Biomedical subjects

Claus Belka

Publications and source records attributed to Claus Belka.

At least 37 records · Page 2Linked to original sources

PubMed2005

Type I and type II reactions in TRAIL-induced apoptosis -- results from dose-response studies.

Death receptor-induced apoptosis is paradigmatically mediated via the recruitment of FADD adapter molecule to the ligand/receptor complex and subsequent activation of caspase-8. However, several observations provided evidence that components of the mitochondrial apoptosis pathway are involved in death receptor-mediated apoptosis. In this regard, caspase-8-mediated activation of Bid induces the release of cytochrome c from the mitochondria, which, in turn, triggers the formation of the apoptosome protein complex, resulting in the activation of caspase-9. Whereas Bax or Bak were shown to be required for the proapoptotic effect of Bid, Bcl-2 was described to interfere with its action. Up to now, contradictory results regarding the role of Bcl-2 in TRAIL-induced apoptosis have been published. In order to study the influence of Bcl-2 on TRAIL-induced cell death more detailed, we utilized a tetracycline-regulated Bcl-2 expression system in Jurkat T cells. After having analysed the dose response for TRAIL-induced activation of caspase-8, -9, -3, breakdown of the mitochondrial membrane potential, and changes in the apoptotic morphology in cells expressing different Bcl-2 levels, we conclude that overexpression of Bcl-2 mediates a partial resistance towards lower doses of TRAIL that can be overcome when higher doses of TRAIL are applied. Thus, the requirement of the mitochondrial pathway for death receptor-induced apoptosis in type II cells should be reconsidered, since the protective effect of Bcl-2 is limited to lower TRAIL doses or early observation time points.

Antineoplastic Agents↗

PubMed2004

Evidence for a protective role of Mcl-1 in proteasome inhibitor-induced apoptosis.

Proteasome inhibitors exhibit antitumor activity against malignancies of different histology. Yet, the mechanisms underlying this effect are poorly understood. Recent evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, possibly reducing their cytotoxicity. These include the Bcl-2 family member Mcl-1, whose down-regulation has been proposed to initiate apoptosis in response to genotoxic stimuli. In this study, we found that proteasome inhibitors release cyotochrome c and second mitochondria-derived activator of caspase (SMAC)/Diablo and trigger the subsequent apoptotic cascade in spite of concomitant Mcl-1 increase. However, our data indicate that subtraction of Mcl-1 during apoptosis, although not required for early release of proapoptotic factors, is probably relevant in speeding up cell demise, since RNA interference-mediated Mcl-1 silencing is lethal in lymphoma cells. Consistent with this, the cytotoxic effects of proteasome inhibitors are enhanced when Mcl-1 increase is impeded. Thus, this study identifies Mcl-1 accumulation as an unwanted molecular consequence of exposure to proteasome inhibitors, which slows down their proapoptotic effects. Pharmacologic or genetic approaches targeting Mcl-1, including therapeutic RNAi, may increase the effectiveness of these compounds.

Acetylcysteine↗

PubMed2004

Adenine deoxynucleotides fludarabine and cladribine induce apoptosis in a CD95/Fas receptor, FADD and caspase-8-independent manner by activation of the mitochondrial cell death pathway.

The adenine deoxynucleosides cladribine (2CdA) and fludarabine (FAraA) are DNA-damaging agents that interfere with DNA repair and induce apoptosis in nonproliferating lymphoid cells. Although both drugs are clinically used for the treatment of indolent lymphoproliferative diseases, the pathways of apoptosis induction remain largely unknown. In the present work, we demonstrate that both drugs induce apoptosis independently of death receptor signaling but activate the mitochondrial cell death pathway. To dissect the signaling pathways, we employed Jurkat cells either deficient for FADD or caspase-8 or overexpressing Bcl-2. In Bcl-2 overexpressing cells, apoptosis and cytochrome c release were blocked whereas processing of caspase-9, -3 and -8 was partially inhibited. In contrast, neither the deficiency of FADD or caspase-8 nor the interference with death receptor signaling by neutralizing anti-CD95/Fas antibodies affected cell death. Inhibitor experiments revealed that caspase-8 is processed by caspase-3-like caspases. Moreover, cytochrome c release and processing of caspase-9 and -3 occurred to an equal extent in wild-type FADD -/- and caspase-8 -/- Jurkat cells. Likewise, apoptosis induction by cladribine or fludarabine was not hampered upon inhibition of caspase-8 in MOLT-3 and MOLT-4 cells or overexpression of a dominant-negative FADD mutant in BJAB cells. Thus, we conclude that apoptosis induced by nucleoside analogues is independent from death receptor signaling as well as from a proposed direct effect on APAF-1, but rather follows the mitochondrial signaling pathway of cytochrome c release and subsequent processing of caspase-9 and -3.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway.

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

Apoptosis↗

PubMed2004

Cyclic exposure to hypoxia and reoxygenation selects for tumor cells with defects in mitochondrial apoptotic pathways.

The negative influence of hypoxia on the outcome of malignant tumors may be caused by direct oxygen effects, and potentially, the selection of resistant tumor cells under repetitive hypoxia. To evaluate whether cyclic hypoxia selects for resistant cells and to analyze the underlying mechanisms, the influence of cyclic hypoxia on intracellular death pathways was determined in tumor cells. It could be demonstrated that cyclic hypoxia selects for cells with increased resistance against hypoxia-induced apoptosis. These cells exhibited a cross-resistance against paradigmatic triggers of mitochondrial apoptotic pathways (ionizing radiation/etoposide). In contrast, TRAIL-receptor mediated apoptosis remained unaffected. Thus, cyclic hypoxia selects for cells with defects of the mitochondrial rather than receptor-mediated pathways. Selection of p53-defective cells has been described as a consequence of cyclic hypoxia; therefore, we evaluated the impact of hypoxic selection on activation of p21 and downstream mediators of p53-dependent apoptosis. p53 function and protein levels of key mediators of mitochondrial apoptosis remained unaffected by hypoxic selection. However, radiation-induced conformational changes of Bax were reduced after cyclic hypoxia. In summary, it could be demonstrated that hypoxic stress confers a selection pressure on mitochondrial apoptotic pathways and, consecutively, to an increased resistance toward mitochondrial death triggers.

Apoptosis↗

PubMed2004

Involvement of tyrosine kinase p56/Lck in apoptosis induction by anticancer drugs.

Induction of apoptosis is a hallmark of the cellular response of human lymphocytes and lymphoma cells to treatment with anticancer drugs and irradiation. Both treatment modalities trigger apoptosis through intrinsic, mitochondrial apoptosis pathways resulting in the activation of caspases. We and others have shown that the tyrosine kinase p56/Lck is involved in the regulation of apoptosis induced by irradiation or treatment with ceramide but dispensable for death receptor triggered cell death. However, the role of p56/Lck for apoptosis induction in response to anticancer drugs is unclear. To elucidate the putative requirement of p56/Lck for apoptosis signaling of cytotoxic drugs, activation of caspases and alteration of mitochondrial functions were determined in Jurkat T cells, the p56/Lck deficient JCaM1.6 cells and the p56/Lck retransfected JCaM1.6/Lck cells in response to chemotherapeutic drugs with different targets of their primary action. Treatment with Doxorubicin, Paclitaxel or 5-Fluorouracil induced a breakdown of the mitochondrial membrane potential and apoptotic cell death in p56/Lck expressing Jurkat and the retransfected JCaM1.6/Lck cells within 48h of treatment. However, almost no mitochondrial alterations and no induction of apoptosis could be detected in the p56/Lck deficient JCaM1.6 cells. Correspondingly, activation of caspases-9, -8, and -3 and cleavage of the caspase-3 substrate PARP (poly-(ADP-ribose)-polymerase) were almost completely absent in JCaM1.6 cells while present in p56/Lck positive Jurkat and JCaM1.6/Lck cells. In contrast, retransfection of the cells with the p56/Lck-related tyrosine kinase Src could not restore sensitivity to the treatment with cytotoxic drugs indicating a specific role of the tyrosine kinase p56/Lck in apoptosis signaling. Importantly, kinase-activity of p56/Lck may be dispensable for its pro-apoptoptic action since preincubation with the Src-kinase inhibitor PP2 did not reduce apoptosis induced by cytotoxic drugs. In conclusion, the tyrosine kinase p56/Lck is essential for apoptosis induction by Doxorubicin, Paclitaxel and 5-Fluorouracil regulating early steps of the mitochondrial apoptosis signaling cascade, including alteration of mitochondrial functions and caspase-activation.

Antineoplastic Agents↗

PubMed2004

Molecular ordering of hypoxia-induced apoptosis: critical involvement of the mitochondrial death pathway in a FADD/caspase-8 independent manner.

Dys-regulated growth and improper angiogenesis commonly lead to areas of hypoxia in human tumors. Hypoxia is known to be associated with a worse outcome since a lack of oxygen interferes with the efficacy of chemotherapy or radiotherapy. In parallel, hypoxia-induced apoptosis may also impose a selection pressure favoring growth of more resistant tumor cells. However, the mechanisms of hypoxia-induced apoptosis and the relative contribution of intrinsic and extrinsic apoptotic pathways are not understood. Therefore, Jurkat cell lines with defined defects in the extrinsic or intrinsic signaling cascades were used to evaluate the role of either pathway for induction of apoptosis under hypoxic conditions. Jurkat cells were incubated in hypoxia and the rate of apoptosis induction was determined by Western blotting, fluorescence microscopy and flow cytometry. Hypoxia-induced apoptosis was not affected by lack of caspase-8 or FADD, whereas overexpression of Bcl-2 or expression of dominant-negative caspase-9 mutant rendered the cells resistant to hypoxia-induced apoptosis. These results suggest that hypoxia-induced apoptosis mainly relies on intrinsic, mitochondrial pathways, whereas extrinsic pathways have no significant implications in this process. Thus, in human tumors, hypoxia will mainly lead to the selection of hypoxia-resistant cells with defects in mitochondrial apoptosis signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Influence of hypoxia on TRAIL-induced apoptosis in tumor cells.

PURPOSE: Tumor hypoxia reduces the efficacy of radiotherapy, many types of chemotherapy, and tumor necrosis factor-alpha (TNF-alpha). TRAIL (TNF-alpha-related apoptosis-inducing ligand) is a ligand for death receptors of the TNF superfamily shown to be selectively toxic for tumor cells and thereby a promising antineoplastic tool. The impact of hypoxia on TRAIL-induced apoptosis was examined in this study. METHODS AND MATERIALS: Apoptosis induction and growth rates of various tumor cell lines under hypoxia were evaluated in vitro. Biologically effective induction of hypoxia was verified by determination of hypoxia-inducible factor-1 (HIF-1) activation. The efficacy of TRAIL- and radiation-induced apoptosis under different oxygen conditions was quantified in vitro. The impact of Bcl-2 on TRAIL-induced apoptosis under hypoxia or normoxia was evaluated by comparing cells expressing Bcl-2 with a vector control. RESULTS: Moderate hypoxia caused no growth retardation or apoptosis, but led to activation of HIF-1 as a prerequisite of hypoxic gene induction. Cellular responses to TRAIL differed considerably among the cell lines tested. Hypoxia reduced radiation-induced, but not TRAIL-induced, apoptosis in the tested cell lines. Hypoxia did not induce Bcl-2 expression. Bcl-2 had a minor impact on the efficacy of TRAIL-induced apoptosis. CONCLUSION: Taken together, the data indicate that TRAIL is clearly effective under conditions of proven hypoxia.

Antineoplastic Agents↗

PubMed2004

Apoptosis-modulating agents in combination with radiotherapy-current status and outlook.

PURPOSE: To increase the therapeutic efficacy of ionizing radiation or to reduce radiation-mediated side effects, diverse research centers for translational radiation oncology have headed for a specific modulation of defined cellular death pathways. In this regard, several signaling systems have proved to be of high potential value. RESULTS: It has previously been shown that apoptotic pathways induced by ionizing radiation are distinct from death pathways triggered by death ligands such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The combination of both radiation and TRAIL was highly efficient in vitro and in preclinical mouse models. However, several aspects of normal tissue toxicity have not been solved, and no Phase I data are available yet. A second approach tested in a Phase I trial is based on the observation that synthetic phospholipid derivatives (alkyllysophospholipids and alkylphosphocholines) strongly enhance apoptotic effects by modulating the balance among the mitogenic, anti-apoptotic MAPK, phosphatidylinositol 3'-kinase (PI3K)/Akt, and the pro-apoptotic SAPK/JNK signaling pathways. Furthermore, others have provided evidence that inhibition of anti-apoptotic signals generated by mitogenic stimuli may increase radiation responses. In this context, controversial data are available regarding the influence of a pharmacologic abrogation of MEK1, Erk1/2 signaling on apoptotic sensitivity but no Phase I trials of MEK inhibitors either alone or in combination with radiation have yet been published. However, inhibition of the PI3K/Akt survival pathway using compounds such as the protein kinase C (PKC) inhibitor PKC412 has been shown to induce apoptosis or to increase the apoptotic sensitivity of tumor cells. Therefore, these drugs may be used alone or in combination with radiation to increase tumor control; however, Phase I data are lacking. Several other drugs, including cyclooxygenase-2 inhibitors, betulinic acid, and proteasome inhibitors, have been shown to interact with apoptotic signal transduction. Again, most of the drugs have not been tested in combination with radiation in vivo or-in the case of cyclooxygenase-2 inhibitors-exert pleiotropic effects. CONCLUSION: Although the examples do not reflect all available strategies, it is clear that several promising approaches targeting defined cell death pathways have been developed and entered into clinical trials. The use of synthetic phospholipid derivatives in a Phase I trial is an important example, proving that basic research in radiation biology finally guides the development of new treatment strategies. This, and other approaches, will hopefully increase tumor control rates and reduce side effects in the future.

Apoptosis↗

PubMed2004

Palliative percutaneous radiotherapy in non-small-cell lung cancer.

Percutaneous radiotherapy is an effective tool for the palliative treatment of patients with non-small-cell lung cancer (NSCLC). About two thirds of patients experience a notably improvement of symptoms after palliative radiotherapy. A whole variety of very different radiation schedules like a single fraction of 10 Gy, 2 fractions of 8.5 Gy, 10 fractions of 3 Gy, 25 fractions of 2 Gy, and others have been used for palliation. The effects of these different schedules have been compared in a total of 11 randomized trials of which 10 reported survival data and form subject of this review. According to these studies, an increase in total irradiation dose does not substantially prolong median survival, but results in a significant better 1-year survival. A comprehensive review of the data reveals that patients with poor performance status (ECOG score > or = 3) do not benefit from higher doses, but patients with good performance status do benefit. Patients with poor performance status, and patients with large distant tumour burden regardless of their performance status, are efficiently treated by a short course of relatively low dose radiotherapy. Schedules like 2 x 8.5 Gy and 4 x 5 Gy are most appropriate in this situation. For patients with good performance status the choice of the optimal radiation schedule is less clear. Schedules with total doses between 30 and 45 Gy in 2.5-3.0 Gy fractions should be preferred in these situations.

Carcinoma, Non-Small-Cell Lung↗

PubMed2003

Celecoxib activates a novel mitochondrial apoptosis signaling pathway.

The cyclooxygenase (COX)-2 inhibitor Celecoxib may inhibit cancer cell growth independently of its capacity to block the COX-2 enzyme. The growth inhibitory effect had been attributed to its pro-apoptotic effects. However, the molecular details of Celecoxib-induced apoptosis have not been analyzed yet. To differentiate between death receptor and mitochondrial signaling pathways, induction of apoptosis upon treatment with Celecoxib was tested in Jurkat T- and BJAB B-lymphoma cell lines with defects in either pathway. Celecoxib-induced dose- and time-dependent apoptosis in Jurkat and BJAB cells involving i) activation of caspases-9, -8, and -3, ii) cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNAase, iii) breakdown of the mitochondrial membrane potential, and iv) release of cytochrome c. Lack of Fas-associated death domain protein (FADD), overexpression of a dominant negative FADD, lack of caspase-8, and treatment with caspase-8-specific inhibitors had no influence on Celecoxib-induced apoptosis. In contrast, overexpression of a dominant negative caspase-9 or pharmacological inhibition of caspase-9 strongly interfered with Celecoxib-induced cell death. Furthermore, expression of Apaf-1 was required for Celecoxib-induced apoptosis. Importantly, Bcl-2 overexpression did not abrogate caspase activation, mitochondrial alterations, and apoptosis upon Celecoxib treatment while inhibiting radiation induced apoptosis. In conclusion, Celecoxib induces apoptosis via a novel apoptosome-dependent but Bcl-2-independent mitochondrial pathway.

Animals↗

PubMed2003

Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal.

Efficient engulfment of the intact cell corpse is a critical end point of apoptosis, required to prevent secondary necrosis and inflammation. The presentation of "eat-me" signals on the dying cell is an important part of this process of recognition and engulfment by professional phagocytes. Here, we present evidence that apoptotic cells secrete chemotactic factor(s) that stimulate the attraction of monocytic cells and primary macrophages. The activation of caspase-3 in the apoptotic cell was found to be required for the release of this chemotactic factor(s). The putative chemoattractant was identified as the phospholipid, lysophosphatidylcholine. Further analysis showed that lysophosphatidylcholine was released from apoptotic cells due to the caspase-3 mediated activation of the calcium-independent phospholipase A(2). These data suggest that in addition to eat-me signals, apoptotic cells display attraction signals to ensure the efficient removal of apoptotic cells and prevent postapoptotic necrosis.

Animals↗

PubMed2003

Intracellular mediators of erucylphosphocholine-induced apoptosis.

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFalpha-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Experimental thermoradiotherapy in malignant hepatocellular carcinoma.

PURPOSE: The human liver is known to be a relatively radiosensitive organ that develops clinically relevant late radiation hepatitis subsequent to whole liver treatment with total doses above 30 Gy in conventional fractionation. Experimental data, as well as clinical series, have demonstrated that hyperthermia of solid tumors in addition to radiotherapy enhances tumor growth inhibition and tumor control probability. We therefore developed an experimental model for combined radiotherapy and hyperthermia of the liver in transplantable rat Morris hepatoma 3924A. METHODS AND MATERIALS: A cube of approximately 8 mm(3) was implanted subcapsularly into the middle liver lobe of 59 male syngenic ACI rats weighing approximately 180-200 g. On Day 16 after tumor implantation, irradiation of the tumor-bearing liver with either 0 Gy/25 Gy/35 Gy/45 Gy total dose in 10 fractions +/- hyperthermia (target temperature 40-42 degrees C) twice a week was initiated. Energy deposition was monitored by temperature probes in the liver and esophagus of the rats. Determination of tumor volume with magnetic resonance imaging was performed 2 to 5 weeks after the end of therapy. The tumor growth rates could be estimated for 44 rats. If the growth rate was positive (37 rats), the inverse of the growth rate was interpreted as the time to 10-fold tumor volume. Otherwise the maximum observation time was considered as a censored value in a parametric survival analysis. RESULTS: Intrahepatic temperature probes showed a temperature plateau of greater than 40 degrees C after 5 to 8 min subsequent to initiation of hyperthermia. The target temperatures could be maintained for at least 22 min > or =40 degrees C and 10 min > or =41 degrees C, respectively. Median plateau temperature in liver, esophagus, and epicutaneously was 41.2 degrees C (standard deviation [SD] 0.7 degrees C; range 38.2 to 43.3 degrees C), 40.4 degrees C (SD 1.08 degrees C; range 38.9 to 41.8 degrees C), and 40.8 degrees C (SD 0.8 degrees C; range 38.2 to 42.7 degrees C), respectively. Elevation of the temperature in the esophagus correlated with intrahepatic temperatures in the range of 39-42 degrees C, r = 0.957. The increase in time to 10-fold tumor volume for each step of irradiation dosage was by 34% (95% confidence interval [CI] 20% to 49%) without hyperthermia and by 60% (95% CI 47% to 80%) with hyperthermia (p < 0.0001). CONCLUSION: Treatment outcome after experimental percutaneous thermoradiotherapy in intrahepatically implanted Morris hepatoma 3924A was related to total dose of irradiation and concurrently administered regional hyperthermia. An increased radiosensitivity due to hyperthermia (<42 degrees C) has to be assumed.

Animals↗

PubMed2003

Tributyltin (TBT) induces ultra-rapid caspase activation independent of apoptosome formation in human platelets.

Activation of caspases has been demonstrated to be involved in thrombocytopenia and prolonged storage of platelet concentrates. Platelets represent enucleate cells that comprise all elements of the mitochondrial apoptosis pathway. However, no apoptotic stimuli capable of activating the endogenous caspase cascade have been identified so far. Using tributyltin (TBT) we could identify a compound that is capable of activating caspase-9 and -3 in platelets. Recent studies implicate that TBT induces apoptosis via the mitochondrial signaling pathway that is characterized by the formation of a high-molecular-weight complex (apoptosome) containing the adapter protein Apaf-1 and active caspase-9. Interestingly, addition of TBT induced the activation of caspase-9 in an ultra-rapid kinetic within the first 2 min. In addition, size exclusion chromatography revealed that TBT-mediated processing of caspase-9 occurs in the absence of the apoptosome. Thus, these data implicate that TBT induces the activation of caspase-9 by a mechanism not involving the formation of the apoptosome.

Apoptosis↗

PubMed2003

The tyrosine kinase Lck is involved in regulation of mitochondrial apoptosis pathways.

The induction of apoptosis requires the activation of a highly coordinated signaling network ultimately leading to the activation of caspases. In previous experiments we and others have shown that the tyrosine kinase Lck is required for adequate apoptosis induction in response to ionizing radiation, ceramide incubation and overexpression of the HIV-TAT protein. However, the position of Lck within given apoptotic signaling cascades remains unclear. We therefore aimed to define the role of Lck during radiation-induced apoptosis. Apoptosis induction in response to ionizing radiation, CD95 or TRAIL receptor stimulation was determined in Jurkat T-cells, the Lck-deficient Jurkat clone JCaM1.6- and Lck-retransfected JCaM1.6/Lck. No apoptosis, release of cytochrome c, breakdown of the mitochondrial potential were detectable during the first 48 h after irradiation of JCaM1.6 cells. In parallel, no activation of caspase-9, -8 and -3 was detectable. Since mitochondrial apoptosis pathways act within a feedback mechanism during death-receptor-mediated apoptosis, the influence of the Lck defect on CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was also tested. Both stimuli induced apoptosis in Lck-deficient cells. However, the kinetics of apoptosis induction determined by caspase-8, -9 and -3 activation as well as deltapsi(m) breakdown was slowed. We conclude that the Lck deficiency influences early steps during radiation-induced mitochondrial alterations.

Apoptosis↗

PubMed2003

[Death inducing ligands in combination with ionizing radiation: objective and current knowledge].

BACKGROUND: Apart from optimization of the radiation technology, future new strategies in radiation oncology will focus on the biological optimisation leading to improved adaptation of the tumor therapy on each tumor entity. In this regard, different areas of biological research may be distinguished: prediction, development of new cytotoxic agents, improvement of the tolerance of normal tissue and the optimisation of radiochemotherapy. MATERIAL AND METHOD: For the development of new strategies the knowledge of molecular mechanisms of radiation induced cell death is essential. In the present article a novel biological strategy for optimisation of radiotherapy is introduced. RESULTS: Distinct pathways mediate apoptosis in response to death receptor stimulation and ionizing radiation although an overlapping set of molecules is involved. TRAIL (TNF alpha-related apoptosis inducing ligand) is a type-II membrane protein belonging to the TNF family, which preferentially induces apoptotic cell death in a wide variety of tumor cells but not in normal cells. Based on the assumption of distinct signalling pathways, combination of TRAIL and ionizing radiation seems ideal to increase the therapeutic efficacy. Our study presents the rationale, own data, data of other groups as well as the current status of the latest findings regarding the function and physiological role of the TRAIL death ligand. CONCLUSIONS: Apoptosis resistance towards ionising radiation is not associated with a general apoptosis resistance. Death ligands, for example TRAIL trigger apoptosis even in cells not undergoing apoptosis in response to radiation. A combination of both modalities induces additive or synergistic effects in regard to apoptosis induction and eradication of clonogenic tumor cells. Thus, the combination of TRAIL with radiation proofs that novel treatment strategies may be developed on the basis of molecular or cell biological research.

Animals↗

PubMed2003

Molecular requirements for the combined effects of TRAIL and ionising radiation.

BACKGROUND AND PURPOSE: Previously it was shown that combination of death ligand TRAIL and irradiation strongly increases cell kill in several human tumour cell lines. Since Bcl-2 overexpression did not strongly interfere with the efficacy, components of the mitochondrial death pathway are not required for an effective combined treatment. In the present study the minimal molecular prerequisites for the efficacy of a combined treatment were determined. MATERIALS AND METHODS: Apoptosis induction in control, caspase-8 and FADD negative Jurkat cells, BJAB control and FADD-DN cells was analysed by FACS. Activation of caspase-8, -10 and -3 and cleavage of PARP was determined by immunoblotting. TRAIL receptors were activated using recombinant human TRAIL. Surface expression of TRAIL receptors DR4 and DR5 was analysed by FACS. RESULTS: Jurkat T-cells express the agonistic DR5 receptor but not DR4. Presence of FADD was found to be essential for TRAIL induced apoptosis. Caspase-8 negative cells show very low rates of apoptosis after prolonged stimulation with TRAIL. No combined effects of TRAIL with irradiation could be found in FADD-DN overexpressing and FADD deficient cells. However, the combination of TRAIL and irradiation clearly lead to a combined effect in caspase-8 negative Jurkat cells, albeit with reduced death rates. In these cells activation of the alternative initiator caspase-10 could be detected after combined treatment. CONCLUSION: Our data show that a combined therapy with TRAIL and irradiation will only be effective in cells expressing at least one agonistic TRAIL receptor, FADD and caspase-8 or caspase-10.

Adaptor Proteins, Signal Transducing↗