Gene Therapy (1999) 6, 1952–1959  1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt therapy using a pro-apoptotic gene, -3

K Yamabe1, S Shimizu1,2, T Ito1, Y Yoshioka1, M Nomura1, M Narita1, I Saito3, Y Kanegae3 and H Matsuda1 1The First Department of Surgery, 2Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Suita; and 3Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Caspase-3 is a member of the cysteine protease family, 3, but not the empty vector, induced extensive which plays a crucial role in apoptosis. We applied the and reduced tumor volume when combined with etoposide human caspase-3 gene as a novel form of anticancer gene administration. However, this effect was not observed with therapy. Overexpression of human caspase-3 alone could a Bcl-2 overexpressing tumor. In conclusion, caspase-3 not induce apoptosis of tumor lines, but apoptosis was gene transduction accompanied by an additional death markedly enhanced by the addition of etoposide. In an stimulus may be a useful method of anticancer gene AH130 liver tumor model, transduction of human caspase- therapy, except for Bcl-2 overexpressing tumors.

Keywords: caspase-3 gene; anticancer gene therapy; Bcl-2; recombinant adenovirus vector

Introduction advantages of high transduction efficiency, loss of episo- mal adenoviral DNA after several cycles of cell prolifer- 1–3 Caspase-3 (also called CPP32 or apopain) is a member ation without any effect on other foreign , and the of the cysteine protease family, which plays an important ability to prepare high titer (Ͼ108 p.f.u./ml) viral stock 4 role in apoptosis. Apoptotic signals activated by various solutions. stimuli converge towards a common pathway, in which We previously showed that transduction of human have a signaling role. More than 10 caspases caspase-3 gene into HeLa cells induced little apoptosis, 5 have been identified in mammals. Among them, cas- whereas apoptosis was substantially induced by sub- pase-3 has been implicated in cell death, based upon sequent addition of anti-Fas .7 Accordingly, the 6 abnormalities found in knockout mice and the inhibition present study investigated the effect of human caspase-3 7,8 of cell death by a caspase-3 inhibitor. Caspase-3 is syn- gene transduction combined with exposure to chemo- thesized as an inactive precursor of 32 kDa that is first therapy using several cancer cell lines and a liver tumor processed into subunits (P20 and P12), and finally into model. Our results suggested the possibility of using the active subunits (P17 and P12) when a death-inducing sig- human caspase-3 gene for anticancer gene therapy. nal is applied.1–3 Therefore, expression of caspase-3 pre- cursor (pro-caspase-3) alone cannot induce apoptosis and its activation requires a death-inducing stimulus, such as Results cancer chemotherapy agents.7 Several death-inducing genes have been reported to be Effect of caspase-3 gene transduction on apoptosis in useful for anticancer gene therapy, and some of them, vitro including the thymidine kinase gene, require additional To evaluate the effect of human caspase-3 (hcasp-3) and stimuli to kill cancer cells. Such regulation could have human caspase-1 (hcasp-1) gene transduction on apoptosis, great advantages in cancer therapy, since targeting of HepG2 cells (a human hepatoma cell line) were transi- additional stimuli could allow these genes selectively to ently transduced with an hcasp-3- and hcasp-1-containing kill transduced cells. In this context, the caspase-3 gene adenovirus vector (Figure 1A) or with the empty vector should be eminently suitable for anticancer gene therapy, (as a control) together with a ␤-gal-containing adenovirus because (1) it has a strong effect when activated, and (2) vector (as a reporter gene). As shown in Figure 1B (a–c), cell death is not induced by gene transduction alone. almost all the HepG2 cells were positively stained with As the gene delivery system we used a recombinant X-gal at 10 multiplicities of infection (MOI), indicating 9,10 inactivated adenovirus vector with an expression efficient expression of the transduced genes. There were ␤ cosmid consisting of the chicken -actin promoter and some variations in the intensity of staining, possibly 11 the cytomegalovirus enhancer. This method has the caused by differences in the number of copies of the transduced genes. The extent of apoptosis was estimated by TUNEL staining. As shown in Figure 1B (d–f) and C, Correspondence: S Shimizu, The First Department of Surgery, Osaka the proportion of apoptotic cells rapidly increased to 80% University Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan on 1 day after transduction with 10 MOI of the hcasp-1 Received 1 February 1999; accepted 15 July 1999 adenovirus, whereas transduction of hcasp-3 adenovirus Caspase-3 gene therapy K Yamabe et al 1953

Figure 1 Low induction of apoptosis in hcasp-3-transduced HepG2 cells. (A) Structure of recombinant adenovirus with the caspase-1 and caspase-3 expression units (AxCAOYCaspase-1, and -3). AxCAOYCaspase-1 and -3 have the cytomegalovirus enhancer (CMV.E.), chicken ␤-actin promotor, and human caspase-1 and -3, respectively, at the E1 region. (B) Representative appearance of transduced HepG2 cells. HepG2 cells were transiently transduced with hcasp-3, hcasp-1, or the empty vector (10 MOI) together with the lacZ gene-containing adenovirus vector (10 MOI). Cells were fixed and stained with X-gal buffer (X-gal), the TUNEL method (TUNEL), and Hoechst33342 (Hoechst) at 1 day after transduction. (C and D) Induction of apoptosis in hcasp-1-transduced HepG2 cells, but little effect of hcasp-3. Similar experiments to those in (B) were performed, and the proportion of apoptotic cells was estimated by TUNEL staining (C) or nuclear fragmentation (D) at the indicated times (C) and at 1 day after transduction (D). Data are expressed as the mean ± s.d. of four independent experiments. Caspase-3 gene therapy K Yamabe et al 1954 or the vector alone induced a slight and no apoptosis and AH130 cells were treated with hcasp-3 and etoposide after 3 days, respectively, findings consistent with pre- (data not shown). These findings indicated that addition vious observations.7 Similar results were also obtained by of etoposide caused marked caspase-3 activation in hcasp- assessing cells with nuclear fragmentation after staining 3 gene-transduced cells, resulting in the induction of using Hoechst33342 (Figure 1B (g–i) and D). Further- apoptosis. more, similar results were noted when HeLa cells (a human cervical carcinoma cell line) or AH130 cells (a rat Effect of hcasp-3 gene transduction plus etoposide on hepatoma cell line) were used (data not shown). These solid tumors findings indicated that hcasp-1 expression, but not hcasp- To determine whether hcasp-3 transduction was also 3 expression, could induce apoptosis by itself. The clinical effective against solid tumors, we used a liver tumor application of hcasp-1 seemed to be difficult because it is model that was created by injecting AH130 cells (a Don- not easy to regulate its apoptosis-inducing ability, so we ryu rat ascites hepatoma cell line) into the livers of Don- focused on hcasp-3. ryu rats. At 3 days after injection, tumors with a volume of about 1 cm3 were formed. Then adenovirus (5 × 108 Efficient expression of hcasp-3 in an inactive form p.f.u.) transduction was done by the direct injection Since hcasp-3 transduction caused little apoptosis, inef- method. Four days after transduction, etoposide (0.2 ficient expression of caspase-3 was a possibility. As mg/100 g body weight) was injected via the dorsal vein shown in Figure 2, however, expression of caspase-3 pro- of the penis. As shown in Figure 4A (a–d), caspase-3 was form (P32) in hcasp-3-transduced cells was four-fold efficiently expressed in hcasp-3-transduced, but not vec- higher than in non-transduced or vector-transduced cells. tor-transduced tumors, as assessed by immunohisto- We could not detect P20 (active P17 plus the prodomain), chemistry using an anti-human caspase-3 antibody that which is the product of caspase activation, in hcasp-3- did not cross-react with rat caspase-3. Consistent with the transduced cells after culture for 3 days, indicating that results obtained using HepG2 cells (Figure 1), hcasp-3 hcasp-3 gene transduction alone did not cause caspase transduction alone could not induce apoptosis as activation, a result that was consistent with the slight detected by the TUNEL assay (Figure 4Ae), whereas sub- induction of apoptosis (Figure 1). These findings indi- sequent addition of etoposide induced extensive cated that hcasp-3 gene transduction using an adenovirus apoptosis of hcasp-3-transduced tumor cells (Figure 4Ag), vector at 10 MOI resulted in efficient production of but not of vector-transduced cells (Figure 4Ah). At this inactive caspase-3 and did not induce apoptosis. dose of etoposide, there was rapid growth of the vector- transduced tumors, but growth was significantly reduced Effect of etoposide on hcasp-3-transduced cells in hcasp-3-transduced tumors (Figure 4B), possibly due to Since caspase-3 activation requires a death-inducing the induction of apoptosis. Transduction of hcasp-3 alone stimulus, we added 100 ␮m etoposide (a chemotherapy did not reduce the tumor volume (Figure 4B). These agent) to cultures from 1 day after adenovirus transduc- findings indicated that transduction of human caspase-3 tion. At this concentration, production of processed cas- was effective for inducing apoptosis and reducing tumor pase-3 (P20) was observed in hcasp-3-transduced cells but volume in a liver tumor model when applied together not in vector-transduced cells (Figure 3A). The increase with etoposide. of pro-caspase-3 during etoposide treatment of hcasp-3- transduced cells may have been due to the sustained pro- duction of pro-caspase-3. Consistent with the results of Effect of hcasp-3 transduction plus etoposide on Bcl-2- caspase-3 activation, numerous hcasp-3-transduced cells, overexpressing AH130 tumors but few vector-transduced cells, underwent apoptosis Bcl-2 is known as an anti-apoptotic ,12–14 and is after addition of etoposide to cultures as assessed by cell reported to function upstream of caspase-3,15,16 so we shrinkage (Figure 3B) and nuclear fragmentation (Figure next examined the effect of hcasp-3 gene transduction on 3B and C). Similar results were also obtained when HeLa Bcl-2-overexpressing tumors. After injecting AH130-Bcl- 2 cells, which constitutively overexpressed human Bcl- 2,17 into the livers of Donryu rats, Bcl-2-overexpressing AH-130 tumors were created. Then hcasp-3 or the vector was transduced. The level of caspase-3 expression in AH130 Bcl-2 tumors was equal to that in AH130 tumors as assessed by immunohistochemistry (Figures 4Aa, c and 5Aa, c), indicating that production of caspase-3 was not affected by the presence of Bcl-2. However, there was markedly less apoptosis of AH130 Bcl-2 tumors than AH130 tumors after administration of etoposide (Figures 4Ag and 5Ag). Consistently, tumor volume increased when hcasp-3 was transduced into AH130 Bcl-2 tumors (Figure 5B). These results were also consistent with the Figure 2 Efficient expression of pro-caspase-3 in hcasp-3-transduced previous observation that Bcl-2 inhibits caspase-3 acti- HepG2 cells. HepG2 cells were transiently transduced with hcasp-3 vation ,15,16 and indicated that hcasp-3 gene therapy (hcasp-3) or the empty vector (vector) using 10 MOI of adenovirus. At the would be less effective for Bcl-2-overexpressing tumors. indicated times, cell lysates were collected and the expression of caspase-3 Taken together, our findings suggested that hcasp-3 gene was analysed by Western blotting using an anti-human caspase-3 mono- clonal antibody. ‘no’ means non-transduced HepG2 cells. Each sample transduction using an adenovirus vector plus adminis- contained 30 ␮g of protein. Data are representative of three inde- tration of etoposide may be effective for solid tumors, pendent experiments. except for Bcl-2-overexpressing tumors. Caspase-3 gene therapy K Yamabe et al 1955

Figure 3 Induction of apoptosis by etoposide in hcasp-3-transduced, but not vector-transduced, HepG2 cells. (A) Activation of caspase-3 by etoposide in hcasp-3-transduced, but not vector-transduced, HepG2 cells. HepG2 cells were transduced with hcasp-3 (10 MOI) (hcasp-3) or the empty vector (10 MOI) (vector) together with the lacZ gene-containing adenovirus vector (10 MOI). One day after gene transduction, cells were treated with etoposide (100 ␮m) for the indicated times, and were lysed. Expression of pro-caspase-3 (P32) and its active form (P20) was assessed by Western blotting using an anti-human caspase-3 antibody (Transduction Lab). ‘no’ means non-transduced HepG2 cells. Each sample contained 30 ␮g of protein. Data are representative of three independent experiments. (B and C) Induction of apoptosis by etoposide in hcasp-3-transduced, but not vector-transduced, HepG2 cells. HepG2 cells were treated as described in (A), and were fixed and stained with X-gal buffer (X-gal) and Hoechst33342 (Hoechst) at the indicated times. Representative appearance of transduced HepG2 cells is shown in (B). The percentage of apoptotic cells was measured as nuclear fragmented cells per total cells after staining with Hoechst33342 at the indicated times (C). Data represent the mean ± s.d. (n = 4). #P Ͻ 0.001 versus transduction of the empty vector. Caspase-3 gene therapy K Yamabe et al 1956

Figure 4 Effect of hcasp-3 transduction plus etoposide on AH130 tumors. (A) Induction of apoptosis by etoposide in hcasp-3-transduced, but not vector- transduced, AH130 tumors. AH130 tumors were directly transduced with hcasp-3 (hcasp-3) or the empty vector using adenovirus at 5 × 108 p.f.u. Four days after gene transduction, 0.2 mg/100 g body weight of etoposide (VP16(+)) or saline (VP16(−)) was injected. At 7 days after gene transduction, human caspase-3 expression (hcasp-3) and apoptosis (TUNEL) were assessed by immunohistochemistry and the TUNEL assay, respectively. (B) Reduction of tumor volume by etoposide treatment of hcasp-3-transduced, but not vector-transduced, AH130 tumors. AH130 tumors were treated as described in (A), and tumor volume was measured on the indicated days. hCasp-3 (circles) and the empty vector (squares) were transduced on day 0, and etoposide (open symbols) and saline (closed symbols) were injected on day 4. All data represent the mean ± s.d. (n = 7). #P Ͻ 0.001 versus vector transduction plus etoposide. *P Ͻ 0.001 versus no treatment.

Discussion machinery causing programmed cell death.4,18 Among the known mammalian caspases, we focused on caspase- We showed that the combination of hcasp-3 gene trans- 3, because this seemed to be the most important caspase duction and administration of etoposide could efficiently based on the observations that: (1) it is certainly involved induce apoptosis in solid tumors and could reduce the in apoptosis;6,18 (2) it is expressed in many tissues;1–3 and tumor volume. Caspase-3 is a member of the cysteine (3) it resembles Ced-3 (the C. elegans cell death gene) with 4 protease family, which is a crucial component of the regard to sequence homology and substrate specifity.5 Caspase-3 gene therapy K Yamabe et al 1957

Figure 5 Weak effect of hcasp-3 transduction on AH130-Bcl-2 tumors. (A) Inefficient induction of apoptosis in both hcasp-3-transduced and vector- transduced AH130-Bcl-2 tumors. AH130-Bcl-2 tumors were treated as described in Figure 4A. At 7 days after gene transduction, human caspase-3 expression (hcasp-3) and apoptosis (TUNEL) were assessed by immunohistochemistry and the TUNEL assay, respectively. (B) Weak effect of hcasp-3 transduction on the growth of AH130-Bcl-2 tumors. AH130-Bcl-2 tumors were treated as described in Figure 4B, and tumor volume was measured on the indicated days. hCasp-3 (circles) and the empty vector (squares) were transduced on day 0, and etoposide (open symbols) and saline (closed symbols) were injected on day 4. All data represent the mean ± s.d. (n = 7).

We also considered that this gene has the advantages of pase-1 possesses a long prodomain that allows oligo- a strong death effect and induction of typical apoptosis, merization and self-activation through its prodomain, which seemed to be important so as not to damage the resulting in apoptosis, whereas caspase-3 possesses a surrounding normal tissues. Unlike caspase-1, the trans- short pro-domain and shows low self-activation ability.19 duction of caspase-3 is itself insufficient to induce Since hcasp-3 transduction alone did not induce apoptosis (Figure 1), which is a great advantage for can- apoptosis (Figures 1 and 4), we used etoposide as a cer gene therapy. This difference probably depends on death-triggering factor. Etoposide targets DNA topoiso- the length of the N-terminal prodomain region, since cas- merase II and induces DNA damage, which triggers the Caspase-3 gene therapy K Yamabe et al 1958 machinery of apoptosis. In this mechanism, Bcl-2 family Hamamatsu, Japan), according to our institutional guide- act as signal regulators, and the caspases func- lines. Expression of Bcl-2 protein was confirmed by West- tion as executors. When cells were exposed to a low con- ern blot analysis, as reported previously.17 centration of etoposide, the apoptotic stimulus was weak and there was little caspase activation (Figure 3A), Recombinant adenovirus transduction and apoptosis resulting in low levels of apoptosis (Figure 3B and C). In assay the case of caspase-3 transduction, there is a low level of Recombinant adenoviruses containing the human caspase- initial activation subsequently caspase-3 activation shows 3 or caspase-1 gene (AxCAOYCaspase-3 and AxCAOYCa- self-amplification (Figure 3A), resulting in substantial spase-1, respectively) and the lacZ gene (AxCAISLacZ) induction of apoptosis (Figure 3B and C). Thus, etoposide were constructed as described elsewhere.9,10 In brief, the plus caspase-3 transduction might show a synergistic expression cosmid cassette was introduced into the viral effect. Triggering of caspase-3 activation is not restricted by homologous recombination, and the resultant to etoposide, and various stimuli including other cancer virus was propagated using human embryo kidney 293 chemotherapy agents, irradiation, and cytokines4,5 should cells that stably expressed the proteins encoded by the be effective. Anticancer therapy with hcasp-3 gene trans- E1 region. We purified the adenovirus by sequential cen- duction has the following advantages: (1) radiation- trifugation on double CsCl step gradients. The concen- insensitive or chemotherapy-insensitive tumors might trated virus was dialyzed against phosphate-buffered become sensitive; and (2) induction of extensive saline with 10% glycerol. The titer of the virus stock sol- apoptosis by a weak death signal which does not affect ution was assessed by a plaque-forming assay using non-transduced cells. Accordingly, the dose of chemo- human embryo kidney 293 cells. A recombinant aden- therapy agents or radiation could possibly be reduced by ovirus that lacked any expression unit in the E1 deletion using this gene, leading to effective cancer therapy with region (Adex1w1) was also prepared as a control. minimal side-effects. HepG2 cells (2 × 105 cells) were transduced with cas- The present study also revealed that a Bcl-2-over- pase-3 or the empty vector using recombinant adenovirus expressing tumor showed resistance to apoptosis after at 10 MOI (p.f.u. per cells). At this dose transduction hcasp-3 transduction and exposure to etoposide (Figure efficiency was almost 100% and transduction toxicity was 5). This result was consistent with our previous finding weak. After incubation for 1 h at 37°C, cells were washed that Bcl-2 prevented apoptosis upstream of caspase-3 twice with phosphate buffer to remove free virions or activation in cell lines.16 Some tumors show considerable liposomes. Transduced HepG2 cells were stained with X- expression of Bcl-2, and caspase-3 gene transduction gal, the TUNEL method, and Hoechst33342 at the indi- seems unlikely to be efficient for these tumors. However, cated times, and apoptosis was measured as the percent- since the extent of Bcl-2 expression varies, and the age of TUNEL-positive or nuclear fragmented cells per expression of other apoptosis-related proteins also dif- total cells using fluorescence microscopy (Olympus BX50, fers, Bcl-2 expression will not always invalidate caspase- Tokyo, Japan). 3 gene therapy. The ultimate efficacy may depend on the nature of the death-triggering agent and on the level of Western blotting Bcl-2 expression. HepG2 cells were homogenized in buffer containing 50 In conclusion, our study raised the possibility of a new mm Tris-Cl (pH 8.0), 20 mm EDTA, 1% SDS and 100 mm form of anticancer gene therapy by combining etoposide NaCl. The lysates were cleared by centrifugation, and administration with hcasp-3 gene transduction using an caspase-3 precursor (P32) and its active form (P20; P17 + adenovirus vector. the prodomain) were detected by Western blot analysis using an anti-human caspase-3 antibody.

Materials and methods Liver tumor model AH130-vector cells or AH130-Bcl-2 cells (5 × 105) were Chemicals injected into the livers of Donryu male rats to form solid Anti-caspase-3 antibody was obtained from Transduction tumors. Three days later, 5 × 108 p.f.u. of adenovirus was Lab (Lexington, KY, USA). Apoptag was from Oncor directly injected into the solid tumors. Four days after (Gaithersburg, MD, USA). Caspase-1 and caspase-3 cDNA gene transduction, etoposide (0.2 mg/100 g body weight) was kindly provided by Drs J Yuan (Harvard Medical was injected via the dorsal vein of the penis. At 0, 4 and School, Boston) and VM Dixit (Genentech, Inc., South San 7 days after gene transduction, the rats were anesthetized Francisco), respectively. Other chemicals were purchased for laparotomy and the tumors were measured using cal- from Wako (Tokyo, Japan). ipers. Almost all the tumors were globular and the vol- ume was calculated by the equation V = ab2/2, where a Cell lines is the longest diameter and b is the shortest diameter of HepG2 cells20 (a human hepatoma cell line) were main- the tumor. At 11 day after transduction, tumors were tained in RPMI 1640 medium supplemented with 2 mm excised, fixed and embedded in paraffin. Expression of l-glutamine, 1 mm sodium pyruvate, 0.1 mm non-essen- caspase-3 was detected by immunohistochemistry using tial amino acids, 10 mm Hepes/Na+ (pH 7.4), 0.05 mm 2- an anti-human caspase-3 antibody. Apoptosis was mercaptoethanol, 100 U/ml penicillin, 100 ␮g/ml strep- assessed by TUNEL staining using ApopTaq (Oncor). tomycin, and 10% fetal calf serum (FCS). AH130 (a rat ascites hepatoma cell line) derivatives17 (AH130-Bcl-2 and Statistical analysis AH130-V cells with stable transduction of human bcl-2 or Statistical evaluation was performed by the paired t test the empty vector, respectively) were maintained intra- or analysis of variance. Scheffe’s test was used for indi- peritoneally in 200–250 g Donryu male rats (Japan SLC, vidual comparisons of groups when a significant differ- Caspase-3 gene therapy K Yamabe et al 1959 ence was observed by analysis of variance. A P value of using adenovirus DNA-terminal protein complex and a cosmid less than 0.01 was considered to indicate statistical bearing the full-length virus genome. Proc Natl Acad Sci USA significance. 1996; 93: 1320–1324. 10 Kanegae Y et al. Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre Acknowledgements recombinase. Nucleic Acids Res 1995; 23: 3816–3821. 11 Niwa H, Yamamura K, Miyazaki J. 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