Apoptosis - the regulated destruction of a cell - is a complicated process. The decision to die cannot be taken lightly, and the activity of many genes influence a cell's likelihood of activating its self-destruction programme. Once the decision is taken, proper execution of the apoptotic programme requires the coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the main pathways that are used to activate cell death.

The biochemistry of apoptosis

http://web.stanford.edu/group/brunet/Cell1999

Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor

Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis, the cell suicide program critical for development, tissue homeostasis, and protection against pathogens. Those most similar to Bcl-2 promote cell survival by inhibiting adapters needed for activation of the proteases (caspases) that dismantle the cell. More distant relatives instead promote apoptosis, apparently through mechanisms that include displacing the adapters from the pro-survival proteins. Thus, for many but not all apoptotic signals, the balance between these competing activities determines cell fate. Bcl-2 family members are essential for maintenance of major organ systems, and mutations affecting them are implicated in cancer.

https://science.sciencemag.org/content/sci/281/5381/1322.full.pdf

The Bcl-2 Protein Family: Arbiters of Cell Survival

Cell Death: Critical Control Points

Cleavage of BID by Caspase 8 Mediates the Mitochondrial Damage in the Fas Pathway of Apoptosis

We have identified two cell types, each using almost exclusively one of two different CD95 (APO‐1/Fas) signaling pathways. In type I cells, caspase‐8 was activated within seconds and caspase‐3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for ∼60 min. However, both type I and type II cells showed similar kinetics of CD95‐mediated apoptosis and loss of mitochondrial transmembrane potential (ΔΨ m ). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl‐2 or Bcl‐x L overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl‐2 or Bcl‐x L blocked caspase‐8 and caspase‐3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase‐8 by the death‐inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase‐8 and caspase‐3 occurred following the loss of ΔΨ m . Overexpression of caspase‐3 in the caspase‐3‐negative cell line MCF7‐Fas, normally resistant to CD95‐mediated apoptosis by overexpression of Bcl‐x L , converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl‐x L . In summary, in the presence of caspase‐3 the amount of active caspase‐8 generated at the DISC determines whether a mitochondria‐independent apoptosis pathway is used (type I cells) or not (type II cells).

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Two CD95 (APO-1/Fas) signaling pathways

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.

Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells.

Anticancer agents have been shown to trigger apoptosis in chemosensitive tumors such as neuroblastomas. We previously identified activation of the CD95 system as one of the key mechanisms for doxorubicin-induced apoptosis in leukemic T cells. Here, we report that therapeutic concentrations of doxorubicin, cisplatinum, and VP-16 led to induction of CD95 receptor and CD95 ligand (CD95-L) that mediated cell death in chemosensitive neuroblastoma cells. Using F(ab′)2 anti-CD95 antibody fragments to interfere with CD95-L-receptor interaction markedly reduced apoptosis induced by those drugs in vitro. Cyclosporin A inhibited induction of CD95 mRNA and CD95-L mRNA and blocked drug-mediated apoptosis. Drug-induced apoptosis involved activation of caspases (interleukin 1β-converting enzyme/Ced-3-like proteases) and processing of the prototype caspase substrate PARP and was completely blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a peptide inhibitor of caspases. In addition, neuroblastoma cells that were resistant to CD95-triggered apoptosis also displayed cross-resistance to chemotherapeutic agents. These data provide new clues for understanding the molecular requirements for drug-induced apoptosis in chemosensitive neuroblastoma cells by demonstrating that cell death was mediated via the CD95-L-receptor system and may open new avenues for targeting drugresistance of neuroblastoma.

https://cancerres.aacrjournals.org/content/canres/57/17/3823.full.pdf

The CD95 (APO-1/Fas) System Mediates Drug-induced Apoptosis in Neuroblastoma Cells

Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-XL conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.

https://cancerres.aacrjournals.org/content/canres/57/21/4956.full.pdf

Betulinic Acid Triggers CD95 (APO-1/Fas)- and p53-independent Apoptosis via Activation of Caspases in Neuroectodermal Tumors

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-XL 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.

Claudia Friesen

Papers

Two CD95 (APO-1/Fas) signaling pathways

Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells.

The CD95 (APO-1/Fas) System Mediates Drug-induced Apoptosis in Neuroblastoma Cells

Betulinic Acid Triggers CD95 (APO-1/Fas)- and p53-independent Apoptosis via Activation of Caspases in Neuroectodermal Tumors

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