Induce apoptosis with staurosporine immunochemistry. M staurosporine was markedly potentiated by cycloheximide. Since staurosporine is a strong apoptosis inducer and hela cells closely resemble the target cells of natural chlamydial infection, this system was used in the. A proapoptotic effect of the cdk inhibitor p57kip2 on staurosporine. L12100 shows a delayed response to staurosporineinduced apoptosis. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in. Cells may also become damaged or undergo some type of infection. Exposure of murine cortical cell cultures to 30100 nm staurosporine induced concentrationdependent selective neuronal degeneration over the following day. What are the effects of pkc inhibition using go6983 on hela cells. This concentration works well for inducing many cell lines including hl60 and jurkat cells when using a 45 hour 37c incubation period. The results indicated that hela cells underwent apoptosis in dose and timedependent manners when treated with dioscin. The mechanisms of apoptosis induction are diverse and may depend on the cell type. Staurosporine is a cell permeable alkaloid isolated from streptomyces staurosporeus exhibiting anticancer activity. This agent induces apoptosis by an undetermined mechanism.
This change in the caspase cascade suggests cdv induces apoptosis via the intrinsic pathway, excluding the need for the initiator caspase8. Both p21cip1 and p27kip1 have been shown to protect cells from staurosporine induced apoptosis in hela cells 7. Rickettsia rickettsii infection protects human microvascular. Ijms free fulltext the apoptotic volume decrease is an. Since heat shock can activate apoptosis, we tested for a possible antiapoptotic behavior of us11. The model faithfully reproduces key elements of apoptosis, including chromatin condensation, dna fragmentation. Role of bovine bcl2a1 gene in staurosporineinduced. Protection against heat and staurosporine mediated apoptosis by. Response to staurosporine and expression of pro and antiapoptotic genes in endothelial cells infected withr.
Fkbp51 protects 661w cell culture from staurosporineinduced apoptosis donald raymond daudt, thomas yorio department of pharmacology and neuroscience university of north texas health science center fort worth, tx purpose. Intracellular alkalinization was accompanied by translocation of bax to the mitochondria, cytochromec release, and cell death. One way to remove these cells without causing harm to other cells is for your body to initiate apoptosis. Apoptosis is initiated by various kinds of stimuli, including infections, radiation, etc. Staurosporine is often used as a general method for inducing apoptosis 1. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. By itself, dox did not induce apoptosis in hela cells, mock transfected control cells, or kn1 and. Apoptosis time lapse imaging of u2os cells treated with. Online monitoring of apoptosis in insulinsecreting cells.
Aug 21, 2000 the sensitivity of kn1 and knx2 cells to staurosporine was also evaluated by tunel analysis fig. Morphological and biochemical analysis of staurosporineinduced apoptosis in l1210s cells in the presence of zvadfmk. Mechanisms of staurosporine induced apoptosis in a human. Staurosporine transiently increased the phosphotransferase activity of cjun nterminal kinase1 jnk1, which in turn may activate the transcriptional activity of activating protein1 ap1. Research paper staurosporine induced apoptosis may.
Spatiotemporal activation of caspasedependent and independent. Jun 21, 2000 mitochondrial changes during apoptosis. A proapoptotic effect of the cdk inhibitor p57 kip2 on staurosporineinduced apoptosis in hela cells article in biochemical and biophysical research communications 2963. Staurosporine, a microbial alkaloid, is a strong inhibitor of protein kinases.
Induction of apoptosis in hela cells with staurosporine produced a rise in the intracellular ph ph i. It is a potent and cellpermeable competitive inhibitor for protein kinase c ic50 0. It is commonly used to induce apoptosis in experimental settings, although the mechanism is not fully understood. Staurosporineinduced apoptosis in chang liver cells is. Tafani m1, cohn ja, karpinich no, rothman rj, russo ma, farber jl. There are other forms such as necroptosis or autophagy, which share some of the characteristics of either form. Staurosporineinduced apoptosis was investigated on both cell lines l1210s and l12100 cells exposed for. Hydrogen peroxide induces apoptosis in hela cells through mitochondrial pathway mayank singh, himani sharma, neeta singh department of biochemistry, all india institute of medical science, new delhi 110029, india. To dissect the molecular events leading to neuronal cell death, we have developed a cellfree model of neuronal apoptosis. Staurosporine can induce death of hela cells via a cytochrome c caspase9caspase3 mitochondrialdependent apoptotic pathway. The adhesion of h295r cells to type v collagen induced a significant increase of apoptosis up to 52 %. Nov 04, 2016 this video will show you how to perform an apoptosis assay on the celigo image cytometer using caspase 37 and hoechst reagents.
Staurosporine induces apoptosis in human neuroblastoma cells and chick embryonic neurons 3,4. This video will show you how to perform an apoptosis assay on the celigo image cytometer using caspase 37 and hoechst reagents. Cells free fulltext apoptotic volume decrease avd is. The apoptosis of mcf7 cells induced by staurosporine can be enhanced by the expression of functional caspase3 via caspase8 activation and bid cleavage. In the present study, we investigated the relationship between the avd induction and the stressresponsive mapk cascade activation during the apoptosis process induced by staurosporine sts in hela cells. Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanismleading to programmed cell death. For online detection of apoptosis using the fret technique, rinm5f cells were transiently transfected with plasmids pfretkeaf or pfretdevd. When apoptosis was induced by staurosporine in hela cells, sp2 protein suppressed nuclear fragmentation and apoptotic body formation. Induction of apoptosis in hela cells by transactivationdeficient p53 ygal haupt, 1 sheldon rowan, 1 eitan shaulian, karen h. Changes in endoplasmic reticulum luminal environment affect. Staurosporine induced apoptosis of endothelial cells involves activation of caspase3, and could be a useful model to study strategies of cell death inhibition. Protection against heat and staurosporine mediated apoptosis. Infectious diseases modulate apoptosis, and this contributes to disease pathogenesis. Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes.
Establishment of a cellfree system of neuronal apoptosis. Staurosporine and etoposide induce apoptosis in cancer cells. Store at 20c staurosporine kda 200 140 100 80 60 50 cleaved parp 0 1 0. It has been found that one way in which staurosporine induces apoptosis is by activating caspase3. At lower concentration, depending on the cell type, staurosporine induces specific cell cycle effects arresting cells either in g 1 or in g 2 phase of the cell cycle. Inhibition of apoptosis in chlamydiainfected cells. Cells may recognize viruses and gene mutations and can induce death to prevent the damage from spreading. Vousden, 2 and moshe oren 14 department of chemical immunology, the weizmann institute of science, rehovot 76100, israel. H295r cells grown on plastic showed a low responsiveness to staurosporine, with an apoptotic rate of 24 %, as compared to breast cancer mcf7 cells, with an apoptotic rate of 60 %. Cells were incubated in the presence filled squares or absence open triangles of 2. Staurosporine induced apoptosis may activate cancer stemlike.
Jun 22, 2001 l12100 shows a delayed response to staurosporine induced apoptosis. For example, treatment with staurosporine, a protein kinase c inhibitor, but not with antifas mab induces apoptosis in hela cells, which are fas receptordeficient. Lipidomic analysis of apoptotic hela cells induced by. Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the. On the other hand, activation of the map kinase mapk cascade is also known to play a critical role in apoptosis. The staurosporine treated hela lysate is designed for use as a western blot positive control for antibodies against apoptosis specific proteins or for experimental treatments to induce apoptosis. The apoptotic volume decrease is an upstream event of map kinase activation during staurosporineinduced apoptosis in hela cells. Apoptosis is a fundamental process required for normal development of the nervous system and is triggered during neurodegenerative disease. Protection against heat and staurosporine mediated. In the present study, we compare the sensitivity of caski and hela cells hpv positive, wildtype p53 and c33a cells hpv negative, mutated p53 to a protein. Staurosporine induced apoptosis was investigated on both cell lines l1210s and l12100 cells exposed for 3 h to the drug 5.
The clickit tunel assays have been tested in hela, a549 and cho k1 cells with a variety of reagents that induce apoptosis, including staurosporine, and multiplexed with antibodybased detection of other apoptosis biomarkers such as cleaved polyadpribose polymerase parp, cleaved caspase3 and phosphohistone 2b. Measure apoptosis using suspension jurkat cells treated with 3. Regulation of intracellular ph mediates bax activation in hela cells treated with staurosporine or tumor necrosis factoralpha. Hela cell apoptosis caused by cdv follows a different mechanism than that in vero cell lines. We induced apoptosis in murine osteoblast mc3t3e1 cells by exposure to the staurosporine. Zvadfmk was administrated to the cells 1 h prior the addition of staurosporine.
Role of bovine bcl2a1 gene in staurosporineinduced apoptotic. Apr 28, 20 ericminikel biose16 these are notes from lecture 11 of harvard extensions cell biology course. The extracellular release of hmgb1 during apoptotic cell. Here, we show that, in hela cells, us11 expression strongly reduced heat induced apoptosis, a phenomenon independent of hsp expression and characterized by a delayed cytochrome c ef. Na zhou 1, rong wang 1, yizhuang zhang 1, zhen lei 2, xuehui zhang 1, ruobi hu 1, hui li 1, yiqing mao 1, xi wang 1, david m. These data indicate that orp150 has an important cytoprotective role in hypoxiainduced cellular perturbation and that orp150associated inhibition of apoptosis may. These results showed clearly that phospholipids and related phospholipases played important roles in paclitaxelinduced apoptosis in hela cells. May 01, 2009 response to staurosporine and expression of pro and antiapoptotic genes in endothelial cells infected withr.
However, apoptosis induced by cdv in hela cells does not involve the initiator protein caspase8. Apoptosis and necrosis represent two extremes of cell death, with a full range of variations in between. However, the processing of caspase 3 induced by staurosporine in chlamydia. Dohaekseungkitang extract induced apoptosis in human cervical carcinoma hela cells. The mechanism of how it mediates this is not well understood. There are other forms such as necroptosis or autophagy, which share some of. In keeping with the annexinv binding results described above, we found that doxdependent overexpression of calreticulin increased the sensitivity of kn1 cells to apoptosis fig. Moreover, us11 expression also protected against staurosporine induced apoptosis. Staurosporineinduced apoptosis of hpv positive and negative. Staurosporine induced neuronal death was accompanied by cell body shrinkage, chromatin condensation, and dna laddering. A proapoptotic effect of the cdk inhibitor p57 kip2 on.
Separasetriggered apoptosis enforces minimal length of. I am using two different inhibitors staurosporine and go6983 for hela cells. Staurosporineinduced neuronal apoptosis sciencedirect. Spike cell cultures at a staurosporine concentration of 1 m in the cell culture media. Fkbp51 protects 661w cell culture from staurosporine. Staurosporine, bradykinin and thapsigargin were from sigma. Increased apoptosis may assist the dissemination of intracellular pathogens or induce immunosuppression. Induction of apoptosis in hela cells by transactivation. Staurosporine induces different cell death forms in. Staurosporineinduced apoptosis in chang liver cells is associated with downregulation of bcl2 and bclxl.
Dec 20, 2002 regulation of intracellular ph mediates bax activation in hela cells treated with staurosporine or tumor necrosis factoralpha. M staurosporine triggers apoptosis in nearly 40% of the cells after 12 hours, rapidly activates caspase3, and induces parp cleavage. Staurosporineinduced neuronal death was accompanied by cell body shrinkage, chromatin condensation, and dna laddering. Cells were treated for 12 h at 37c with staurosporine 5. Nci staurosporine is an indolocarbazole alkaloid and an organic. Hydrogen peroxide induces apoptosis in hela cells through. Regulation of intracellular ph mediates bax activation in. A well recognized inducer of apoptosis in hek cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wildtype and antisense cells.
Jul 10, 2014 h295r cells grown on plastic showed a low responsiveness to staurosporine, with an apoptotic rate of 24 %, as compared to breast cancer mcf7 cells, with an apoptotic rate of 60 %. Apoptosis of cultured human endothelial cells was induced by the mycotoxin staurosporine, which has been shown to induce apoptosis in a wide variety of cell types. Our study also demonstrates that lipidomics provide a powerful tool for better understanding anticancer mechanisms of chemotherapeutic agents and for biomarker screening. Caspase inhibition prevents staurosporineinduced apoptosis. This equates to a 1 l spike of the 1 mm staurosporine stock per ml of the cell culture suspension. Changes in mitochondrial membrane potential during. The sensitivity of kn1 and knx2 cells to staurosporine was also evaluated by tunel analysis fig. Here, we observed that avd is induced not only by a mitochondrial apoptosis inducer, staurosporine sts, in mouse b lymphoma wehi231 cells, but also by ligation of the death receptor fas in human b lymphoblastoid skw6. Some methods such as treatment with kinase inhibitors, exposure to ultraviolet uv light or xray irradiation are more. Measurements are expressed as a percentage of control incubations at time zero. Apoptosis is a process that plays a critical role in the elimination of infected cells. Molecular mechanism of staurosporineinduced apoptosis in.
Staurosporine induces apoptosis through both caspase. Add 1 m final concentration staurosporine eg, sigma s6942 to cell suspension e. To determine whether sequencespecific transactivation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of hela cells. However, this activity was not observed in wsl and vero cell cultures. Measure apoptosis using suspension jurkat cells treated with. Apr 10, 2018 cells may also become damaged or undergo some type of infection. Staurosporine is a potent, nonselective inhibitor of protein kinases, including protein kinase c.