We are starting our blog with a review of cell death assays for drug discovery. In this posting I will describe different modes of cell death as well as background information necessary for better understanding of cell death assays. All assays in our blog are available as a service from Alera Labs. Please contact us at (919) 228-8209 or click here to learn more about our assays services.
Cell death and toxicity issues are critically important to all drug discovery projects and culminate in a Clinical Trial Phase I study primarily designed to establish safety and tolerability of a drug.
Different cell death modes play an important role in multiple diseases including stroke, heart attack, Parkinson’s, Alzheimer’s, Huntington’s, toxic and infectious syndromes. Impaired cell death is usually associated with cancer and autoimmune disorders.
For the purposes of drug discovery it is safe to assume that cell death is caused by either apoptosis or necrosis.
Apoptosis is a programmed form of cell death that manifests itself with cell detachment from the substrate, cell shrinkage, chromatin condensation and nuclear fragmentation and finally cell fragmentation into small apoptotic bodies which are cleared by phagocytes in vivo.
Apoptosis can be caused by a diverse range of cell signals, that may include toxins, hormones, growth factors, cytokines, heat, radiation, nutrient deprivation, viral infection, or damage to the membrane. In many cases apoptosis can be viewed as an ultimate but unsuccessful attempt by cells to cope with stress and maintain homeostasis.
In contrast to apoptosis, necrosis is almost always a traumatic cell death caused by external toxins, infection or trauma. Chemical or toxin burns come to mind as an appropriate example of necrosis. Rupture of cell membrane and disorganised release of cellular contents are the most obvious signs of necrosis. Also, while apoptosis is accompanied by cell shrinkage (pyknosis), necrosis is usually accompanied by cell swelling (oncosis).
An important point is that in cell culture ALL cell death modalities including apoptosis will turn into necrosis if allowed to proceed for long enough time. This is because cell culture usually lacks professional phagocytes that are responsible for clearing apoptotic bodies in vivo. This fact also makes it quite challenging to distinguish cell death modalities using assays because all signs of late apoptosis are shared with signs of necrosis in cell culture. Therefore it is critical to follow the timing of marker appearance in cell culture to distinguish apoptosis fromm necrosis.
There are several other forms of cell death such as lysosomal degradation of cytoplasm structure (autophagy), programmed necrosis (necroptosis), and detachment of cells from extracellular matrix (anoikis) that are encountered in several specialized situations and that are rarely encountered in the context of drug discovery. This might change as autophagy inducers and inhibitors are starting to be considered as attractive drug candidates in cell death syndromes such as myocardial infarction, ischemia, cancer, and neurodegenerative disease (See Ref 2 for more information).
It is therefore important not only to measure cell death but also determine cell death modality responsible for the effect.
We will discuss available cell death assays in the next article.