Bax activation by engagement with, then release from, the BH3 binding site of Bcl-xL.

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.

Positive regulation of apoptosis by HCA66, a new Apaf-1 interacting protein, and its putative role in the physiopathology of NF1 microdeletion syndrome patients.

As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment.

We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.

Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak.

The pro-apoptotic protein Bak is converted from a latent to an active form by damage-induced signals. This process involves an early exposure of an occluded N-terminal epitope of Bak in intact cells. Here we report a subsequent damage-induced change in Bak, detected using an antibody to the central BH-1 domain. Bak co-immunoprecipitated with Bc1-x(L) both in undamaged cells and early after damage, when the N-terminal epitope was exposed but the BH-1 epitope remained occluded. A subsequent decrease in binding of Bak to Bc1-x(L) correlated with exposure of an epitope in the Bak BH-1 domain. Overexpression of Bc1-x(L) did not affect the kinetics of exposure of the Bak N-terminal epitope but delayed exposure of the BH-1 domain. Cytochrome c release from mitochondria facilitates the activation of apoptotic caspases. The majority of cells with exposed Bak BH-1 domains contained cytosolic cytochrome c. However, a small proportion of cells exhibited exposed Bak BH-1 domains that co-localized with mitochondrial cytochrome c. The data are consistent with a two-step model for the activation of Bak by drug-induced damage signals where dissociation of Bc1-x(L) from the BH-1 domain of Bak occurs immediately prior to or concomitantly with cytochrome c release.

Induction of cyclin E and inhibition of DNA synthesis by the novel acronycine derivative S23906-1 precede the irreversible arrest of tumor cells in S phase leading to apoptosis.

S23906-1 is a diester derivative of 1,2-dihydrobenzo[b]acronycine with an unknown mechanism of action. This cytotoxic compound was 20-fold more potent than acronycine in inhibiting the proliferation of six tumor cell lines. Using a clonogenic assay of cell survival, the HT29 human colon carcinoma cell line was 100-fold more sensitive to S23906-1 than acronycine. Cell cycle analysis, by flow cytometry, showed that S23906-1 induced a partially reversible arrest of HT29 cells in G2+M at 1 microM and below and an irreversible arrest in S phase at 2.5 microM and above. These cell cycle effects were followed by cell death through apoptosis, quantified by annexin-V labeling. Inhibition of DNA synthesis was observed by complete prevention of bromodeoxyuridine (BrdU) incorporation after only 4 h of incubation with 5 microM S23906-1. Interestingly, under the same experimental conditions, a significant increase of cyclin E protein level was observed without any modification of cyclins D1, D2, D3, or A. This overexpressed cyclin E protein was not complexed with Cdk2, as shown by western blotting for Cdk2 in immunoprecipitates of cyclin E. Similar inhibition of BrdU incorporation and elevation of cyclin E protein were observed after treatment with cytosine arabinoside, which reversibly inhibited progression into S phase, but not after DNA damage induced by cisplatin. S23906-1 thus has a novel mechanism of action. A cell line resistant to S23906-1 showed that overexpression of cyclin E was implicated in the novel cytotoxic activity of this compound.

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate.

Sequential steps in the activation of the pro-apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH-EP1 and SH-SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N-terminal epitope of Bax. This was reversible and occurred before the translocation of cytosolic Bax to mitochondria. The N-terminal change in Bax, its subsequent movement to mitochondria and its dimerization/complex formation were insufficient for commitment to death, occurring in the same proportion of cells that either maintained (SH-SY5Y) or lost (SH-EP1) clonogenic survival after taxol treatment. Suppression of taxol-induced apoptosis occurred upstream of cytochrome c release from mitochondria in SH-SY5Y cells. The data suggest that a further drug damage-induced event occurs after Bax dimerization/complex formation but prior to cytochrome c release. This event was absent in the taxol-resistant cells.

Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity.

Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylglycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action.

Disruption of focal adhesions mediates detachment during neuronal apoptosis.

Apoptosis participates in the development of the nervous system and in neurodegeneration. The aim of this study was to investigate the mechanisms of detachment of neuronal cells from the extracellular matrix (ECM) during apoptosis. Detachment of Ntera2 neuronal cells was accompanied by decreased surface expression of the beta1 integrin and redistribution of proteins from focal adhesions (FA). FA proteins were cleaved in a discrete sequence: p130cas, then paxillin, then talin. Caspase inhibition prevented detachment and cleavage of paxillin and p130cas, whilst calpain inhibition blocked talin cleavage. Neuronal cells therefore detach as a result of redistribution and caspase-dependent cleavage of focal adhesion proteins. Cleavage occurs sequentially such that critical ECM-integrin survival signalling cascades are severed before disruption of focal adhesion-cytoskeletal links.

MDM2 mediated nuclear exclusion of p53 attenuates etoposide-induced apoptosis in neuroblastoma cells.

The p53 gene in neuroblastoma tumors (NB) is rarely mutated but the protein accumulates in the cytoplasm. Because p53 can mediate the cytotoxic effects of chemotherapeutic agents, it is important to determine whether accumulation of p53 in the cytoplasm impairs p53 function. Data presented here indicate that hyperactive nuclear export of p53 suppresses etoposide-induced apoptosis but does not prevent growth arrest. We compared p53 function in a pair of NB subclones. Our data show etoposide induces complete trans-location of p53 to the nucleus and activation of apoptosis in the neuroblastic NB cell line SH-SY5Y (N-type), which expresses low levels of MDM2. However, in Schwann cell-like SH-EP1 cells (S-type), which have up to 10-fold higher levels of MDM2, p53 accumulates in the cytoplasm and the cells are extremely resistant to etoposide-induced apoptosis. Notably, when MDM2 expression is inhibited in S-type cells, with a phosphorothioated antisense oligonucleotide (AS5), then p53 accumulates in the nucleus and the SH-EP1 cells undergo apoptosis. Surprisingly, induction of p21 and G1-arrest are not attenuated in S-type cells, despite the predominantly cytoplasmic location of p53. Whereas, G1-arrest is attenuated in the SH-SY5Y cells, which have high levels of nuclear p53. Taken together, these findings suggest attenuation of G1-arrest is related to the differentiation status of neuroblastomas and occurs downstream of p53 nuclear accumulation. These results demonstrate for the first time that hyperactive nuclear export of p53 attenuates chemotherapy-induced apoptosis in NB cells, and our findings suggest that inhibitors of MDM2 may enhance the therapeutic efficacy of etoposide by promoting apoptosis rather than trans-differentiation.

The importance of p53-independent apoptosis in the intestinal toxicity induced by raltitrexed (ZD1694, Tomudex): genetic differences between BALB/c and DBA/2 mice.

The thymidylate synthase inhibitor raltitrexed (ZD1694, Tomudex) induces greater intestinal toxicity, manifested as diarrhea and weight loss, in BALB/c than in DBA/2 mice. No convincing pharmacokinetic or pharmacodynamic reason for this strain difference has been established. We have investigated whether this strain difference in response to raltitrexed is related to differential susceptibilities of intestinal mucosae to undergo apoptosis and also whether p53 expression, a critical factor in 5-fluorouracil-induced intestinal apoptosis and toxicity, modulates this response. Ten mg/kg or 100 mg/kg raltitrexed were administered as single or double i.p. injections 24 h apart to BALB/c, DBA/2, and p53-/- mice. Apoptosis, mitosis, and tissue damage were assessed in intestinal epithelium, and animal weight was recorded. BALB/c mice developed diarrhea and weight loss following 100 mg/kg x2 raltitrexed, whereas DBA/2 mice did not. BALB/c mice were more sensitive than DBA/2 to induction of small-intestinal and colonic apoptosis 24 h following 100 mg/kg raltitrexed. Inhibition of mitosis was equivalent in both strains. Both strains showed histopathological damage to the small intestine after 100 mg/kg x2 raltitrexed, but only BALB/c mice demonstrated colonic damage. p53-null mice showed the same level of small intestinal apoptosis as their wild-type counterparts 24 h after 100 mg/kg x1 raltitrexed and also the same levels of intestinal toxicity 3, 5, and 7 days after 100 mg/kg x2 raltitrexed. Thus, BALB/c mice were more susceptible to induction of intestinal apoptosis by raltitrexed than DBA/2 mice and also demonstrated more histopathological damage in the colon correlating with the induction of diarrhea and weight loss. In contrast to 5-fluorouracil, the intestinal apoptosis and toxicity induced by raltitrexed were p53-independent.

Apoptosis and cancer chemotherapy.

Apoptosis is a fundamental mechanism of cell death that can be engaged by a range of cellular insults. One of the major modes of action of chemotherapeutic drugs may be via the activation of apoptosis. Understanding how the cell death program is engaged following an insult, and hence why it fails to be engaged in certain settings, offers a novel approach to overcoming the clinical problem of drug resistance. The tumour suppressor gene p53 and its downstream effector genes p21, mdm-2, and gadd45 seem to be important in the cellular response to genotoxic drug induced damage. Considerable evidence has accrued about the effect of mutations of this pathway on drug sensitivity and this is discussed. The expanding Bcl-2 family of proteins also play an important role in the cell death program. Evidence suggests that these proteins may function as integrators of damage signals, and may be the final decision point as to whether a cell lives or dies. These proteins may thus represent a logical target for new approaches to overcoming drug resistance.

Bcl-w is an important determinant of damage-induced apoptosis in epithelia of small and large intestine.

The potential role of the bcl-2 relative bcl-w as a physiological regulator of apoptosis in intestinal epithelia has been investigated. Immunoblots for bcl-w with new monoclonal antibodies revealed that it was expressed in the small intestine and colon, among other murine tissues, as well as in six human tumour cell lines of epithelial origin, including two colon carcinoma lines. To assess whether bcl-w regulates either spontaneous or damage-induced apoptosis in the small intestine or colon, apoptosis in intestinal crypts of bcl-w -/- and wild-type mice was quantified microscopically on a cell positional basis. Spontaneous apoptosis within crypt epithelia was not significantly increased by loss of bcl-w, in either the small intestine or midcolon. However, after treatment with the cytotoxic drug 5-fluorouracil or with gamma-radiation, the bcl-w-null animals exhibited substantially more apoptosis than their wild-type counterparts in both tissues. The greatest enhancement of apoptosis attributable to the absence of bcl-w (up to sixfold) occurred in the small intestine. Hence, bcl-w is an important determinant of damage-induced apoptosis in intestinal epithelia, and unlike bcl-2, which regulates only colonic apoptosis, plays a major role in small intestinal epithelium.

Epigenetic determinants of resistance to etoposide regulation of Bcl-X(L) and Bax by tumor microenvironmental factors.

BACKGROUND: Epigenetic factors (i.e., alterations of gene activity not involving mutations), as well as genetic changes in surviving cancer cells, may play an important role in drug resistance following cancer chemotherapy-a common cause of tumor relapse. Bcl-2 family proteins are central to the regulation of apoptotic cell death and modulate drug sensitivity. We investigated how survival signals in the cellular microenvironment affect the expression, protein conformation, and protein-protein interactions of the Bcl-2 family proteins Bax and Bcl-x(L) and how changes in response to microenvironmental signals alter the response of cancer cells to the drug etoposide. METHODS: JLP119 human B-lymphoma cells were treated with etoposide (40 microM) and then cultured in the presence of an activating anti-CD40 antibody, vascular cellular adhesion molecule-1 (VCAM-1)-to activate VLA-4 (alpha4beta1) integrin, and interleukin 4. Cell fate was monitored after etoposide treatment with or without these microenvironmental signals. Bcl-x(L) gene transcription and protein levels of Bcl-x(L) and Bax were measured by northern and western blotting, respectively. Nuclear translocation of transcription factor NF-kappaB was monitored by immunofluorescence and inhibited by (E)-capsaicin. Bax conformation and Bax-Bcl-x(L) interactions were monitored by immunofluorescence and immunoprecipitation, respectively. RESULTS: Microenvironmental survival signals produced statistically significant reductions in etoposide-induced apoptotic cell death, from 84.6% (95% confidence interval [CI] = 76.7%-92.4%) to 21.3% (95% CI = 19.5%-23.0%); P<.001. Activation of surface protein CD40 increased Bcl-x(L) protein levels via an (E)-capsaicin-inhibitable activation of NF-kappaB; i.e. , (E)-capsaicin restored etoposide sensitivity. Interleukin 4 had no effect on Bcl-x(L) protein levels but accelerated the increase in Bcl-x(L) protein associated with CD40 activation. VCAM-1- and interleukin 4-mediated signals diminished conformational changes in Bax protein and prevented the etoposide-induced disruption of constitutive Bax-Bcl-x(L) binding. CONCLUSIONS: Microenvironmental factors reduce the sensitivity of a B-cell lymphoma to etoposide in vitro by modulating the expression and functions of Bax and Bcl-x(L). This interaction may provide a paradigm for epigenetically induced drug resistance in other tumors.

Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo.

The influence of bcl-2 and bax expression on apoptotic cell death in mouse intestinal epithelia was assessed using homozygously null mice. Apoptosis was induced in vivo by the enterotoxin 5-fluorouracil (5FU) or by gamma-irradiation and its cell positional incidence was assessed. 5FU and gamma-radiation treated bax-null mice surprisingly showed no reductions in apoptotic yield in the small intestine or midcolon at 4.5 h at cell positions in which both agents had previously been shown to strongly induce p53 protein expression. The colonic epithelia of 5FU treated bcl-2-null mice showed elevated levels of apoptosis at 4.5 h: from 48 apoptotic events in wild-type mice to 273 in the nulls, scoring 200 half crypts. The increase occurred specifically in the cell positions considered to harbour colonic stem cells, at the base of crypts, where there is selective expression of bcl-2. There was a modest but significant increase in apoptosis in the small intestine of the bcl-2-null mice although the epithelia of wild-type mice here are not immunohistochemically positive for bcl-2 protein. These findings show that bcl-2 plays a key role in determining the sensitivity of colonic stem cells to damage-induced death but that bax is not responsible for the p53-dependent induction of apoptosis in this context.

Nucleolar segregation during apoptosis of haemopoietic stem cell line FDCP-Mix.

The programmed elimination of cells during apoptosis is distinct from necrosis both morphologically and biochemically. Currently, the morphological description of apoptosis discriminates between the segregation of the nucleolus and the so called 'chromatin condensation'. The latter originates from observations of electron dense material adjacent to the nuclear envelope of apoptotic nuclei. Although there is ample evidence for an involvement of DNA in electron dense marginations, their true nature is still unknown. By studying apoptosis in FDCP-Mix, a pluripotent murine haemopoietic stem cell line, we found morphological and histochemical evidence that electron dense material at the nuclear envelope (NE) has emerged as a result of the segregation of nucleoli in association with the nuclear membrane. The remaining electron dense and homogenous bulk of the nucleolus labels for RNAse-gold, but even more intensely for DNAse-gold, and therefore could possibly be mistaken as 'condensed chromatin' in the light microscope. The labelling of the electron dense material for DNase-gold in FDCP-Mix could be explained by a migration of DNA into the bulk of the nucleoli at an early stage of cell death.

Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland.

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.

Bcl-2 overexpression results in reciprocal downregulation of Bcl-X(L) and sensitizes human testicular germ cell tumours to chemotherapy-induced apoptosis.

Testicular germ cell tumours are hypersentive to chemotherapy and cell lines derived from these tumours are chemosensitive in vitro. We have previously shown that these cell lines express undetectable levels of the suppressor of apoptosis Bcl-2 and relatively high levels of the apoptosis inducer Bax (Chresta et al., 1996). To determine whether the absence of Bcl-2 in these cell lines makes them highly susceptible to drug-induced apoptosis, Bcl-2 was expressed ectopically in the 833K testicular germ cell tumour cell line. Stable overexpressing clones were isolated and three clones were studied further. Surprisingly, Bcl-2 overexpressing cells were sensitized to chemotherapy-induced apoptosis compared to the parental and vector control cells. Analysis of potential mechanisms of sensitization revealed there was a reciprocal downregulation of the endogenously expressed Bcl-X(L) in the Bcl-2 overexpressing clones. Downregulation of Bcl-X(L) to the same extent using antisense oligonucleotides enhanced etoposide-induced apoptosis by twofold. Our results indicate that Bcl-2 and Bcl-X(L) have different abilities to protect against chemotherapy-induced apoptosis in testicular germ cell tumours. In contrast to findings in some tumour cell types, Bcl-2 did not act as a gatekeeper to prevent entry of p53 to the nucleus.