JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tatsuta, T.
Right arrow Articles by Mountz, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tatsuta, T.
Right arrow Articles by Mountz, J. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 275, Issue 19, 14248-14254, May 12, 2000


The Prodomain of Caspase-1 Enhances Fas-mediated Apoptosis through Facilitation of Caspase-8 Activation*

Toru TatsutaDagger §, Akio ShiraishiDagger , and John D. Mountz§

From the Dagger  Neuroscience and Immunology Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa, Tokyo 140-8710, Japan and the § University of Alabama at Birmingham and the Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 35294-0007

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Caspase-1 (interleukin-1beta converting enzyme) is produced in the form of a latent precursor, which is cleaved to yield a prodomain in addition to the p20 and p10 subunits. It has been established that the (p20/p10)2 heterotetramer processes the latent precursor of interleukin-1beta into an active form during apoptosis, but the function of the residual prodomain of caspase-1 (Pro-C1) has not been established. To evaluate the involvement of Pro-C1 in apoptosis, a Pro-C1 expression vector was transfected into the HeLa cell line, which is susceptible to Fas-mediated apoptosis. Expression of recombinant Pro-C1 in HeLa cells enhanced apoptosis mediated by Fas, but not etoposide-induced apoptosis. This enhancement of Fas-mediated apoptosis was abolished by inhibitors of caspase-8 (Ile-Glu-Thr-Asp-fluoromethyl ketone) and caspase-3 (Asp-Glu-Val-Asp-aldehyde) but was only slightly diminished by an inhibitor of caspase-1 (acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone). During apoptosis induced by an agonistic anti-Fas antibody, the activation of caspase-8 and caspase-3 was more pronounced and occurred more rapidly in HeLa/Pro-C1 cells than in the empty vector transfectant (HeLa/vec) cells; in contrast, caspase-1 was not activated in either HeLa/Pro-C1 or HeLa/vec cells. These results demonstrate an additional and novel function for caspase-1 in which Pro-C1 acts to enhance Fas-mediated apoptosis, most probably through facilitation of the activation of caspase-8.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fas (apo-1/CD95) can induce apoptosis and is a member of an extensive family of cell surface molecules involved in the signaling of cell death including TNF-R1,1 TNF-R2, the neutrophin receptor, CD27, CD30, CD40, OX40, death receptor 3, and death receptor 4 (1-13). The binding of Fas ligand to Fas induces aggregation of its intracellular death domains leading to the recruitment of several important signaling proteins, such as the Fas-associated death domain (FADD) protein (14). Through bridging by FADD, caspase-8 is recruited to the cytoplasmic death domain of Fas and then activated (15-17). Caspase-8 is a member of a growing family of cysteine proteases that are involved in Fas-mediated apoptosis as well as apoptosis triggered by other mechanisms (18). Caspase-1 (interleukin-1 beta  (IL-1beta ) converting enzyme) is a member of this family and most likely is involved in the apoptosis pathway downstream of caspase-8. Although the details of the molecular processes downstream of FADD and caspase-8 have not been elucidated fully, it is likely that upstream caspases, such as caspase-8, induce sequential activation of the downstream caspases, which include caspase-1 and caspase-3 (19, 20). Activated caspase-3 cleaves numerous life-sustaining intracellular proteins leading to the morphologic changes and disruption of cell nuclei that are characteristic of apoptosis (20-23).

The caspases can be divided into three groups: those with large prodomains without death effector domains (caspases 1, 2, and 9), those with large prodomains with death effector domains (caspases 8 and 10), and those with small prodomains (caspases 3, 6, and 7) (24, 25). It is the prodomain of caspase-8 that mediates the recruitment of this molecule to the adapter molecules, such as FADD or CRADD/RAIDD, that interact with the receptor (14, 26-29). The prodomain of caspase-9 is necessary for its activation through binding of Apaf-1, which then associates with cytochrome c (30, 31). It also has been reported that several inhibitory molecules of the FADD-death effector domain homology regions, such as FLAME-1 and p14.1 of adenovirus, enable extensive recruitment of apoptotic effector molecules and apoptosis (32, 33). Thus, the prodomains of the caspases can function to physically link the death receptors or intracellular apoptotic mediators to downstream caspase activation pathways.

Caspase-1, first known as IL-1beta converting enzyme, is a member of the group of caspases with large prodomains. The prodomain of caspase-1 (Pro-C1) represents the 11.5-kDa amino acid terminal portion of the 45-kDa caspase-1 precursor. Pro-C1 is produced as a residual component after proteolytic cleavage of the precursor generates the functional caspase-1 subunits of molecular masses 20 and 10 kDa, known as the p20 and p10 subunits, respectively (Fig. 1). Active caspase-1, a (p20/p10)2 tetramer, is necessary and sufficient for cleavage of precursor IL-1beta as well as for induction of apoptosis in some cell lines (34, 35). Although the role of p20/p10 subunits has been well characterized, the function of the residual 11.5-kDa Pro-C1 has not been clarified.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1.   Structure of caspase-1. The human precursor caspase-1 (45-kDa) is cleaved at four distinct sites. Cleavage at sites I and II generates the 11.5-kDa Pro-C1 consisting of residues 1-119 and a 30-kDa protein (p20-p10). Successive cleavage at sites III and IV generates a 20-kDa protein (p20) consisting of residues 120-297 and a 10-kDa protein (p10) consisting of residues 317-404. The p20 and p10 subunits form a tetramer, (p20/p10)2, which is responsible for proteolytic maturation of the precursor of IL-1beta . ICE, IL-1beta converting enzyme.

In this study, using a yeast two-hybrid system, Pro-C1 is shown to exhibit self-association but does not appear to associate with the other subunits of caspase-1 or caspase-3. Pro-C1 facilitates Fas apoptosis signaling and activation of caspase-8 and caspase-3 but does not affect activation of caspase-1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Treatment-- Jurkat (human T cell lymphoma), HeLa (human cervical carcinoma), and HL-60 (human promyelocytic leukemia) cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Cansera International Inc., Canada), 100 units/ml of penicillin, and 100 µg/ml of streptomycin (Life Technologies, Inc.).

Establishment of Recombinant Human Pro-C1 Transfectant Cells-- The recombinant human Pro-C1 genes were constructed as follows. The full-length human Pro-C1 cDNA fragments were amplified by 35 cycles of the reverse transcriptase polymerase chain reaction of total RNA isolated from HL-60 cells. Each cycle consisted of denaturation at 94 °C for 1 min, annealing at 60 °C for 1.5 min, and extension at 72 °C for 1 min. The primers used to amplify Pro-C1 were 5'-CGGAATTCATGGCCGACAAGGTCCTG-3' (nucleotides 1-18 with EcoRI linker) and 5'-GCGTCGACTTAGTCTTGCATATTAAGGTAATTTCCAGA-3' (complementary to nucleotides 283-309 with SalI linker). The amplified Pro-C1 cDNA fragments with EcoRI/SalI linkers were subcloned directly into the pCRII vector (Invitrogen, San Diego, CA) following the procedures recommended by the manufacturer. DNA sequencing analysis revealed that the inserted cDNA sequence was identical to the published sequence. The insert was then excised using EcoRI and subcloned into the EcoRI site of the pcDNA3 vector (pcDNA3/Pro-C1) in the correct orientation. The HeLa cells used for transfection were harvested during the logarithmic growth phase and washed with transfection buffer consisting of 21 mM HEPES (pH 7.1) containing 10 µg/ml pcDNA3 or pcDNA3/Pro-C1, 145 mM NaCl, and 125 mM CaCl2 for 4 h. The cells were incubated in 1 ml of 21 mM HEPES (pH 7.1) containing 15% glycerol for 3 h at room temperature and resuspended in growth medium. The transfectant cells were selected after incubation with 200 µg/ml of geneticin (Sigma) for 3 weeks and cloned by limiting dilution. A clone with high levels of Pro-C1 expression was identified by reverse transcriptase-polymerase chain reaction analysis and used for further experiments.

Analysis of Apoptosis Responses, Fas Expression, and Caspase Activity-- Apoptosis was induced in HeLa, HeLa/Pro-C1, and Jurkat cells by treatment with mouse anti-human Fas antibody (CH-11) (MBL Co. Ltd, Nagoya, Japan), recombinant human TNF-alpha (Genzyme, Cambridge, MA), or etoposide (Sigma) in the presence or absence of the caspase inhibitors Ac-YVAD-cmk (Bachem, Switzerland), DEVD-cho (Calbiochem), IETD-fmk (CLONTECH Laboratories Inc. Palo Alto, CA), or zVAD-CH2DCB (Phoenix Pharmaceuticals, Inc., Mountain view, CA) during culture in 200 µl of growth medium in 96-well plates in a humidified atmosphere of 5% of CO2 for 72 h. Cell viability was determined by XTT analysis (Sigma). The number of viable cells was determined by Trypan blue exclusion analysis (Life Technologies, Inc.). The cell viability at each concentration of CH-11, etoposide, or TNF-alpha is indicated as the percentage of the control. Flow cytometric analysis of cell surface Fas expression was accomplished using HeLa cells (1.0 × 106) or HeLa/Pro-C1 cells (1.0 × 106) harvested during logarithmic growth after incubation with 1 ml of phosphate-buffered saline containing 0.1% EDTA for 30 min at 4 °C. After washing with phosphate-buffered saline at 4 °C, the cells were incubated in 50 µl of FACS buffer (phosphate-buffered saline containing 2.5% fetal bovine serum, 0.1% NaN3) containing 1 µl of fluorescein isothiocyanate-conjugated mouse anti-human Fas antibody (UB2) (MBL Co. Ltd.) or fluorescein isothiocyanate-conjugated isotype control (Pharmingen, San Diego, CA) for 1 h at 4 °C. The cells were washed three times with FACS buffer and analyzed using a Cyto ACE-150 flow cytometer (JASCO Corp., Tokyo, Japan) with a logarithmic scale. The activity of the caspases was determined in cell lysates. The caspase-8 colorimetric assay kit (MBL Co. Ltd.) with the synthetic colorimetric peptidyl substrate IETD-pNA and the ApoAlert caspase-3 colorimetric assay kit (CLONTECH Laboratories Inc.) with the synthetic colorimetric peptidyl substrate DEVD-pNA were used to determine the activity of caspase-8 and caspase-3, respectively. The activity of caspase-1 was measured as described previously (36) using the synthetic colorimetric peptidyl substrate YVAD-pNA (Biomol Research Laboratories Inc., Plymouth Meeting, PA). The concentration of protein in the cell lysates was measured using the BCA protein assay reagent kit (Pierce). The activities of each caspase were calculated as the amount (picomoles) of colorimetric peptide substrate cleaved by 1 milligram of protein in 1 min.

Two-hybrid Analysis of the Interaction between Caspase-1 Components-- The full-length human Pro-C1, p20, and p10 and caspase-3 p17 and p12 cDNA fragments were amplified by reverse transcriptase-polymerase chain reaction, as described above, from total RNA isolated from HL-60 cells. The primers used to amplify Pro-C1 were 5'-CGGAATTCATGGCCGACAAGGTCCTG-3' (nucleotides 1-18) and 5'-GCGTCGACTTAGTCTTGCATATTAAGGTAATTTCCAGA-3' (complementary to nucleotides 283-309). The primers used to amplify caspase-1 p20 were 5'-CGGAATTCAACCCAGCTATGCCCACA-3' (nucleotides 358-375) and 5'-GCGTCGACTTAATCTTTAAACCACACCACACC-3' (complementary to nucleotides 871-891). The primers used to amplify caspase-1 p10 were 5'-CGGAATTCGCTATTAAGAAAGCCCACATA-3' (nucleotides 949-969) and 5'-GCGTCGACTTAATGTCCTGGGAAGAGGTA-3' (complementary to nucleotides 1192-1212). The primers used to amplify caspase-3 p17 were 5'-CGGAATTCATGGAGAACACTGAAAAC-3' (nucleotides 85-102) and 5'-GCGTCGACTTAGTCTGTCTCAATGCCACAGTC-3' (complementary to nucleotides 505-525). The primers used to amplify caspase-3 p12 were 5'-CGGAATTCATGGCGTGTCATAAAATA-3' (nucleotides 545-562) and 5'-GCGTCGACTTAGTGATAAAAATAGAGTTC-3' (complementary to nucleotides 811-831). Each amplified component cDNA fragment with EcoRI/SalI linkers was subcloned directly into the pCRII vector (Invitrogen) following the procedures recommended by the manufacturer. DNA sequencing analysis revealed that the inserted cDNA sequences were identical to the published sequences. The inserts were then excised by EcoRI and SalI and inserted into the hybrid vectors for two-hybrid analysis (CLONTECH). Hybrid vectors encoding the Gal4 binding domain/caspase subunit and Gal4 activation domain/caspase subunit were co-transfected into yeast SFY526 cells. beta -Galactosidase activity in transformants was measured using o-nitrophenyl beta -D-galactopyranoside (Sigma) as the substrate according to the procedures recommended by the manufacturer. The Gal4 binding domain/p53 and Gal4 activation domain/SV40 large T antigen were used as positive controls.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pro-C1 Exhibits Self-binding Activity and Does Not Bind to Capase-1 or Caspase-3 Subunits-- The molecular interactions of Pro-C1 were tested using a yeast two-hybrid system to analyze the binding of Pro-C1 to the other components of caspase-1 or caspase-3 (Fig. 2). The predominant binding activity of Pro-C1 was to itself. Somewhat surprisingly, binding to the other subunits of caspase-1, p10 or p20, or to the subunits of caspase-3, p17, and p12, was not observed. Moreover, Pro-C1 exhibited only marginal levels of binding to CrmA, although both the caspase-1 subunits displayed binding to CrmA as anticipated (37). These studies of the binding activity of Pro-C1 suggested to us that Pro-C1 may have distinct biologic properties that are independent of its binding or association with the active portions of caspase-1 or caspase-3.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2.   The prodomain of caspase-1 associates with itself but not with the p10 or p20 subunits of caspase-1 or the p17 and p12 subunits of caspase 3. The binding affinity of caspase-1 components was evaluated in a semiquantitative beta -galactosidase assay after hybrid vectors encoding the Gal4 activation domain (GAL4 ad)/caspase-1 components or the Gal4 binding domain (GAL4 bd)/caspase-1 components were co-transfected into yeast SFY526 cells. beta -Galactosidase activity was measured using a liquid culture assay with o-nitrophenyl beta -D-galactopyranoside as the substrate. The data are expressed as the mean ± S.D. (bars) of triplicate determinations. Pro-C1, caspase-1/Pro-C1; p20, caspase-1/p20; p10, caspase-1/p10; Cp17, capsase-3/p17; Cp12, caspase-3/p12; CrmA, cowpox viral CrmA; p53, murine p53(72-390); and SV40LT, SV40 large T-antigen(84-708).

Transfection of HeLa Cells with Pro-C1 Increases Their Susceptibility to Fas-mediated Apoptosis but Does Not Affect the Response to Etoposide or TNF-alpha -- To evaluate the involvement of Pro-C1 in Fas-mediated apoptosis, the pcDNA3/Pro-C1 expression vector was transfected into HeLa cells. Several transfectant clones were obtained, and the experiments described here were carried out with the clone expressing the highest levels of Pro-C1 (HeLa/Pro-C1). The cell viability of HeLa, HeLa/Pro-C1, and HeLa/vec (vector transfectant) cells decreased in a dose-dependent manner in response to treatment with an agonistic anti-Fas antibody, CH-11 (Fig. 3A). Interestingly, the viability of HeLa/Pro-C1 cells treated with higher doses of CH-11 was significantly lower than that in either HeLa or HeLa/vec cells. After treatment with CH-11 at 1000 ng/ml for 72 h, the viability of the HeLa/Pro-C1 cells was 33% compared with 64% for the HeLa/vec cells and 74% for the HeLa cells. In contrast, there was no significant difference in the viability of the three cell lines after treatment with different concentrations of etoposide (Fig. 3B). TNF-alpha is not cytotoxic for HeLa cells, and transfection with Pro-C1 did not promote TNF-alpha susceptibility on the cells (Fig. 3C). Spontaneous apoptosis in the absence of Fas signaling did not occur in HeLa/Pro-C1 and HeLa/vec cells (data not shown), indicating that neither expression of Pro-C1 nor transfection with vectors renders HeLa cells more susceptible to spontaneous apoptosis. To further characterize the effect of overexpression of Pro-C1 on Fas-mediated apoptosis in HeLa cells, we determined the time course of Fas-mediated apoptosis induced by CH-11 at 1000 ng/ml using the Trypan blue exclusion assay of viable cells (Fig. 4). The time at which 50% cell death (t1/2) was induced in HeLa/Pro-C1 cells was 19 h compared to 79 h in HeLa/vec cells and 2.4 h in Jurkat cells. This more rapid decrease in the numbers of viable HeLa/Pro-C1 cells than in HeLa/vec cells indicated that the HeLa/Pro-C1 cells are more susceptible to Fas-mediated apoptosis than HeLa/vec cells. As the increased susceptibility to Fas-mediated apoptosis could simply reflect an effect of Pro-C1 on the expression of Fas antigen, we compared the expression of Fas antigen in HeLa and HeLa/Pro-C1 cells by flow cytometry. HeLa/Pro-C1 cells showed a similar staining profile to that of HeLa cells (Fig. 5). Thus, neither transfection nor expression of Pro-C1 increased the expression of Fas antigen. Taken together, these studies suggest that the expression of Pro-C1 does not induce but does specifically enhance Fas-mediated apoptosis of HeLa cells and that this enhancement of Fas-mediated apoptosis in HeLa/Pro-C1 cells is brought about at a point downstream of ligation of Fas antigen.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Transfection with Pro-C1 enhances the susceptibility of HeLa cells to apoptosis triggered by engagement of Fas but does not enhance susceptibility to apoptosis triggered by etoposide or TNF-alpha . Shown is the effect of graded concentrations of (A) mouse anti-human Fas antibody (CH-11), (B) etoposide, and (C) recombinant human TNF-alpha on HeLa (closed circles), its empty vector transfectant (HeLa/vec; open circles), and Pro-C1 transfectant cells (HeLa/Pro-C1; closed squares). Cell viability was determined after 72 h using XTT analysis and calculated as the percentage of untreated control cells (percentage of control). The data are expressed as the mean ± S.D. (bars) of triplicate determinations. The absence of error bars on data points indicates that the error associated with the replicated experiment is within the range covered by the symbols.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4.   The time course of Fas-mediated apoptosis is more rapid in HeLa cells transfected with Pro-C1. Shown, is the viability of Jurkat (closed circles), HeLa empty vector transfectant (HeLa/vec) (open circles), and HeLa Pro-C1 transfectant cells (HeLa/Pro-C1) (closed squares), at different time points after incubation with mouse anti-human Fas antibody (CH-11) at 1000 ng/ml. The number of viable cells was determined by Trypan blue exclusion analysis and calculated as the percentage of viable cells relative to the untreated control cells (percentage of control). The data are expressed as mean ± S.D. (bars) of triplicate determinations. The absence of error bars on data points indicates that the errors associated with the replicated experiments are within the range covered by the symbols.


View larger version (56K):
[in this window]
[in a new window]
  		Fig. 5.   The expression of Fas antigen on Pro-C1 transfectant cells is unaltered. HeLa cells (A) and Pro-C1 transfectant (HeLa/Pro-C1) cells (B) were immunostained with fluorescein isothiocyanate-conjugated anti-human Fas antibody (UB2) or fluorescein isothiocyanate-conjugated isotype control. The percentage of Fas antigen positive cells was analyzed by flow cytometry using a logarithmic scale.

The Susceptibility of Fas-mediated Apoptosis Is Inhibited by Inhibitors of Caspase-8 and Caspase-3 but Not Caspase-1-- One of the most important intracellular events in apoptosis signal transduction is the sequential activation of caspases. Fourteen caspases have shown to be involved in apoptosis signal transduction. Of these, it has been reported that caspase-8 and caspase-1 are activated at an early stage of Fas-mediated apoptosis, whereas caspase-3 is activated at a later stage (19, 20). To determine which, if any, of the caspases are associated with the enhancement of Fas-mediated apoptosis by Pro-C1, Fas-mediated apoptosis was examined in the presence or absence of specific inhibitors of caspase-1 (Ac-YVAD-cmk), caspase-3 (DEVD-cho), and caspase-8 (IETD-fmk) (Fig. 6). We also used zVAD-CH2DCB, which exhibits a broad spectrum of inhibition affecting apoptosis-inducing factor (38) as well as several caspases, including caspase-1, caspase-3, and caspase-8. In the absence of inhibitors, the percentage of viable HeLa/Pro-C1 cells remaining after treatment with CH-11 (1000 ng/ml, 72 h) was 26 ± 1%, which was significantly lower than the viability of similarly treated HeLa/vec cells (69 ± 1%). In the presence of DEVD-cho, IETD-fmk, or zVAD-CH2DCB at concentrations of 10 µM, the viability of HeLa/Pro-C1 cells treated with CH-11 at 1000 ng/ml was significantly higher (95 ± 2%, 87 ± 2%, and 87 ± 1%, respectively), suggesting that these inhibitors protect HeLa/Pro-C1 cells from Fas-mediated apoptosis. In contrast, Ac-YVAD-cmk (400 µM) exhibited only a marginal protective effect against Fas-mediated apoptosis in HeLa/Pro-C1 cells. These four inhibitors either failed to produce an inhibitory effect or produced only a weak effect on etoposide-induced apoptosis in HeLa/vec and HeLa/Pro-C1 cells. The concentrations of inhibitors used in this study (400 µM for Ac-YVAD-cmk, 10 µM for the others) were optimal for maximum protection against Fas-mediated apoptosis in HeLa cells (data not shown). These results suggest that the increased susceptibility to Fas-mediated apoptosis in HeLa/Pro-C1 cells can be inhibited by blocking caspase-3 and caspase-8 activity but is not dependent on caspase-1 activity.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 6.   The enhancement of Fas-mediated apoptosis in Pro-C1 transfectants is abolished by inhibition with inhibitors of caspase-8 and caspase-3 activity but not by inhibitor of caspase-1. HeLa empty vector transfectant (HeLa/vec), HeLa Pro-C1 transfectant cells (HeLa/Pro-C1), and Jurkat cells were incubated with mouse anti-human Fas antibody (CH-11) at 1000 ng/ml (A) or etoposide at 100 µM (B) in the absence or presence of caspase inhibitors Ac-YVAD-cmk (400 µM), DEVD-cho (10 µM), IETD-fmk (10 µM), or zVAD-CH2-DCB (10 µM) for 72 h. After incubation, the cell viability was determined by XTT analysis. The viability of the treated cells is indicated as a percentage of the control. The data are expressed as the mean ± S.D. (bars) of triplicate determinations.

Pro-C1 Accelerates Activation of Caspase-3 and Caspase-8, but Not Caspase-1-- To further confirm that Pro-C1 plays a role in regulation of caspase-3 and caspase-8 activation during Fas-mediated apoptosis, we used caspase-specific colorimetric peptidyl substrates and investigated the time course of caspase activation in HeLa/vec and HeLa/Pro-C1 cells during Fas-mediated apoptosis. The activity of caspase-3 (Fig. 7B) and caspase-8 (Fig. 7C) but not caspase-1 (Fig. 7A) increased significantly in all three cell lines after treatment with CH-11. The increase in activity of caspase-8 in HeLa/Pro-C1 cells occurred shortly after treatment with CH-11 and reached a peak at 120 min. A similar time course was observed for the activity of caspase-3 in HeLa/Pro-C1 cells. In HeLa/vec cells, there was significantly less activity of caspase-3 and caspase-8 compared with HeLa/Pro-C1 cells. The peak activity of caspase-3 in HeLa/Pro-C1 cells at 120 min was 410 ± 20 pmol/min/mg of protein; at this time point the value for HeLa/vec cells was 57 ± 1 pmol/min/mg of protein. Similarly, at 120 min, the activity of capsase-8 was 120 ± 20 pmol/min/mg of protein in HeLa/Pro-C1 cells and only 6 ± 4 pmol/min/mg of protein in HeLa/vec cells. The more rapid activation of caspase-3 and caspase-8 in HeLa/Pro-C1 (Fig. 7) is consistent with the evidence that HeLa/Pro-C1 cells undergo Fas apoptosis more rapidly than HeLa/vec cells (Fig. 4).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 7.   The activity of caspase-8 and caspase-3, but not caspase-1, is enhanced during Fas-mediated apoptosis in Pro-C1 transfectants. HeLa empty vector transfectant (HeLa/vec) (closed circles), HeLa Pro-C1 transfectant cells (HeLa/Pro-C1) (open circles), and Jurkat cells (closed squares) were incubated with mouse anti-human Fas antibody (CH-11) at 1000 ng/ml. After indicated time periods, cell-free extract was prepared from each cell line. The activity of caspase-1 (A), caspase-3 (B), and caspase-8 (C) in the cell-free extract was measured using colorimetric peptide substrates (YVAD-pNA for caspase-1, DEVD-pNA for caspase-3, and IETD-pNA for caspase-8). Activity in each cell is indicated as the amounts (picomole) of colorimetric peptide substrates cleaved by 1 mg of protein in 1 min. The data are expressed as the mean ± S.D. of duplicate determinations.

To determine whether Pro-C1 promotes activation of both caspase-3 and caspase-8 directly, we examined activation of caspase-3 and caspase-8 in HeLa/vec or HeLa/Pro-C1 cells treated with CH-11 in the presence of DEVD-cho or IETD-fmk (Fig. 8). In both cell lines, activation of caspase-3 was inhibited by the caspase-8 selective inhibitor, IETD-fmk, whereas activation of caspase-8 was not inhibited by the caspase-3 selective inhibitor, DEVD-cho. Neither caspase-3 nor caspase-8 was activated in the presence of a combination of these inhibitors. These results suggest that the activation of caspase-3 requires the activation of caspase-8 and that Pro-C1 promotes the activation of caspase-8 during Fas-mediated apoptosis in HeLa cells.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 8.   Caspase-8 and caspase 3 are activated sequentially during Fas-mediated apoptosis in Pro-C1 transfectants. HeLa empty vector transfectant (HeLa/vec) (A) and HeLa Pro-C1 transfectant (HeLa/Pro-C1) (B) cells were incubated in the absence or presence of caspase inhibitors DEVD-cho (10 µM) and IETD-fmk (10 µM) for 2 h. After the addition of mouse anti-human Fas antibody (CH-11) at 1000 ng/ml, the cells were further incubated for 4 h. The cell-free extract was prepared from each cell line. The activity of caspase-3 (filled columns) and caspase-8 (open columns) in the cell-free extract was measured using colorimetric peptide substrates (DEVD-pNA for caspase-3 and IETD-pNA for caspase-8). Activity in each cell was indicated as percentage of activity of the untreated control cells (percentage of control). The data are expressed as the mean ± S.D. of triplicate determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been established that the prodomains of caspase-8 and caspase-9 are functional; we therefore undertook a study of the function of Pro-C1. Because of the key role of the caspases in apoptosis, we investigated the role of Pro-C1 in apoptosis using HeLa cells that were transfected with Pro-C1 (HeLa/Pro-C1 cells). We found that HeLa cells transfected with Pro-C1 (HeLa/Pro-C1) are more susceptible to Fas-mediated apoptosis than HeLa or HeLa/vec cells and that this enhanced susceptibility to apoptosis in HeLa/Pro-C1 cells is specific for apoptosis triggered by Fas because apoptosis triggered by etoposide was unaffected and the cells remained resistant to TNF-alpha -induced cytotoxicity. Overexpression of Pro-C1 did not induce apoptosis in the absence of a trigger, suggesting that Pro-C1 does not act as an initiator of apoptosis but rather facilitates Fas-mediated apoptosis specifically.

It has been reported previously that, in some cell lines, susceptibility to Fas-mediated apoptosis is correlated with the amount of Fas antigen expressed on the cell surface (39). There was, however, no significant difference in the levels of Fas expression in HeLa/Pro-C1 cells and HeLa cells, suggesting that Pro-C1 acts to enhance the apoptotic process by affecting a component of the apoptotic pathway that lies downstream of Fas ligation.

Prime candidates for the regulation of apoptosis are the caspases that play a critical role in the apoptosis signal transduction cascade and the execution of apoptosis-related functions. Notably, caspase-1 was not activated in either the transfected cell line or the vector control cell line. In contrast, the activity of caspase-3 and caspase-8 was significantly higher in HeLa/Pro-C1 cells than in HeLa/vec cells, suggesting that the facilitation of Fas apoptosis by Pro-C1 depends on the activation of caspase-3 and caspase-8 but not caspase-1. This was substantiated by the finding that DEVD-cho or IETD-fmk, inhibitors for caspase-3 and caspase-8, almost completely abolished the enhancement of Fas-mediated apoptosis in HeLa/Pro-C1 cells, whereas YVAD-cmk, an inhibitor for caspase-1, did not affect the enhancement of Fas-mediated apoptosis. Although the activation of caspase-3 and caspase-8 is known to be important in apoptotic signal transduction (40), the hierarchy, interactions, and regulation of the caspases have not been elucidated fully. In some cell lines, activation of caspase-8 and caspase-1 precedes that of caspase-3, whereas caspase-3 and caspase-8 appear to be activated simultaneously during apoptosis in other cell lines receiving the same stimuli (20, 40). It also has been reported that caspase-8 activates caspase-3 through proteolysis (27, 28), and several intracellular components have been identified as substrates for caspase-3 (25). Taken together, these reports suggest that caspase-8 plays a role in the initial amplification of caspase activation, whereas caspase-3 plays a pivotal role during the final execution of apoptosis. We were therefore able to analyze the potential interactions of Pro-C1 by comparing the time course of activation in the presence and absence of specific caspase inhibitors in the transfected cells. We found that the activation of caspase-3 and caspase-8 occurred more rapidly in the Pro-C1/HeLa cells. The activation of caspase-3 was inhibited by the inhibitor of caspase-8 (IETD-fmk), whereas the activation of caspase-8 was not inhibited by the inhibitor of caspase-3 (DEVD-cho) in HeLa/Pro-C1 cells treated with CH-11. These results suggest that caspase-8 is activated upstream of caspase-3 in Fas-mediated apoptosis of HeLa cells and that Pro-C1 accelerates the activation of caspase-8 in signal transduction of Fas-mediated apoptosis.

The present experiments indicate that overexpression of Pro-C1 specifically enhances Fas apoptosis signaling as well as activation of caspase-8 and, thereby, caspase-3 activation but not caspase-1 activation in HeLa cells. The specificity for Fas apoptosis signaling is likely because of unique interactions of Pro-C1 not with the TNF receptor 1-associated death signaling complex but with the Fas-associated death signaling complex. Caspase-1 is synthesized initially as a single inactive polypeptide zymogen consisting of the p20 and p10 subunits connected to a long N-terminal prodomain of 119 amino acids, which is cleaved off before or during the formation of the active protease tetramer (p10/p20)2 (25, 34, 41). It has been proposed that the prodomains of caspases could prevent premature protease activation or participate in the complex process of cis- and trans-cleavage at internal Asp residues resulting in the release of the small and large subunits and their realignment to form the active tetramer (25, 42). Overexpression of caspase-7 that lacks the short prodomain causes apoptosis, whereas expression of pro-caspase-7 does not, implying that this prodomain may play a role in silencing the caspase activity (43). Here, we have shown that Pro-C1 exhibits binding affinity for itself, but not for the other components of either caspase-1 or caspase-3 and that facilitation of Fas apoptosis by Pro-C1 is independent of the activity of caspase-1. These were surprising findings as the amino acid sequences that are known to be necessary for association with the other caspases or regulatory molecules, such as caspase-8 or FADD, have not been identified in Pro-C1 (26-29), which would suggest that the most likely mechanism by which Pro-C1 would affect the activation of caspase-1 would be through its ability to self-associate. Thus, the ability of Pro-C1 to act by indirectly enhancing the activity of caspase-8 differs from the previously described activities of the caspase prodomains.

The prodomain of caspase-1 has been shown to play a role in nuclear translocation of caspase-1 with the amino acids 4-11 of this domain representing the KVLKEKRK nuclear localization signal. Increased expression of the Pro-C1 resulted in activation of caspase-1 and apoptosis of 293 T cells, whereas expression of p20-IL-1beta -converting enzyme or p10-IL-1beta -converting enzyme did not induce apoptosis (44). Thus, it is possible that Pro-C1-mediated apoptosis in 293 T cells may involve the sequential activation of caspases including caspase-1 as well as caspase-3 and caspase-8. It is not clear, however, whether the nuclear localization of Pro-C1 is related to the activation of caspase-8 in apoptosis signaling.

The question remains as to how Pro-C1 facilitates the activation of caspase-8. It has been reported that a novel protein with caspase recruitment domains interacts with components of death receptor signaling pathways, such as TRAIL receptor 1 (DR4) and TRADD, and activates nuclear factor-kappa B (45). Thus, it is possible that Pro-C1 may suppress anti-apoptotic signaling, such as that mediated by activation of nuclear factor-kappa B, and then facilitates activation of caspase-8 through interaction with regulatory molecules.

In conclusion, we have demonstrated that the prodomain of caspase-1 specifically enhances Fas-mediated apoptosis through facilitated activation of caspase-8 and caspase-3. An understanding of the function of Pro-C1 as an accelerator of apoptosis, as well as an improved understanding of the functions of the prodomains of the other caspases, could provide new insights into the molecular mechanisms involved in signal transduction of apoptosis.

    ACKNOWLEDGEMENTS

We are sincerely grateful to Dr. Masahiko Ohtsuki, Dr. Kazuki Hirahara, and Dr. Martin Fleck for helpful discussions. We greatly appreciate the editorial assistance provided by Dr. Fiona Hunter.

    FOOTNOTES

* This work was supported by funds from the Sankyo Co., Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-3492-3131; Fax: 81-3-5436-8560; E-mail: tatuta@shina.sankyo.co.jp.

    ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; FADD, Fas-associated death domain; IL, interleukin; Pro-C1, prodomain of caspase-1; Ac-YVAD-cmk, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone; DEVD-cho, Asp-Glu-Val-Asp-aldehyde; IETD-fmk, Ile-Glu-Thr-Asp-fluoromethyl ketone; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide; zVAD-CH2DCB, carbobenzoxy-Val-Ala-Asp-CH2-2,6-dichlorobenzonate; YVAD-pNA, Tyr-Val-Ala-Asp-p-nitroanilide; DEVD-pNA, Asp-Glu-Val-Asp-aldehyde-p-nitroanilide; IETD-pNA, Ile-Glu-Thr-Asp-p-nitroanilide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Yonehara, S., Ishii, A., and Yamamoto, M. (1989) J. Exp. Med. 169, 1747-1756[Abstract/Free Full Text]
2. Trauth, B. C., Klas, C., Peters, A. M. J., Matzku, S., Moller, P., Falk, W., Debatin, K. M., and Krammer, P. H. (1989) Science 245, 301-305[Abstract/Free Full Text]
3. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S-I., Samashima, M., Hase, A., Seto, Y., and Nagata, S. (1991) Cell 66, 233-243[CrossRef][Medline] [Order article via Infotrieve]
4. Suda, T., Takahashi, T., Goldstein, P., and Nagata, S. (1993) Cell 75, 1169-1178[CrossRef][Medline] [Order article via Infotrieve]
5. Cheng, G., Cleary, A. M., Ye, Z. S., Hong, D. I., Lederman, S., and Baltimore, D. (1995) Science 267, 1494-1498[Abstract/Free Full Text]
6. Kitson, J., Raven, T., Jiang, Y. P., Goeddel, D. V., Giles, K. M., Pun, K. T., Grinham, C. J., Brown, R., and Farrow, S. N. (1996) Nature 384, 372-375[CrossRef][Medline] [Order article via Infotrieve]
7. Chinnaiyan, A. M., O'Rouke, K., Yu, G. L., Lyons, R. H., Garg, M., Duan, D. R., Xing, L., Gentz, R., Ni, J., and Dixit, V. M. (1996) Science 274, 990-992[Abstract/Free Full Text]
8. Gedrich, R. W., Gifillan, M. C., Duckett, C. S., Van Dongen, J. L., and Thompson, C. B. (1996) J. Biol. Chem. 271, 12852-12858[Abstract/Free Full Text]
9. Lee, S. Y., Park, C. G., and Choi, Y. (1996) J. Exp. Med. 183, 669-674[Abstract/Free Full Text]
10. Marsters, S. A., Sheridan, J. P., Donahue, C. J., Pitti, R. M., Gray, C. L., Goddard, A. D., Bauer, K. D., and Ashkenazi, A. (1996) Curr. Biol. 6, 1669-1676[CrossRef][Medline] [Order article via Infotrieve]
11. Pan, G., O'Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and Dixit, V. M.. (1997) Science 276, 111-113[Abstract/Free Full Text]
12. Bodmer, J. L., Burns, K., Schneider, P., Hofmann, K., Steiner, V., Thome, M., Bornand, T., Hahne, M., Schroter, M., Becker, K., Wilson, A., French, L. E., Browning, J. L., MacDonald, H. R., and Tschopp, J. (1997) Immunity 6, 79-88[CrossRef][Medline] [Order article via Infotrieve]
13. Hsu, H., Solovyev, I., Colombero, A., Elliot, R., Kelley, M., and Boyle, W. J. (1997) J. Biol. Chem. 272, 13471-13474[Abstract/Free Full Text]
14. Chinnaiyan, A. M., O'Rourke, K., Tewari, M., and Dixit, V. M. (1995) Cell 81, 505-512[CrossRef][Medline] [Order article via Infotrieve]
15. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815[CrossRef][Medline] [Order article via Infotrieve]
16. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, K. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[CrossRef][Medline] [Order article via Infotrieve]
17. Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Mann, M., Krammer, P. H., and Peter, M. E. (1997) EMBO J. 15, 2794-2804[CrossRef]
18. Henkart, P. A. (1996) Immunity 4, 195-201[CrossRef][Medline] [Order article via Infotrieve]
19. Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1995) Nature 380, 723-726
20. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675-1687[CrossRef][Medline] [Order article via Infotrieve]
21. Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) Cell 89, 175-184[CrossRef][Medline] [Order article via Infotrieve]
22. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43-50[CrossRef][Medline] [Order article via Infotrieve]
23. Mukae, N., Enari, M., Sakahira, H., Fukuda, Y., Inazawa, J., Toh, H., and Nagata, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9123-9128[Abstract/Free Full Text]
24. Hofmann, K., Bucher, P., and Tschopp, J. (1997) Trends. Biochem. Sci. 22, 155-156[CrossRef][Medline] [Order article via Infotrieve]
25. Cohen, G. M. (1997) Biochem. J. 326, 1-16
26. Ahmad, H., Srinivasula, S. M., Wang, L., Talanian, R. V., Litwack, G., Fernandes-Alnemri, T., and Alnemri, E. S. (1997) Cancer Res. 57, 615-619[Abstract/Free Full Text]
27. Duan, H., and Dixit, V. M. (1997) Nature 385, 86-89[CrossRef][Medline] [Order article via Infotrieve]
28. Muzio, M., Stockwell, B. R., Stennicke, H. R., Salvesen, G. S., and Dixit, V. M. (1998) J. Biol. Chem. 273, 2926-2930[Abstract/Free Full Text]
29. Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., and Nicholson, D. W. (1997) J. Biol. Chem. 272, 17907-17911[Abstract/Free Full Text]
30. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413[CrossRef][Medline] [Order article via Infotrieve]
31. Hu, Y., Benedict, M. A., Wu, D., Inohara, N., and Nunez, G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4386-4391[Abstract/Free Full Text]
32. Srinivasula, S. M., Ahmad, M., Ottilie, S., Bullrich, F., Banks, S., Wang, Y., Fernandes-Alnemri, T., Croce, C. M., Litwack, G., Tomaselli, K. J., Armstrong, R. C., and Alnemri, E. S. (1997) J. Biol. Chem. 272, 18542-18545[Abstract/Free Full Text]
33. Li, Y., Kang, J., and Horwitz, M. S. (1997) J. Virol. 71, 1576-1582[Abstract]
34. Alnemri, E. S., Fernandes-Alnemri, T., and Litwack, G. (1995) J. Biol. Chem. 270, 4312-4317[Abstract/Free Full Text]
35. Tatsuta, T., Cheng, J., and Mountz, J. D. (1996) J. Immunol. 157, 3949-3957[Abstract]
36. Bump, N. J., Hackel, M., Hugunin, M., Seshagiri, S, Brady, K., Chen, P., Frentz, C., Franklin, S., Ghayur, T., Li, P., Licari, P., Mankovich, J., Shi, L., Greenberg, A. H., Miller, L. K., and Wong, W. W. (1995) Science 269, 1885-1888[Abstract/Free Full Text]
37. Zhou, Q., Snipas, S., Orth, K., Mizio, M., Dixit, V. M., and Salvesen, G. S. (1997) J. Biol. Chem. 272, 7797-7800[Abstract/Free Full Text]
38. Susin, S. A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Dasgas, E., Geuskens, M., and Kroemer, G. (1996) J. Exp. Med. 184, 1331-1341[Abstract/Free Full Text]
39. Sumimoto, S., Ishigami, T., Horiguchi, Y., Yonehara, S., Kanazashi, S., Heike, T., Katamura, K., and Mayumi, M. (1994) Cell Immunol. 153, 184-193[CrossRef][Medline] [Order article via Infotrieve]
40. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., and Yuan, J. (1993) Cell 75, 653-660[CrossRef][Medline] [Order article via Infotrieve]
41. Nicholson, D. W., All, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Nunday, N. A., Raju, R. M., Smulson, M. E., Yamin, T. T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37-43[CrossRef][Medline] [Order article via Infotrieve]
42. Gu, Y., Wu, J., Faucheu, C., Lalanne, J-L., Diu, A., Livingston, D. J., and Su, M.-S. (1995) EMBO J. 14, 1923-1931[Medline] [Order article via Infotrieve]
43. Duan, H., Chinnaiyan, A. M., Hudson, P. L., Wing, J. P., He, W.-W., and Dixit, V. M. (1996) J. Biol. Chem. 271, 1621-1625[Abstract/Free Full Text]
44. Mao, P-L., Jiang, Y., Wee, B-Y., and Porter, A. G. (1998) J. Biol. Chem. 273, 23621-23624[Abstract/Free Full Text]
45. Costanzo, A., Chantal, G., and Vito, P. (1999) J. Biol. Chem. 274, 20127-20132[Abstract/Free Full Text]

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
M. Sarai, D. Hartung, A. Petrov, J. Zhou, N. Narula, L. Hofstra, F. Kolodgie, S. Isobe, S. Fujimoto, J.-L. Vanderheyden, et al.
Broad and Specific Caspase Inhibitor-Induced Acute Repression of Apoptosis in Atherosclerotic Lesions Evaluated by Radiolabeled Annexin A5 Imaging
J. Am. Coll. Cardiol., December 11, 2007; 50(24): 2305 - 2312.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
F. M. Syed, H. S. Hahn, A. Odley, Y. Guo, J. G. Vallejo, R. A. Lynch, D. L. Mann, R. Bolli, and G. W. Dorn II
Proapoptotic Effects of Caspase-1/Interleukin-Converting Enzyme Dominate in Myocardial Ischemia
Circ. Res., May 27, 2005; 96(10): 1103 - 1109.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Selleri, J. P. Maciejewski, N. Montuori, P. Ricci, V. Visconte, B. Serio, L. Luciano, and B. Rotoli
Involvement of nitric oxide in farnesyltransferase inhibitor-mediated apoptosis in chronic myeloid leukemia cells
Blood, August 15, 2003; 102(4): 1490 - 1498.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
C. Choi, O. Kutsch, J. Park, T. Zhou, D.-W. Seol, and E. N. Benveniste
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells
Mol. Cell. Biol., February 1, 2002; 22(3): 724 - 736.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
C. A. Martin and A. Panja
Cytokine regulation of human intestinal primary epithelial cell susceptibility to Fas-mediated apoptosis
Am J Physiol Gastrointest Liver Physiol, January 1, 2002; 282(1): G92 - G104.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
T. E. Hansen-Hagge, U. Trefzer, A. S. zu Reventlow, K. Kaltoft, and W. Sterry
Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)
Nucleic Acids Res., February 15, 2001; 29(4): e20 - e20.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. N. Winter, A. Kramer, A. Borkowski, and N. Kyprianou
Loss of Caspase-1 and Caspase-3 Protein Expression in Human Prostate Cancer
Cancer Res., February 1, 2001; 61(3): 1227 - 1232.
[Abstract] [Full Text]

Home page
 Cancer Res.Home page
N. Droin, M. Beauchemin, E. Solary, and R. Bertrand
Identification of a Caspase-2 Isoform that Behaves as an Endogenous Inhibitor of the Caspase Cascade
Cancer Res., December 1, 2000; 60(24): 7039 - 7047.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend