Apoptosis Apoptosis, the process of programmed cell death, involves complex signaling pathways and cascades of molecular events. This process is needed for proper development during embryonic and fetal growth where there is destruction and reconstruction of cellular structures. In adult organisms, apoptosis is needed to maintain differentiated tissue by striking the balance between proliferation and cell death. It is known that intracellular proteases called caspases degrade the cellular contents of the cell by proteolysis upon activation of the death pathway. Mammalian cells have two main pathways that lead to apoptosis. 1. Extrinsic Pathway: Initiated by extrinsic ligands binding to death receptors on the surface of the cell. An example of this is the binding of tumour necrosis factor-alpha (TNF-alpha) to TNF-alpha receptor. An example of a TNF receptor is Fas (CD95), which recruits activator caspases like caspase-8 upon binding TNF at the cell surface. The activation of the initiator caspases then initiates a downstream cascade of events that results in the induction of effector caspases that function in apoptosis. 2. Intrinsic Pathway: This pathway is initiated by intracellular or environmental stimuli. It is focused on detecting the improper functioning of the mitochondria in the cell and, as a result, activates signaling pathways to commit suicide. The membrane permeability of the mitochondria increases and particular proteins are released into the cytoplasm that facilitates the activation of initiator caspases. The particular protein released from the mitochondria is cytochrome c. Cytochrome c then binds to Apaf-1 in the cytosol and results in the activation of initiator caspase-9. The activation of the initiator caspases then initiates a downstream cascade of events that results in the induction of effector caspases that function in apoptosis. One family of proteins called IAPs plays a role in regulating cell death by inhibiting the process. IAPs like<unk> , inhibit apoptosis by physically binding to and inhibiting proper caspase function. The function of IAPs is evolutionarily conserved as Drosophila homologues of IAPs have been shown to be essential for cell survival. IAPs have been implicated in studies to have a regulatory effect on cell division. Yeast cells with knock-outs of certain IAP genes did not show problems associated with cell death, but showed defects in mitosis characterized by improper chromosome segregation or failed cytokinesis. Deletion of particular IAPs does not seem to have a profound effect on the cell-death pathway as there is a redundancy of function by the many IAPs that exist in a cell. They have been implicated, however, to play a role in maintaining an anti-apoptotic environment intracellularly. Changing the expression of particular IAPs has shown an increase in spontaneous cell death induction or increased sensitivity to death stimuli. Mechanism of action Inhibition of Bax and Fas-induced apoptosis Tamm et al. have shown that<unk>  inhibits both Bax and Fas-induced apoptotic pathways. The experiment involved transfecting HEK 293 cells with a Bax-encoding plasmid, which resulted in an increase in apoptosis (~7 fold) as measured by DAPI staining. They then contransfected the 293 cells with Bax-encoding plasmid and<unk> -encoding plasmids. They observed that cells transfected along with the<unk>  showed a significant decrease in apoptosis (~3 fold). A similar result also showed for cells transfected with the Fas-overexpressing plasmid. Immunoblots were performed and confirmed that<unk>  does not inhibit by mechanism of preventing Bax or Fas protein from being made into fully functional proteins. Therefore,<unk>  should be acting somewhere downstream of the Bax or Fas signaling pathway to inhibit apoptosis through these pathways. Interaction with caspase-3 and -7 In this part of the experiment, Tamm et al. transfected 293 cells with<unk>  and lysed them to obtain cell lysate. The lysates were incubated with different caspase forms and<unk>  was immunopercipitated with anti-survivin antibody. The idea behind this is that, if<unk>  binds physically with the caspase it is incubated with, it will be co-precipitated along with the<unk>  while everything else in the lysate is washed away. The immunoprecipitates were then run on SDS-PAGE and then immunoblotted for detection of the desired caspase. If the caspase of interest was detected, it meant that it was bound to<unk>  in the immunoprecipitation step implicating that<unk>  and the particular caspase had bound beforehand. Active caspase-3 and -7 coimmunoprecipitated with<unk> . The inactive proforms of caspase-3 and -7 did not bind<unk> .<unk>  also does not bind to active caspase-8. Caspase-3 and -7 are effector proteases whereas caspase-8 is an initiator caspase that sits more upstream in the apoptotic pathway. These results demonstrate<unk> 's capability to bind with particular caspases in vitro, but may not necessarily translate over to actual physiological conditions. Later, a 2001 study confirmed that human<unk>  tightly binds caspase-3 and -7 when expressed in E. coli. Further evidence to support the idea that<unk>  blocks apoptosis by directly inhibiting caspases was given by Tamm et al. 293 cells were transfected with either overexposed caspase-3 or -7 encoding plasmid and with<unk> . They showed that<unk>  inhibited processing of these two caspases into their active forms. While<unk>  has been shown as mentioned above to bind to only the active forms of these caspases, it is likely here that<unk>  inhibits the active forms of the caspases resulting from cleaving and activating more of its own proforms. Thus,<unk>  acts possibly by preventing such a cascade of cleavage and activation amplification from happening resulting in decreased apoptosis. In similar manner, looking at the mitochondrial pathway of apoptosis, cytochrome c was transiently expressed in 293 cells to look at the inhibitory effects<unk>  had on this pathway. Although the details are not here,<unk>  was shown to also inhibit cytochrome c and caspase-8-induced activation of caspases. Regulation of cytokinesis While the mechanism by which<unk>  may regulate cell mitosis and cytokinesis is not known, the observations made on its localization during mitosis suggests strongly that it is involved in some way in the cytokinetic process. Proliferating Daoy cells were placed on a glass coverslip, fixed and stained with fluorescent antibodies for<unk>  and alpha-tubulin. Immunoflourescence using confocal microscopy was used to look at the localization of<unk>  and tubulin during the cell-cycle to look for any patterns of<unk>  expression.<unk>  was absent in interphase, but present in the G2-M phase. During the different stages of mitosis, one could see that<unk>  follows a certain localization pattern. At prophase and metaphase,<unk>  is mainly nuclear in location. During prophase, as the chromatin condenses so that it is visible under the microscope,<unk>  starts to move to the centromeres. At prometaphase when the nuclear membrane dissociates and spindle microtubules cross over the nuclear region,<unk>  stays put at the centromeres. At metaphase, when the chromosomes align at the middle plate and are pulled with high tension to either pole by the kinetochore attachments,<unk>  then associates with the kinetochores. At anaphase as separation of the chromatids happens, the kinetochore microtubules shorten as the chromosomes move towards to the spindle poles and<unk>  also moves along to the midplate.<unk>  thus accumulates at the midplate at telophase. Finally,<unk>  localizes to the midbody at the cleavage furrow. Interaction and localization to the mitochondria It has been shown that<unk>  can heterodimerize individually with the two splice variants<unk> -2B and<unk> -deltaEx3. Evidence of the heterodimerization of<unk>  splice variants with<unk>  was shown with co-immunoprecipitation experiments after cotransfection with the respective<unk>  variants with<unk> . To determine the localization of exogenously expressed<unk> -2B and<unk> -deltaEx3, fusion constructs of the proteins were made with GFP and HcRed respectively and Daoy cells were transfected with the plasmid constructs.<unk>  was also tagged with a fluorescent protein. The fusion of the<unk>  variants with the fluorescent molecules allows for simple detection of cellular location by fluorescence microscopy.<unk> -2B by itself, localized to both nuclear and cytoplasmic compartments whereas<unk> -deltaEx3 localized only in the nucleus. The localization of the three variants<unk> ,<unk> -2B, and<unk> -deltaEx3) differ, however, when cotransfected together rather than individually. To see which subcellular compartments contained the<unk>  splice variants complexes, fluorescent antibody markers for different organelles in the cell were employed. The assumption is that, under fluorescence microscopy, if the particular<unk>  complex is located in that particular cell compartment, one would observe an overlap from the fluorescence given off by the tagged<unk>  complex and the tagged compartment as well. Different color fluorescence is used to distinguish compartment from<unk> . * Endoplasmic reticulum and lyosomes: no colocalization * Mitochondria and golgi: both<unk><unk> -2B and<unk><unk> -deltaEx3 colocalize To verify these observations, they fractionated the subcellular compartments and performed western blot analysis to definitively say that<unk>  complexes did indeed localize at these compartments. Role in cancer Expression in different carcinomas<unk>  is known to be expressed during fetal development and across most tumour cell types, but is rarely present in normal, non-malignant adult cells. Tamm et al. showed that<unk>  was expressed in all 60 different human tumour lines used in the National Cancer Institute's cancer drug-screening program, with the highest levels of expression in breast and lung cancer
Survivin
Tetherin
Perospirone
Adhyperforin
Diamphotoxin
Lectin
Suramin
Visine