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Synaptogenesis in humans occurs in the last trimester of gestation and in the first few years of life whereas it occurs in the postnatal period in rodents. A single exposure of neonatal rodents to ethanol during this period evokes extensive neuronal apoptosis. Previous studies indicate that ethanol triggers the intrinsic apoptotic pathway in neurons and that this requires the multi-BH domain, pro-apoptotic Bcl-2 family member Bax. To define the upstream regulators of this apoptotic pathway, we examined the possible roles of p53 and a subclass of pro-apoptotic Bcl-2 family members (i.e. the BH3 domain-only proteins) in neonatal wild-type and gene-targeted mice that lack these cell death inducers. Acute ethanol exposure produced greater caspase-3 activation and neuronal apoptosis in wild type mice than in saline-treated littermate controls. Loss of Puma resulted in marked protection from ethanol-induced caspase-3 activation and apoptosis. Although Puma expression has been reported to be regulated by p53, p53-deficient mice exhibited a similar extent of ethanol-induced caspase-3 activation and neuronal apoptosis as wild-type mice. Mice deficient in other pro-apoptotic BH3-only proteins, including Noxa, Bim or Hrk, showed no significant protection from ethanol-induced neuronal apoptosis. Collectively, these studies indicate a p53-independent, Bax- and Puma-dependent mechanism of neuronal apoptosis and identify Puma as a possible molecular target for inhibiting the effects of intrauterine ethanol exposure in humans.
Intrauterine exposure of the human fetus to alcohol causes the dysmorphogenic fetal alcohol syndrome (1–4). The full syndrome includes craniofacial malformations, a reduction in brain mass and an array of neuro-behavioral abnormalities (5, 6). The feto-toxic effects of ethanol can also manifest as neurobehavioral disturbances that are unaccompanied by craniofacial dysmorphogenesis (7). The term “alcohol related neurodevelopmental disorder” refers to partial syndromes that primarily affect the central nervous system (CNS) (8).
Ethanol is a complex neurotoxin; the precise mechanisms by which it causes neuropathological changes are not clearly defined. Ethanol may have differential effects on immature neurons; it may act as a stimulant on them, rather than as a generalized CNS depressant (9). Extensive studies have shown that neurons develop resistance to the effects of ethanol as they mature (10, 11). Other reports have shown that administration of doses of ethanol that would cause extensive neuronal loss in neonatal mice are not associated with loss of neurons in the adult brain (12, 13). The effects of ethanol are partly due to a direct cytotoxic effect on immature neurons that are particularly sensitive to ethanol during the synaptogenesis period of brain development (14). This developmental period occurs in the third trimester and in the first few years of life in humans, whereas it occurs between approximately 3 to 10 days after birth in rodents (15, 16). Ethanol appears to exert its neurotoxic effects through the blockade of N-methyl-D-aspartic acid (NMDA) glutamate receptors and hyperactivation of γ-aminobutyric acidA (GABAA) receptors (17). The cell death process after exposure of neonatal mice to ethanol occurs over a period of 4 to 16 hours (15, 18–20); it is accompanied by marked caspase-3 activation; and it exhibits classical ultrastructural features of apoptosis (21–24).
We have previously reported that Bax-deficient mice are markedly protected from ethanol-induced caspase-3 activation and neuronal loss (25). The upstream molecular events that trigger Bax activation following ethanol exposure are, however, largely unknown. During apoptosis, Bax undergoes a conformational change and is translocated from the cytosol to the outer mitochondrial membrane where it triggers cytochrome c release and apoptosome formation leading to the activation of the initiator caspase, caspase-9 and the effector caspase, caspase-3 (26). In response to some cytotoxic stimuli such as DNA damage, Bax activation requires a p53-induced expression of the pro-apoptotic BH3-only Bcl-2 family members puma (p53 upregulated mediator of apoptosis) and, albeit to a lesser extent, noxa (27). In response to other death stimuli, such as growth factor deprivation or death receptor signaling, other BH3-only proteins (i.e. Bim and Bid, respectively) are critical for Bax/Bak-dependent cell death (28).
Puma has classically been considered to be a p53-inducible regulator of cell death (29–31) but it also plays a critical role in p53-independent cell death pathways, such as those induced by growth factor deprivation or treatment with glucocorticoids (27, 32, 33) The mechanism of the pro-apoptotic action of Puma has been ascribed to its ability to bind with high affinity and consequently inhibit all anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1 (34–36). It has also been reported that Puma can activate the intrinsic apoptotic pathway by directly binding to Bax (37–39). To investigate the role of BH3-only proteins in regulating ethanol-induced neurodegeneration, we performed a series of in vivo studies by administering ethanol to neonatal wild-type mice and gene-targeted mice that are deficient for different BH3-only proteins. We found that Puma critically regulates ethanol-induced, Bax-dependent, caspase-3 activation and neuronal loss by a mechanism independent of p53.
Seven-day-old C57BL/6J mice were used in all experiments. Mice were housed and cared for according to the NIH Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care Committee of the University of Alabama at Birmingham. The generation of mice deficient for Bax, Puma, Noxa, Bim, Bad, Bid or Hrk has previously been previously described (27, 40–45). p53−/− mice were purchased from Taconic (Germantown, NY). All mice were backcrossed for at least 6 generations onto the C57BL/6J background. Genotyping of the gene-targeted mice was performed by PCR using DNA extracted from tail clips.
Wild-type and gene-targeted neonatal mice were injected subcutaneously with a solution of 20% ethanol in 0.9% saline administered as 2 doses (at 0 and 2 hours) of 2.5 g/kg of body weight, as previously described (46). This is equivalent to a volume of 15.9 μl per gram body weight of the 7-day-old neonatal mouse when administered as a 20% solution in saline. This dosing regimen produces a blood alcohol concentration above 200 mg/dl for at least 8 hours; this concentration is needed to trigger a maximally robust apoptotic response (22, 23, 47, 48). Littermate controls received an equivalent volume of saline. Animals were killed after 6 hours and their brains were harvested, as previously described (24, 46). One hemisphere of each brain was frozen at −80°C for biochemical studies and the other half was immersed in Bouin’s fixative overnight at 4°C, followed by 3 washes in 70% ethanol. Fixed tissues were processed; embedded in paraffin and cut into 5-μm-thick sagittal sections.
Immunohistochemical (IHC) staining was performed as previously described (49). Briefly, sections were deparaffinized and antigen retrieval was performed by incubation in a 10% solution of DAKO high pH target antigen retrieval solution (Dako Corporation, Carpinteria, CA) in phosphate buffered saline (10 mM PBS; pH 7.2), for 30 minutes in a steamer. After a 20-minute cool down period, the sections were incubated in 3% H2O2 in PBS to decrease endogenous peroxidase activity, followed by washes in water and PBS. Nonspecific antibody binding was inhibited by incubating sections in PBS-blocking buffer (PBS-BB; PBS containing 1% bovine serum albumin, 0.2% nonfat powdered milk, and 0.3% Triton X-100) for 30 minutes at room temperature. Slides were then incubated overnight at 4°C with a primary polyclonal rabbit antibody specific for the 17-kDa fragment of cleaved (i.e. active) caspase-3 (Cell Signaling Technology, Beverly, MA) diluted in 1:1000 in PBS-BB. The sections were then washed 3 times with PBS and incubated for 1 hour at room temperature with SuperPicTure Polymer Detection kit, a goat-anti-rabbit IgG secondary antibody conjugated to a horseradish peroxide polymer (diluted 1:50 in PBS-BB; Zymed Laboratories, Invitrogen Corporation, Carlsbad, CA). 1Antigen-antibody complexes were subsequently detected by tyramide signal amplification (TSA) using cyanine-3 conjugated tyramide according to the manufacturer’s recommendations (TSA PLUS; Perkin-Elmer Life Sciences Products, Boston, MA). Cell nuclei were visualized by staining sections for 10 minutes with 0.2 μg/ml bisbenzamide (Hoechst 33258; Sigma, St. Louis, MO). Fluorescent staining was visualized with a Zeiss-Axioskop microscope equipped with epifluorescence.
To quantify the cleaved caspase-3-like immunoreactivity, the total number of labeled cells in 4 contiguous 40x fields of the cerebral cortex immediately superior to the hippocampal formation were counted by an observer blinded to the treatment and the genotype of the mice from which the sections were derived. The total area of each cortical section was determined with Axiovision software and the data were expressed as numbers of positive cells per mm2 of cortical area. For quantification of caspase-3-positive cells, the mean and standard deviation were determined from 3 independent experiments, with an “n” of one representing one gene-disrupted mouse treated with ethanol accompanied by its wild-type and heterozygous littermates treated with ethanol. The effect of genotype on the number of caspase-3-positive cells was analyzed for significance by one-way ANOVA followed by Dunnett’s multiple comparison Test using Graph Pad Prism (Version 4.0) software. In all cases, a p value ≤ 0.05 was considered significant.
Preparation of tissue lysates was performed as follows. Hemi-brains were suspended and homogenized in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% TritonX-100 and 10% Glycerol) containing 1% phenylmethylsulfonylfluoride (PMSF; a serine protease inhibitor), 1% protease inhibitor cocktail (Sigma) and 1% phosphatase inhibitor cocktail (Sigma).
The samples were incubated on ice for 30 minutes with vortexing every 10 minutes. Samples were then centrifuged at 13,000 g for 15 minutes to obtain a clear supernatant. The supernatant was then transferred to a fresh tube and the protein concentrations determined using the Pierce BCA assay kit (Pierce, Rockford, IL).
Equal amounts of whole tissue lysates were resolved using SDS-PAGE and then transferred onto PVDF membranes. Blots were blocked for 1 hour at room temperature in 5% milk in wash buffer (200 mM Tris Base, 1.37 M NaCl, 1% Tween 20, pH 7.6) and then incubated with primary antibodies overnight at 4°C. Blots were probed for cleaved caspase-3 (Cell Signaling Technologies, Danvers, MA) and Puma (ProSci Inc., Poway, CA). β-tubulin detection (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a loading control. After incubation with the primary antibodies, all blots were washed with 1x TBS containing 1% Tween 20, then incubated for 1 hour with a goat anti-rabbit IgG secondary antibody (BioRad, Hercules, CA) at room temperature. The blots were subsequently washed and the signal detected using Supersignal Chemiluminiscence (Pierce), or ECL (Amersham, Fairfield, CT). Western blots were scanned into Adobe Photoshop and digitized with UN-SCAN-IT software, Version 6.1 (Orem, Utah).
Previous studies have shown that administration of ethanol to neonatal mice triggers caspase-3 activation and massive neuronal cell death that meets the ultrastructural criteria for apoptosis (22). As previously reported (21), we observed a robust increase in cleaved caspase-3 protein levels by Western blot analysis; increased levels are detectable after 6 hours, they peak at 12 hours and then they decrease at 24 hours (Fig. 1A). After 6 hours the activation of caspase-3 can also be observed by IHC detection of cleaved (i.e. active) caspase-3 immunoreactivity (Fig. 1B). The time course for caspase-3 activation differs among different neuronal populations, however (21). To monitor the pathway and minimize the temporal effects of ethanol in different brain regions, we chose the 6-hour time point when activated caspase-3 was detectable both by IHC and Western blot analysis. As previously reported, saline-treated wild-type mice contained only very few scattered cortical neurons undergoing cell death that were detected by IHC of cleaved caspase-3. In contrast, 6 hours after treatment with ethanol a large number of cleaved caspase-3-immunoreactive cells were found in the brains of wild-type mice (Fig. 1C).
We previously reported that Bax-deficient mice are significantly protected from ethanol-induced caspase-3 activation and neuronal cell death (25). Ethanol-induced neuronal apoptosis involves the translocation of Bax to the mitochondrial membrane causing cytochrome c release and activation of the initiator caspase, caspase-9, and then the effector caspase, caspase-3 (21). Loss of Bax protects neurons from both acute and delayed neuronal degeneration following ethanol exposure. Consistent with previous results, Bax-deficient mice showed minimal changes in cleaved caspase-3 levels in response to ethanol, as monitored by Western blotting (Fig. 2A) or IHC. Comparison of numbers of cleaved caspase-3-immunoreactive cells between brain sections from wild-type and bax−/− mice showed a highly significant protective effect of Bax deficiency (Fig. 2B).
Compared to brains from ethanol-treated wild-type mice, brains from ethanol-treated mice heterozygous for puma showed a significant decrease in cleaved caspase-3 protein levels on Western blot analysis, which was further decreased with a homozygous deficiency of puma (Fig. 3A). Accordingly, by IHC there was a significant decrease in numbers of cleaved caspase-3-immunoreactive cells in both ethanol treated puma−/− and puma+/− mice compared to their wild-type littermates (Fig. 3B).
Puma is a pro-apoptotic BH3-only gene that is a direct transcriptional target of the tumor suppressor p53, which is activated by DNA damage, oncogene-induced stress and certain other cytotoxic stimuli (50, 51). Puma, however, has also been reported to be activated by other transcription factors such as FOXO3a, p73, HIF1α and E2F1 (52–55). Puma can also activate Bax either directly or indirectly (37, 39, 56). Therefore, Puma is an initiator of apoptosis that functions upstream of Bax/Bak and the activation of caspase-3. Puma in this model may regulate apoptosis by either direct Bax activation or by an indirect mechanism involving binding and inhibition of the pro-survival Bcl-2 family members in the cell, thereby liberating Bax to cause mitochondrial outer membrane permeabilization and consequent activation of the effector phase of apoptosis.
Since puma may be regulated by p53 (27, 32), we determined whether this occurs during ethanol-induced neuronal cell apoptosis by examining the effects of loss of p53 on this cell death process. Interestingly, p53−/− mice were not protected against the acute toxic effects of ethanol since Western blotting showed that the brains from these mice exhibited an increase in cleaved caspase-3 protein levels that was comparable to the levels seen in brains from wild-type and p53+/− mice (Fig. 4A). These in vivo results are consistent with our previous in vitro studies in which the death of internal granule cerebellar neurons in response to ethanol, MK801 (an NMDA antagonist) and diazepam (a GABAA agonist) was independent of p53 (57). Quantification of cleaved caspase-3-immunoreactive cells in response to ethanol treatment confirmed the Western blot data showing no significant decrease in dying neurons in ethanol-injected p53−/− animals compared to wild-type or p53+/− mice (Fig. 4B).
We next examined whether ethanol injection caused a change in Puma protein levels in the brains of neonatal mice. We administered ethanol to wild-type mice and sacrificed them 3, 6, 12 or 24 hours later and performed Western blot analysis to compare the Puma protein levels in their brains to those in saline-treated littermate controls. Quantification of Puma protein levels in 4 independent experiments in littermate pairs that received saline or ethanol 6 hours earlier showed no differences in Puma protein levels (Fig. 5A).
We next examined the effects of Bax deficiency on Puma protein levels and found that Bax-deficient mice had the same levels of Puma protein as the wild-type mice or bax+/− mice after treatment with saline or ethanol (Fig. 5B). We also compared basal levels of Puma between the brains of wild-type and p53-deficient mice by Western blotting and found comparable levels. As in wild-type mice, ethanol exposure had no effect on Puma levels in the p53-deficient mouse brains (Fig. 5C). The anti-Puma antibody was specific for recognition of murine Puma protein since the brains from puma−/− mice had no detectable protein levels at the appropriate molecular weight of Puma on Western blot analysis (Fig. 5D). These results indicate that baseline Puma expression is independent of p53 but is sufficient and essential for Bax-induced caspase-3 activation and apoptosis in neurons of ethanol-treated mice. Although Puma protein levels do not change markedly in brains in response to ethanol treatment, Puma-deficient mice are unable to activate Bax due to a loss in this baseline expression, indicating that Puma is a critical upstream mediator of ethanol-induced Bax activation in neurons.
We next examined the effects of acute ethanol administration in neonatal mice deficient for certain other BH3-only proteins to assess their possible role in the regulation of ethanol-induced caspase-3 activation. Noxa is another pro-apoptotic BH3-only protein that can be regulated by p53; it contributes to DNA damage-induced, p53-mediated apoptosis (58, 59). Ethanol administration in neonatal mice deficient for Noxa, however, resulted in no difference in cleaved caspase-3 protein levels by Western blotting compared to wild-type or noxa+/− mice (Fig. 6A). The numbers of cleaved caspase-3-immunoreactive cells showed a statistically insignificant decrease in the noxa−/− animals compared to wild-type or noxa +/− mice (Fig. 6B).
Noxa cooperates with Puma in some models of apoptosis (60). Consistent with this, we observed that combined Noxa and Puma deficiency afforded neurons with the greatest protection from ethanol-induced caspase-3 activation (Fig. 7). Although the major inhibitory effect could still be explained by Puma deficiency, a combined deficiency of Puma and Noxa caused an even further decrease in cleaved caspase-3 protein levels compared to Puma deficiency alone. We previously reported that other BH3-only proteins, such as Bad and Bid, that were reported to regulate neuronal cell death in response to certain cytotoxic stimuli did not protect neurons in vitro from the toxic effects of ethanol, MK 801 or diazepam (57). Accordingly, administration of ethanol to bid−/− and bad−/− neonatal mice elicited a robust activation of caspase-3 in vivo that was comparable to the response seen in wild-type animals (data not shown).
Deficiency of Bim and Hrk/Dp5, 2 other BH3-only proteins that have been shown to exert neuroprotective effects in vivo, either singularly or in collaboration (61), also failed to show any protective effects against ethanol-induced caspase-3 activation (data not shown). Puma is thus the most critical regulator of ethanol-induced, Bax-mediated neuronal apoptosis with Noxa playing a lesser but complementary role.
The complex death-promoting effects of ethanol on the developing CNS are most pronounced during the synaptogenesis period in the developing brain. The most severe consequences of ethanol result from chronic maternal ingestion, although transient intrauterine exposure to ethanol can also have profound effects (62). Indeed, a transient increase in blood alcohol concentration in the infant mouse brain can trigger widespread apoptosis (63). This neurotoxic effect of ethanol may be a major contributing factor to the pathogenesis of human fetal alcohol syndrome.
Apoptosis is regulated by 2 major pathways: the death receptor-mediated (also called extrinsic) pathway and the mitochondrial (also called intrinsic or Bcl-2-regulated) pathway. We previously demonstrated that ethanol does not act via activation of caspase-8, a critical component of the death receptor pathway but rather through the Bax-dependent release of cytochrome c and the intrinsic apoptotic pathway (25). To characterize the upstream regulators of acute ethanol-induced, Bax-dependent apoptosis in vivo, we examined the effects of neonatal ethanol administration in various gene-disrupted mice.
As previously reported, ethanol administration triggered a robust and time-dependent increase in cleaved (active) caspase-3 levels that preceded the death of the neurons. Caspase-3 processing was significantly elevated at 6 hours following ethanol injection as detected both by Western blotting and IHC. In wild-type mice, the apoptotic cell morphology is easily detectable by light microscopy in susceptible regions of the brain within 8 to 10 hours of ethanol injection and becomes abundant at approximately 16 hours post-treatment. The neurons progress to the terminal stages of apoptosis between 16 to 24 hours and are subsequently engulfed and degraded by phagocytes (47). Caspase-3 activation can be detected relatively early in the cell death process when the affected neurons are still of normal size. The time course for caspase-3 activation differs among different neuronal populations. At 4 hours after ethanol administration, many neurons across the entire cerebro-cortical mantle and other affected brain regions show caspase-3 activation that is readily detectable by IHC. Several other brain regions, particularly the anterior thalamic nuclei, show only a small response at 4 to 6 hours but a robust response peaking at approximately 8 hours. In all brain regions, caspase-3 activation is a transitory phenomenon that in a given neuron lasts for ~2 to 3 hours and runs its course for a given nuclear group of similar neurons over a 6 to 8 hours period (21). We previously reported that the loss of caspase-3 alters the time course and morphological characteristics of ethanol-induced neuronal cell death but does not prevent ethanol-induced neurodegeneration, indicating that the point of cell death commitment is upstream of the caspase-3 activation step and/or that other effector caspases, such as caspases -6 and -7, can compensate for the loss of caspase-3 (25).
We identified Puma as a critical initiator of ethanol-induced, Bax-dependent neuronal apoptosis since Puma-deficient neurons are markedly protected from ethanol-induced caspase-3 activation. Puma was originally defined as a direct transcriptional target of p53 that is activated by this tumor suppressor in response to various stimuli, particularly genotoxic injury (27). Puma, however, can be regulated by p53-independent death stimuli, such as cytokine deprivation or treatment with glucocorticoids (27, 32, 33). Since p53-deficient mice were not protected from ethanol-induced caspase-3 activation, Puma appears to be regulated by a mechanism that is p53-independent in this model. In addition to p53, several other transcription factors, including
Forkhead box O3 (FOXO3a), hypoxia-inducible factor-1α (HIF1α), E2F transcription factor 1 (E2F1) and the p53 homolog p73, induce the expression of puma in the CNS and in non-CNS cells (52–55). In particular, p73 is known to induce apoptosis via the mitochondrial pathway by causing transcriptional activation of puma (64), making it a potential candidate for the regulation of Puma in our model. Surprisingly, however, Puma protein levels were not elevated in response to treatment with ethanol in wild-type, Bax- or p53-deficient mice. A possible explanation for this could be that Puma is only upregulated in a minority of the cells in the brain and that therefore Western blotting is not sufficiently sensitive to detect this change. This might be resolved by IHC but currently available antibodies to Puma are not suitable for these applications. We also examined Bax levels in response to ethanol and did not observe a noticeable change (Supplemental Fig. 1). Since Puma triggers apoptosis by activating Bax/Bak either directly or indirectly by binding to the anti-apoptotic Bcl-2 family members (36, 37, 39, 56), it is theoretically also possible that ethanol administration somehow causes a post-translational activation of Puma.
Noxa is another BH3-only pro-apoptotic molecule that can also be activated by p53. The mouse but not the human form of Noxa harbors 2 BH3 domains. Over-expression of Noxa induces cytochrome c release, caspase-9 activation and apoptosis in several human tumor-derived cell lines (65, 66). Embryonic fibroblasts and keratinocytes (59) from Noxa-deficient mice show resistance to a range of DNA damage inducing stimuli (58). It has also been proposed that p53 induces apoptosis largely via Puma in several cell types but that Noxa can play a complementary role (60). Accordingly, we tested whether Puma and Noxa cooperated in our model and found that Noxa deficiency by itself provided no significant protection from ethanol-induced caspase-3 activation. Mice that were deficient for both Puma and Noxa, however, showed the greatest level of protection from ethanol-induced caspase-3 activation, suggesting that these 2 BH3-only proteins do indeed cooperate in this cell death pathway.
It has been reported that Bad, a BH3-only molecule can be de-phosphorylated in some cell types in response to certain apoptotic stimuli thereby promoting its interaction with certain pro-survival Bcl-2 family members and causing subsequent Bax-dependent cytochrome c release and caspase activation (67, 68). Bid, another BH3-only protein, is activated by caspase-8 in response to death receptor signaling and is thought to trigger apoptosis, like Puma, by direct or indirect activation (via binding and inhibition of pro-survival Bcl-2-like proteins) of Bax/Bak (69, 70). Consistent with our previously reported studies (57), however, loss of Bad or Bid did not protect neurons from the effects of ethanol.
The BH3-only proteins Bim and Hrk/Dp5, which also trigger apoptosis through Bax/Bak activation, have both been implicated in trophic factor withdrawal- and potassium deprivation-induced neuronal cell apoptosis (71). In these conditions these BH3-only proteins are upregulated by p53-independent mechanisms. In culture, Bim- and Hrk-deficient neurons show protection from a variety of death inducing stimuli (72, 73). Loss of either Bim, Hrk/Dp5 or a combined deficiency of both did not protect neurons from the toxic effects of ethanol, however.
In summary, this study identifies the pro-apoptotic BH3-only, Bcl-2 family member Puma as the critical initiator of acute ethanol-induced neurodegeneration. Puma activates Bax and caspase-3 through a mechanism that is independent of p53 in this model. Although other BH3-only proteins do not appear to play a major role, Noxa plays a lesser but complementary role to Puma in the initiation of this process. Further characterization of this pathway identifying the signaling cascade and the molecular events regulating Puma- and Bax-mediated apoptotic cell death may assist in the development of future therapies for fetal alcohol syndrome.
The authors would like to thank the UAB Neuroscience Core Facilities (NS 47466 and NS 57098) and Cecelia B. Latham (University of Alabama at Birmingham) for technical assistance. The authors would also like to thank Dr. Andreas Villunger (Innsbruck Medical University) for providing the puma mutant mice.
This work was supported by grants from the National Institutes of Health (NS 35107 and NS 41962). A.S is supported by the National Health and Medical Research Council (Canberra, Australia) and the Leukemia and Lymphoma Society of America.