Apoptosis, a physiological process for killing cells, is critical for the normal development and function of multicellular organisms. Abnormalities in cell death control can contribute to a variety of diseases, including cancer, autoimmunity, and degenerative disorders. Signaling for apoptosis occurs through multiple independent pathways that are initiated either from triggering events within the cell or from outside the cell, for instance, by ligation of death receptors. All apoptosis signaling pathways converge on a common machinery of cell destruction that is activated by a family of cysteine proteases (caspases) that cleave proteins at aspartate residues. Dismantling and removal of doomed cells is accomplished by proteolysis of vital cellular constituents, DNA degradation, and phagocytosis by neighboring cells. This article reviews current knowledge of apoptosis signaling, lists several pressing questions, and presents a novel model to explain the biochemical and functional interactions between components of the cell death regulatory machinery.

Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-x L and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.

Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade.

The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ϵ proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. Cancer Res; 74(12); 3238–47. ©2014 AACR.

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

BackgroundAmplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum.MethodsTargeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software “HaplotypR” was developed for data analysis.Results Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold.ConclusionsTo reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10′000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4260-y) contains supplementary material, which is available to authorized users.