Michael O. Hengartner

The biochemistry of apoptosis

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 cells 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.

Programmed cell death: Alive and well in the new millennium

Research performed over the past decade has transformed apoptosis from a distinctive form of cell death known only by its characteristic morphology and genomic destruction to an increasingly well understood cellular disassembly pathway remarkable for its complex and multifaceted regulation. Here, we summarize current understanding of apoptotic events, note recent advances in this field and identify questions that might help guide research in the coming years.

Finding function in novel targets: C. elegans as a model organism

Despite its apparent simplicity, the nematode worm Caenorhabditis elegans has developed into an important model for biomedical research, particularly in the functional characterization of novel drug targets that have been identified using genomics technologies. The cellular complexity and the conservation of disease pathways between C. elegans and higher organisms, together with the simplicity and cost-effectiveness of cultivation, make for an effective in vivo model that is amenable to whole-organism high-throughput compound screens and large-scale target validation. This review describes how C. elegans models can be used to advance our understanding of the molecular mechanisms of drug action and disease pathogenesis.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

A Conserved Checkpoint Pathway Mediates DNA Damage–Induced Apoptosis and Cell Cycle Arrest in C. elegans

To maintain genomic stability following DNA damage, multicellular organisms activate checkpoints that induce cell cycle arrest or apoptosis. Here we show that genotoxic stress blocks cell proliferation and induces apoptosis of germ cells in the nematode C. elegans. Accumulation of recombination intermediates similarly leads to the demise of affected cells. Checkpoint-induced apoptosis is mediated by the core apoptotic machinery (CED-9/CED-4/CED-3) but is genetically distinct from somatic cell death and physiological germ cell death. Mutations in three genes--mrt-2, which encodes the C. elegans homolog of the S. pombe rad1 checkpoint gene, rad-5, and him-7-block both DNA damage-induced apoptosis and cell proliferation arrest. Our results implicate rad1 homologs in DNA damage-induced apoptosis in animals.

miRNAs and apoptosis: RNAs to die for

MicroRNAs (miRNAs) are small non-coding RNAs of about 18–24 nucleotides in length that negatively regulate gene expression. Discovered only recently, it has become clear that they are involved in many biological processes such as developmental timing, differentiation and cell death. Data that connect miRNAs to various kinds of diseases, particularly cancer, are accumulating. miRNAs can influence cancer development in many ways, including the regulation of cell proliferation, cell transformation, and cell death. In this review, we focus on miRNAs that have been shown to play a role in the regulation of apoptosis. We first describe in detail how Drosophila has been utilized as a model organism to connect several miRNAs with the cell death machinery. We discuss the genetic approaches that led to the identification of those miRNAs and subsequent work that helped to establish their function. In the second part of the review article, we focus on the involvement of miRNAs in apoptosis regulation in mammals. Intriguingly, many of the miRNAs that regulate apoptosis have been shown to affect cancer development. In the end, we discuss a virally encoded miRNA that influences the cell death response in the mammalian host cell. In summary, the data gathered over the recent years clearly show the potential and important role of miRNAs to regulate apoptosis at various levels and in several organisms.

Programmed cell death in Caenorhabditis elegans

Programmed cell death in the nematode Caenorhabditis elegans requires the activities of the genes ced-3 and ced-4 and is antagonized by the activity of the gene ced-9. Cloning of these C. elegans genes has shown that two of them encode proteins with similarity to vertebrate cell death genes and has revealed that nematodes and mammals share a common pathway for programmed cell death.

Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis

BACKGROUND Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. RESULTS We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. CONCLUSIONS BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.

Candidate Adaptor Protein CED-6 Promotes the Engulfment of Apoptotic Cells in C. elegans

The rapid engulfment (phagocytosis) of cells undergoing programmed cell death (apoptosis) is a fundamental biological process that is not well understood. Here we report the cloning and functional characterization of ced-6, a gene specifically required for the engulfment of apoptotic cells in the nematode C. elegans. The CED-6 protein contains a phosphotyrosine binding domain at its N terminus and a proline/serine-rich region in its C-terminal half. Genetic mosaic analysis demonstrates that ced-6 acts within engulfing cells. We also show that ced-6 can promote the engulfment of cells at both early and late stages of apoptosis. Our data suggest that CED-6 is an adaptor molecule acting in a signal transduction pathway that specifically mediates the recognition and engulfment of apoptotic cells.

Developmental apoptosis in C. elegans: a complex CEDnario

Apoptosis, an evolutionarily conserved programme of cellular self-destruction, is essential for the development and survival of most multicellular animals. It is required to ensure functional organ architecture and to maintain tissue homeostasis. During development of the simple nematode Caenorhabditis elegans, apoptosis claims over 10% of the somatic cells that are generated ? these cells were healthy but unnecessary. Exciting insights into the regulation and execution of apoptosis in C. elegans have recently been made. These new findings will undoubtedly influence our perception of developmental apoptosis in more complex species, including humans.