apoptotic and necrotic cells. There are also various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow cytometry), cellular markers such as DNA fragmentation (flow cytometry), caspase activation, Bid cleavage, and cytochrome c release (Western blotting). Supernatant screening for caspases, HMGB1, and cytokeratin 18 release can identify primary from secondary necrotic cells. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic markers (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). A selection of techniques that can be used to distinguish apoptosis from necroptotic cells could be found in these references. Implication in disease Defective pathways The many different types of apoptotic pathways contain a multitude of different biochemical components, many of them not yet understood. As a pathway is more or less sequential in nature, removing or modifying one component leads to an effect in another. In a living organism, this can have disastrous effects, often in the form of disease or disorder. A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each one is the same: The normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis. This results in a cell that lives past its "use-by date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell's becoming cancerous or diseased. A recently described example of this concept in action can be seen in the development of a lung cancer called NCI-H460. The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in cells of the H460 cell line. XIAPs bind to the processed form of caspase-9 and suppress the activity of apoptotic activator cytochrome c, therefore overexpression leads to a decrease in the number of proapoptotic agonists. As a consequence, the balance of anti-apoptotic and proapoptotic effectors is upset in favour of the former, and the damaged cells continue to replicate despite being directed to die. Defects in regulation of apoptosis in cancer cells occur often at the level of control of transcription factors. As a particular example, defects in molecules that control transcription factor NF-κB