Cytotoxic drugs used in chemotherapy of leukemias and solid tumors cause apoptosis in target cells. In lymphoid cells the CD95 (APO-1/Fas)/CD95 ligand (CD95-L) system is a key regulator of apoptosis. Here we describe that doxorbicin induces apoptosis via the CD95/CD95-L system in human leukemia T-cell lines. Doxorubicin-induced apoptosis was completely blocked by inhibition of gene expression and protein synthesis. Also, doxorbicin strongly stimulates CD95-L messenger RNA expression in vitro at concentrations relevant for therapy in vivo. CEM and jurkat cells resistant to CD95-mediated apoptosis were also resistant to doxorbicin-induced apoptosis . Furthermore, doxorbicin-induced apoptosis was inhibited by blocking F(ab')2 anti-APO-1 (anti-CD95) antibody fragments. Expression of CD95-L mRNA and protein in vitro was also stimulated by other cytotoxic drugs such as methotrexate. The finding that apoptosis caused by anticancer drugs may be mediated via the CD95 system provides a new molecular insight into resistance and sensitivity toward chemotherapy in malignancies.

The major regulators of the c‐jun promoter are ATF‐2 and c‐Jun. They act as pre‐bound heterodimers on two ‘AP‐1‐like’ sites, and are preferentially addressed by different types of extracellular signals. The transactivating potential of ATF‐2 is stimulated to a higher extent than that of c‐Jun by a broad group of agents causing DNA damage and other types of cellular stress, such as short‐wavelength UV, or the alkylating compounds N‐methyl‐N’‐nitro‐N‐nitroso‐guanidine (MNNG) or methylmethanesulphonate (MMS). In contrast, treatment with the phorbol ester TPA preferentially enhances c‐Jun‐dependent transactivation but does not affect ATF‐2. Accordingly, UV and MMS but not TPA induce c‐jun transcription in F9 cells, which express ATF‐2, but not c‐Jun. Stimulation of ATF‐2‐dependent transactivation by genotoxic agents requires the presence of threonines 69 and 71 located in the N‐terminal transactivation domain. These sites are the target of p54 and p46 stress‐activated protein kinases (SAPKs) which bind to, and phosphorylate ATF‐2 in vitro. However, p46 and p54 kinase activity is not increased by phorbol ester, which strongly suggests that the protein kinase phosphorylating c‐Jun in response to TPA is distinct from SAPKs and does not act on ATF‐2. Our data demonstrate that distinct signal transduction pathways converge at c‐Jun/ATF‐2, whereby each subunit is individually addressed by a specific class of protein kinases. This allows fine tuned modulation of c‐jun expression by a large spectrum of extracellular signals.

IntroductionIn an overall scenario, the development of malignant tumors results from deregulated proliferation or an inability of cells to undergo apoptotic cell death. 1,2 Anticancer drugs inhibit proliferation and induce apoptosis in sensitive tumor cells. 3,4 The cellular targets for different cytotoxic agents are diverse. Thus, anticancer drugs are classified as DNA-damaging agents (cyclophosphamide, cisplatin, doxorubicin), antimetabolites (methotrexate, 5-fluorouracil), mitotic inhibitors (vincristine), nucleotide analogs (6-mercaptopurine), or inhibitors of topoisomerases (etoposide). The common underlying mechanism for chemotherapy-induced apoptosis might be damage to DNA, lipid components of cell membranes, and cellular proteins causing an imbalance of the cellular homeostasis commonly designated as cellular stress. This in turn initiates a complex cascade of stress-inducible signaling molecules in an attempt to return the cell to its previous equilibrium. As for the response to DNA damage, this may include cell-cycle regulation and repair mechanisms. The type and dose of stress within the cellular context appears to dictate the outcome of the cellular response, which is intimately converted to complex pathways mediating cell-cycle control or cell death. Apoptosis seems to be induced if damage exceeds the capacity of repair mechanisms. Here, we review mechanisms of cellular stress signaling with respect to their integration into apoptosis pathways. The cell death machinery Caspases as death effectorsApoptosis signaling induced by anticancer drugs converges in the activation of intracellular caspases and their modification of protein substrates within the nucleus and cytoplasm (Figure 1). Currently more than 14 caspases have been cloned and partially characterized in mammals, some of which are not involved in apoptosis but rather mediate cytokine processing. Caspases are cysteine proteases produced as inactive zymogens that cleave their substrates at aspartic acid residues contained within a tetrapeptide recognition motif. Activation of initiator caspases (procaspase-8, -9, -10) leads to the proteolytic activation of downstream effector caspases (caspase-3, -6, -7) that cleave specific substrates. For example, cleavage of the nuclear lamin is required for nuclear shrinking and budding. Loss of overall cell shape is probably caused by the cleavage of cytoskeletal proteins, such as fodrin, gelsolin, plectin, actin, and cytokeratin. DNA fragmentation is due to cleavage and inactivation of ICAD, the initiator of CAD (caspase-activated DNase). In addition, the activation of several kinases by caspase cleavage, including PAK2-a member of the p21-activated kinase family-and the Ste20-related kinases MST1 and SLK, contributes to the membrane remodeling and active blebbing observed in apoptotic cells. [5][6][7] Two independent initiator pathways lie immediately upstream of these effector events: cross-linking of death receptors by their ligands and the release of apoptogenic factors from mitochondria. 5,8,9 Mitoc...

Autophagy or ''self eating'' is frequently activated in tumor cells treated with chemotherapy or irradiation. Whether autophagy represents a survival mechanism or rather contributes to cell death remains controversial. To address this issue, the role of autophagy in radiosensitive and radioresistant human cancer cell lines in response to ;-irradiation was examined. We found irradiation-induced accumulation of autophagosomes accompanied by strong mRNA induction of the autophagy-related genes beclin 1, atg3, atg4b, atg4c, atg5, and atg12 in each cell line. Transduction of specific targetsiRNAs led to down-regulation of these genes for up to 8 days as shown by reverse transcription-PCR and Western blot analysis. Blockade of each autophagy-related gene was associated with strongly diminished accumulation of autophagosomes after irradiation. As shown by clonogenic survival, the majority of inhibited autophagy-related genes, each alone or combined, resulted in sensitization of resistant carcinoma cells to radiation, whereas untreated resistant cells but not sensitive cells survived better when autophagy was inhibited. Similarly, radiosensitization or the opposite was observed in different sensitive carcinoma cells and upon inhibition of different autophagy genes. Mutant p53 had no effect on accumulation of autophagosomes but slightly increased clonogenic survival, as expected, because mutated p53 protects cells by conferring resistance to apoptosis. In our system, short-time inhibition of autophagy along with radiotherapy lead to enhanced cytotoxicity of radiotherapy in resistant cancer cells. [Cancer Res 2008;68(5):1485-94]

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31 þ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs.

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