Elsevier

Cytokine & Growth Factor Reviews

Volume 18, Issues 5–6, October–December 2007, Pages 435-440
Cytokine & Growth Factor Reviews

Programmed cell death: From novel gene discovery to studies on network connectivity and emerging biomedical implications

https://doi.org/10.1016/j.cytogfr.2007.06.004Get rights and content

Introduction

During the early 1990s it was believed that programmed cell death is mainly driven by proteolytic cascades activated and executed by members of the caspase family of proteases. The caspases were mapped along two major pathways, the so called ‘extrinsic’ and the mitochondrial-based ‘intrinsic’ pathways. This simple view was based on the initial genetic screens in C. elegans and the subsequent intensive search for the corresponding mammalian ortologues and interacting proteins. Yet, being aware of the phenotypic complexity of the death process in mammals, we hypothesized many years ago that the molecular basis of programmed cell death may extend beyond such simplistic linear pathways and a more complicated network model should be constructed. To address this working hypothesis, which was against the consensus at that time, we embarked upon an ambitious direction aimed at performing high throughput genome wide screens in mammalian cell cultures exposed in vitro to a death signal (e.g., a killing cytokine; interferon-γ was finally chosen due to several technical advantages). To this end, we first developed a strategy of random knock down of gene expression (TKO) using anti-sense cDNA libraries carried by episomal vectors ([1], [2]; reviewed by [3], [4]). We assumed that the selective reduction in the expression of a gene which positively contributes to the final system performance should attenuate the cell death responses in cells which are continuously exposed to the initial death signal, and thus should provide a forward selection to rescue the relevant genes. In retrospect, the large number of novel death promoting genes that we identified by this function-based strategy confirmed our initial hypothesis on the network's complexity. In addition, the technique was elaborated in a way that we could eventually select individual anti-sense cDNA fragments which displayed different degrees of death protection, suggesting for the first time that different components of the death machinery differ in their impact on system performance (i.e., differ in their ‘functional weight’—a theme which was first discussed in [5]).

We pioneered this function-based genetic screen in a very successful manner as detailed below. Later on the technology was adapted by many research groups and currently it provides one of the most powerful strategies in the developing field of “functional genomics’ in mammalian cells (reviewed by [4]; see also [6]). In recent years, once small interfering RNAs were introduced into mammalian cell studies, functional screens with siRNA or shRNA whole genome libraries also started to be conducted based on the same main TKO principles discussed above. Here I wish to detail step by step how the use of this high throughput function-based approach shaped our global view on the composition, topology and performance of molecular networks moving from the initial gene discovery stage towards global views of network's performance. Based on this work, novel targets for therapeutic intervention in pathologies associated with accelerated cell death and new cancer related prognostic tools have been and will be continued to be discovered as detailed below (reviewed by [7], [8]).

Section snippets

DAP gene discovery

As predicted by our initial working hypothesis, the repertoire of genes that were selected by our screen was very large and included many new components of the cell death machinery which were not discovered by the C. elegans screens. This means that the molecular basis of cell death is larger than initially thought. Most of the death protective anti-sense cDNA fragments which we isolated knocked down the expression of genes which were unknown at that time and therefore we named them death

Clinical implications in cancer

Obviously, one of the challenges in the study of the DAP genes was to find out whether one or more of these pro-cell death genes is a potential tumor suppressor subjected to loss or inactivation in cancer. To this end, several independent directions were undertaken in our laboratory over the past years, including experimental systems which assess the effects of loss of DAPk on tumorigenesis, as well as initial screens of human tumors (reviewed in [7], [14], [37]). The experimental approaches

Implications in pathological cell death: designing novel death protective drugs based on inhibitors of DAP-kinase

The other side of the coin concerning DAPk's structure/function analysis relates to its hyper-activation or gain of function abnormalities, which might lead to excessive cell death in some human pathologies associated with cell loss. Of special interest is the DAPk involvement in neuronal cell death. Mature neurons in adult organisms undergo cell death in response to a variety of stress conditions, including lack of neurotrophic factors, anoxia, excitotoxicity, traumatic injury and

Network characteristics of programmed cell death that emerged from DAP gene discovery

Programmed cell death displays several cellular phenotypes affecting various intracellular organelles, membranes, and nuclei. For example, the well characterized chromatin condensation, nuclear and cytoplasmic fragmentations, membrane blebbing, and mitochondrial membrane permeabilization—are part of what is classified as type I apoptotic cell death. The autophagosome and autolysosome formation, and mitochondrial fragmentation followed by engulfment by autophagosomes are characteristics of type

Conclusions and perspectives

In conclusion, our genome wide genetic screens added a collection of new genes and proteins to the field of programmed cell death resulting in the establishment of novel mechanisms and unexpected concepts. The initial set of anti-sense cDNA fragments, which corresponded to unknown genes when they were functionally selected, led to the characterization of the DAP proteins at the structural, biochemical, and cellular levels. This was followed by wiring the DAP proteins to their upstream and

Acknowledgments

This work was supported by the Kahn Center for System Biology and from the Center of Excellence grant from Flight Attendant Medical Research Institute (FAMRI); A.K. is the incumbent of the Helena Rubinstein Chair of Cancer Research.

Adi Kimchi is a full professor at the Weizmann Institute in the Department of Molecular Genetics which she chaired over the last six years. Her group has developed high throughput genetic screens in mammalian cell cultures, which were successfully used for the isolation of pro-death genes. This led to the discovery of a group of novel death-promoting genes (the DAP genes) and through their study to the establishment of mechanistic views on autophagic cell death and on switches between different

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    Adi Kimchi is a full professor at the Weizmann Institute in the Department of Molecular Genetics which she chaired over the last six years. Her group has developed high throughput genetic screens in mammalian cell cultures, which were successfully used for the isolation of pro-death genes. This led to the discovery of a group of novel death-promoting genes (the DAP genes) and through their study to the establishment of mechanistic views on autophagic cell death and on switches between different forms of cell death. Kimchi is a member of EMBO, and was a member of the Council for Higher Education in Israel between 1996 and 2003. Kimchi was the Director of the Leo and Julia Forchheimer Center for Molecular Genetics between 2001 and 2007. She currently serves in many institutional, national and international scientific committees. Among her list of awards is the Milstein Award for Excellency in Cytokine Research (1999), the Landau Award for Excellency in Biology and Biotechnology (1999), the Seroussi Award for Cancer Research (2002), and the Lombroso Prize for Cancer Research (2006).

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