Staurosporine apoptosis hela cells book

Cholix exhibits high structural homology to eta, which was shown to cause caspase3 and 8dependent apoptosis in human mast cells. Cell death plays an essential role in the development of tissues and organisms, the etiology of disease, and the responses of cells to therapeutic drugs. Induction of apoptosis in hela cells with staurosporine produced a rise in the intracellular ph ph i. Individual cells within a population undergo apoptosis at distinct, apparently random time points. Analysis of the underlying signaling pathway reveals that apoptosis induction. Staurosporine induces apoptosis through both caspase. 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 %. Hsp105 family proteins suppress staurosporineinduced. First it was found that cholixtreated hela cells exhibited caspasedependent apoptosis, whereas intestinal cells. Staurosporine is a broadspectrum protein kinase inhibitor that has been used extensively to induce apoptosis in a variety of cells including tumor cells, lymphocytes, neurons and other primary. Aug 31, 2017 caspase 37 apoptosis assay staurosporine cool science. For example, treatment with staurosporine, a protein kinase c inhibitor, but not with antifas mab induces apoptosis in hela cells, which are fas receptordeficient. There are at least two known processes that may result in the avd. A549 parental cells and bcl2overexpressing clones bb and b1s 5 410 cells were incubated with 3mm staurosporine for 3hours.

Spike cell cultures at a staurosporine concentration of 1 m in the cell culture media. Apoptosis time lapse imaging of u2os cells treated with staurosporine duration. Induction of apoptosis with staurosporine resulting in activation of the icenirf probe. Staurosporineinduced apoptosis in chang liver cells is associated with downregulation of bcl2 and bclxl. In the present study, we have examined the apoptosis inducing potential of staurosporine in cultured melanoma cell lines and dissected the staurosporine induced apoptotic signaling pathway. By analyzing cellular mitotic history, we identified that sibling hela cell pairs, in contrast to. In etatreated mef cells, mompdependent apoptosis is induced.

Gli36 cells were treated with 50 m staurosporine for 24 hours ac or with the same percentage of dmso 0. Moreover, us11 expression also protected against staurosporine induced apoptosis. Hela cell apoptosis caused by cdv follows a different mechanism than that in vero cell lines. Protection against heat and staurosporine mediated. Add 1 m final concentration staurosporine eg, sigma s6942 to cell suspension e. Staurosporineinduced apoptosis in chang liver cells is. Cells were pelleted then lysed with lysis buffer 50 mm trishcl ph 7.

Staurosporine treated 3 hours, showing parp cleavage as evidence of induction of apoptosis. Activation of mapk family in hela cells by h 2o 2 mapk pathway is another pathway that mediates signals stimulated by ros. Role of bovine bcl2a1 gene in staurosporineinduced. Since staurosporine is a strong apoptosis inducer and hela cells closely resemble the target cells of. Staurosporine antibiotic am2282 or sts is a natural product originally isolated in 1977 from the bacterium streptomyces staurosporeus. Wt1 is an inhibitor of apoptosisa u2os cells were tra openi. Increased apoptosis may assist the dissemination of intracellular pathogens or induce immunosuppression. One way to remove these cells without causing harm to other cells is for your body to initiate apoptosis. The binding of ucn01 to the active site of pdpk1 has been studied by xray crystallography and compared to that of staurosporine, indicating the importance of the hydroxy group in the former. Apoptosis very probably plays a key part in endothelial cell loss during corneal storage in organ culture as well as hypothermic storage.

Conducted an independent research project investigating the mechanism of staurosporineinduced apoptosis in hela cells utilized uvvis and. Separasetriggered apoptosis enforces minimal length of. However, the mechanisms underlying endothelial apoptosis are poorly understood. Inhibition of the devdcleaving activity by a synthetictetrapeptide inhibitor devdcho, blocked staurosporine induced downstream apoptotic phenotypes, suchas morphological characteristics and dna fragmentation. Although avd has traditionally been attributed to water loss, direct evidence for that is often lacking. Apoptosis time lapse imaging of u2os cells treated with. Unlike these reports, we found that cholixinduced hela cell. The response of a human corneal endothelial cell hcec line to staurosporine, a known inducer of apoptosis, was. Role of bovine bcl2a1 gene in staurosporineinduced apoptotic.

Staurosporine 1 m induced caspase37 activity more than 15fold within 8 h. These cells were then exposed to the apoptosisinducing agent staurosporine. Infectious diseases modulate apoptosis, and this contributes to disease pathogenesis. Morphological and biochemical analysis of staurosporineinduced apoptosis in l12100 and l1210s cells. Intracellular alkalinization was accompanied by translocation of bax to the mitochondria, cytochromec release, and cell death. Apr 10, 2018 cells may also become damaged or undergo some type of infection. Monitoring of cellular dna fragmentation revealed that incubation with staurosporine at a concentration of 2 m results in a sufficient level of apoptosis induction in hela, l11, wsl cell cultures after 6 hours and in vero cell cultures after 12 hours. Staurosporine and conventional anticancer drugs induce. Y haupt, s rowan, e shaulian, k h vousden, and m oren. A proapoptotic effect of the cdk inhibitor p57kip2 on staurosporine. Ijms free fulltext the apoptotic volume decrease is an. It is a potent and cellpermeable competitive inhibitor for protein kinase c ic50 0. Induction of apoptosis in hela cells with staurosporine produced a rise in the intracellular ph phi.

M staurosporine triggers apoptosis in nearly 40% of the cells after 12 hours, rapidly activates caspase3, and induces parp cleavage. Apoptosis is initiated by various kinds of stimuli, including infections, radiation, etc. Staurosporine showed a wide range of growth inhibition effects against cultured cells and was extremely cytotoxic in some cases 4pm, hela s3, 72 hr exposure. 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.

Cells may also become damaged or undergo some type of infection. Staurosporine induced apoptosis as do anticancer drugs, but via partly distinct pathways. Inhibition of the devdcleaving activity by a synthetictetrapeptide inhibitor devdcho, blocked staurosporineinduced downstream apoptotic phenotypes, suchas morphological characteristics and dna fragmentation. Regulation of intracellular ph mediates bax activation in. Dec 20, 2002 induction of apoptosis in hela cells with staurosporine produced a rise in the intracellular ph phi. Of these, staurosporine falls within the most common subgroup, called indolo 2,3apyrrole 3,4ccarbazoles. The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell. Staurosporine transiently increased the phosphotransferase activity of cjun nterminal kinase1 jnk1, which in turn may activate the transcriptional activity of activating protein1 ap1. Cholix toxin cholix is a novel adpribosylating cytotoxin produced by vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and pseudomonas exotoxin a. The chemical structure of staurosporine was elucidated by xray analysis of a single crystal and the absolute stereochemical configuration by the same.

Research paper staurosporine induced apoptosis may. Apoptosis was induced in hela cells treated with staurosporine, a compound know to induce apoptosis by activation of caspase 3, at concentrations greater than 0. Blended phasecontrast and redgreen images taken at 20x illustrate homogenous express of red nuclear signal and activation of caspase37 green. Staurosporine induced neuronal death was accompanied by cell body shrinkage, chromatin condensation, and dna laddering. This equates to a 1 l spike of the 1 mm staurosporine stock per ml of the cell culture suspension. Exposure of murine cortical cell cultures to 30100 nm staurosporine induced concentrationdependent selective neuronal degeneration over the following day. The structure and stereochemistry of staurosporine were deduced by xray. Hd phase images provide a qualitative assessment of morphological correlates to apoptosis left. Characterization of cholix toxininduced apoptosis in hela cells. Staurosporine has long been used in vitro as an initiator of apoptosis in many different cell types, but the mechanism involved remains poorly understood. In this study, we quantified intracellular water in. Staurosporine, an indolo2,3acarbazolole alkaloid that showed the potent protein kinase inhibiting activiry, was first isolated from streptomyces staurosporeus am2282 in 1977 1 and subsequently from other actinomycetes, e.

Ijms free fulltext the apoptotic volume decrease is. However, apoptosis induced by cdv in hela cells does not involve the initiator protein caspase8. 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 %. Apoptotic cells were determined by the propidium iodide method as. Induction of apoptosis by staurosporine involves the inhibition of. Cellplayer 96well kinetic caspase37 apoptosis assay. Staurosporine am2282 is a potent atpcompetitive kinase inhibitor with ic50 of 0. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal dna fragmentation, and global vague mrna decay. The response of a human corneal endothelial cell hcec line to staurosporine, a known inducer of apoptosis, was investigated to gain insights into the intracellular. These fall into two classes halogenated chlorinated and nonhalogenated. Apoptosis was induced in hela cells treated with staurosporine, a compound know to induce apop tosis by activation of caspase 3, at concentrations greater than 0.

Subsequently, cells were stained with hoechst 33258 at a concentration of 2mm for 20minutes. Buy pkc inhibitor staurosporine am2282 from abmole bioscience. Mediates the killing of hela cells by staurosporine induction of the. Jun 22, 2001 morphological and biochemical analysis of staurosporineinduced apoptosis in l12100 and l1210s cells. Hydrogen peroxide induces apoptosis in hela cells through. Wt1 is an inhibitor of apoptosisa u2os cells were tra. Fluorescent detection of apoptotic cells using a family of. Research paper staurosporine induced apoptosis may activate.

Staurosporine induced apoptotic water loss is cell and. Apoptosis is a process that plays a critical role in the elimination of infected cells. Intracellular alkalinization was accompanied by translocation of bax to the mitochondria, cytochrome c release, and cell death. An indolocarbazole that is a potent protein kinase c inhibitor which enhances camp mediated responses in human neuroblastoma cells. Wholecell extracts were prepared and immunoblotted with the antibodies indicated. Apoptotic volume decrease avd is a characteristic cell shrinkage observed during apoptosis. Methioninase gene therapy with selenomethionine induces. Staurosporineinduced neuronal apoptosis sciencedirect. Store at 20c staurosporine kda 200 140 100 80 60 50 cleaved parp 0 1 0. Caspase inhibition prevents staurosporineinduced apoptosis. Molecular mechanism of staurosporineinduced apoptosis in. Identification and characterization of novel regulators in mitochondria related cell. At 24 hr later, the cells were treated with 100 nm staurosporine for the indicated times. Dynamics of outer mitochondrial membrane permeabilization.

Right hela nuclight red cells treated with 300 nm staurosporine. 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. Confluent hela cells were treated with either the vehicle dmso, 0. Inhibition of apoptosis in chlamydiainfected cells. Staurosporine induces apoptosis of melanoma by both caspase. 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. Hela, l11, vero and wsl cell lines were used to investigate apoptosis induced by staurosporine. However, the molecular mechanisms by which hsp105alpha regulates stressinduced apoptosis are not fully understood.

We induced apoptosis in murine osteoblast mc3t3e1 cells by exposure to the staurosporine. Department of chemical immunology, weizmann institute of science, rehovot, israel. 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. Caspase 37 apoptosis assay staurosporine cool science. Adhesion to type v collagen enhances staurosporineinduced. Cells were pelleted then lysed with lysis buffer 50. Staurosporine is often used as a general method for inducing apoptosis 1. Western blot analysis of extracts from hela cells, untreated or. In this study, we analyzed the mechanism of cholixinduced cytotoxicity in hela cells. Staurosporine showed a wide range of growth inhibition effects against cultured cells and was extremely cytotoxic in some cases 4pm, helas3, 72 hr exposure. 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. Characterization of cholix toxininduced apoptosis in hela. Cytosolic fractions were analysed by immunoblotting for cytochrome c.

Staurosporineinduced neuronal apoptosis article pdf available in experimental neurology 52. Fkbp51 protects 661w cell culture from staurosporine. It belongs to the most frequently isolated group of indolocarbazoles. The human androgendependent prostate cancer cell line lncap, which is. Here, we established hela cells that overexpress either hsp105alpha or hsp105beta by removing doxycycline and examined how hsp105 modifies staurosporine stsinduced apoptosis in hela cells. We discuss the delay that follows exposure of cells to prodeath stimuli, control of mitochondrial. Staurosporine induced apoptosis may activate cancer stemlike.

Staurosporine is an indolocarbazole alkaloid and an organic heterooctacyclic compound. Staurosporine induces apoptosis in human neuroblastoma cells and chick embryonic neurons 3,4. Cell viability was determined using a calcein ampropidium iodide assay. On the other hand, activation of the map kinase mapk cascade is also known to play a critical role in apoptosis. The chloride channel inhibitor furosemide prevented intracellular alkalinization, bax translocation, cytochrome c release, and. Mechanisms of staurosporine induced apoptosis in a human.

Wt1 is an inhibitor of apoptosisa u2os cells were transfected with a control sirna or sirna that targets wt1. Induction of apoptosis in hela cells by transactivationdeficient p53. The data document the central role of the mpt in the killing of hela cells by str. Induce apoptosis with staurosporine immunochemistry. The results indicated that hela cells underwent apoptosis in dose and timedependent manners when treated with dioscin. These results suggest that staurosporine inducedapoptosis in chok1 cells is mediated through the cpp32caspase3like cysteine proteases. Induction of apoptosis in hela cells by transactivation. B m15 cells were analyzed as in a except that the sirnas were mouse specific. Staurosporine induced apoptosis may activate cancer stem. The adhesion of h295r cells to type v collagen induced a significant increase of apoptosis up to 52 %. This concentration works well for inducing many cell lines including hl60 and jurkat cells when using a 45 hour 37c incubation period. Protection against heat and staurosporine mediated apoptosis.

Cells may recognize viruses and gene mutations and can induce death to prevent the damage from spreading. Changes in mitochondrial membrane potential during. We discuss the delay that follows exposure of cells to prodeath stimuli, control of. Biochemical events lead to characteristic cell changes and death. Based on morphological and biochemical criteria, staurosporine induced apoptosis in a human corneal endothelial cell line has been clearly demonstrated in this study. Protein levels and activation of nuclear factor kappalightchainenhancer of activated b cells nf. Some methods such as treatment with kinase inhibitors, exposure to ultraviolet uv light or xray irradiation are more. It was the first of over 50 alkaloids to be isolated with this type of bisindole chemical structure. Cell fate profiles of hela k cells expressing the indicated mcl1 fragments and cultured in the presence of sirhoechst and a fluorogenic caspase 37 reporter. These results suggest that staurosporineinducedapoptosis in chok1 cells is mediated through the cpp32caspase3like cysteine. Cnfy induces apoptosis in lncap prostate cancer cells mdpi. 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.

206 381 578 400 1155 482 596 1468 1532 216 319 1090 46 1031 1100 1038 455 185 1505 1200 1428 915 1171 496 1210 1274 468 751 868 786 1615 1464 932 963 816 1033 559 1139