Apoptosis and Cell Proliferation, страница 2

Understanding the normal regulation of cell death and cell proliferation will becritical e.g., for the development of new and more successful therapies for preventing and treating cancer and for the screening of new anti-cancer compounds.Many assays exist to measure cell death and cell proliferation. However, if you have only recently become interested in celldeath or cell proliferation, you may find the diversity of such assays bewildering. You may not be able to determine whateach assay measures nor decide which assays are best for your purposes.

This guide is designed to help you make such decisions. It presents a brief overview of cell death and cell proliferation, along with the major assays currently available to measure each. In addition, it clearly lists the advantages and the disadvantages of these assays.For those who want to eliminate radioactivity from their laboratories, this review also describes a number of non-radioactiveassays that can serve as alternatives to radioactive assays. Wherever possible, the review will compare the sensitivity of theradioactive and non-radioactive assays.What is new in this second edition?Since the first edition of this guide appeared in 1995, apoptosis research has made much progress.

Apoptosis now is recognized as an essential mechanism of physiological cell death. The basic mechanisms of apoptosis have been clarified.This second edition of the guide reflects that progress in apoptosis research. It contains more information on apoptosis anddescribes more Roche Molecular Biochemicals products to make apoptosis research easier and faster.This edition of the guide also describes new kits for the field of cell proliferation, which continues to be an important researcharea.Some of the highlights of this edition are:P Several new products for the measurement of apoptosis such as a Caspase Assay, Annexin, Anti-Fas and Anti-PARP.P An apoptosis pathways chart, which summarizes information from many laboratories, and a brief literature guide forthose interested in learning more about apoptosis research (see Section 1.1.3, on page 4)P Method selection guides at the beginning of the apoptosis section and the cell proliferation chapter, to help you quicklyfind the Roche Molecular Biochemicals product that best fits your research needs (see Section 1.2., page 7, and Section2.1, page 68)P A separate section, within the cell death chapter, which spotlights those kits that can be used to measure cytotoxicity,regardless of whether the measured cell death is due to apoptosis or necrosis (see Section 1.3, page 50)P More information on the use of flow cytometry to answer questions about cell death and cell proliferationP An appendix, which presents supplementary technical information on such important techniques as TUNEL (TdTmediated X-dUTP nick end labeling)P An introduction to the Apoptosis Chapter by Professor Andrew H.

Wyllie, co-author of the first publication onapoptosis.As we added new information, however, we always kept the original purpose of the guide in mind. As with the first edition,this second edition is still designed to answer one question: What is the best way for you to get the answers you need in yourapoptosis or cell proliferation research?To answer that question, we have retained the features that users told us they liked, such as the flow charts which give anoverview of each assay and numerous examples of “typical assay results”.

We have also added a summary of the main characteristics of each assay and more references to literature describing applications of the assay.VCELL DEATH by Andrew H. WyllieOver the past five or six years there has been a near-exponential increase in publications on apoptosis. Around 30new molecules have been discovered whose knownfunctions are exclusively to do with the initiation or regulation of apoptosis. A further 20 molecules at least, althoughalready associated with important roles in signalling orDNA replication, transcription or repair, have been recognised as affecting the regulation of apoptosis.

This articleis dedicated to young scientists thinking of entering this exploding area of biology, and to those more mature ones whohappened to be looking elsewhere when the blast reachedthem, and consequently are in need of a rapid introductionto the present state of affairs.The term apoptosis first appeared in the biomedical literature in 1972, to delineate a structurally-distinctive mode ofcell death responsible for cell loss within living tissues1.

Thecardinal morphological features are cell shrinkage, accompanied by transient but violent bubbling and blebbing from thesurface, and culminating in separation of the cell into a cluster of membrane-bounded bodies. Organellar structure isusually preserved intact, but the nucleus undergoes a characteristic condensation of chromatin, initiated at sublamellarfoci and often extending to generate toroidal or cap-like,densely heterochromatic regions.

Changes in several cellsurface molecules also ensure that, in tissues, apoptotic cellsare immediately recognised and phagocytosed by their neighbours. The result is that many cells can be deleted from tissues in a relatively short time with little to show for it inconventional microscopic sections.This remarkable process is responsible for cell death in development, normal tissue turnover, atrophy induced by endocrine and other stimuli, negative selection in the immunesystem, and a substantial proportion of T-cell killing. It alsoaccounts for many cell deaths following exposure to cytotoxic compounds, hypoxia or viral infection.

It is a majorfactor in the cell kinetics of tumors, both growing andregressing. Many cancer therapeutic agents exert their effectsthrough initiation of apoptosis, and even the process of carcinogenesis itself seems sometimes to depend upon a selective, critical failure of apoptosis that permits the survival ofcells after mutagenic DNA damage. Apoptosis probablycontributes to many chronic degenerative processes, including Alzheimer’s disease, Parkinson’s disease and heartfailure. So how does it work?Molecular genetic studies on the hard-wired developmentalcell death programme of the nematode Caenorhabditis elegans led to discovery of a set of proteins, widely representedby homologues in other species, and responsible for turningon or off the final commitment to death2. In the nematodethese proteins include the products of the ced3 and ced4genes (which initiate cell suicide), ced9 (which prevents it)and a series of some seven genes involved in recognition andphagocytosis of the doomed cell.VICED3 is the prototype of a family of around a dozen mammalian proteases, called caspases because of the obligatorycysteine in their active site and their predilection for cuttingadjacent to aspartate residues.

Mammalian caspases appearto constitute an autocatalytic cascade, some members (notably caspase 8 or FLICE) being “apical” and more susceptibleto modification by endogenous regulatory proteins, whilstothers (notably caspase 3 – also called CPP32, Yama andapopain) enact the final, irreversible commitment to death.Study of caspase substrates is providing interesting insightsinto the ways in which cells dismantle their structure andfunction. Such substrates include – not surprisingly – cytoskeletal proteins such as actin and fodrin and the nuclearlamins, but also an array of regulatory and chaperone-likeproteins whose function is altered by cleavage in subtle andsuggestive ways3. A recent example is the nuclease chaperone ICAD, whose cleavage permits nuclear entry by a distinctive apoptosis nuclease responsible for chromatin cleavage to oligonucleosome fragments4.Caspases appear to be present in most if not all cells in inactive pro-enzyme form, awaiting activation by cleavage.

Oneof the killing mechanisms of cytotoxic T cells is a protease,granzyme B, that is delivered to the target cell by the T cellgranules and triggers these latent pro-enzymes. There areendogenous triggers also, and the first to be discovered – theC. elegans CED4 protein and its mammalian homologue – isparticularly intriguing because of its mitochondrial origin5.Thus CED4 could be the signal that initiates apoptosis underconditions of shut-down of cellular energy metabolism, orwhen there is a critical level of cell injury affecting mitochondrial respiration. In this way CED4 may act as thelink between agents long known to be associated with mitochondrial injury, such as calcium and reactive oxygen species, and the initiation of apoptosis.A second mitochondrial protein of enormous significance inapoptosis is BCL2, a mammalian homologue of the nematode CED9 protein.

BCL2 has the tertiary structure of abacterial pore-forming protein, and inserts into the outermembrane of mitochondria. It abrogates apoptosis, probably through binding CED4 and another protein BAX,with which it forms heterodimers and which, like CED4, isalso a “killer” protein6. Both BCL2 and BAX have severalstructurally and functionally similar homologues and someof this family at least also tap into other cell membranes suchas the outer nuclear membrane and the endoplasmic reticulum.So are there other sources of death transducers, activatingthe caspase cascade because of injury to or signals arising inother parts of the cell than mitochondria? There are alreadyexamples that show that the answer is yes. Thus, the oncosuppressor protein p53 is activated following some types ofDNA damage and can trigger apoptosis. One way – but onlyone of several – whereby this happens is through transcrip-tional activation of BAX7.