follows a certain localization pattern. at prophase and metaphase, survivin is mainly nuclear in location. during prophase, as the chromatin condenses so that it is visible under the microscope, survivin starts to move to the centromeres. at prometaphase when the nuclear membrane dissociates and spindle microtubules cross over the nuclear region, survivin 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, survivin 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 survivin also moves along to the midplate. survivin thus accumulates at the midplate at telophase. finally, survivin localizes to the midbody at the cleavage furrow. interaction and localization to the mitochondria it has been shown that survivin can heterodimerize individually with the two splice variants survivin-2b and survivin-deltaex3. evidence of the heterodimerization of survivin splice variants with survivin was shown with co-immunoprecipitation experiments after cotransfection with the respective survivin variants with survivin. to determine the localization of exogenously expressed survivin-2b and survivin-deltaex3, fusion constructs of the proteins were made with gfp and hcred respectively and daoy cells were transfected with the plasmid constructs. survivin was also tagged with a fluorescent protein. the fusion of the survivin variants with the fluorescent molecules allows for simple detection of cellular location by fluorescence microscopy. survivin-2b by itself, localized to both nuclear and cytoplasmic compartments whereas survivin-deltaex3 localized only in the nucleus. the localization of the three variants (survivin, survivin-2b, and survivin-deltaex3) differ, however, when cotransfected together rather than individually. to see which subcellular compartments contained the survivin splice variants complexes, fluorescent antibody markers for different organelles in the cell were employed. the assumption is that, under fluorescence microscopy, if the particular survivin complex is located in that particular cell compartment, one would observe an overlap from the fluorescence given off by the tagged survivin complex and the tagged compartment as well. different color fluorescence is used to distinguish compartment from survivin. endoplasmic reticulum and lyosomes: no colocalization mitochondria and golgi: both survivin/survivin-2b and survivin/survivin-deltaex3 colocalize to verify these observations, they fractionated the subcellular compartments and performed western blot analysis to definitively say that survivin complexes did indeed localize at these compartments. expression in different carcinomas survivin 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 survivin 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 lines and the lowest levels in renal cancers. knowing the relative expression levels of survivin in different tumour types may prove helpful as survivin-related therapy may be administered depending on the expression level and reliance of the tumour type on survivin for resistance to apoptosis. as an oncogene survivin can be regarded as an oncogene as its aberrant overexpression in most cancer cells contributes to their resistance to apoptotic stimuli and chemotherapeutic therapies, thus contributing to their ongoing survival. genomic instability most human cancers have been found to have gains and losses of chromosomes that may be due to chromosomal instability (cin). one of the things that cause cin is the inactivation of genes that control the proper segregation of the sister chromatids during mitosis. in gaining a better understanding of survivin's function in mitotic regulation, scientists have looked into the area of genomic instability. it is known that survivin associates with microtubules of the mitotic spindle at the start of mitosis. it has been shown in the literature that knocking out survivin in cancer cells will disrupt microtubule formation and result in polyploidy as well as massive apoptosis. it has also been shown that survivin-depleted cells exit mitosis without achieving proper chromosome alignment and then reforms single tetraploid nuclei. further evidence also suggests that survivin is needed for sustaining mitotic arrest upon encounter with mitosis problems. the evidence mentioned above implicates that survivin plays an important regulatory role both in the progression of mitosis and sustaining mitotic arrest. this seems strange, as survivin is known to be highly upregulated in most cancer cells (that usually contain chromosome instability characteristics), and its function is that which promotes proper regulation of mitosis. regulation by p53 p53 inhibits survivin expression at the transcriptional level wild-type p53 has been shown to repress survivin expression at the mrna level. using an adenovirus vector for wild-type p53, human ovarian cancer cell line 2774qw1 (which expresses mutant p53) was transfected. mrna levels of survivin were analyzed by real-time quantitative pcr (rt-pcr) and showed time-dependent down regulation of survivin mrna levels when the cells were infected with wild-type p53. a 3.6 fold decrease of survivin mrna level was observed 16 hours after infection initiation and decreased 6.7 fold 24 hours after infection. western blot results do show that there is indeed the p53 from the adenoviral vector was being expressed in the cells using antibody specific for p53. the expression of p53 levels indicative of its role in survivin repression shows that p53 started to be expressed 6 hours into infection and had its highest level at 16–24 hours. to further confirm that endogenous wild-type p53 is really causing the repression of survivin gene expression, the authors induced a549 (human lung cancer cell line with wild-type p53) and t47d (human breast cancer cell line with mutant p53) cells with dna-damaging agent adriamycin to trigger the physiological p53 apoptotic response in these cancer cells and compare the survivin levels measured to the same cells without dna damage induction. the a549 line, which intrinsically has functioning wild-type p53, showed significant reduction in survivin levels compared to non-induced cells. this same effect was not seen in t47d cells that carry mutant inactive p53. p53's normal function is to regulate genes that control apoptosis. as survivin is a known inhibitor of apoptosis, it can be implied that p53 repression of survivin is one mechanism by which cells can undergo apoptosis upon induction by apoptotic stimuli or signals. when survivin is over-expressed in the cell lines mentioned in the previous paragraph, apoptotic response from dna-damaging agent adriamycin decreased in a dose-dependent manner. this suggests that down-regulation of survivin by p53 is important for p53-mediated apoptotic pathway to successfully result in apoptosis. it is known that a defining characteristic of most tumors is the over-expression of survivin and the complete loss of wild-type p53. the evidence put forth by mirza et al. shows that there exists a link between survivin and p53 that can possibly explain a critical event that contributes to cancer progression. p53 suppression of survivin expression in order to see whether p53 re-expression in cancer cells (that have lost p53 expression) has the suppressive effect on the promoter of the survivin gene, a luciferase reporter construct was made. the isolated survivin promoter was placed upstream of the luciferase reporter gene. in a luciferase reporter assay, if the promoter is active, the luciferase gene is transcribed and translated into a product that gives off light that can measured quantitatively and, thus, represents the activity of the promoter. this construct was transfected into cancer cells that had either wild-type or mutant p53. high luciferase activity was measured in the cells with mutant p53 and significantly lower luciferase levels were measured for cells with wild-type p53. transfection of different cell types with wild-type p53 was associated with a strong repression of the survivin promoter. transfection with mutant p53 was not shown to strongly repress the survivin promoter. more luciferase constructs were prepared with varying degrees of deletion from the 5' end of the survivin promoter region. at one point, there was deletion that caused the survivin levels to be indifferent to the presence of the p53 over-expression plasmid, indicating that there is a specific region proximal to the transcription start site that is needed for p53 suppression of survivin. although it has been found that two p53 binding sites are located on the survivin gene promoter, analysis using deletions and mutations has shown that these sites are not essential to transcriptional inactivation. instead, it is observed that modification of the chromatin inside of the promoter region may be responsible for the transcriptional repression of the survivin gene. this is explained below in the epigenetic regulation section. cell cycle regulation survivin is shown to be clearly regulated by the cell cycle, as its expression is found to be dominant only in the g2/m phase. this regulation exists at the transcriptional level, as there is evidence of the presence of cell-cycle-dependent element/cell-cycle gene homology region (cde/chr)boxes located in the survivin promoter region. further evidence to support this mechanism of regulation includes the evidence that surivin is poly-ubiquinated and degraded by proteasomes during interphase of the cell cycle. moreover, survivin has been shown to localize to components of the mitotic spindle during metaphase and anaphase of mitosis. physical association between polymerized tubulin and survivin have been shown in vitro as well. it is also shown that post-transcriptional modification of survivin involving the phosphorylation of thr34 leads to increased protein stability in the g2/m phase of the cell cycle. it is known from mirza et al. that repression of survivin by p53 is not a result of any cell cycle progressive regulation. the same experiment by mirza et al. with regard to determining p53 suppression of survivin at the transcriptional level was repeated, but this time for cells arrested in different stages of the cell cycle. it was shown that, although p53 arrests the numbers of cells to different extents in different phases, the measured level of survivin mrna and protein levels were the same across all the samples transfected with the wild-type p53. this shows that p53 acts in a cell-cycle independent manner to inhibit survivin expression. epigenetic and genetic regulation as observed through the literature, survivin is found to be over-expressed across many tumour types. scientists are not sure of the mechanism that causes this abnormal over-expression of survivin; however, p53 is downregulated in almost all cancers, so it is tempting to suggest that survivin over-expression is due to p53 inactivity. wagner et al. investigated the possible molecular mechanism involved with the over expression of survivin in acute myeloid leukemia (aml). in their experiments, they did both an epigenetic and a genetic analysis of the survivin gene promoter region in aml patients and compared the observations to what was seen in peripheral blood mononuclear cells (pbmcs) that have been shown to express no survivin. assuming that the molecular mechanism of survivin re-expression in cancerous cells is at the transcriptional level, the authors decided to look at particular parts of the promoter region of survivin in order to see what happens in cancer cells that does not happen in normal cells that causes such a high level of survivin to be expressed. with regards to an epigenetic mechanism of survivin gene regulation, the authors measured the methylation status of the survivin promoter, since it is accepted that methylation of genes plays an important role in carcinogenesis by silencing of certain genes or vice versa. the authors used methylation specific polymerase chain reaction with bisulfite sequencing methods to measure the promoter methylation status in aml and pbmcs and found unmethylated survivin promoters in both groups. this result shows that dna methylation status is not an important regulator of survivin re-expression during leukemogenesis. however, de carvalho et al. performed a dna methylation screening and identified that dna methylation of irak3 plays a key role in survivin up-regulation in different types of cancer, suggesting that epigenetic mechanisms plays an indirect role on abnormal over-expression of survivin. with regard to genetic analysis of the survivin promoter region, the isolated dna of aml and pbmcs were treated with bisulfite, and the survivin promoter region sequence was amplified out with pcr and sequenced to look for any particular genetic changes in the dna sequence between the two groups. three single-nucleotide polymorphisms (snps) were identified and were all present both in aml patients and in healthy donors. this result suggests that the occurrence of these snps in the promoter region of the survivin gene also appears to be of no importance to