BACKGROUND
calcium  is a universal signaling molecule regulating many aspects of cellular function and is one of the key elements of apoptotic signaling pathways  <cit> .
some agents, including glucocorticoids, the endoplasmic reticulum  ca2+-atpase inhibitor thapsigargin and various cancer therapeutic drugs, mobilize ca2+ stores and trigger apoptosis by early transient elevation of intracellular free calcium .
however, elevation of i has been described also at later stages of the apoptotic process  <cit> .
a number of calcium-mediated apoptosis signaling mechanisms have been described.
mitochondria are located in microdomains close to ca2+ channels of the er where cytosolic ca2+ concentrations may become high on channel opening  <cit> .
mitochondrial ca2+ uptake may result in mitochondrial permeability transition, leading to mitochondrial swelling and apoptosis.
the calpains, calcium-activated cysteine proteases, have been implicated in apoptosis induction by some stimuli  <cit> .
calpain cleaves, and thereby activates, a number of molecules that have important functions in the apoptosis process, including caspase- <dig>  <cit> , bax  <cit>  and bid  <cit> .
calmodulin  is the major ca2+ sensor of nonmuscle cells  <cit>  and signaling involving calmodulin has been implicated in apoposis  <cit> .
different signaling mechanisms down-stream from cam are involved in various types of apoptotic responses, including pathways involving calcineurin, dap kinase and calmodulin kinases.
calmodulin-dependent kinase ii  has been found to be both pro-apoptotic  <cit>  and anti-apoptotic  <cit>  in different studies.
the use of libraries of agents with diverse biological mechanisms allows for elucidation of new biological targets as well as mechanisms relevant to tumor cell viability  <cit> .
many previous studies have identified compounds that prevent the proliferation of tumor cells and several active compounds have been developed into clinically effective anticancer drugs.
we here identified apoptosis-inducing agents in a compound library prescreened for agents that inhibit tumor cell proliferation.
the objective of our study was to use the different agents as bioprobes to investigate the role of different calcium signaling mechanisms for apoptosis signaling.
apoptosis induction was analyzed in the presence and absence of a number of agents that influence ca2+ signaling.
the results show that most, but not all, apoptotic drugs examined require calcium and that camkii is an important mediator of apoptotic signaling.
RESULTS
identification of apoptosis-inducing agents by screening
the aim of this study was to investigate the importance of different calcium pathways for induction of apoptosis of tumor cells.
since our study required a wide variety of apoptotic stimuli, we first set out to identify apoptosis-inducing agents by screening a chemical library.
the nci mechanistic drug set, which contains  <dig> compounds selected from approximately  <dig>  compounds based on different mechanisms of action with regard to cell growth inhibition, was screened to identify mechanistically diverse apoptosis-inducing agents.
apoptosis-inducing agents were identified using an assay which measures the accumulation of a caspase-cleavage product of cytokeratin- <dig>  in cultures containing dying and dead cells  <cit> ; see experimental procedures).
importantly, this assay provides an integrative measure of apoptosis and facilitates end-point measurements after treatment with collections of drugs that induce apoptosis with different kinetics.
we used the human colon carcinoma cell line hct <dig> as a screening target .
forty agents  that induce strong caspase-cleavage activity were chosen based on representation of seven of the nine different response areas of the 3d mind self-organizing maps  <cit> .
agents that segregate to the two remaining response areas  were not identified.
the  <dig> bioprobes were used at the lowest possible concentration required for apoptosis induction.
we first profiled the pattern of signaling induced by these bioprobes using four hct <dig> cell lines which stably express different β-lactamase reporters for  <dig> pathways relevant to apoptosis signaling: p <dig>  ap- <dig>  nfat and nf-κb .
ap- <dig> reporter induction was most frequently observed , whereas nfat induction was most infrequent .
a positive correlation was observed between induction of the ap- <dig> and nf-κb reporters .
we conclude that the bioprobes chosen on the basis of different mechanisms of growth inhibition also induce a diverse pattern of signaling.
examination of the role of calcium in apoptosis
we used the fluo- <dig> fluorescent calcium indicator to examine whether the bioprobes induced ca2+ fluxes in hct <dig> cells.
twenty-seven of  <dig>  bioprobes induced >50% increases in ca2+ after  <dig> hours of exposure .
smaller increases in ca2+ were observed after  <dig> and  <dig> hours of drug exposure .
to examine the importance of calcium signaling for induction of apoptosis, cells were treated with the bioprobes in the presence or absence of the ca2+ chelator bapta-am  followed by determination of caspase-cleaved ck <dig>  bapta-am inhibited the generation of caspase-cleaved ck <dig> by the majority of the bioprobes .
a median level of inhibition of approximately 60% was observed ).
caspase-cleavage activity induced by some agents were not affected at all, showing that ca2+ is not required for all types of apoptotic stimuli.
furthermore, induction of caspase-cleavage activity by thapsigargin  was inhibited by 85%.
bapta-am could be added as late as  <dig> hours after drug addition and still provide significant protection from apoptosis .
apoptosis was inhibited by blocking the ryanodine receptor  and inositol  <dig> ,5-triphosphate  receptors using ryanodine and dantrolene, respectively .
these results show that ca2+ is required for full induction of caspase-cleavage activity by many bioprobes but that ca2+ is not a mandatory requirement for all apoptotic signals.
results were validated by analysis of caspase- <dig> activity , loss of mitochondrial membrane potential  and activation of the conformation of the pro-apoptotic mitochondrial protein bak .
many apoptotic signals involve the mitochondria, leading to release of molecules such as cytochrome c and aif from mitochondria to the cytosol.
the bak and bax proteins are essential regulators of mitochondrial permeabilization.
activation of bak during apoptosis results in an altered molecular conformation, leading to altered conformation of the n-terminus of the protein  <cit> .
bapta-am was found to inhibit the conformational change of bak after addition of  <dig> agents that showed calcium-dependent apoptosis induction .
we conclude from these experiments that calcium signaling is required at a late stage during apoptosis, but prior to conformational activation of bak.
importance of calmodulin for drug-induced apoptosis
we examined the relative roles of three processes known to be potentially involved in calcium-regulated apoptosis: mitochondrial calcium uptake, activation of the protease calpain and signaling involving calmodulin.
ruthenium red is a non-competitive inhibitor of the mitochondrial ca2+ uniporter  <cit> , used to inhibit mitochondrial calcium uptake in cells  <cit> .
ruthenium red was found to inhibit apoptosis induced by some agents, but did not affect apoptosis induced by the majority of bioprobes .
calpain is a ca2+-activated protease involved in apoptosis signaling  <cit> .
calpeptin is a membrane-permeable inhibitor of both μ-calpain and m-calpain  <cit> .
calpeptin showed some inhibitory activity of apoptosis , and increased calpain activity was accordingly detected after treatment with calpeptin-sensitive agents .
however, the median level of inhibition of caspase-cleaved ck <dig> was only one fourth of that observed using bapta-am .
we finally examined calmodulin, a central regulator of cellular ca2+ responses.
we found that the calmodulin inhibitor w <dig> was more effective than calpeptin in inhibiting apoptosis, generating a median level of inhibition of 24% .
relative importance of calmodulin-regulated pathways
the finding that w <dig> inhibited apoptosis by several drugs prompted us to examine the potential involvement of different calmodulin-regulated pathways.
calcineurin  is a ca2+/calmodulin activated serine/threonine-specific phosphatase implicated in apoptotic signaling  <cit> .
calcineurin dephosphorylates nfat, resulting in nuclear translocation and gene activation  <cit> .
the antibiotic fk <dig> binds fkbp- <dig> and the resulting  complex inhibits calcineurin activity  <cit> .
fk <dig> did not significantly inhibit apoptosis induction by the bioprobe set .
we also examined the possible role of death-associated protein kinase , a ca2+/calmodulin-regulated protein kinase implicated in cell death  <cit> .
down-regulation of dapk expression by sirna did not significantly affect apoptosis induced by the bioprobe set , consistent with previous findings  <cit> .
other calmodulin-induced signaling pathways linked to apoptosis signaling involve calmodulin kinases.
camkk-β was recently implicated in induction of macro-autophagy  <cit> .
the camkk inhibitor sto- <dig> had a modest inhibitory effect on apoptosis .
camkii has been implicated in apoptosis by some stimuli  <cit> .
interestingly, the camkii inhibitor kn- <dig> reduced apoptosis to a similar degree as w <dig> .
furthermore, treatment with camkii sirna inhibited apoptosis induced by some examined compounds .
increases in camkii activity were observed after treatment by a number of compounds at  <dig> hours of treatment .
these results suggested that a pathway involving camkii is important in calcium-mediated apoptotic signaling down-stream of cam.
role of camkii in apoptotic signaling
the experiments presented so far used the entire set of  <dig> bioprobes to examine to which extent different inhibitors affected apoptosis induction.
this approach could not be used to specifically study a single pathway, since many of the compounds would be irrelevant.
to specifically study camkii dependent apoptosis we chose the plant sesquiterpene lactone helenalin  which showed kn- <dig> sensitive apoptosis and that induced camkii activity.
the selection was also supported by a previous report that helenalin causes increases in i in 3t <dig> cells  <cit> .
importantly, in contrast to other candidates considered, helenalin has not been reported to induce reactive oxygen species or dna damage.
others have, however, described helenalin as a specific inhibitor of nf-κb at  <dig> μm  <cit> .
we here used helenalin at a concentration of  <dig> μm .
calcium signaling via camkii activates apoptosis signaling kinase  <dig>   <cit> .
ask- <dig> is a serine/threonine protein kinase capable of activating the pro-apoptotic jnk pathway.
hct <dig> cells were treated with  <dig> μm helenalin and ask- <dig> kinase activity was measured using an in vitro immunokinase assay with p <dig> as a substrate.
stimulation of ask- <dig> activity was observed at  <dig> hours of treatment .
ask- <dig> has been reported to be activated by calcium signaling  <cit> , but there are few reports linking calcium signaling to jnk activation during apoptosis.
we found that helenalin had induced jnk activity at  <dig> hours of treatment, assessed by an immunokinase assay .
in order to determine whether ask- <dig> is required for helenalin-induced apoptosis, hct <dig> cells were transfected with a dominant negative mutant of ask- <dig>  strong inhibition of apoptosis was observed .
furthermore, the jnk inhibitor sp <dig> inhibited helenalin-induced apoptosis.
addition of the jnk inhibitor at  <dig> hours of drug treatment was sufficient for inhibition of apoptosis .
the observation of a late requirement for jnk is consistent with a late requirement for calcium and of an apoptosis-specific role for sustained jnk.
helenalin did not induce phosphorylation of p <dig> and apoptosis was not inhibited by the p <dig> kinase inhibitor sb <dig> .
the levels of ask- <dig> and jnk kinase activation by helenalin were moderate and the precision of the in vitro kinase assays was not found sufficient to reliably determine whether these kinases were inhibited by bapta-am and kn- <dig>  we therefore used the ap- <dig> reporter hct <dig> cell line described in fig.
<dig>  stimulation with phorbol ester and thapsigargin induced a reproducible increase in reporter activity .
helenalin induced a reproducible 2-fold induction of the ap- <dig> reporter at  <dig> hours of treatment .
reporter activity represents jnk activity since stimulation was decreased by the jnk inhibitor sp <dig>  but not by the erk inhibitor pd <dig>  using this assay we found that both bapta-am and kn- <dig> inhibited helenalin-induced activation of the ap- <dig> reporter , showing that helenalin-induced ap- <dig> activity is dependent on calcium signaling.
discussion
single-drug studies are limited with regard to the information about the relative contribution of various pathways to apoptosis.
we here used a collection of  <dig> small molecular agents to induce apoptosis.
these agents were selected from a larger drug set compiled on the basis of different mechanisms of growth inhibition of the nci  <dig> cell line panel.
studies using a set of reporter cell lines confirmed that these compounds induced different patterns of signaling.
this approach allowed a comprehensive evaluation of the role of calcium signaling in apoptosis.
the data show that for many types of stimuli, calcium is generally required late during apoptosis induction.
calcium was likely to be released from intracellular stores, as evidenced by our finding that apoptosis was inhibited by blocking the ryanodine receptor  and the inositol  <dig> ,5-triphosphate  receptor.
of the calcium-regulated mechanisms examined, calmodulin-regulated pathways appeared to be most important for induction of apoptosis.
calmodulin has previously been implicated in apoptosis, both as a pro-survival  <cit>  and as a pro-apoptotic factor  <cit> .
camkii is a major mediator of cellular ca2+ effects and inhibition of this kinase was here found to inhibit apoptosis to a greater extent than inhibition of other candidates such as calpain and calcineurin/pp2b.
the biological actions of camkii have been studied in most detail in intermediate metabolism and neural signaling  <cit> .
camkii has been reported to be activated downstream of protease activation in apoptosis induced by uv light and tnf  <cit> .
induction of apoptotic cell death in hepatocytes by the toxin microcystin can be blocked by camkii inhibitors  <cit> .
furthermore, cadmium induces camkii and cadmium-induced apoptosis is dependent on this kinase  <cit> .
our data support these previous studies and suggest a common role for camkii in apoptosis by a number of different stimuli.
other calmodulin-regulated pathways that were considered involved calcineurin and death-associated protein kinase .
calcineurin is a widely expressed protein phosphatase regulated by ca2+/calmodulin.
calcineurin dephosphorylates the nfat transcription factor, resulting in nuclear translocation and gene activation  <cit> .
overexpression of calcineurin leads to apoptosis in cells deprived of growth factors  <cit> .
calcineurin dephosphorylates the pro-apoptotic bcl- <dig> family protein bad, leading to translocation of bad to mitochondria  <cit> .
calcineurin has been implicated in p53-mediated apoptosis of colon carcinoma cells as down-stream mediator of calcium released from intracellular stores through the generation of reactive oxygen species  <cit> .
calcineurin did not appear to an important role for apoptosis induced by the set of bioprobes studies here, and we only found weak induction of nfat.
dapk is a ca2+/calmodulin-regulated protein kinase that mediates cell death  <cit> .
there is little evidence in the literature that dapk signaling is important for drug- or radiation-induced apoptosis of tumor cells  <cit> .
we did not find any significant effect of dapk down-regulation by sirna on apoptosis induced by the bioprobe set.
sustained jnk activation is recognized as a hallmark of many apoptotic processes  <cit> .
sustained jnk activation has been demonstrated to be associated with repression of the jnk-phosphatase mkp <dig>  <cit>  but is also believed to be due to increased de novo phosphorylation by uncharacterized signaling pathways  <cit> .
it is known that the calcium ionophore ionomycin induces jnk activity  <cit> , but a role of calcium signaling for sustained jnk activation is not generally accepted.
it is conceivable that calcium signaling contributes to apoptosis-specific sustained jnk activation via camkii.
of the different inhibitors that we have tested for inhibition of apoptosis by the current bioprobe set, bapta-am and the jnk inhibitor sp <dig> have provided the strongest degree of protection .
other agents that we have been tested are the superoxide scavenger tiron and the p <dig> kinase inhibitor sb <dig>  both of which were found to be less effective .
jnk activation and calcium signaling pathways are likely to be of fundamental importance for apoptosis induced by many signals.
that calcium signaling may be important for jnk activation is therefore an attractive possibility, but has not been as extensively studied as for example jnk activation by reactive oxygen/nitrogen species  <cit> .
small molecules are powerful tools for charting signaling pathways.
it is, however, important to bear in mind that small molecules rarely affect single targets.
this complicates the interpretation of the mechanism of action of e.g.
anticancer drugs.
in the present study, we used the plant sesquiterpene helenalin to study the role of camkii in apoptosis and found that helenalin induced camkii, ask- <dig> and jnk and that inhibiton of these kinases inhibits apoptosis.
helenalin may, however, have other effects and has in fact previously been described as a specific inhibitor of nf-kb when used at  <dig> μm  <cit> .
we here used helenalin at  <dig> μm and did not observe nf-κb inhibition at this concentration .
in fact, we have earlier found that when used at  <dig> μm, the response to helenalin was found to shift from apoptosis to necrosis  <cit> .
CONCLUSIONS
a number of calcium-regulated apoptosis pathways have been identified in various studies.
using a chemical biology approach with high throughput potential we have here both demonstrated a strategy for pathway charting as well as demonstrated an important role for camkii in apoptosis signaling in a human carcinoma cell line.
our data raise the possibility that calcium signaling is involved in sustained jnk activation during apoptosis induced by different stimuli.

 calcium is a universal signaling molecule regulating many aspects of cellular function and is one of the key elements of apoptotic signaling pathways. some agents, including glucocorticoids, the endoplasmic reticulum ca2+-atpase inhibitor thapsigargin and various cancer therapeutic drugs, mobilize ca2+ stores and trigger apoptosis by early transient elevation of intracellular free calcium. however, elevation of i has been described also at later stages of the apoptotic process. a number of calcium-mediated apoptosis signaling mechanisms have been described. mitochondria are located in microdomains close to ca2+ channels of the er where cytosolic ca2+ concentrations may become high on channel opening. mitochondrial ca2+ uptake may result in mitochondrial permeability transition, leading to mitochondrial swelling and apoptosis. the calpains, calcium-activated cysteine proteases, have been implicated in apoptosis induction by some stimuli. calpain cleaves, and thereby activates, a number of molecules that have important functions in the apoptosis process, including caspase-, bax and bid. calmodulin is the major ca2+ sensor of nonmuscle cells and signaling involving calmodulin has been implicated in apoposis. different signaling mechanisms down-stream from cam are involved in various types of apoptotic responses, including pathways involving calcineurin, dap kinase and calmodulin kinases. calmodulin-dependent kinase ii has been found to be both pro-apoptotic and anti-apoptotic in different studies. the use of libraries of agents with diverse biological mechanisms allows for elucidation of new biological targets as well as mechanisms relevant to tumor cell viability. many previous studies have identified compounds that prevent the proliferation of tumor cells and several active compounds have been developed into clinically effective anticancer drugs. we here identified apoptosis-inducing agents in a compound library prescreened for agents that inhibit tumor cell proliferation. the objective of our study was to use the different agents as bioprobes to investigate the role of different calcium signaling mechanisms for apoptosis signaling. apoptosis induction was analyzed in the presence and absence of a number of agents that influence ca2+ signaling. the results show that most, but not all, apoptotic drugs examined require calcium and that camkii is an important mediator of apoptotic signaling.  identification of apoptosis-inducing agents by screening the aim of this study was to investigate the importance of different calcium pathways for induction of apoptosis of tumor cells. since our study required a wide variety of apoptotic stimuli, we first set out to identify apoptosis-inducing agents by screening a chemical library. the nci mechanistic drug set, which contains compounds selected from approximately compounds based on different mechanisms of action with regard to cell growth inhibition, was screened to identify mechanistically diverse apoptosis-inducing agents. apoptosis-inducing agents were identified using an assay which measures the accumulation of a caspase-cleavage product of cytokeratin- in cultures containing dying and dead cells; see experimental procedures). importantly, this assay provides an integrative measure of apoptosis and facilitates end-point measurements after treatment with collections of drugs that induce apoptosis with different kinetics. we used the human colon carcinoma cell line hct as a screening target. forty agents that induce strong caspase-cleavage activity were chosen based on representation of seven of the nine different response areas of the 3d mind self-organizing maps. agents that segregate to the two remaining response areas were not identified. the bioprobes were used at the lowest possible concentration required for apoptosis induction. we first profiled the pattern of signaling induced by these bioprobes using four hct cell lines which stably express different β-lactamase reporters for pathways relevant to apoptosis signaling: p ap- nfat and nf-κb. ap- reporter induction was most frequently observed, whereas nfat induction was most infrequent. a positive correlation was observed between induction of the ap- and nf-κb reporters. we conclude that the bioprobes chosen on the basis of different mechanisms of growth inhibition also induce a diverse pattern of signaling. examination of the role of calcium in apoptosis we used the fluo- fluorescent calcium indicator to examine whether the bioprobes induced ca2+ fluxes in hct cells. twenty-seven of bioprobes induced >50% increases in ca2+ after hours of exposure. smaller increases in ca2+ were observed after and hours of drug exposure. to examine the importance of calcium signaling for induction of apoptosis, cells were treated with the bioprobes in the presence or absence of the ca2+ chelator bapta-am followed by determination of caspase-cleaved ck bapta-am inhibited the generation of caspase-cleaved ck by the majority of the bioprobes. a median level of inhibition of approximately 60% was observed ). caspase-cleavage activity induced by some agents were not affected at all, showing that ca2+ is not required for all types of apoptotic stimuli. furthermore, induction of caspase-cleavage activity by thapsigargin was inhibited by 85%. bapta-am could be added as late as hours after drug addition and still provide significant protection from apoptosis. apoptosis was inhibited by blocking the ryanodine receptor and inositol,5-triphosphate receptors using ryanodine and dantrolene, respectively. these results show that ca2+ is required for full induction of caspase-cleavage activity by many bioprobes but that ca2+ is not a mandatory requirement for all apoptotic signals. results were validated by analysis of caspase- activity, loss of mitochondrial membrane potential and activation of the conformation of the pro-apoptotic mitochondrial protein bak. many apoptotic signals involve the mitochondria, leading to release of molecules such as cytochrome c and aif from mitochondria to the cytosol. the bak and bax proteins are essential regulators of mitochondrial permeabilization. activation of bak during apoptosis results in an altered molecular conformation, leading to altered conformation of the n-terminus of the protein. bapta-am was found to inhibit the conformational change of bak after addition of agents that showed calcium-dependent apoptosis induction. we conclude from these experiments that calcium signaling is required at a late stage during apoptosis, but prior to conformational activation of bak. importance of calmodulin for drug-induced apoptosis we examined the relative roles of three processes known to be potentially involved in calcium-regulated apoptosis: mitochondrial calcium uptake, activation of the protease calpain and signaling involving calmodulin. ruthenium red is a non-competitive inhibitor of the mitochondrial ca2+ uniporter, used to inhibit mitochondrial calcium uptake in cells. ruthenium red was found to inhibit apoptosis induced by some agents, but did not affect apoptosis induced by the majority of bioprobes. calpain is a ca2+-activated protease involved in apoptosis signaling. calpeptin is a membrane-permeable inhibitor of both μ-calpain and m-calpain. calpeptin showed some inhibitory activity of apoptosis, and increased calpain activity was accordingly detected after treatment with calpeptin-sensitive agents. however, the median level of inhibition of caspase-cleaved ck was only one fourth of that observed using bapta-am. we finally examined calmodulin, a central regulator of cellular ca2+ responses. we found that the calmodulin inhibitor w was more effective than calpeptin in inhibiting apoptosis, generating a median level of inhibition of 24%. relative importance of calmodulin-regulated pathways the finding that w inhibited apoptosis by several drugs prompted us to examine the potential involvement of different calmodulin-regulated pathways. calcineurin is a ca2+/calmodulin activated serine/threonine-specific phosphatase implicated in apoptotic signaling. calcineurin dephosphorylates nfat, resulting in nuclear translocation and gene activation. the antibiotic fk binds fkbp- and the resulting complex inhibits calcineurin activity. fk did not significantly inhibit apoptosis induction by the bioprobe set. we also examined the possible role of death-associated protein kinase, a ca2+/calmodulin-regulated protein kinase implicated in cell death. down-regulation of dapk expression by sirna did not significantly affect apoptosis induced by the bioprobe set, consistent with previous findings. other calmodulin-induced signaling pathways linked to apoptosis signaling involve calmodulin kinases. camkk-β was recently implicated in induction of macro-autophagy. the camkk inhibitor sto- had a modest inhibitory effect on apoptosis. camkii has been implicated in apoptosis by some stimuli. interestingly, the camkii inhibitor kn- reduced apoptosis to a similar degree as w. furthermore, treatment with camkii sirna inhibited apoptosis induced by some examined compounds. increases in camkii activity were observed after treatment by a number of compounds at hours of treatment. these results suggested that a pathway involving camkii is important in calcium-mediated apoptotic signaling down-stream of cam. role of camkii in apoptotic signaling the experiments presented so far used the entire set of bioprobes to examine to which extent different inhibitors affected apoptosis induction. this approach could not be used to specifically study a single pathway, since many of the compounds would be irrelevant. to specifically study camkii dependent apoptosis we chose the plant sesquiterpene lactone helenalin which showed kn- sensitive apoptosis and that induced camkii activity. the selection was also supported by a previous report that helenalin causes increases in i in 3t cells. importantly, in contrast to other candidates considered, helenalin has not been reported to induce reactive oxygen species or dna damage. others have, however, described helenalin as a specific inhibitor of nf-κb at μm. we here used helenalin at a concentration of μm. calcium signaling via camkii activates apoptosis signaling kinase. ask- is a serine/threonine protein kinase capable of activating the pro-apoptotic jnk pathway. hct cells were treated with μm helenalin and ask- kinase activity was measured using an in vitro immunokinase assay with p as a substrate. stimulation of ask- activity was observed at hours of treatment. ask- has been reported to be activated by calcium signaling, but there are few reports linking calcium signaling to jnk activation during apoptosis. we found that helenalin had induced jnk activity at hours of treatment, assessed by an immunokinase assay. in order to determine whether ask- is required for helenalin-induced apoptosis, hct cells were transfected with a dominant negative mutant of ask- strong inhibition of apoptosis was observed. furthermore, the jnk inhibitor sp inhibited helenalin-induced apoptosis. addition of the jnk inhibitor at hours of drug treatment was sufficient for inhibition of apoptosis. the observation of a late requirement for jnk is consistent with a late requirement for calcium and of an apoptosis-specific role for sustained jnk. helenalin did not induce phosphorylation of p and apoptosis was not inhibited by the p kinase inhibitor sb. the levels of ask- and jnk kinase activation by helenalin were moderate and the precision of the in vitro kinase assays was not found sufficient to reliably determine whether these kinases were inhibited by bapta-am and kn- we therefore used the ap- reporter hct cell line described in fig. stimulation with phorbol ester and thapsigargin induced a reproducible increase in reporter activity. helenalin induced a reproducible 2-fold induction of the ap- reporter at hours of treatment. reporter activity represents jnk activity since stimulation was decreased by the jnk inhibitor sp but not by the erk inhibitor pd using this assay we found that both bapta-am and kn- inhibited helenalin-induced activation of the ap- reporter, showing that helenalin-induced ap- activity is dependent on calcium signaling. discussion single-drug studies are limited with regard to the information about the relative contribution of various pathways to apoptosis. we here used a collection of small molecular agents to induce apoptosis. these agents were selected from a larger drug set compiled on the basis of different mechanisms of growth inhibition of the nci cell line panel. studies using a set of reporter cell lines confirmed that these compounds induced different patterns of signaling. this approach allowed a comprehensive evaluation of the role of calcium signaling in apoptosis. the data show that for many types of stimuli, calcium is generally required late during apoptosis induction. calcium was likely to be released from intracellular stores, as evidenced by our finding that apoptosis was inhibited by blocking the ryanodine receptor and the inositol,5-triphosphate receptor. of the calcium-regulated mechanisms examined, calmodulin-regulated pathways appeared to be most important for induction of apoptosis. calmodulin has previously been implicated in apoptosis, both as a pro-survival and as a pro-apoptotic factor. camkii is a major mediator of cellular ca2+ effects and inhibition of this kinase was here found to inhibit apoptosis to a greater extent than inhibition of other candidates such as calpain and calcineurin/pp2b. the biological actions of camkii have been studied in most detail in intermediate metabolism and neural signaling. camkii has been reported to be activated downstream of protease activation in apoptosis induced by uv light and tnf. induction of apoptotic cell death in hepatocytes by the toxin microcystin can be blocked by camkii inhibitors. furthermore, cadmium induces camkii and cadmium-induced apoptosis is dependent on this kinase. our data support these previous studies and suggest a common role for camkii in apoptosis by a number of different stimuli. other calmodulin-regulated pathways that were considered involved calcineurin and death-associated protein kinase. calcineurin is a widely expressed protein phosphatase regulated by ca2+/calmodulin. calcineurin dephosphorylates the nfat transcription factor, resulting in nuclear translocation and gene activation. overexpression of calcineurin leads to apoptosis in cells deprived of growth factors. calcineurin dephosphorylates the pro-apoptotic bcl- family protein bad, leading to translocation of bad to mitochondria. calcineurin has been implicated in p53-mediated apoptosis of colon carcinoma cells as down-stream mediator of calcium released from intracellular stores through the generation of reactive oxygen species. calcineurin did not appear to an important role for apoptosis induced by the set of bioprobes studies here, and we only found weak induction of nfat. dapk is a ca2+/calmodulin-regulated protein kinase that mediates cell death. there is little evidence in the literature that dapk signaling is important for drug- or radiation-induced apoptosis of tumor cells. we did not find any significant effect of dapk down-regulation by sirna on apoptosis induced by the bioprobe set. sustained jnk activation is recognized as a hallmark of many apoptotic processes. sustained jnk activation has been demonstrated to be associated with repression of the jnk-phosphatase mkp but is also believed to be due to increased de novo phosphorylation by uncharacterized signaling pathways. it is known that the calcium ionophore ionomycin induces jnk activity, but a role of calcium signaling for sustained jnk activation is not generally accepted. it is conceivable that calcium signaling contributes to apoptosis-specific sustained jnk activation via camkii. of the different inhibitors that we have tested for inhibition of apoptosis by the current bioprobe set, bapta-am and the jnk inhibitor sp have provided the strongest degree of protection. other agents that we have been tested are the superoxide scavenger tiron and the p kinase inhibitor sb both of which were found to be less effective. jnk activation and calcium signaling pathways are likely to be of fundamental importance for apoptosis induced by many signals. that calcium signaling may be important for jnk activation is therefore an attractive possibility, but has not been as extensively studied as for example jnk activation by reactive oxygen/nitrogen species. small molecules are powerful tools for charting signaling pathways. it is, however, important to bear in mind that small molecules rarely affect single targets. this complicates the interpretation of the mechanism of action of e.g. anticancer drugs. in the present study, we used the plant sesquiterpene helenalin to study the role of camkii in apoptosis and found that helenalin induced camkii, ask- and jnk and that inhibiton of these kinases inhibits apoptosis. helenalin may, however, have other effects and has in fact previously been described as a specific inhibitor of nf-kb when used at μm. we here used helenalin at μm and did not observe nf-κb inhibition at this concentration. in fact, we have earlier found that when used at μm, the response to helenalin was found to shift from apoptosis to necrosis.  a number of calcium-regulated apoptosis pathways have been identified in various studies. using a chemical biology approach with high throughput potential we have here both demonstrated a strategy for pathway charting as well as demonstrated an important role for camkii in apoptosis signaling in a human carcinoma cell line. our data raise the possibility that calcium signaling is involved in sustained jnk activation during apoptosis induced by different stimuli. 
a set of agents that induce apoptosis was first identified by screening of a chemical library. intracellular free calcium is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. using p ap- nfat and nf-κb reporter cell lines, these bioprobes were verified to induce different patterns of signaling. our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a camkii/ask signaling mechanism is important for sustained jnk activation and apoptosis by some types of stimuli. further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. inhibition of calmodulin kinase ii resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/pp2b or dap kinase. we here used a chemical biology strategy to elucidate the relative importance of such different pathways. experiments using the calcium chelator bapta-am showed that ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that ca2+ was in general required several hours into the apoptosis process.
 intracellular free calcium is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. we here used a chemical biology strategy to elucidate the relative importance of such different pathways.  a set of agents that induce apoptosis was first identified by screening of a chemical library. using p ap- nfat and nf-κb reporter cell lines, these bioprobes were verified to induce different patterns of signaling. experiments using the calcium chelator bapta-am showed that ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that ca2+ was in general required several hours into the apoptosis process. further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. inhibition of calmodulin kinase ii resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/pp2b or dap kinase. we used one of the bioprobes, the plant alkaloid helenalin, to study the role of camkii in apoptosis. helenalin induced camkii, ask and jun-n-terminal kinase activity, and inhibition of these kinases inhibited apoptosis.  our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a camkii/ask signaling mechanism is important for sustained jnk activation and apoptosis by some types of stimuli. 