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