resulting from inefficient gene transfer or alternatively, from intrinsic resistance of the tumor in vivo. Herpes simplex virus thymidine kinase/ganciclovir (TK/GCV), a paradigmatic suicide gene therapy system, has been described to exert its cytotoxic effect, at least in part, by inducing apoptosis in target cells. Here, we report that mitochondria amplify TK/GCV-induced apoptosis by regulating p53 accumulation and the effector phase of apoptosis. Treatment with TK/GCV led to mitochondrial perturbations including loss of the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol, inducing caspase activation and nuclear fragmentation. Inhibition of TK/GCV-induced mitochondrial perturbations by Bcl-2 overexpression or by the mitochondrion-specific inhibitor bongkrekic acid also strongly inhibited TK/GCV-induced activation of caspases and apoptosis. TK/GCV-induced mitochondrial perturbations depended on caspase activity possibly initiated by death receptor signaling. Perturbation of mitochondrial function mediated accumulation of wild-type p53 protein, since Bcl-2 overexpression, bongkrekic acid, or inhibition of mitochondrial protein synthesis with chloramphenicol strongly reduced TK/GCV-induced accumulation of wild-type p53 protein. These findings suggest that TK/GCV therapy may be less efficient in tumors in which the mitochondrial amplification of TK/GCV-induced apoptosis is blocked, e.g., by Bcl-2 overexpression. Given the low efficacy of currently used gene therapy systems, our data on molecular mechanisms that regulate sensitivity or resistance toward TK/GCV-induced cytotoxicity might have important implications to improve the clinical application of suicide gene therapy.

Anti-Bacterial Agents↗

PubMed2000

Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells.

Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)

Apoptosis↗

PubMed2000

MycN sensitizes neuroblastoma cells for drug-triggered apoptosis.

BACKGROUND: Amplification of the MYCN gene is found in a large proportion of neuroblastomas and is associated with a poor prognosis. PROCEDURE: To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs, we used a human neuroblastoma cell line with tetracycline-controlled expression of MycN. RESULTS: Neither conditional expression of MycN alone nor low drug concentrations induced apoptosis. However, MycN and cytotoxic drugs cooperated to induce cell death. Apoptosis triggered by MycN and doxorubicin was mediated by cleavage of caspases and involved activation of the CD95 system. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression and to trigger mitochondrial permeability transition and cytochrome c release. CONCLUSION: In that amplification of MYCN is considered an adverse prognostic factor, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.

Apoptosis↗