Ganciclovir-Induced Apoptosis in HSV-1 Thymidine Kinase Expressing Cells: Critical Role of DNA Breaks, Bcl-2 Decline and Caspase-9 Activation

Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation

Maja T Tomicic1, Rudolf Thust2 and Bernd Kaina*,1

1Division of Applied Toxicology, Institute of Toxicology, Medical Faculty, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany; 2Institute for Antiviral Chemotherapy, Medical Faculty, University of Jena, Winzerlaer Str. 10, D-07745 Jena, Germany

Although ganciclovir (GCV) is most often used in suicide ganciclovir (GCV). The HSVtk expressing cells become anticancer gene therapy, the mechanism of GCV-induced speci®cally sensitive to GCV and die by apoptosis cell killing and apoptosis is not fully understood. We (Moolten, 1986). Moreover, not only cells expressing analysed the mechanism of apoptosis triggered by GCV the transgene but also neighboring non-transduced using a model system of CHO cells stably transfected tumor cells can undergo apoptosis. This so-called with HSV-1 thymidine kinase (HSVtk). GCV-induced `bystander killing' is due to intercellular transfer of apoptosis is due to incorporation of the drug into DNA the activated pro-drug (i.e. GCV-triphosphate) via gap resulting in replication-dependent formation of DNA junctions and is restricted to dividing cells (Hamel et double-strand breaks and, at later stages, S and G2/M al., 1996; Mesnil et al., 1996). The most profound arrest. GCV-provoked DNA instability was likely to be results thus far have been collected in clinical trials on responsible for the observed initial decline in Bcl-2 level the assessment of HSVtk/GCV gene therapy for the and caspase-9/-3 activation. Further decline in the Bcl-2 treatment of brain tumors (Old®eld et al., 1993; level was due to cleavage of the protein by caspase-9, as Klatzmann et al., 1996; Izquierdo et al., 1996). demonstrated by use of caspase inhibitors and transfec- However, the therapy suers from various limitations tion with trans-dominant negative caspase expression such as low target speci®city and low transfection/ vectors. Bcl-2 cleavage resulted in the appearance of a transduction eciency of target cells. Also, because of pro-apoptotic 23 kDa Bcl-2 fragment and in excessive lack of knowledge of GCV action, the optimal protocol cytochrome c release, dephosphorylation of BAD, of GCV administration is not fully established. Despite cleavage of PARP and ®nally DNA degradation. Since this, GCV is still being used in anti-cancer gene Fas/CD95 and caspase-8 were only slightly activated we therapy. To improve HSVtk/GCV therapy, a more conclude GCV-induced apoptosis to occur in this cell detailed insight into the molecular mechanism of GCV- system mainly by activating the mitochondrial damage induced cell killing is required which is a prerequisite pathway. This process is independent of p53 for which for optimized application of GCV. the cells are mutated. Caspase-9 mediated cleavage of Apart from utilization in suicide gene therapy, GCV Bcl-2 accelerates the apoptotic process and may explain is frequently used as a classical antiviral agent in the the high potential of GCV to induce apoptosis. Data are therapy of human cytomegalovirus (HCMV) diseases also discussed as to implications for HSVtk gene therapy in immunocompromised individuals, such as cancer or utilizing GCV. AIDS patients and transplant recipients (Crumpacker, Oncogene (2002) 21, 2141 ± 2153. DOI: 10.1038/sj/ 1996). The wide application of the drug raises the onc/1205280 question as to its genotoxic potential. GCV was found to be a potent recombinogenic and chromosomal Keywords: apoptosis; Bcl-2 cleavage; caspase-9; ganci- breakage-inducing agent in metabolically competent clovir; DNA breakage; suicide gene therapy cells expressing HSVtk (Thust et al., 2000a). Regarding the biochemical and molecular mechanism involved in GCV-induced genotoxicity, evidence has been provided Introduction that GCV is incorporated into the cellular DNA, thus causing sister-chromatid exchanges (SCEs) and chro- The concept of HSVtk/GCV gene therapy is based on mosomal aberrations, reproductive cell death and transduction of tumor cells with a `suicide gene' apoptosis (Rubsam et al., 1998; Wei et al., 1998; Thust encoding the thymidine kinase of herpes simplex virus et al., 2000a,b). Whether the genotoxic eects and type 1 (HSVtk) or by administration of HSVtk stably apoptosis induced by GCV are inter-related is an transfected cells followed by systemic treatment with interesting issue that waits to be clari®ed. In this work, we investigated the mechanism of GCV-induced apoptosis in order to explore cellular processes evoked by GCV both in herpes virus-infected *Correspondence: B Kaina, E-mail: [email protected] Received 6 September 2001; revised 17 December 2001; accepted 19 cells and in antiviral or HSVtk suicide gene therapy. December 2001 To this end we used Chinese hamster ovary (CHO) Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2142 cells stably transfected with HSV-1 thymidine kinase electrophoresis showing the typical internucleosomal (HSV ± TK). We chose CHO cells as a model system fragmentation pattern (Figure 1D). for these investigations because they have most widely been used for cyto- and genotoxicity studies and are GCV causes cell cycle delay in the second post-exposure well-characterized in terms of mutagenic, cytotoxic and cycle due to S- and G2/M-arrest genotoxic responses to a broad range of genotoxic agents (for alkylating drugs see Kaina et al., 1997). We Since GCV-induced apoptosis in CHO ± HSV ± TK should also note that CHO cells are, contrary to most cells is a late response starting not earlier than one tumor cell lines, karyotypically quite stable and day of treatment with GCV, it indicates that post- represent a homogenous cell population facilitating exposure cell cycle events are involved. To elucidate chromosomal and cell cycle studies. The cells are this process in more detail we followed cell cycle mutated for p53 (Lee et al., 1997). With this cell system progression upon GCV exposure. Growth curves (data the molecular mechanism of p53-independent apoptosis not shown) and ¯ow cytometry (Figure 2A) revealed due to non-repaired alkylation and UV-C induced that exponentially growing cells exposed to GCV (for DNA damage has recently been analysed (Ochs and 14 h) were not immediately blocked in division but Kaina, 2000; Dunkern et al., 2001). Here we show that started to accumulate after the ®rst post-exposure GCV-induced apoptosis is due to incorporation of cycle (412 h post-exposure) in S and G2/M phase. GCV into the cellular DNA. GCV-induced apoptosis is Thus, 24 h after GCV treatment *40 and 25% of a replication-dependent process which is associated cells were arrested in S and G2/M, respectively, with Bcl-2 decline and ampli®ed by caspase-9-driven whereas in the untreated sub-con¯uent control the cleavage of Bcl-2. The positive ampli®cation loop of percentage of cells in S and G2/M was 10 and 16%, Bcl-2 degradation provokes GCV-exposed HSVtk- respectively. At 24 h after GCV exposure, cells started transduced tumor cells to accelerate cell killing and to undergo apoptosis, as shown in the histograms by may explain the high eciency of GCV to induce an increase in the sub-G1 peak. The time-course of apoptosis. To our knowledge, this is the ®rst report appearance of the sub-G1 population was consistent demonstrating GCV to cause DNA breakage that with the induction of apoptosis determined by annexin triggers caspase-9-driven cleavage of Bcl-2 resulting in V assay (see Figure 1C). After a post-incubation time p53-independent apoptosis. Since resistance of tumor of 36 and 48 h, cells accumulated predominantly in cells is often associated with abnormal production of G2/M and stopped division. The cell cycle distribution Bcl-2 protein (Gallo et al., 1999; Feinmesser et al., of control cells (CHO-neo) was unaected under the 1999) and mutations in the gene encoding caspase-9 same conditions of treatment and no apoptotic have been reported (Srinivasula et al., 1999; Seol and changes were detected (data not shown). Overall, the Billiar, 1999), the data bare implications for HSVtk/ data show that during the GCV exposure cycle and GCV gene therapy. the ®rst post-exposure cycle, CHO ± HSV ± TK cells are not signi®cantly delayed in cell cycle progression. They were, however, severely blocked thereafter, in the second post-exposure cell cycle, where they started Results to die by apoptosis. Hypersensitivity of CHO ± HSV ± TK cells to the cytotoxic effect of GCV Induction of DNA double-strand breaks by GCV precedes induction of apoptosis The killing response of HSVtk-transfected CHO cells and control cells (transfected with the neo gene only) Previously we showed that GCV becomes incorpo- treated for one cell cycle (i.e. 14 h) with GCV is rated into DNA. The incorporated GCV however shown in Figure 1A. A concentration of 0.5 and 1 mM was not genotoxic by its own, but appeared to GCV reduced survival (colony formation) to 60 and generate critical DNA lesions in the post-exposure less than 1% respectively. The CHO-neo control was cell cycle triggering genotoxicity (such as SCEs and completely insensitive to GCV in this concentration aberrations) and apoptosis (Thust et al., 2000a). range. Cytotoxicity caused by GCV in HSVtk- These secondary DNA lesions were hypothesized to transfected cells was mainly due to apoptosis, which be DNA double-strand breaks (DSBs) that were was quanti®ed and compared to the induction of previously shown to trigger genotoxicity (Obe et al., necrosis (see Materials and methods) by ¯ow 1985) and apoptosis (Lips and Kaina, 2001). To cytometry. When exponentially growing cells were prove whether GCV treatment results in DNA treated with GCV for the duration of one cell cycle breakage, we determined the frequency of DSBs by (14 h), the frequency of apoptosis increased as a single cell gel electrophoresis (SCGE). As shown in function of dose (Figure 1B). Time-course experiments Figure 2B, DSBs were induced as early as 18 h after revealed that the induction of apoptosis was a late GCV exposure, reaching a maximum at 36 h after response, starting between 24 ± 36 h after treatment treatment. Since signi®cant increase of apoptosis was with GCV. The frequency of necrosis remained nearly observed at times 436 h after GCV treatment, the unaected (Figure 1C). Induction of apoptosis by induction of DSBs clearly preceded apoptosis GCV in CHO ± HSV ± TK cells was con®rmed by gel following GCV exposure.

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2143

Figure 1 Cell survival and apoptosis after exposure of CHO ± HSV ± TK cells to GCV. (A) Reproductive cell death was determined by colony formation. Two independent experiments in duplicates were performed. Fraction of survived colonies is expressed as percentage of control. (B) Induction of apoptosis as a function of GCV concentration was quanti®ed 72 h after treatment. (C) Induction of apoptosis as a function of time was measured after exposure of exponentially growing cells for 14 h to 1 mM GCV. Apoptosis and necrosis were quanti®ed by annexin V and propidium iodide staining via ¯ow cytometry. (D) Internucleosomal fragmentation after exposure to 1 mM GCV. HSV ± TK cells show a typical DNA laddering pattern (48 and 72 h post-exposure). CHO-neo cells and the untreated HSV ± TK control show no fragmentation. As a control of DNA damage-induced apoptotic fragmentation, CHO-neo cells were exposed to 15 mM MNNG as a positive control of DNA fragmentation

up to 60 h. Caspase-8 was activated early (12 h post- Caspase activation and cleavage of PARP incubation) and remained constantly enhanced (1.9- To analyse the involvement of caspases in GCV- fold) throughout the whole post-exposure incubation induced apoptosis, we determined the time-course of period. caspase activation upon GCV exposure. As shown in To assess the role of individual caspases in triggering Figure 3A, a 1.6-fold increase of caspase-9 activity was apoptosis, GCV-exposed cells were treated with speci®c detected already 12 h after GCV treatment. This caspase inhibitors. The general caspase inhibitor activity strongly increased 24 h after treatment and (zVAD) eciently blocked apoptosis by 75%. The remained at a high level up to 60 h thereafter. same strong blockage eect was observed with the Induction of caspase-3 was ®rst noticed 24 h after caspase-9 inhibitor (zLEHD), while caspase-3 GCV exposure and thus occurred signi®cantly later (zDEVD) and caspase-8 (zIETD) inhibitors were rather than the induction of caspase-9. High level induction of ineective in blocking apoptosis (Figure 3B). The eect caspase-3 was observed 48 h later, remaining constant of the inhibitors on GCV-induced caspase activity was

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2144

Figure 3 Determination of caspase activity and modulation of apoptosis by caspase inhibitors. (A) Cytosolic extracts of CHO ± HSV ± TK cells were assayed for caspase-3, -8 and -9 activity as a function of time after exposure to 1 mM GCV. The relative caspase activity is represented as fold-increase of the untreated control. (B) Apoptosis was modulated by caspase inhibitors. CHO ± HSV ± TK cells were pre-treated for 10 h with 40 and Figure 2 Cell cycle progression and induction of DNA double- 100 mM of the appropriate inhibitor and thereafter exposed to strand breaks after exposure to GCV. (A) The ¯ow histograms 1 mM GCV for 60 h and subjected to annexin V staining. zVAD, showing cell cycle progression of CHO ± HSV ± TK cells with the pan caspase inhibitor; zDEVD, zIETD and zLEHD, caspase-3, -8 sub-G1 DNA content at dierent post-exposure times were and -9 inhibitor, respectively. Data (relative apoptosis expressed determined by ¯ow cytometry. The sub-G1 fraction representing in % of GCV control) are the mean of three independent the apoptotic population is indicated by `Apo'. Abscissa, relative experiments+s.d. (C) Eect of various caspase inhibitors on DNA content; ordinate, relative cell number. Untreated cells caspase activity. Caspase inhibitors were administered at a (control) and cells exposed to 1 mM GCV for 14 h were assayed at concentration of 100 mM and were shown to act speci®cally. the indicated time points (0 ± 72 h) after drug exposure. (B) Signi®cant undesired cross-inhibitory eects were not detected Induction of DNA double-strand breaks as a function of post- exposure time, as revealed by neutral SCGE. The relative comet length (`Olive tail movement') was de®ned as the product of the quantity of DNA in the comet tail and the distance between the mass centers of the comet head and comet tail. Fifty comets on zDEVD and zIETD did not exert a cross-reactive eect each slide were randomly evaluated. The diagram shows the mean on any examined caspases, whereas zLEHD reduced of three independent experiments+s.d. caspase-3 activity by *20% indicating that, in CHO ± HSV ± TK cells after GCV treatment, activation of caspase-3 was under the control of caspase-9. Inhibi- determined in control experiments, con®rming the tion of caspase-9 activity did not lead to inhibition of inhibitors to be active and speci®c at the concentration caspase-8, implicating caspase-8 to be an initiator used (100 mM), i.e. they did not have undesired cross- caspase in our cell system. All assayed caspase inhibitory eects on other caspases (Figure 3C). Thus, activities were signi®cantly down-modulated by zVAD.

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2145 Overall, the data indicate that caspase-9 plays a pression level of Bcl-2 (data not shown). This decisive regulatory role in GCV-induced apoptosis in supports the view that Bcl-2 decline is a post- CHO cells expressing HSV ± TK. translational phenomenon (see below and Discus- A speci®c endogenous substrate of caspases is sion). Bcl-xL and Bax were expressed during the PARP. Cleavage of PARP was observed as early as whole post-exposure period without signi®cant 14 h after GCV exposure and continued at later times changes either in CHO ± HSV ± TK or in control (Figure 4A). Since caspase-9 activity was detectable cells. BAD was expressed as a double-band with already 12 h after GCV treatment and clearly preceded some increase in expression of the unphosphorylated the rise in caspase-3 activity, it was obvious that at that pro-apoptotic form and simultaneous decrease in the time point cleavage of PARP was mediated by caspase- expression of the phosphorylated anti-apoptotic 9. This was veri®ed by utilization of caspase inhibitors, form, 48 ± 72 h after GCV exposure of CHO ± showing that zVAD and zLEHD prevented formation HSV ± TK cells, which was not observed in the of the 85 kDa PARP cleavage fragment whereas control (Figure 5D). zDEVD was barely eective (Figure 4B). This supports the hypothesis that caspase-9 rather than caspase-3 is CD95 receptor-mediated apoptosis is a minor pathway an executive caspase during GCV-triggered apoptosis. during GCV-induced apoptosis in CHO ± HSV ± TK cells Since caspase-8, which is downstream of Fas, was Decline in Bcl-2 level is not due to changes in transcription found to be (slightly) activated after GCV treatment or de novo protein synthesis (see Figure 3A) and its inhibition (by zIETD) To elucidate whether the mitochondrial damage resulted in *25% reduction of apoptosis, the pathway is involved in GCV-induced apoptosis, the expression of Fas (CD95/APO-1) receptor (FasR) expression of proteins of the Bcl-2 family was and Fas ligand (FasL) was examined. There was no assessed. As shown in Figure 5A, the level of Bcl- signi®cant change in the expression of FasR, whereas 2 protein decreased 24 ± 72 h after GCV treatment, a twofold induction of FasL was observed during the which was not observed in control (CHO-neo) cells. time period of 12 ± 72 h after GCV treatment (Figure The timing of Bcl-2 decline was related to the 5E). This may be taken to indicate that the Fas induction of apoptosis (Figure 5B). The amount of system only plays a minor role in GCV-induced bcl-2 mRNA did not change, as shown by RT ± PCR apoptosis. It has been reported that the activation of analysis of RNA isolated from control and GCV- this pathway is regulated by p53 (Muller et al., 1998). treated cells (Figure 5C). Also, treatment of GCV- We should note that CHO cells are mutated for p53 exposed cells with inhibitors of protein synthesis (Lee et al., 1997) and the cells we were working with (cycloheximide+anisomycin) did not aect the ex- do not show p53-driven promoter activation (Dun- kern and Kaina, unpublished data).

Initial release of cytochrome c is independent of caspase activation To further prove that mitochondrial damage is associated with GCV-induced apoptosis, we determined the release of cytochrome c (cyt c) from mitochondria into the cytosol (Figure 6A,B). cyt c release occurred early (i.e. 12 h post-exposure). It paralleled caspase-9 activation (see Figure 3A) and was detectable before the decline in the Bcl-2 level. 72 h after exposure to GCV, the cytosolic fraction was highly enriched with cyt c (Figure 6B). Interestingly, neither zVAD nor zLEHD blocked the release of cyt c (Figure 6C) which indicates that the initial cyt c release was independent of caspase activation.

Cleavage of Bcl-2 by caspase-9 To analyse whether Bcl-2 is degraded by caspases Figure 4 Expression of PARP upon GCV treatment. (A) cleavage of PARP (p113) as a function of time after exposure upon GCV exposure, we performed Western blot of cells to 1 mM GCV. The relative increase of the PARP fragment experiments using two dierent cross-reactive anti- (p85) was quanti®ed by densitometry (Bioimage). (B) Inhibition bodies: anti-Bcl-2 mAb and pAb (both epitopes of PARP cleavage in the presence of caspase inhibitors. CHO ± corresponding to amino acids 1-205 of human Bcl- HSV ± TK cells were pre-incubated with an appropriate caspase 2). The mAb detected the intact Bcl-2 protein of inhibitor for 10 h and exposed to 1 mM GCV for 60 h. The membranes were stained with Poencaue R to con®rm equal 26 kDa (Bcl-2 ± p26), whose quantity signi®cantly loading decreased after GCV exposure (see Figure 5A). The

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2146

Figure 5 Expression of apoptosis-regulating proteins after exposure to GCV. (A) Incubation with anti-Bcl-2 mAb (epitope 1 ± 250) revealed unique signal of intact Bcl-2 (p26). For equal loading, the membrane was stained with Poencaue R. (B) Inverse correlation of Bcl-2 decrease and induction of apoptosis as a function of post-exposure time. (C) RT ± PCR ampli®cation of the 580 bp bcl-2 fragment after exposure to 1 mM GCV; gapdh (glyceraldehyde phosphodehydrogenase) was used as a loading control. (D) Expression of Bax, Bcl-xL and BAD in extracts of CHO ± HSV ± TK cells and the CHO-neo control after exposure to 1 mM GCV. Expression of BAD in extracts of GCV-treated CHO ± HSV ± TK cells; lower band, unphosphorylated form; upper band (designated as BAD-p), phosphorylated form. (E) Expression of Fas receptor (FasR) and Fas ligand (FasL) as a function of post-exposure time. The membranes were incubated with anti-ERK2 antibody as a loading control

pAb gave only a weak signal for the intact Bcl-2 (Figure 7B). Interestingly, the generation of Bcl-2 ± p23 protein, but detected a Bcl-2 fragment of 23 kDa (Bcl- was almost completely abolished by either zVAD or 2 ± p23). This fragment appeared ®rst 24 h after GCV zLEHD. The caspase-3 inhibitor (zDEVD) was clearly treatment and accumulated up to 50-fold in the less eective in preventing the cleavage. This indicates further post-incubation period. Bcl-2 ± p23 was not that in GCV-treated CHO ± HSV ± TK cells the observed in CHO-neo control cells (Figure 7A). Since proteolytic cleavage of Bcl-2 is mainly accomplished caspases were activated within this time period, it was by caspase-9. The appearance of Bcl-2 ± p23 24 h after reasonable to assume that the Bcl-2 p23 fragment GCV exposure and its subsequent accumulation resulted from proteolytic cleavage of Bcl-2 by correlated with the kinetics of the induction of caspases. To prove this, the expression level of both apoptosis (see Figure 1C) indicating cleavage of Bcl- Bcl-2 p26 and p23 was determined in GCV-treated 2 by caspase-9 to contribute signi®cantly to GCV- cells in the presence and absence of caspase inhibitors induced apoptosis.

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2147 Trans-dominant inhibition of caspase-9 but not of caspase-3 abrogates cleavage of Bcl-2 and overall apoptosis following exposure to GCV To further elucidate the importance of caspase-9 in GCV- induced apoptosis, we modulated caspase-9 activity by transient transfection of either wild-type or a dominant- negative caspase-9 expression construct in CHO ± HSV ± TK cells. We also transfected the cells with a dominant- negative caspase-3 construct and compared the eect of expression with the induction of apoptosis by GCV. Wild-type caspase-9 (here designated as casp9-wt) was in this experiment only moderately expressed (Figure 8A) and did not signi®cantly increase GCV-induced apoptosis (Figure 8B). On the other hand, high expression of dominant-negative caspase-9 (here designated as casp9- DN; for Western blot analysis, see Figure 8A) led to a signi®cant decrease in the induction of apoptosis (Figure 8B). A strong expression of dominant-negative caspase-3 (designated as casp3-DN; see Figure 8A, low panel) resulted in only marginal reduction of apoptosis (Figure Figure 6 Expression of cytochrome c (cyt c) and eect of 8B). This was in accordance with the weak reduction of caspase inhibitors on its release upon exposure to GCV. (A) apoptosis observed after treatment with zDEVD. The Decrease in the amount of cyt c in the mitochondrial fraction (mCytc) and (B) increase in the amount of cyt c in the cytosol modulation of caspase activities upon transient transfec- (cCytc), measured in parallel to (A). (C) Release of cyt c from tion of the transdominant negative vectors (noted above) mitochondria after exposure to 1 mM GCV in the presence of the is shown in Figure 8C, demonstrating a signi®cant broad caspase inhibitor (zVAD) and the speci®c caspase-9 reduction of endogenous caspase-9 and -3 activities upon inhibitor (zLEHD). For control of loading, the membranes were transfection and GCV treatment. either incubated with anti-ERK2 or stained with Poencaue R To determine whether the expression of casp9-DN and casp3-DN exerts an eect on Bcl-2 cleavage, we analysed Bcl-2 expression in transiently transfected CHO ± HSV ± TK cells upon GCV treatment. The amount of Bcl-2 ± p23 was signi®cantly reduced in cells in which caspase-9 activity was down-modulated by transfection of casp9-DN but not in cells transfected with casp3-DN (Figure 8D). These results support the conclusion that caspase-9 mediates GCV-induced apoptosis via cleavage of Bcl-2; it is therefore an important execution caspase in this process. To further assess the involvement of caspase-9 in Bcl-2 cleavage, we imitated the apoptotic process evoked by GCV in CHO ± HSV ± TK cells. For this purpose we over- expressed the active form of caspase-9 (casp9-wt) and determined the cleavage of Bcl-2 in the presence and absence of the caspase-3-like inhibitor zDEVD. As shown in Figure 8E, the transiently expressed active wild-type caspase-9 cleaved Bcl-2, both in the absence and presence of zDEVD. Under the conditions of total caspase-3 inhibition, the activity of caspase-7 was also blocked (data not shown) which makes it unlikely that it participated in cleavage of Bcl-2. Thus this data con®rm that Bcl-2 is cleaved by caspase-9 in vivo without the requirement of caspase-3. Figure 7 Cleavage of Bcl-2 and modulation of the cleavage by caspase inhibitors. (A) Incubation with anti-Bcl-2 pAb (epitope 1 ± 250); upper band, a weak signal for intact Bcl-2 p26; lower band, a strong signal for the 23 kDa Bcl-2 cleavage product (Bcl- Discussion 2 p23). CHO-neo cells treated with GCV were negative for the cleavage. (B) Eect of caspase inhibitors on Bcl-2 cleavage in the GCV has been used for a decade in the therapy of presence of 1 mM GCV, as shown by immunoblotting with anti- cytomegalovirus infections, which are a life-threatening Bcl-2 mAb and pAb. CHO ± HSV ± TK cells exposed to 15 mM MNNG were used for comparison. The membranes were stained complication in immunocompromised patients. Upon with Poencaue R to con®rm equal loading metabolization to the corresponding triphosphate,

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2148

Figure 8 Modulation of apoptosis by transient transfection of caspase-9 and -3 expression constructs. (A) Expression of the human Flag-tagged wild-type caspase-9 (casp9-wt), dominant-negative caspase-9 (casp9-DN), and AU1-tagged dominant-negative caspase-3 (casp3-DN) was determined by Western blot analysis. (B) Modulation of GCV-triggered apoptosis (60 h after exposure to 1 mM GCV) by transient transfection with dierent expression constructs. Control, pcDNA3/pcDNA3.1-transfected cells. The expression of casp9-DN signi®cantly reduced apoptosis. Expression of casp3-DN had only an insigni®cant eect. (C) Expression of caspase activity after transient expression of trans-dominant negative constructs and GCV exposure. (D) Modulation of Bcl-2 cleavage carried out in the transfectants treated (+) and not treated (7) with 1 mM GCV. Reduction of Bcl-2 cleavage was detected only in cells expressing casp9-DN. ERK2 was used as a loading control. Data of two separate experiments are shown. (E) Transient transfection of CHO ± HSV ± TK cells with the active form of human wild-type caspase-9 (casp9-wt) in the presence and absence of the caspase-3 inhibitor (zDEVD), without exposure to GCV

GCV is incorporated into the virus DNA thus blocking alterations per se. Chromosomal changes are rather a its replication. GCV becomes also incorporated into consequence of secondary DNA alterations arising the genomic DNA of mammalian cells during replica- from replication of a template containing GCV in the tion (Rubsam et al., 1998; Thust et al., 2000a; Tomicic post-exposure cell cycles (Thust et al., 2000a). and Kaina, unpublished data). Unlike acyclovir (ACV), The mechanism of GCV-induced cell death down- which is an absolute chain terminator, GCV only stream of the induced DNA damage is far less poorly inhibits replication of cellular DNA in the understood. GCV-induced cytotoxicity is largely due exposure cell cycle (Thust et al., 2000a). GCV to apoptosis. Apoptotic cell death increased with the incorporation into DNA does not lead to genotoxic dose of GCV, whereas the frequency of necrotic cells

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2149 (as measured by annexin V/propidium iodide double- cantly inhibit the activation of caspase-8 by transfec- staining) remained fairly constant below 10%. When tion of dominant-negative FADD (Dunkern et al., exponentially growing cells were exposed to GCV for a 2001). Therefore, Fas receptor-triggered pathways seem period of one cell cycle, apoptotic cells appeared ®rst at to play only a marginal role in this cell system. *24 h after treatment with the drug and signi®cantly Activation of caspase-8 was shown to be dependent on increased further at later time points (36 ± 72 h). Cell CD95 ligand-mediated oligomerization of CD95 re- cycle analysis revealed that cells passed both the ceptor (Srinivasula et al., 1996). CD95 receptor/ligand- incorporation and the ®rst post-exposure cell cycle independent caspase-8 activation and apoptosis have without much delay, whereas they were severely also been described (Wesselborg et al., 1999; Ferreira et inhibited in the next cell cycle (i.e. the second post- al., 2000). It has been reported that GCV induces exposure cycle). At this stage the cells became arrested apoptosis via caspase-8 activation in p53 pro®cient in S and G2/M phase and were triggered to die. The HSVtk-expressing neuroblastoma cells, due to p53- induction of S and G2/M arrest seems to be a general mediated translocation of the CD95 receptor from the feature of GCV-treated cells. It was also observed in cytosol to the membrane without aecting the CD95 other systems, such as human glioblastoma and murine ligand (Beltinger et al., 1999). Involvement of FasR/ melanoma cells upon GCV treatment (Rubsam et al., FasL pathway in GCV-induced apoptosis was also 1998; Wei et al., 1998). It has been proposed that the demonstrated in dierent tumor cell lines, all of which irreversible G2/M-arrest is associated with cytoskeletal possessed functional p53 (Wei et al., 1999; Beltinger et reorganization leading to cell death (Halloran and al., 2000). Since in our cell system expression of FasR Fenton, 1998). We favor the hypothesis that the cell was unaltered and caspase-8 was initially activated but cycle arrest is a late eect of GCV-induced DNA not further induced, we assume that Fas/caspase-8- damage. GCV incorporated into DNA leads to driven apoptosis plays a minor role in GCV treated replication-dependent induction of DNA double-strand cells not expressing wild-type p53. Overall it appears breaks (DSBs) that were observed in the post-exposure that, depending on the cell type, GCV can induce replication cycle. It is pertinent to conclude that these apoptosis both via a p53-dependent and p53-indepen- DSBs ®nally trigger apoptosis because: (i) DSBs dent pathway. generated within cellular DNA by electroporation of A hallmark of GCV-induced p53-independent apop- restriction enzyme eciently trigger apoptosis but not tosis in HSV ± TK expressing cells was decline in Bcl-2 necrosis (Lips and Kaina, 2001); (ii) DSBs are likely that correlated with the appearance of apoptotic cells. the most critical lesion inducing apoptosis upon Decrease in the Bcl-2 protein level was not due to a ionizing radiation (Radford and Murphy, 1994; Lips change in bcl-2 transcription or mRNA stability. Also, and Kaina, 2001); and (iii) DSBs are formed upon inhibitors of protein synthesis did not aect the decline treatment of cells with chemical mutagens and UV-C of Bcl-2 (data not shown), indicating that the light and are supposed to trigger apoptosis (Ochs and regulation of Bcl-2 activity after GCV exposure is Kaina, 2000; Dunkern and Kaina, unpublished data). dependent on post-translational events. We also Since DSBs formation requires replication of DNA observed that Bax and Bcl-xL expression was not containing GCV, the data provide at the same time an altered upon GCV exposure. This is, for instance, in explanation for the ®nding that GCV-induced apopto- contrast to increased levels of Bax in rat glioma cells sis is replication-dependent (Hamel et al., 1996). upon GCV treatment (Craperi et al., 1999). In It is important to note that CHO-9 cells possess metabolically competent CHO ± HSV ± TK cells, BAD mutated p53 (Orren et al., 1995; Lee et al., 1997; Hu et was expressed in the pro-apoptotic hypophosphory- al., 1999) and do not show transactivation of a p53- lated form. In the CHO-neo control, which was not driven promoter (Dunkern and Kaina, unpublished aected by GCV, BAD was hyper-phosphorylated, data). Also, p21 was constitutively expressed in the which is known to be mediated by the protein kinase cells without any further rise after GCV treatment Akt (Datta et al., 1997). (data not shown). It thus appears that apoptosis To elucidate in more detail the mechanism of Bcl-2 induced by GCV in CHO ± HSV ± TK cells does not decline and the involvement of caspases in GCV- require p53/p21. The involvement of a p53/p21- triggered apoptosis, we investigated the eect of independent pathway in GCV-induced apoptosis is of caspase inhibitors. The caspase-3 inhibitor zDEVD special interest in view of the application of HSVtk/ only partially prevented Bcl-2 and PARP cleavage and GCV suicide gene therapy for tumors that do not insigni®cantly reduced apoptosis. This indicates that in express functional p53. It has been shown that p53 can CHO ± HSV ± TK cells caspase-3 does not play a major activate the gene encoding CD95 (Fas/Apo-1) receptor role in mediating cell killing. On the other hand, the in response to DNA damage by anticancer drugs caspase-9 inhibitor zLEHD and the broad caspase (Muller et al., 1998). In our cell system, the expression inhibitor zVAD were ecient in suppressing GCV- level of the CD95 receptor was unaltered, and induced PARP/Bcl-2 cleavage and apoptosis. This *twofold induction of the CD95 ligand was observed suggests that caspase-9 activation is an early event in which was accompanied by *twofold induction of GCV-induced cell killing, PARP cleavage and apopto- caspase-8 activity. We should also note that we were sis. It also demonstrates that Bcl-2 is mainly cleaved by unable to stimulate induction of apoptosis in CHO caspase-9 and, to a lesser extent, by caspase-3, thus cells using an agonistic anti-Fas mAb, or to signi®- causing the decline in the cellular Bcl-2 level

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2150 upon GCV exposure. This conclusion was veri®ed by transfection of dominant-negative (DN) caspase-9 and caspase-3 into CHO ± HSV ± TK cells. Expression of DN-caspase-9 strongly prevented Bcl-2 cleavage and apoptosis after GCV treatment, whereas DN-caspase-3 exerted only a slight eect. This supports the critical role of caspase-9 in GCV-induced Bcl-2 cleavage. If caspase-9 is predominantly involved in Bcl-2 cleavage, overexpression of the constitutively active form of caspase-9 should be able to imitate some of the eects brought about by GCV. This was indeed the case, since the transiently transfected active form of human wild- type caspase-9 cleaved Bcl-2 in HSV ± TK expressing cells without GCV treatment, which occurred even in the presence of the inhibitor of caspase-3. A direct interaction between caspase-3 and Bcl-2 has been shown in several cell systems (Cheng et al., 1997; Grangirard et al., 1998; Fujita and Tsuruo, 1998). However, the involvement of caspase-9 in Bcl-2 cleavage has not been demonstrated before. Caspase-9-mediated cleavage of Bcl-2 resulted in a cleavage product of 23 kDa (Bcl-2 ± p23), which accumulated in HSV ± TK expressing cells upon treatment with GCV. The Bcl-2 cleavage fragment was shown to localize in the mitochondrial membrane and to promote the release of cyt c from mitochondria into the cytosol, thus being actively involved in apoptosis (Kirsch et al., 1999). Cleavage of Bcl-2 by caspases occurs at a de®ned cleavage site that was ®rst characterized for caspase-3 in the human and rat protein (Cheng et al., 1997). To determine the putative caspase cleavage site of the hamster Bcl-2 protein, we cloned the hamster bcl-2 cDNA and showed that potential cleavage sites for caspases are highly conserved between hamster and other mammalian species (Tomicic et al., 2000). We also observed that hamster Bcl-2 protein is cleaved in vitro by caspase-9 (Tomicic and Kaina, 2001) resulting in the same 23 kDa fragment that has been detected in Figure 9 A model of induction of apoptosis by GCV. Incorporation of GCV ± TP into DNA leads to generation of vivo. This supports our conclusion that caspase-9 is secondary DNA lesions within the subsequent (post-exposure) majorly involved in cleavage of Bcl-2 upon GCV replication cycle due to errors in replication or repair of GCV- treatment. induced DNA damage. The critical, ultimate lesions are supposed One of the earliest apoptotic features observed in to be DNA double-strand breaks (DSBs) that accumulate and lead to the formation of chromosomal aberrations and S- and CHO ± HSV ± TK cells after GCV treatment was G2/M-arrest. The early release of cytochrome c (cyt c) from release of cyt c. It occurred before signi®cant decline mitochondria is probably due to a slight initial decrease in the of Bcl-2 became detectable. Since the initial cyt c mitochondrial membrane level of Bcl-2. Cyt c (in a complex with release was not blocked by caspase inhibitors, which Apaf-1, dATP and pro-caspase-9) activates caspase-9, which in was also reported by others for chemical agent-induced turn cleaves downstream executive caspases and other substrates such as PARP. Moreover, active caspase-9 and, to a lesser extent, apoptosis (Bossy-Wetzel et al., 1998; Sun et al., 1999), caspase-3, cleave Bcl-2. The product of this cleavage, a pro- cleavage of Bcl-2 by caspase-9 is likely not to be apoptotic Bcl-2 p23 fragment, promotes additional cyt c release responsible for the very early cyt c release but rather and further caspase-9 activation, resulting in an ampli®cation for excessive leakage of mitochondria at later times loop of Bcl-2-driven apoptosis after GCV exposure. Based on these and previous results, the overall scenario of GCV-induced p53- independent cell death is hypothesized as follows (see through a slight initial decrease in the mitochondrial Figure 9). Incorporation of GCV into DNA leads to membrane level of Bcl-2 changing the Bax:Bcl-2 ratio. the formation of secondary DNA lesions within the Binding of cyt C to Apaf-1 activates caspase-9 which in subsequent (post-exposure) DNA replication cycle due turn activates downstream executive caspases (Li et al., to errors in replication or repair of GCV-induced DNA 1997; Slee et al., 1999). Moreover, active caspase-9 damage. The critical ultimate lesions are supposed to cleaves Bcl-2. The product of this cleavage, a pro- be DSBs which trigger, by a yet unknown mechanism, apoptotic Bcl-2 fragment (Bcl-2 ± p23), promotes the early release of cyt C from mitochondria, probably further cyt c release and caspase-9 activation. This

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2151 positive ampli®cation loop appears to be a speci®c vectors as described (Thust et al., 2000a). For transient feature of GCV-induced apoptosis in HSV ± TK transfection, 26105 CHO ± HSV ± TK cells were transfected expressing cells and may explain the exceptional high with 2 mg of the test vector using lipofectine according to eciency of GCV to trigger apoptosis independently of the manufacturer's instructions (Gibco Life Technologies). p53. It remains to be seen whether this process is a Otherwise, the cells were transiently transfected using eectine protocol (Qiagen). The test vectors, ICE ± LAP6 ± general characteristic of p53 mutated tumor cells which FLAG (casp9-wt), ICE ± LAP6mt ± FLAG (casp9-DN) and respond to GCV by activating the mitochondrial AU1 ± Yama-mt (casp3-DN) were already described (Duan damage pathway. et al., 1996). After 16 ± 20 h transfection, the fresh medium The ®nding that GCV must be incorporated into containing serum and GCV (1 mM) was added. Sixty hours DNA and cells need to replicate further in order to later, the cells were subjected to annexin V staining to become apoptotic suggests that slowly replicating determine apoptosis. tumor cells are likely not accessible to GCV if the drug is administered only for a short period of time. Semi-quantitative RT ± PCR Since DNA breaks appear to be an ultimate trigger of GCV-induced apoptosis it would also be interesting to Total RNA was used as template for both the cDNA synthesis and PCR according to TitanTM One Tube RT ± PCR System see whether a combination of GCV and a DNA (Roche). Oligonucleotide primers for ampli®cation of 580 bp breaking agent such as bleomycin or ionizing bcl-2 fragment were 5'-GGAAGGATGGCGCAAGCCGG- radiation (using a dose not blocking replication) GAG-3' (forward) and 5'-CCCAGCCTCCGTTATCCTG- enhances the killing eect. Finally, the ®nding that GATC-3' (reverse). The RT ± PCR program used was as caspase-9 is essential for GCV-induced apoptosis in described (Tomicic et al., 2000). p53-de®cient cells may be useful to improve suicide HSVtk/GCV gene therapy. Thus, transfer of caspase-9 Clonogenic survival and determination of apoptosis along with the HSVtk gene may enhance the therapeutic eect of GCV by increasing apoptosis in Reproductive cell death was assayed by measuring colony tumor cells transduced with HSVtk or targeted by formation with and without GCV treatment as described (Thust et al., 2000a). Drug-induced apoptosis was quantita- metabolic cooperation. This protocol might be of tively and qualitatively determined. For quanti®cation of particular bene®t for tumor cells that exhibit intrinsic apoptosis and necrosis, cells were stained with annexin V-FITC resistance due to overexpression of anti-apoptotic (Pharmingen) and propidium iodide (PI), and subjected to ¯ow proteins (e.g. Bcl-2, Bcl-xL) or due to a defect in cytometry, as described (Vermes et al., 1995). For qualitative caspase-9 expression or its regulation. An enhanced determination, internucleosomal fragmentation was deter- killing response of tumor cells might allow to reduce mined as described (Ioannou and Chen, 1996). the dose of GCV to be administered which may diminish systemic toxicity of the agent, allowing Cell cycle analysis patients a better recovery after the therapy. Cell cycle analysis was performed as described (Nicoletti et al., 1991). Brie¯y, after 14 h-exposure to GCV, cells were trypsinized and washed twice with cold PBS. Cell pellets were re-suspended in PBS and ®xed by the addition of 70% Materials and methods ethanol overnight at 7208C and processed for ¯ow cytometry. Cells within dierent cell cycle phases and the Reagents and antibodies fraction of cells exhibiting a DNA content lower than G1 Ganciclovir (9-[1,3-dihydroxy-2-propoxymethyl]-guanine, were quanti®ed using CellQuest (Becton Dickinson). CymeveneTM) was a product of Syntex Arzneimittel GmbH (Aachen, Germany). zVAD, zDEVD, zLEHD and zIETD Neutral single cell gel electrophoresis were irreversible cell permeable ¯uoromethyl ketone (fmk) modi®ed caspase inhibitors and purchased from Calbiochem, The originally described procedure for the neutral single cell R&D Systems and Enzyme Systems Products. Mouse anti- gel electrophoresis (SCGE) for detection of DNA double- Bcl-2 monoclonal antibody (mAb) as well as rabbit anti-Bcl- strand breaks (DSBs) (Klaude et al., 1996) was slightly 2, anti-Bax, anti-Fas, anti-PARP, anti-cytochrome c and anti- modi®ed. At various times after GCV treatment, sub- ERK2 polyclonal antibody (pAb) were from Santa Cruz, and con¯uent cells were trypsinized, washed with cold PBS, mouse anti-BAD mAb, as well as rabbit anti-Bcl-xL pAb and kept on ice until assayed. Cells were embedded in 0.1% were from Transduction Laboratories. Mouse anti-FasL low melting point-agarose, and microscope slides were mAb used was either from Santa Cruz or Transduction immersed in ice-cold lysis solution. Slides were incubated Laboratories. Mouse anti-Flag M2 and anti-AU1 mAb were in the lysis buer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, products of Stratagene, and BabCo, respectively. Horseradish 1% Na-laurylsarcosine (pH 7), 1% Triton X-100, 10% peroxidase-coupled secondary anti-mouse and anti-rabbit DMSO were added freshly) for 1 h at 48C. After lysis, IgG antibodies were from Amersham. electrophoresis (25 V) was carried out for 15 min at 48C (90 mM Tris, 90 mM boric acid, 2 mM EDTA). The ®xed and ethidium bromide-stained slides were analysed using Cell lines and transfection experiments ¯uorescence microscope. Analysis of DNA migration CHO-9 cells were routinely cultivated in DMEM/Ham's F12 (related to the induction of DNA DSBs) was performed medium supplemented with 5% inactivated (30 min, 568C) by image analysis system (Kinetic Imaging Ltd.; Komet fetal bovine serum (Gibco Life Technologies). The cells were 4.0.2; Optilas) using de®ned standard and expressed as Olive co-transfected with pMCI-tk and pSV2neo expression tail moment (Olive et al., 1991).

Oncogene Caspase-9-mediated cleavage of Bcl-2 by GCV MT Tomicic et al 2152 fractions of 10 ± 40 mg protein were separated by 10 ± 12% Determination of caspase activity SDS ± PAGE, electroblotted onto nitrocellulose membrane Caspase Colorimetric Assay (R&D Systems) was used (Schleicher and Schuel, Germany), that were incubated with according to the manufacturer's protocol. Brie¯y, at various antibodies as described (Tomicic et al., 1997). The used post-exposure times, cells were trypsinized, counted, and antibodies were mostly diluted (1 : 500) in 5% non-fat dry collected by centrifugation. The cell pellet was lyzed on ice milk, 0.2% T-PBS. Protein-antibody complexes were visua- and centrifuged. The supernatant was transferred and kept on lized by enhanced chemiluminiscence (ECL Kit, Amersham). ice. The enzymatic reaction was carried out in 96-well microplate (405 nm, 378C, 1 ± 2 h) with addition of reaction buer and appropriate caspase substrate supplied with the kit.

Acknowledgments Preparation of cell extracts and immunoblotting We are grateful to Dr Vincenz (Univesity of Michigan, Whole-cell extracts were prepared by lysis in ice-cold sample USA) for the generous gift of the ICE ± LAP6 ± Flag, ICE ± buer (25 mM Tris-HCl (pH 6.8), 5% glycerol, 2.5% 2- LAP6 ± mt-Flag and AU1 ± Yama-mt constructs. We thank mercaptoethanol; fresh PMSF), followed by soni®cation Dr Markus Christmann for helpful discussion and critical (Branson soni®er, 30 KHz, 3610 s) on ice. The mitochon- reading of the manuscript. This work was supported by the drial and cytosolic extracts were isolated by dierential Deutsche Forschungsgemeinschaft (grants KA 724/7-1 and centrifugation as described (Wang and Studzinski, 1997). 7-3 to B Kaina and TH 670/1-2 to R Thust) and the Protein extracts were quanti®ed (Bradford, 1976) and Stiftung Rheinland-Pfalz.

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Oncogene