Caspase-9 and Apaf-1 Are Expressed and Functionally Active in Human Neuroblastoma Tumor Cell Lines with 1P36 LOH and Ampli®Ed MYCN

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Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and ampli®ed MYCN

Tal Teitz1,3, Tie Wei1,2,3, Dong Liu1, Virginia Valentine1, Marcus Valentine1, Jose Grenet1, Jill M Lahti1 and Vincent J Kidd*,1

1Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, TN 38105, USA

Important roles have been suggested for caspase-8, Introduction caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methy- Neuroblastoma is one of the most common pediatric lation and deletion of Apaf-1 and CASP8 results in the solid tumors, and it is commonly classi®ed by the stage loss of their expression in melanoma and neuroblastoma, of tumor development (Brodeur et al., 1992). Early respectively, while CASP9 localization to 1p36.1 stage neuroblastoma tumors (i.e., stages I, II and III) suggests it is a good candidate tumor suppressor. The are readily treatable with chemotherapeutic drugs and status of CASP9 and Apaf-1 expression in numerous irradiation, and many will even spontaneously regress. neuroblastoma cell lines with/without ampli®ed MYCN However, the prognosis for late stage tumors, es- and chromosome 1p36 loss-of-heterozygosity (LOH) was pecially stage IV tumors, and stage IV tumors that therefore examined to test the hypothesis that one or overexpress the oncoprotein N-Myc due to ampli®ca- both of these genes are tumor suppressors in neuro- tion of the corresponding MYCN gene, is not favorable blastoma. Although CASP9 is included in the region (Caron, 1995). While these late stage tumors may encompassing 1p36 LOH in all neuroblastoma cell lines initially respond positively to treatment, tumors from examined, the remaining CASP9 allele(s) express a this group are particularly aggressive since they often functional caspase-9 enzyme. Apaf-1 is also expressed in become increasingly resistant to chemotherapeutic all neuroblastoma tumor cell lines examined. Thus, the drugs and irradiation-induced apoptosis following their CASP9 or Apaf-1 genes do not appear to function as initial exposure to these agents and relapse is common. tumor suppressors in MYCN ampli®ed neuroblastomas. The prognosis for treatment and potential recovery of However, *20% of the neuroblastoma cell lines with patients with this sub-group of neuroblastoma tumors methylated CASP8 alleles are also highly resistant to is therefore very poor. A ®fth stage of neuroblastoma staurosporine (STS)- and radiation-induced cell death, tumor, stage IVS, represents a disseminated form of presumably because cytochrome c is not released from the disease involving multiple organs/tissues, including mitochondria. This suggests that a second, smaller sub- the liver, skin, and bone marrow, and its prognosis is group of MYCN ampli®ed neuroblastoma tumors exists much better than stage IV disease (Brodeur et al., with defect(s) in apoptotic signaling components up- 1992). Knudson and Meadows (1980) suggested that stream of caspase-9 and Apaf-1. Since no consistent stage IVS neuroblastoma represents a collection of dierences in Bcl-2, Bcl-xL or Bax expression were seen non-malignant neural crest cells containing a mutation in the STS- and radiation-resistant neuroblastomas, it that interferes with their ability to undergo normal suggests that a unique mitochondrial signaling factor(s) dierentiation. is responsible for the defect in cytochrome c release in In an attempt to identify potential tumor suppressor this sub-group of tumors. genes in the stage IV neuroblastomas that contribute to Oncogene (2002) 21, 1848 ± 1858. DOI: 10.1038/sj/ the more aggressive tumor cell phenotype, we recently onc/1205180 analysed the expression and function of a large number of apoptotic genes in both tumor cell lines and patient Keywords: tumor suppressors; apoptosis; caspases; tumor samples. We found that a high percentage of neuroblastoma; MYCN; Apaf-1; cytochrome c stage IV neuroblastoma cell lines with MYCN ampli®cation (i.e., 15/16 or 94%), as well as stage IV primary patient tumor samples with MYCN ampli®cation (i.e., 10/16 or 63%), did not express caspase-8 *Correspondence: VJ Kidd, Department of Tumor Cell Biology, St. mRNA (Teitz et al., 2000; Takita et al., 2001). Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Caspase-8 is a cysteine protease that has an essential Tennessee, TN 38105, USA; E-mail: [email protected] and non-redundant role in receptor-mediated cell death 2Current address: Department Experimental Hematology, St. Jude (Scadi et al., 1997). This protease also appears to be Children's Research Hospital, Memphis, TN 38105, USA an important component of some, but not all, 3These two authors contributed equally to this work Received 23 March 2001; revised 8 November 2001; accepted 13 chemotherapeutic drug-mediated apoptotic pathways November 2001 in certain solid tumor cell types (Belka et al., 2000; Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1849 Engels et al., 2000; Kuwana et al., 1998). The latter the cytosol (see review in refs. Gastman et al., 2000; studies, as well as a separate study demonstrating that Kuida et al., 1998; Soengas et al., 1999). Upon the the cytolinker plectin is a major caspase-8 substrate release of cytochrome c into the cytosol, procaspase-9 (Stegh et al., 2000), suggest that caspase-8 can is activated through the formation of a multi-protein function as both an initiator and eector caspase. complex, termed an `apoptosome', by the binding of Perhaps more importantly, we also found that the cytochrome c to the activating factor Apaf-1 (Saleh et complete loss of caspase-8 mRNA was primarily al., 1999). Once the apoptosome is formed and caspase- restricted to stage IV neuroblastomas with ampli®ed 9 activated, downstream eector caspases, such as MYCN in the patients studied (9/10 were stage IV, caspase-3, can then be activated to promote the cell while the remaining patient tumor was stage II). All death program. It has also been reported that stage IV and IVS patients without ampli®ed MYCN procaspase-9 can be activated without modi®cation from this study may have had a single methylated due to proteolytic cleavage (Stennicke et al., 1999). allele, but the remaining allele continued to express Considering several recent studies (Soengas et al., caspase-8 (Teitz et al., 2000). Similar observations 1999), CASP9 seemed a promising candidate tumor were made in an independent genetic study of a suppressor gene in neuroblastomas with ampli®ed cohort of Japanese neuroblastoma patient tumors MYCN. First, human CASP9 is localized to chromo- (Takita et al., 2001). some 1 band 34-36.1 (Soengas et al., 1999). Numerous The CASP8 gene was either deleted or silenced by reports have shown that the loss of heterozygosity methylation in the neuroblastoma cell lines, while (LOH) of chromosome region 1p36 frequently occurs CASP8 was primarily silenced through the methylation in MYCN ampli®ed neuroblastomas, suggesting that of its promoter sequences in the primary patient tumor the loss of a tumor suppressor gene, or genes, within samples (Teitz et al., 2000). The programmed re- this chromosome region is necessary for the survival of expression of caspase-8 in the neuroblastoma cell lines tumor cells overexpressing N-Myc (Caron et al., 1993). resensitized the tumor cells to death receptor-mediated, Second, Soengas et al. (1999) found that Casp97/7 drug-induced and survival factor-dependent apoptotic mouse embryo ®broblast (MEF) cells are no longer signals. Additional studies veri®ed that CASP8 sensitized to the c-myc-induced apoptotic signal, and methylation resulted in the loss of caspase-8 expression that Casp97/7 MEFs co-expressing c-Myc and Ras in neuroblastoma cell lines as well as in primitive exhibited enhanced tumorigenicity when compared to neuroectodermal brain tumors (PNETs) (Eggert et al., c-Myc/Ras MEFs in immunocompromised mice. And 2001; Grotzer et al., 2000). In separate, and more third, Gerard Evan and colleagues have shown that c- recent, experiments it has been shown that Apaf-1 Myc-induced sensitization to apoptotic signals is expression is lost in a group of melanoma tumor cell mediated through the inappropriate release of cyto- lines and patient samples due to methylation and chrome c from the mitochondria into the cytosol (Juin deletion of the Apaf-1 gene (Soengas et al., 2001). et al., 1999). The potential signi®cance of these ®ndings These data suggest that a common mechanism regarding a possible role for caspase-9 in controlling involving epigenetic gene silencing may be involved in the development of childhood neuroblastoma tumors the speci®c loss of certain apoptotic regulators. Of with 1p36 LOH and MYCN gene ampli®cation is possible signi®cance is the fact that both neuroblasto- therefore clear. ma and melanoma tumors are derived from the same neural crest progenitor cells (LaBonne and Bronner- Fraser, 1999). We therefore decided to analyse the Results status of caspase-9 and Apaf-1 expression and activity, as well as mitochondrial release of cytochrome c, using CASP9 gene deletion in human neuroblastoma cell lines the neuroblastoma cell lines from our previous study, with, and without, 1p36 LOH to determine whether these genes are also inactivated in neuroblastoma. Speci®cally, is caspase-9 and/or Apaf-1 In an eort to determine whether the CASP9 locus was expression or activity aected within the larger sub- altered in neuroblastoma, particularly in those tumors group of neuroblastomas with ampli®ed MYCN and with 1p36 LOH and ampli®ed MYCN, we isolated methylated CASP8 (i.e., 94% of the cell lines and 63% three human CASP9 BAC clones for use in ¯uores- of the patient material)? Alternatively, are defects in cence in situ hybridization (FISH) experiments (Teitz et their expression or activity restricted to the smaller al., 2000). After con®rming that these BACs contained sub-group (i.e., 6% of the cell lines and 37% of the the CASP9 gene by DNA sequencing, FISH analysis patient samples) of tumors that do not completely was performed. The results con®rmed that CASP9 was, silence CASP8 via methylation? The inactivation of indeed, localized to the 35 ± 36 region of the short (p) CASP9 by deletion, mutation and/or methylation arm of chromosome 1 (data not shown). To delineate might help to explain the defects in apoptotic signaling the size of the deletion in this region of chromosome 1 observed in the 37% of tumors that did not silence a separate set of FISH experiments was performed CASP8. using a TNFR2 BAC probe that is slightly more Caspase-9 is an initiator caspase that plays a crucial centromeric than CASP9,oraCDC2L1 BAC probe role in many forms of drug-induced cell death triggered that is more telomeric than CASP9. By conducting by release of cytochrome c from the mitochondria into fractional length measurements of 10 hybridized

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1850 chromosomes is from normal lymphocytes we found analysis of reverse transcriptase-polymerase chain that CASP9 is located at position 1p36.13 ± 1p36.21 reaction (RT ± PCR) products corresponding to cas- (data not shown). Additional FISH experiments were pase-9 mRNA from neuroblastoma cell lines, including then performed on the neuroblastoma cell lines that NB15, with, and without, 1p36 deletions and MYCN had been previously characterized with regard to their gene ampli®cation was performed to determine whether CASP8 status, using both a Casp9 BAC probe and a functionally relevant point mutations were present in chromosome 1 centromere repeat probe. Similar to the CASP9 gene. This RT ± PCR analysis of caspase-9 CDC2L1 and TNFR2, which are frequently deleted/ mRNA demonstrated that its cognate sequence was translocated in neuroblastoma cell lines with ampli®ed normal (Table 1). Finally, Apaf-1 expression was MYCN (Teitz et al., 2000), one allele of CASP9 was analysed by immunoblotting all NB cell lines with an either deleted or translocated to another chromosome hApaf-1-speci®c antibody (Figure 1b). Immunoblot in tumor cells with MYCN ampli®cation (Table 1). In analysis of the untreated and STS-treated cell lysates addition, one allele of CASP9 was deleted or was performed, using cytosolic proteins from cells translocated in several neuroblastoma cell lines that treated with this drug in at least three dierent did not contain ampli®ed MYCN (Table 1). experiments, to determine whether cytochrome c was released appropriately from the mitochondria of these neuroblastoma cells after STS treatment. In all but Caspase-9 and Apaf-1 expression and activity in human three of the neuroblastoma cell lines examined (i.e., neuroblastoma cell lines NB12, NB13 and NB15), cytochrome c was, indeed, To ascertain whether the remaining CASP9 allele(s) released from the mitochondria appropriately following were producing an active enzyme, equal amounts of the exposure to 1.0 mM STS (Figure 1c). To control for lysates from untreated and staurosporine (STS)-treated loading of mitochondrial proteins that might contam- cells were examined by immunoblotting using a inate the cytoplasm due to mitochondrial rupture, a caspase-9 antibody as previously described by this cytochrome oxidase antibody was used as described by laboratory (Tang et al., 1999, 2000). Activation of the our laboratory previously (data not shown) (Tang et procaspase-9 enzyme by autoproteolysis generates a al., 1999, 2000). p17 subunit (Saleh et al., 1999). This subunit was We next determined whether downstream eector readily detected by immunoblotting in all of the caspases were activated and if their cellular substrates neuroblastoma cell lines (Figure 1a and Table 1), were being targeted. Processing of procaspase-3 into its although the level of this subunit was greatly reduced active heteromeric subunits was observed, as well as in NB12, NB13 and NB15. These variations in the level the processing of the 116 kDa form of poly(ADP- of intensity of the caspase-9 p17 subunit in these cell ribose) polymerase (PARP) (Figure 1d). PARP is lines (data not shown) suggest that they may be haplo- known to be cleaved downstream of caspase-3 insucient for this enzyme. Furthermore, caspase-9 activation to generate a smaller 85 kDa protein only RNase protection analysis revealed equivalent levels of after either procaspase-8 is activated via death mRNA in all NB cell lines (Table 1). DNA sequence receptors or procaspase-9 is processed and the active

Table 1 Analysis of CASP9, APAF-1 and p53 in human neuroblastoma cell lines g-irradiation Cell CASP9 Casp9 Casp9 p17 CASP9 Cytochrome c Apaf-1 STS-induced induced Caspase-8 p53 MYCN line (FISH) mRNA a subunit mutation b release protein apoptosis apoptosis protein c status d status c

NB1 +/7 Y Y ND Ylow Y + ++ N Wild-type 2 ± 6 NB2 +/7 YY N Y Y+/7 +/7 N Wild-type 420 NB4 +/7 YY N Y Y+/7 +/7 N Mutant, V173M 420 NB5 +/7 Y Y ND Ylow Y+/7 ++ Y Wild-type 2 ± 5 NB6 +/+t YY N Y Y+/7 +/7 N Mutant, R282W 420 NB7 +/7 Y Y ND Y Y ++ +++ N Wild-type 420 NB8 +/+t YY N Y Y+++/7 N Mutant, del 105-125 420 NB10 +/7 YY N Y Y+/7 +/7 N Wild-type 420 NB12 +/7 Y Y N N Y - - N Mutant, V173M 420 NB13 +/7 Y Y N N Y - - N Mutant, V173M 420 NB14 +/7 Y Y ND Y Y ++ ++ N Mutant, del 126-132 420 NB15 +/7 Y Y N N Y - - Y Mutant, R248W 420 NB16 +/7t Y Y N Y Y ++ +++ Y Wild-type 2 ± 4 NB17 +/7 Y Y ND Y Y ++ ++ N Wild-type 420 NB19 +/+t Y Y ND Y Y ++ ++ N Wild-type 420 NB20 +/7 Y Y ND Y Y ++ ++ N Mutant, R248W 420 NB21 +/7 Y Y ND ND ND ND ND Y Wild-type 2 ± 6 HeLa +/+ Y Y ND Y Y ND ND Y Mutant ND

T = translocated; afrom RNase protection analysis; bfrom the direct DNA sequencing of the CASP9 gene in these cell lines. All exons and their junctions were sequenced from this representative group of NB cell lines. It was not determined in those denoted ND; cfrom Teitz et al. (2000). and references therein; dfrom the direct DNA sequencing of the p53 gene in these cell lines

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1851

Figure 1 Immunoblot analysis of caspase-9, Apaf-1, cytochrome c, and PARP from neuroblastoma cell lines with and without ampli®ed MYCN.(a) Immunoblot analysis of caspase-9 from STS-treated (+) and untreated (7) NB cell lines. The size of the mature procaspase-9 (caspase-9) and one of its active subunits (p17) is indicated to the right. (b) Apaf-1 expression as determined by immunoblotting for each of the NB cell lines. (c) Cytochrome c release from the mitochondria into the cytosol of a group of NB cell lines (required for caspase-9 activation) is shown for cells that have not been (7) or have been (+) exposed to 1.0 mM STS for 2 h. Equal protein loading was determined by actin immunoblot, and for the loading of mitochondrial proteins in panel c, and control for possible mitochondrial rupture, a cytochrome oxidase antibody was used as described by our laboratory previously (Tang et al., 1999, 2000). (d) Cleavage of the nuclear caspase substrate PARP in the presence (+) or absence (7) of STS. The generation of the 85 kDa PARP subunit requires the activation of procaspase-9 and the subsequent activation of downstream eector caspases (e.g., caspase-3)

caspase-9/Apaf-1/cytochrome c apoptosome is formed neuroblastoma cell lines; namely NB12, NB13 and (Gu et al., 1995). Therefore, this immunoblot assay NB15. provided a convenient method that allowed us to ascertain whether caspase-3 was activated appropri- Altered sensitivity to STS- and radiation-induced ately following STS treatment and procaspase-9 apoptosis in a sub-group of human neuroblastoma processing, as well as to determine whether cellular cell lines substrates were being processed correctly. Figure 1(d) shows that PARP was processed appropriately, with Although no abnormalities were detected in procas- the majority of the 116 kDa form of the protein being pase-9 or Apaf-1 expression or activity in any of the cleaved to generate the 85 kDa form in all but three neuroblastoma cell lines, we found that the dierent

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1852 cell lines varied with regard to their sensitivity to STS exposure to 1.0 mM STS (group 3; NB12, NB13 and and g-irradiation (Figure 2 and Table 1). The majority NB15). In addition, exposure to g-irradiation resulted of NB cell lines underwent 70 ± 100% cell death after in a similar ®nding, albeit over a longer time course treatment with 1.0 mM STS within 4 h (group 1; NB1, following initial exposure (Table 1). STS and g- NB5, NB7, NB8, NB14, NB16, NB17, NB19 and irradiation are known to activate apoptosis in cells NB20), while four demonstrated only moderate through the release of cytochrome c from the sensitivity (i.e., 30 ± 45% cell death after 4 h; group 2; mitochondria (Bertrand et al., 1994). If one compares NB2, NB4, NB6 and NB10). However, three of the the amount of cytochrome c present in the cytosol after neuroblastoma cell lines were extremely resistant to the STS treatment (Figure 1c) with the survival data in drug treatment, exhibiting 510% cell death after 4 h Figure 2, only the three highly resistant neuroblastoma

Figure 2 Ecacy of STS-induced apoptosis in the NB cell lines as assessed by the presence of annexin V on the outer surface of their cellular membrane. The various NB cell lines were sub-grouped according to their relative resistance to STS. Group 1 NB cell lines underwent rapid apoptosis after exposure to STS, whereas Group 3 NB cell lines were almost completely resistant to STS- induced apoptosis after 4 h exposure

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1853 cell lines (i.e., NB12, NB13 and NB15) fail to release changes, including the release of cytochrome c, these any cytochrome c after 4 h. This data strongly suggests results immediately suggested that aberrant expression that defect(s) in the mechanism(s) responsible for of a Bcl-2 family member might be involved in cytochrome c release may, at least to some extent, conferring resistance to cell death (Reed, 1997; Gross account for the low level of apoptosis observed in these et al., 1999a). We therefore examined the expression of cell lines. To con®rm this, cell microinjection experi- the anti-apoptotic Bcl-2 and Bcl-xL proteins as well as ments were performed in which exogenous cytochrome the pro-apoptotic family member Bax. Immunoblot c protein and a ¯uorescent marker protein were analysis of lysates from the untreated and STS treated introduced into the cytoplasm of one of the resistant neuroblastoma cell lines from group 3 did not reveal cell lines, NB12, and the cells then exposed to 1.0 mM any conclusive link between the altered expression of STS. These experiments revealed that the NB12 cells these Bcl-2 family members and resistance to STS- were potently resensitized to STS (Figure 3). The NB12 induced apoptosis (Figure 4). While Bcl-2 is expressed cells that were injected with cytochrome c and exposed in many of the neuroblastoma cell lines, it is not to 1.0 mM STS underwent robust apoptosis within 2 ± expressed in all of them (Figure 4, top panel). In 3 h, while the injection of the same concentration of a addition, Bcl-2 levels are actually induced in several of control protein ± bovine serum albumin ± did not cause the STS treated neuroblastoma cell lines (i.e., NB2, cell death. All microinjection experiments were per- NB4 and NB8). The induction of Bcl-2 protein levels in formed a minimum of three dierent times with NB2 and NB4 may contribute to their intermediate identical results (data not shown). Similar microinjec- level of resistance, but its induction in NB8 does not tion experiments were performed using the NB13 and aect STS-induced cell death in this group 1 cell line NB15 cell lines, once again with identical results (data (highly sensitive). No apparent, signi®cant change in not shown). Thus, the apoptotic defect in the STS- and the level of the Bcl-xL protein is seen in any of the radiation-resistant neuroblastoma cell lines appears to neuroblastoma cell lines either (Figure 4, middle involve the caspase-9 pathway, but the defect is located panel). Finally, the Bax protein is induced after STS upstream of caspase-9 and Apaf-1 and the target is treatment in NB17 (a highly sensitive group 1 cell line), most likely involved in triggering or facilitating but not in any of the other cell lines (Figure 4, bottom cytochrome c release from the mitochondria. panel). This might suggest that Bax is functionally relevant for STS-induced apoptosis in NB17, but it is not a generally consistent phenomenon. Expression of both anti- and pro-apoptotic Bcl-2 family It should be emphasized that the speci®c mechan- members in neuroblastoma cell lines ism(s) involved in cytochrome c release is/are not clear Since both the anti- and pro-apoptotic members of the at this time. One possibility is that some unidenti®ed Bcl-2 family have been linked to mitochondrial protein, or group of proteins, is required for

Figure 3 Ability of exogenously introduced cytochrome c to facilitate STS-induced apoptosis in a previously STS-resistant NB cell line. Shown is one example of cytochrome c microinjected into the cytoplasm, along with a rhodamine-labeled dextran marker, of the NB12 cell line, a STS-resistant cell line. As shown, these cells begin to undergo normal apoptosis within 4 h of exposure to 1.0 mM STS. Phase microscopy of the cell ®eld, indirect immuno¯uorescence microscopy of the rhodamine-labeled dextran marker/ cytochrome c, as well as Hoescht staining of nuclei from the same cells, demonstrating the chromatin condensation that is part of the apoptotic cell phenotype, is presented. The arrows indicate the cells being compared between each of the three ®elds shown (phase, immuno¯uorescence, and Hoescht). Finally, a BSA control is included below the NB12 cells microinjected with cytochrome c, with the same three ®elds and arrows delineating the cells being compared between the various ®elds

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1854

Figure 4 Immunoblot analyses of three dierent anti- and pro-apoptotic members of the Bcl-2 protein family, Bcl-2, Bcl-xL, and Bax from the NB cell lines. NB cell lines were treated (+) or untreated (7) with 1.0 mM STS for 4 h. Cell lysates were isolated from these cell lines and then immunoblotted sequentially with anti serum to the anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL or antiserum to the pro-apoptotic Bcl-2 family member Bax (as indicated to the right of each panel). An actin protein loading control immunoblot is shown in the panel at the bottom of this ®gure

cytochrome c release to occur. It is also possible that a AKT (Kennedy et al., 1999; Zhou et al., 2000; Asselin number of dierent proteins can facilitate the release of et al., 2001), as well as the N-myristoylation of the cytochrome c, perhaps in a signal and/or cell-type caspase processed form of Bid, tBid (Zha et al., 2000), speci®c manner. A number of additional proteins also are examples of potential modi®cations which result in in¯uence the release of cytochrome c from mitochon- diminished apoptosis. Both of these modi®cations dria. The anti- and pro-apoptotic Bcl-2 family result in profound changes in the ability of these members (Reed, 1997; Gross et al., 1999a), apoptotic proteins to promote the release of cytochrome c from inducing factor (AIF) (Daugas et al., 2000), and mitochondria. Future studies should address the SMAC/DIABLO, which regulates caspase-9 activity molecular mechanism of the inactivation of this step by its opposing eects on caspase-9 and the caspase-9 in the drug-induced apoptosis pathway within this sub- inhibitor XIAP (Verhagen et al., 2000; Du et al., 2000), group of neuroblastoma tumor cell lines. are examples. In addition, various transporter proteins, such as the voltage-dependent anion channel (VDAC), Status and expression of the p53 tumor suppressor gene in have also been implicated in the release of cytochrome the neuroblastoma cell lines c from mitochondria when associated with pro- apoptotic Bcl-2 family members such as Bax and Bak The tumor suppressor protein p53 is known to sense (Shimizu et al., 2001). Finally, post-translational stress signals and to help propagate drug-induced cell modi®cations (e.g., phosphorylation and dephosphor- death signals, most likely through the release of ylation as well as N-myristoylation) of one or more of cytochrome c from mitochondria (Chen et al., 1996; the proteins capable of forming, or inducing the Lowe et al., 1993; Schuler et al., 2000), although the formation of, functional membrane pores are possible exact mechanism(s) are still not absolutely resolved. It (Zha et al., 2000; Kennedy et al., 1999; Zhou et al., should be noted, however, for the purpose of this study 2000). The phosphorylation of pro-apoptotic Bcl-2- that STS can induce a moderate degree of apoptosis in related family members by the survival protein kinase p537/7 ®broblasts (Slee et al., 2000), clearly indicating

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1855 that p53 is not necessarily required for STS to elicit an (Figures 1c and 2). This is an important point, and apoptotic response. Even so, p53 can modulate and may re¯ect the ability of neuroblastoma tumor cells to enhance the STS-induced apoptotic response to some acquire speci®c mutations that promote their survival. extent. Evidence exists for p53-dependent activation of It appears that p53 is not required for apoptosis the caspase-9 apoptosome (Soengas et al., 1999), as induced by STS in this setting (see Figure 2), well as an essential role for capase-8 in transcription- particularly since ®ve additional neuroblastoma cell independent apoptosis triggered by p53 (Ferreira et al., lines showed moderate to no resistance to STS- 2000). Furthermore, it has recently been demonstrated induced cell death (Figures 2 and 5 and Table 1). that while primary, untreated neuroblastoma tumors Therefore, while it is possible that p53 inactivation rarely harbor p53 mutations, chemotherapeutic drug may play a role in the observed defect in cytochrome treatment can induce p53 gene mutations in neuro- c release in NB12, NB13 and NB15, such inactivation blastoma tumors (Tweddle et al., 2001). In an attempt is de®nitely not sucient by itself to explain the to examine the functional status of the p53 protein in almost complete abrogation of cytochrome c release in the neuroblastoma cell lines being used for these these cell lines. studies, p53 response to irradiation (10 Gy) was looked at by immunoblotting. In 10 of the neuroblastoma cell lines tested we found that the p53 gene product was at Discussion consistent, high levels and that it was not induced by IR-treatment (Figure 5). In the three STS-resistant In summary, our studies appear to have ruled out the neuroblastoma cell lines no correlation between possibility that either the Caspase-9 or Apaf-1 genes are caspase-8 inactivation and p53 status was found (Table common tumor suppressor genes in MYCN ampli®ed 1 and Figure 5). Furthermore, there was no apparent neuroblastomas. Similarly, Bala et al. (2000) have correlation between resistance to g-irradiation-induced shown that Apaf-1 is not mutated in male germ cell apoptosis and p53 status (Table 1 and Figure 5). tumors. Interestingly, Apaf-1 is inactivated in a sub- As additional veri®cation of these results we decided group of melanoma tumors, and the data suggests that to determine the sequence status of p53 from all of the this inactivation also occurs via methylation of the neuroblastoma cell lines used in this study by analysis corresponding gene in the tumor cell (Soengas et al., of RT ± PCR products, as well as through the use of 2001). Importantly, caspase-9 was expressed and active mutant-speci®c p53 antibodies. In eight of the cell in these melanomas as well. Here we have shown that lines we detected point mutations or deletions in the while neither caspase-9 or Apaf-1 is silenced or their DNA-binding domain of p53 that could aect the function signi®cantly altered in 14 MYCN ampli®ed activity of the protein (Table 1). While p53 mutations and two non-MYCN ampli®ed neuroblastomas, a were found in three neuroblastoma cell lines that separate event(s) aecting an upstream regulator, or demonstrated resistance to STS-induced apoptosis regulators, of this pathway is aected in a small sub- (i.e., NB12, NB13 and NB15), microinjection of group of these tumor cell lines. Caspase-8 is also cytochrome c eciently induced apoptosis in all of silenced in this sub-group of tumor cell lines, and it has these cell lines (for an example see Figure 3 and Table been demonstrated that programmed expression of 1). These data indicate that cytochrome c release caspase-8 is, on its own, sucient to re-sensitize these occurs within these cells, in all likelihood, indepen- cells to certain apoptotic insults (Teitz et al., 2000). dently of p53 function. Furthermore, in neuroblasto- Since this current study involves only a large cohort of ma cell lines that contained mutant forms of p53 (e.g., neuroblastoma tumor cell lines, we cannot conclude NB4, NB5, NB6, NB8, NB10, NB14 and NB20) whether similar events involving an upstream regula- cytochrome c release still occurred appropriately tor(s) of the caspase-9 pathway occur in vivo. Based (Figure 1c). In addition, even though cell death was upon the source of the tumor cell lines and patient impaired to some extent in a sub-group of these material that is aected, one might predict that the tumor cell lines (i.e., NB4, NB8 and NB10) it was also inactivation of Apaf-1 and/or CASP8 in human tumors impaired in NB2, which contains wild-type p53 is most likely a very tumor-type speci®c event. Twenty

Figure 5 The various NB cell lines were immunoblotted with a p53 antibody before and after g-irradiation to determine the status of the p53 protein. The same NB cell lines used in the previous studies were also examined with a p53 antibody that detects wild- type and mutant p53 protein of human origin prior to, and after, exposure of these cells to 10 Gy of g-irradiation

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1856 per cent of the neuroblastoma cell lines tested here Biologicals, GA, USA), 2% glutamine (GIBCO BRL) and demonstrated an extremely high resistance to STS. 0.1% gentamicin (GIBCO BRL) in a 378C, 5%CO2, Although these cell lines also contain mutations in p53, humidi®ed incubator. Staurosporine was purchased from they most likely acquired an additional genetic defect Roche, (Mannheim, Germany, cat. no. 1055682). that completely abolishes their ability to release cytochrome c into the cytosol as a result of STS FISH analysis treatment, particularly since STS is capable of inducing FISH analysis of interphase and metaphase cells was 7/7 apoptosis in p53 ®broblasts (Slee et al., 2000). performed as previously described by this laboratory with Finally, analysis of these same neuroblastoma cell lines the following probes isolated from a human BAC genomic with various multi-drug resistance (e.g., MDR1) cDNA library (Genome Systems, Inc., St. Louis, MO, USA): Casp-9 probes did not reveal any signi®cant alteration in their BAC; and previously isolated TNFR2 ± BAC and CDC2L1 ± level of expression (J Sampath, V Kidd and J Schuetz, BAC (Teitz et al., 2000) and a human chromosome 1 unpublished results). centromere repeat probe (Vysis). Of particular interest is the neural crest progenitor cell origin of both the neuroblastoma and melanoma DNA sequence analysis tumors, raising the intriguing possibility that silencing For sequencing caspase-9 mRNA, RT ± PCR was performed of critical apoptotic regulators may be developmen- using a set of six overlapping primer pairs that cover the tally linked. In MYCN ampli®ed neuroblastomas the entire caspase-9 mRNA. The sequence was compared to more common apoptotic gene target for inactivation GenBank accession number U56390, and no amino acid appears to be CASP8 (Teitz et al., 2000), and changes noted. The primers used for the RT ± PCR reactions perhaps secondarily, an upstream regulator(s) that included: primer 9-1 (sense), 5'-CGGAAGCGGACTG- aects the activity of the caspase-9/Apaf-1/cyto- AGGCGGC-3', primer 9-2 (antisense), 5'-CCAATGTC- chrome c apoptosome through its ability to prevent CACTGGTCTGG-3', primer 9-3 (sense), 5'-GCGAACTAA- the release of cytochrome c into the cytosol. Why CAGGCAAGCAG-3', primer 9-4 (antisense), 5'-ACCA- choose dierent genes, and seemingly dierent CGCAGCAGTCCAGAGC-3', primer 9-5 (sense), 5'-CG- CCGCATTTCATGGTGGAG-3' primer 9-6 (antisense), 5'- apoptotic pathways, as targets to inactivate in tumors GTAGGACA-CAAAGATGTCAC-3', primer 9-7 (sense), 5'- of similar progenitor cell origin? That may be one of TCCAGGAAGGTTTGAGGACC-3', primer 9-8 (antisense), the more important questions to have arisen from 5'-TCTGGAAGCTGCTA-AGAGCC-3', primer 9-9 (sense), this and the previous studies. These studies also 5'-GGCTGA-GGCCTGGACTTTCC-3', primer 9-10 (anti- highlight the complexity of apoptotic signaling in sense), 5'-TGATCTATGAGCGA-TACTGG-3', primer 9-11 response to chemotherapeutic drugs and g-irradiation. (sense), 5'-TCTCTAGAGCAGGGA-TTAAC-3' and primer It is also signi®cant to note that the so-called 9-12 (antisense), 5'-CCATATTTGCCTCAGATTTAT-3'. extrinsic, caspase-8 regulated, and intrinsic, caspase- For DNA sequence analysis of the p53 mRNA we 9 regulated, cell death pathways appear to act employed the primer pair (sense) 5'-GATTGGCAGCCA- synergistically to promote cell death (Engels et al., GACTGC'-3', and (antisense) 5'-GGAACAAGAAGTGGA- GAATG'-3'. The sequence was compared to GenBank 2000; Ferreira et al., 2000; Lacour et al., 2001), as accession no K03199. well as promote apoptosis in a more pathway- dependent manner. For example, caspase-9/Apaf-1/ cytochrome c activation following caspase-8 activa- Protein expression analysis tion through mediator-molecules such as BID (Gross For immunoblotting and indirect immuno¯uorescence analy- et al., 1999b) or BAR (Zhang et al., 2000), or sis, the following antibodies were used: anti-cytochrome c alternatively, the activation of caspase-8 following (Pharmingen, USA), PARP (Upstate Biotech, Lake Placid, caspase-9 and caspase-3 activation to augment and NY, USA), anti-caspase 9 (kindly provided by Dr Xiaodang speed cell death (Tang et al., 2000). Collaboration Wang, UTSW), anti-Apaf-1 (Santa Cruz Biotechnology, CA, between the cellular death signals propagated by USA), anti p53 (D0-1, Santa Cruz Biotechnology, CA, USA), anti-Bcl-2 (Santa Cruz Biotechnology, CA, USA), anti-Bcl- caspases-8 and -9 may explain why the inactivation XL (Transduction LAB, USA), and anti-Bax (Santa Cruz of both caspases-8 and -9, or regulators of their Biotechnology, CA, USA). activity such as Apaf-1, could represent a redundant process. Most likely, silencing either of these apoptotic-signaling pathways is sucient to hamper Microinjection with cytochrome c the apoptotic process and promote survival, but how An Eppendorf Transjector system with a Zeiss Axiovert these choices are made may well be cell-type speci®c. 135TV microscope was used to microinject and view the cells after injection. The cytoplasm of 100 ± 150 cells were microinjected with 1.0 mM cytochrome c protein (Sigma), in conjunction with a rhodamine-labeled dextran probe to mark the injected cells (Molecular Probes, USA). The microinjected Materials and methods cells were followed by indirect immuno¯uorescence microscopy for up to 6 h following microinjection and treatment Cell lines and drugs with STS. As a control, BSA fraction V (Sigma) was The NB cell lines used here have been described in detail microinjected into the cytoplasm of the same cells at an previously (Teitz et al., 2000). The cells were grown in RPMI identical concentration along with the rhodamine-labeled 1640 (GIBCO BRL), supplemented with 10% FCS (Atlanta dextran probe.

Oncogene Caspase-9, Apaf-1 and neuroblastoma T Teitz et al 1857 Hoechst 33342 staining to detect chromatin condensation. Irradiation Procedures were followed as previously described in earlier Exponentially growing cells were treated with 10-Gy of g- publications from this laboratory (Tang et al., 2000). irradiation from a 137Cesium gamma irradiator at 1.2 Gy/min and harvested 4 h after irradiation for extraction of cellular proteins and immunoblotting, or at 4, 8 and 24 h to Acknowledgments determine the extent of cell death. Procedures followed were This work was supported by a grant from the NIH (5 RO1 identical to those used in earlier studies (Tang et al., 2000). CA67938) to VJ Kidd, by a Cancer Center Support Grant (5 PO1 CA21765) awarded to St Jude Children's Research Hospital, and by the generous support of the American Apoptosis Lebanese Syrian Associated Charities (ALSAC). Dr Xiao- Apoptosis was measured after cells were treated with 1.0 mM dang Wang kindly provided the human caspase-9 poly- staurosporine by annexin V staining, Tunel assay, and clonal antiserum.

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