the human jurkat t cell line was maintained in rpmi 1640 medium supplemented with 10% fcs , 10   mm hepes , and antibiotics ( all from gibco brl , eggenstein ,   germany ) .
the cd95-resistant jurkat subline jurkat - r was generated by continuous culture in the presence of anti - cd95 mab   ( igg3 , 1 g / ml ; cell diagnostica , mnster , germany ) for 6 mo .   etoposide and mitomycin c
were obtained from the clinical   pharmacy ( medical clinics , tbingen , germany ) .
doxorubicin ,   cycloheximide , and oligomycin were purchased from sigma ( deisenhofen , germany ) and staurosporine from boehringer mannheim gmbh ( mannheim , germany ) .
mitomycin c was dissolved   in methanol , and doxorubicin , etoposide , and staurosporine in   ethanol and kept as stock solutions at 70c .
intracellular atp   was depleted by incubating cells in glucose - free rpmi 1640 medium supplemented with 2 mm pyruvate , 0.1% fcs , and 2.5   m oligomycin , an inhibitor of f0f1-atpases , in order to prevent production of atp from both glycolysis and oxidative phosphorylation ( 25 , 26 ) .
cleavage of caspases and the   caspase substrate poly(adp - ribose)polymerase ( parp ) was detected by immunoblotting .
2  10 cells were seeded in 24-well   plates and treated with the apoptotic stimuli .
after the indicated   time periods , cells were washed in cold pbs and lysed in 1% np-40 , 20 mm hepes , ph 7.9 , 350 mm nacl , 20% glycerol , 1 mm   mgcl2 , 0.5 mm edta , 0.1 mm egta , 0.5 mm dithiothreitol ,   containing 3 g / ml aprotinin , 3 g / ml leupeptin , and 2 mm   pmsf .
subsequently , proteins were separated under reducing   conditions on an sds - polyacrylamide gel and electroblotted to a   polyvinylidene difluoride membrane ( amersham buchler gmbh ,   braunschweig , germany ) .
membranes were blocked for 1 h with   5% nonfat dry milk powder in tbs and then immunoblotted for   1 h with rabbit anti - parp antibody ( 1:2,000 ; boehringer mannheim gmbh ) and mouse mabs directed against caspase-8 ( 1:10   dilution of a hybridoma supernatant ; biomedia , baesweiler , germany ) and caspase-3 ( r&d systems , wiesbaden , germany ) .  
membranes were washed four times with tbs/0.05% tween 20   and incubated with the respective peroxidase - conjugated secondary antibody for 1 h. after extensive washing , the reaction was   developed by enhanced chemiluminescent staining using ecl reagents ( amersham buchler gmbh ) . for analysis of cytochrome c release ,
cells were collected by centrifugation , washed with ice - cold pbs , and resuspended in 5 vol of   buffer a containing 250 mm sucrose , 20 mm hepes , ph 7.5 , 1.5   mm mgcl2 , 10 mm kcl , 1 mm edta , 1 mm egta , 1 mm   dithiothreitol , 0.1 mm pmsf , 10 g / ml leupeptin , and 10 g / ml   aprotinin .
the cells were homogenized with 15 strokes in a   douncer , and the homogenates were centrifuged at 1,000 g to   remove cell nuclei .
the supernatants were transferred to a fresh   tube and centrifuged at 10,000 g for 10 min to deplete mitochondria .
the resulting supernatants , designated as cytosolic s10 fraction ,   from each sample were loaded on a 12%-sds polyacrylamide gel .  
cytochrome c release was analyzed by immunoblotting with the   mouse mab 7h8.2c12 ( pharmingen europe , hamburg , germany ) . to evaluate the ability of cytochrome c to activate   caspases in cytosolic extracts , 150 g of an s10 fraction was incubated with 1.25 m bovine cytochrome c ( sigma ) and 1 mm   datp at 30c in a final volume of 25 l for the indicated time   periods . at the end of the incubation ,
the reaction mixture was   loaded on an sds - polyacrylamide gel and analyzed for caspase-3   and -8 cleavage as described above .
for determination of apoptosis , 3    10 cells per well were seeded in microtiter plates and treated for   the indicated time points with anti - cd95 or the chemotherapeutic agents .
leakage of fragmented dna from apoptotic nuclei   was measured as described previously ( 27 ) . in brief ,
apoptotic nuclei were prepared by lysing cells in a hypotonic buffer ( 1% sodium citrate , 0.1% triton x-100 , 50 g / ml propidium iodide )   and subsequently analyzed by flow cytometry .
nuclei to the left   of the 2n peak containing hypodiploid dna were considered as   apoptotic .
all flow cytometry analyses were performed on   a facscalibur ( becton dickinson gmbh , heidelberg , germany ) using cellquest analysis software .
10 cells   were treated with the proapoptotic stimuli in the presence and   absence of oligomycin .
triton x-100 , 10 mm tris - hcl , ph 7.5 , 1 mm edta and incubated for 10 min on ice .
after removal of cell debris , atp content was measured with a luciferin / luciferase assay using a luminometer ( model ml2200 ;   dynatech deutschland gmbh , denkendorf , germany ) .
the   atp content was calculated using an internal standard , and the   data were calculated as the mean from three experiments .
the human jurkat t cell line was maintained in rpmi 1640 medium supplemented with 10% fcs , 10   mm hepes , and antibiotics ( all from gibco brl , eggenstein ,   germany ) .
the cd95-resistant jurkat subline jurkat - r was generated by continuous culture in the presence of anti - cd95 mab   ( igg3 , 1 g / ml ; cell diagnostica , mnster , germany ) for 6 mo .   etoposide and mitomycin c
were obtained from the clinical   pharmacy ( medical clinics , tbingen , germany ) .
doxorubicin ,   cycloheximide , and oligomycin were purchased from sigma ( deisenhofen , germany ) and staurosporine from boehringer mannheim gmbh ( mannheim , germany ) .
mitomycin c was dissolved   in methanol , and doxorubicin , etoposide , and staurosporine in   ethanol and kept as stock solutions at 70c .
intracellular atp   was depleted by incubating cells in glucose - free rpmi 1640 medium supplemented with 2 mm pyruvate , 0.1% fcs , and 2.5   m oligomycin , an inhibitor of f0f1-atpases , in order to prevent production of atp from both glycolysis and oxidative phosphorylation ( 25 , 26 ) .
cleavage of caspases and the   caspase substrate poly(adp - ribose)polymerase ( parp ) was detected by immunoblotting .
2  10 cells were seeded in 24-well   plates and treated with the apoptotic stimuli .
after the indicated   time periods , cells were washed in cold pbs and lysed in 1% np-40 , 20 mm hepes , ph 7.9 , 350 mm nacl , 20% glycerol , 1 mm   mgcl2 , 0.5 mm edta , 0.1 mm egta , 0.5 mm dithiothreitol ,   containing 3 g / ml aprotinin , 3 g / ml leupeptin , and 2 mm   pmsf .
subsequently , proteins were separated under reducing   conditions on an sds - polyacrylamide gel and electroblotted to a   polyvinylidene difluoride membrane ( amersham buchler gmbh ,   braunschweig , germany ) .
membranes were blocked for 1 h with   5% nonfat dry milk powder in tbs and then immunoblotted for   1 h with rabbit anti - parp antibody ( 1:2,000 ; boehringer mannheim gmbh ) and mouse mabs directed against caspase-8 ( 1:10   dilution of a hybridoma supernatant ; biomedia , baesweiler , germany ) and caspase-3 ( r&d systems , wiesbaden , germany ) .  
membranes were washed four times with tbs/0.05% tween 20   and incubated with the respective peroxidase - conjugated secondary antibody for 1 h. after extensive washing , the reaction was   developed by enhanced chemiluminescent staining using ecl reagents ( amersham buchler gmbh ) .
for analysis of cytochrome c release , cells were collected by centrifugation , washed with ice - cold pbs , and resuspended in 5 vol of   buffer a containing 250 mm sucrose , 20 mm hepes , ph 7.5 , 1.5   mm mgcl2 , 10 mm kcl , 1 mm edta , 1 mm egta , 1 mm   dithiothreitol , 0.1 mm pmsf , 10 g / ml leupeptin , and 10 g / ml   aprotinin .
the cells were homogenized with 15 strokes in a   douncer , and the homogenates were centrifuged at 1,000 g to   remove cell nuclei . the supernatants were transferred to a fresh   tube and centrifuged at 10,000 g for 10 min to deplete mitochondria .
the resulting supernatants , designated as cytosolic s10 fraction ,   from each sample were loaded on a 12%-sds polyacrylamide gel .  
cytochrome c release was analyzed by immunoblotting with the   mouse mab 7h8.2c12 ( pharmingen europe , hamburg , germany ) . to evaluate the ability of cytochrome c to activate   caspases in cytosolic extracts , 150 g of an s10 fraction was incubated with 1.25 m bovine cytochrome c ( sigma ) and 1 mm   datp at 30c in a final volume of 25 l for the indicated time   periods . at the end of the incubation ,
the reaction mixture was   loaded on an sds - polyacrylamide gel and analyzed for caspase-3   and -8 cleavage as described above .
for determination of apoptosis , 3    10 cells per well were seeded in microtiter plates and treated for   the indicated time points with anti - cd95 or the chemotherapeutic agents .
leakage of fragmented dna from apoptotic nuclei   was measured as described previously ( 27 ) . in brief ,
apoptotic nuclei were prepared by lysing cells in a hypotonic buffer ( 1% sodium citrate , 0.1% triton x-100 , 50 g / ml propidium iodide )   and subsequently analyzed by flow cytometry
. nuclei to the left   of the 2n peak containing hypodiploid dna were considered as   apoptotic .
all flow cytometry analyses were performed on   a facscalibur ( becton dickinson gmbh , heidelberg , germany ) using cellquest analysis software .
10 cells   were treated with the proapoptotic stimuli in the presence and   absence of oligomycin .
triton x-100 , 10 mm tris - hcl , ph 7.5 , 1 mm edta and incubated for 10 min on ice .
after removal of cell debris , atp content was measured with a luciferin / luciferase assay using a luminometer ( model ml2200 ;   dynatech deutschland gmbh , denkendorf , germany ) .
the   atp content was calculated using an internal standard , and the   data were calculated as the mean from three experiments .
caspase-8 has been identified as the most proximal   caspase which is recruited to the disc by its unique ded   ( 79 ) . in most cells ,
caspase-8 is synthesized as two isoforms of 55 kd ( caspase-8/a and -8/b ) which , after formation of intermediate cleavage products of 43 and 41 kd ,   are processed to a p18 and p10 heterodimer ( 28 , 29 ) .
as assessed with an anti - p18 antibody , treatment of jurkat t   lymphocytes with agonistic anti - cd95 antibody resulted in   the cleavage of procaspase-8 into its characteristic intermediate fragments and the active p18 subunit ( fig .
1 ) . interestingly , an identical cleavage pattern was obtained after   treatment with other apoptotic stimuli , including the protein kinase inhibitor staurosporine , the anticancer drugs   doxorubicin and mitomycin c , and cycloheximide , an inhibitor of the protein synthesis ( fig .
1 ) . because apoptosis   mediated by anticancer drugs has been proposed to induce   the expression of cd95l and elicit cell death by subsequent cd95 interaction ( 20 , 21 )
, we also used the subclone jurkat - r , which was selected for resistance to cd95   signaling .
incubation with staurosporine , anticancer drugs ,   and cycloheximide induced caspase-8 cleavage to a similar   extent in cd95-resistant and -sensitive cells , whereas virtually no cleavage was observed by anti - cd95 in the   cd95-resistant jurkat - r cell line ( fig .
1 ) . additional experiments revealed that anticancer drugs induced caspase-8   activation with similar kinetics and dose dependency in   both cell types ( data not shown ) .
therefore , these results   suggest that caspase-8 activation is not restricted to apoptosis   mediated by death receptors , but is also induced by other   proapoptotic stimuli in a cd95-independent pathway .  
it has been suggested that the cellular energy charge acts   as control point of cell death by either necrosis or apoptosis , and that atp is necessary for the execution of the apoptotic program ( 25 , 26 , 30 ) . in conjunction with cytochrome c
, atp is required for the function of apaf-1 ,   which initiates caspase activation in the mitochondria - controlled pathway ( 12 , 13 , 15 ) .
therefore , we investigated   whether manipulation of intracellular atp levels can be used   to analyze the involvement of the cytochrome c / apaf-1   pathway during apoptosis triggered by certain stimuli .
atp   levels were depleted in jurkat cells by incubating cells in   glucose - free medium and oligomycin , an inhibitor of mitochondrial f0f1-atpases . under these conditions ,
treatment of jurkat cells   with anti - cd95 induced a marked cleavage of caspase-8   within 1 h that was not affected upon depletion of atp by   oligomycin ( fig .
furthermore , activation of caspase-3   was observed with slightly delayed kinetics in both atp - depleted and nondepleted cells .
we also analyzed the   cleavage of parp , an enzyme involved in dna repair ,   which has been shown to serve as a substrate for caspase-3   ( 31 ) .
2 demonstrates that parp , a 116-kd protein ,   was cleaved into its characteristic 89-kd fragment with   similar kinetics as caspase-3 , regardless of the presence or   absence of atp .  
in contrast to the cd95 pathway , atp depletion   strongly inhibited caspase-8 and -3 activation as well as   parp cleavage in response to the receptor - independent   apoptotic stimuli .
after treatment with staurosporine ,   caspase-8 activation was detectable within 2 h and maximal   after 3 h. inhibition of atp production strongly prevented   caspase-8 activation ( fig .
in addition to staurosporine , caspase activation in   response to chemotherapeutic drugs was almost completely   abolished in atp - depleted cells .
doxorubicin - induced   caspase-8 activation started within 46 h , and was most   pronounced after 10 h ( fig .
interestingly , similar to   cd95 , caspase-3 activation and parp cleavage followed   caspase-8 processing .
however , in contrast to cd95 , activation of both caspases was strongly abrogated by oligomycin .
therefore , the differential requirement of atp for   caspase activation by either cd95 or chemotherapeutic   drugs indicates distinct regulatory pathways for the two   types of apoptosis .
recent evidence has demonstrated that mitochondria   participate in the execution of apoptosis by release of cytochrome c ( 10 , 11 , 1719 ) .
binding of cytochrome c to   apaf-1 results in the cleavage of procaspase-9 or other   caspases , which in turn activate caspase-3 ( 13 ) .
preparation   of cytosolic fractions confirmed that anti - cd95 , staurosporine , etoposide , and cycloheximide ( fig .
3 a ) , as well as the   other chemotherapeutic drugs ( data not shown ) , induced   the redistribution of cytochrome c. it has been shown that   caspase-9 and -3 can be activated in cell - free systems upon   incubation of cytosolic fractions with cytochrome c and   datp ( 12 , 13 , 15 ) . because caspase-8 activation so far has   been observed only after formation of an fadd - containing death receptor complex , we investigated whether   caspase-8 can be also activated in vitro by the addition of   cytochrome c. indeed , incubation of cytosolic extracts with   cytochrome c resulted in a potent processing of caspase-8   that was even more pronounced than caspase-3 cleavage by   cytochrome c ( fig
therefore , the data demonstrate   that the initiator caspase-8 can be activated not only at the   disc level but also by cytochrome c. future studies will   show whether caspase-8 activation is mediated directly by   atp and cytochrome c  bound apaf-1 or by another proximal caspase , such as caspase-9 .  
the above experiments suggested that drug - induced   caspase-8 activation is cd95-independent and entirely   controlled by an atp - dependent step , most probably   apaf-1 activation .
therefore , we investigated the effects of   atp depletion on apoptotic dna fragmentation and cell   death .
stimulation of jurkat cells with anti - cd95 resulted   in the rapid formation of hypodiploid dna , which was   comparable in atp - depleted cells and cells kept under   normal growth conditions ( fig .
, staurosporine - induced nuclear apoptosis was completely abrogated when atp generation was inhibited ( fig . 4 b ) ,
however , atp   depletion did not prevent final cell death , as assessed by   membrane uptake of propidium iodide into cells . instead ,   in the presence of oligomycin , cell death occurred by necrosis .
very similar effects of atp depletion were found   when nuclear apoptosis and cell death in response to chemotherapeutic drugs were analyzed ( data not shown ) .
4 c demonstrates that treatment of cells with oligomycin   resulted in a strong decrease in atp production .
however ,   anti - cd95 and staurosporine themselves only slightly affected intracellular atp levels in early stages of apoptosis .   in summary , manipulation of intracellular atp levels is a   useful means by which to discriminate between the involvement of distinct apoptotic pathways .
although atp   may affect other events , such as chromatin condensation   ( 32 ) , the requirement for atp can be most likely attributed   to its stimulatory function on apaf-1 in the mitochondrial   pathway .
our data reveal that caspase activation induced by   staurosporine and chemotherapeutic drugs is entirely mediated by this signaling pathway and does not require cd95   interaction as previously proposed ( 20 , 21 ) .
in contrast ,   cd95 signaling , although it activates mitochondria ( 33 ,   34 ) , can circumvent this evolutionary conserved pathway   in order to activate caspases . whether this bypass holds true   for all cell types or whether it is dependent on the degree of   disc formation ( 34 )
our findings   convincingly demonstrate that the cytochrome c / apaf-1   pathway and receptor - triggered signaling events are not   necessarily linked to each other .
10 cd95-sensitive jurkat or cd95-resistant jurkat - r   cells were cultured in normal   growth medium and then either   left untreated ( control ) or treated   with anti - cd95 ( 1 g / ml , 3 h ) ,   staurosporine ( stauro ; 2.5 m , 6   h ) , doxorubicin ( doxo ; 2 g / ml ,   12 h ) , mitomycin c ( mito ; 25 g / ml , 8 h ) , or cycloheximide ( chx ;   25 g / ml , 6 h ) .
cellular proteins were separated by sds - page , and   proteolytic processing of caspase-8 was detected by immunoblotting with   an antibody against the caspase-8 p18 subunit .
open arrowheads , the two   isoforms of procaspase-8/a and -8/b , which are cleaved into the intermediate forms p43 and p41 ( filled arrowheads ) and finally processed to the active p18 subunit ( arrow ) .
atp depletion differentially affects caspase activation and   cleavage of the caspase substrate parp in response to anti - cd95 and   drug treatment .
jurkat cells were kept in glucose - free medium in either   the absence or presence of 2.5 m oligomycin , and triggered with different apoptotic stimuli .
anti - cd95 ( a ) , staurosporine ( b ) , doxorubicin   ( c ) , and mitomycin c ( d ) were used at the drug concentrations described   in the legend to fig .
total cell lysates were subjected to 15% sds - page to detect caspase-8 processing , and to 815% gradient sds - page   to detect cleavage of caspase-3 and parp .
separated proteins were immunoblotted with caspase-8 , caspase-3 , and parp - specific antibodies .  
top , caspase-8 cleavage ; only sections of the immunoblots indicating the   cleaved intermediate forms ( p43 and p41 ) of caspase-8/a and -8/b are   shown .
middle , processing of caspase-3 ; the immunoblots denote the position of procaspase-3 and its p17 active subunit .
bottom , parp cleavage ;   filled arrowheads indicate the uncleaved p116 and open arrowheads the   cleaved p89 form of parp .
jurkat cells were either left untreated in normal culture medium or incubated with anti - cd95 ( 1 g / ml , 3 h ) , staurosporine ( stauro ; 2.5 m , 6 h ) ,   etoposide ( etop ; 25 g / ml , 6 h ) , or cycloheximide ( chx ; 25 g / ml , 5 h ) .  
cells were then homogenized , and the s10 fraction depleted of mitochondria was subjected to 12% sds - page .
( b ) in vitro activation of caspases by cytochrome c. aliquots of an s10 fraction ( 150 g )   were incubated for the indicated time with 1.25 m cytochrome c in the   presence of datp .
a control fraction was incubated for 6 h in the absence   of cytochrome c. proteolytic processing of caspase-8 ( left ) and
jurkat cells   were either left untreated ( open   symbols ) or pretreated for 45 min   with oligomycin ( filled symbols )   and then incubated with anti - cd95 ( 1 g / ml ) , staurosporine   ( 2.5 m ) , or medium control   ( dashed line ) .
( a ) formation of apoptotic nuclei was determined by flow cytometry of hypodiploid dna .
( b )   membrane damage was measured by the uptake of propidium iodide into   cells and flow cytometry .
extracts   of control cells and cells treated for 2 h with anti - cd95 or staurosporine   ( stauro ) in the presence and absence of oligomycin were analyzed for   atp content using a bioluminescence assay .
 apoptosis is induced by different stimuli , among them triggering of the death receptor cd95 ,   staurosporine , and chemotherapeutic drugs . in all cases , 
 apoptosis is mediated by caspases , although it is unclear how these diverse apoptotic stimuli cause protease activation . 
 two regulatory pathways have been recently identified , but it remains unknown whether they are functionally independent or linked to each other . 
 one is mediated by recruitment of the proximal   regulator caspase-8 to the death receptor complex . 
 the other pathway is controlled by the release of cytochrome c from mitochondria and the subsequent atp - dependent activation of the   death regulator apoptotic protease - activating factor 1 ( apaf-1 ) . here , we report that both pathways can be dissected by depletion of intracellular atp . 
 prevention of atp production completely inhibited caspase activation and apoptosis in response to chemotherapeutic drugs and   staurosporine . 
 interestingly , caspase-8 , whose function appeared to be restricted to death receptors , was also activated by these drugs under normal conditions , but not after atp depletion . 
 in contrast , inhibition of atp production did not affect caspase activation after triggering   of cd95 . 
 these results suggest that chemotherapeutic drug  induced caspase activation is entirely controlled by a receptor - independent mitochondrial pathway , whereas cd95-induced   apoptosis can be regulated by a separate pathway not requiring apaf-1 function . 