Human Molecular Genetics, 2002, Vol. 11, No. 6 675-687
© 2002 Oxford University Press
The expression of a new variant of the pro-apoptotic molecule Bax, Bax
, is correlated with an increased survival of glioblastoma multiforme patients
1IFR 26, INSERM UMR 419, 9 quai Moncousu, 44035 Nantes Cedex 01, France, 2Clinique Universitaire de Neurochirurgie, Hôpital G and R Laennec, CHU Nantes, Bld J. Monod-St Herblain, 44093 Nantes Cedex 01, France and 3Centre René Gauducheau, Bld J. Monod-St Herblain, 44093 Nantes Cedex 01, France
Received November 20, 2001; Revised and Accepted January 24, 2002.
DDBJ/EMBL/GenBank accession no. AJ417988.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Pro- and anti-apoptotic members of the BCL-2 family play a central role in the implementation of apoptosis. Bax, a pro-apoptotic member of this family, has as such been considered as a potential tumor suppressor. Here, we have examined the expression of Bax in 55 patients with glioblastoma multiforme (GBM), the most common and aggressive form of brain tumors. We report on the existence of a new form of Bax, present in 24% of the patients, which we called Bax
. Bax is a N-terminal truncated form of Bax which results from a partial deletion of the exon 1 of Bax gene. Bax and the wild-type form, Bax
, are encoded by distinct mRNAs, both of which are present in normal tissues. Glial tumors express either Bax or Bax proteins, an apparent consequence of an exclusive transcription of the corresponding mRNAs. The latter feature could be partially linked to distinct methylation profiles of Bax gene in these tumors. The Bax protein is preferentially localized to mitochondria and is a more powerful inducer of apoptosis than Bax. Bax tumors exhibit a slow proliferation in Swiss nude mice and this feature can be circumvented by the co-expression of the Bcl-2 transgene, the functional antagonist of Bax. More importantly, the expression of Bax correlates with a longer survival in patients (18 months versus 10 months for Bax patients). Thus, our results provide the first indication of a beneficial involvement of a variant of the pro-apoptotic protein Bax in tumor progression. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Apoptosis is a cell death program which appears to be associated with various aspects of normal and pathological cell physiology (1,2). In vitro studies suggest that the loss of a fully functional apoptotic program could influence several stages of tumorigenesis (2,3). The complexity of this process, which involves numerous molecular partners in multiple metabolic or signaling pathways, as well as the lack of reliable techniques to visualize cell death can, at least partially, explain the absence of definitive evidence of the implication of apoptosis in tumorigenesis in vivo (2–4). Members of the BCL-2 family are potent regulators of the execution phase of apoptosis (5). Bax, a pro-apoptotic member of this family, plays a central role by mediating the release of apoptogenic proteins, such as cytochrome c, from mitochondria (6). Due to its pro-apoptotic action, Bax has been considered a potential tumor suppressor and indeed, this has been directly shown in animal models of brain and mammary tumorigenesis (7,8). Consistent with this putative role, Bax mutations associated with microsatellite instability, which inactivate its function, enhance tumorigenesis in human hereditary non-polyposis colorectal cancers (9) and increase the resistance to apoptosis in cell lines derived from hematopoietic malignancies (10,11). The expression of Bax has been reported to be under the transcriptional control of p53 in human tumors (12) and also under that of c-myc (13). Thus, in addition to direct inactivation, alterations in the function of these proto-oncogenes, which are often observed in cancers, could also affect the function of Bax. These results suggest that the dysregulation of the function of Bax in malignant tumors could have a selective advantage for the progression of cancers. The Bax gene encodes several variants of Bax but their functions as well as their regulation are not known nor appear to be different from the principal form, Bax
(14). In this work, we report on the existence of a naturally occurring variant of Bax, the expression of which is restricted to tumors of glial origin. We found that this variant is a more potent inducer of apoptosis than Bax and that its expression could be correlated with an increased survival of patients with glioblastoma multiforme (GBM), the leading cause of death in primary human brain cancer (15,16). |
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Immunoblot analysis of Bax in a series of GBM
We have analyzed by immunoblot the expression of Bax in 55 human GBMs (WHO grade IV astrocytoma) (15) resected tumors using several antibodies raised against distinct parts of the human Bax
sequence (Fig. 1A). We have illustrated the results of immunoblots performed on 55 patients with an antibody raised against the amino acid sequence (amino acids 43–61; Fig. 1B). In most tumors (42/55), a single band with an apparent molecular weight of 21 kDa (p21Bax) was detected by this antibody, whereas in two out of the 55 tumors no immunoreactivity toward Bax was found (Fig. 1B). At variance, in 13/55 tumors, a unique immunoreactive band with an apparent molecular weight of 19 kDa (p19) was observed (Fig. 1B). As truncation of the N-terminus of Bax during the terminal phase of apoptosis has been reported (17), we performed immunoblots with the antibody raised against the N-terminal sequence of human Bax (amino acids 3–16) (Fig. 1A). As shown in Figure 1C, this antibody failed to recognize the p19. However, this protein was recognized by an antibody raised against a C-terminal peptide (150–165) of human p21Bax (Fig. 1B), confirming that p19 belonged to the Bax/p21Bax family. On the other hand, no immunoreactivity with the latter antibodies was observed in the two tumors which thus failed to respond to all anti Bax antibodies tested (data not shown).
|
Distinct mRNAs encode for Bax
and BaxWe next analyzed the mRNA structure of p21Bax and p19Bax by RT–PCR using primers listed in Table 1 and schematically located on Bax
mRNA in Figure 2A. The Bax N primer in combination with Bax C was used to amplify Bax mRNAs in tumors harboring either p21Bax or p19Bax. As shown in Figure 2B, no amplification was obtained with mRNAs extracted from p19Bax tumors whereas a 580 bp amplified band, the size expected from the Bax sequence (14), was observed in p21Bax tumors. As a control, we used primers which amplified a region encompassing exons 3 and 4. In the latter case, amplification products with similar and expected sizes were observed in both p21Bax and p19Bax tumors (Fig. 2B). This result suggested that the first nucleotides of Bax mRNA were absent or modified in mRNA(s) encoding p19Bax. The 5' end of Bax mRNAs was analyzed by SMART RT–PCR in p21Bax and p19Bax GBM (see Materials and Methods). In Bax tumors, a single band of 621 bp was obtained (Fig. 2B) and its sequence was in complete accordance with the published sequence of p21Bax/Bax (14) (Fig. 2C). In contrast, in p19Bax tumors, SMART RT–PCR produced a single band of 556 bp (Fig. 2B). Amplification between exon 3 and the SMART 5' sequence confirmed that the deletion was located within the first 3 exons (Fig. 2B). Sequencing of the SMART RT–PCR amplified Bax products, indicated that the p19Bax was encoded by a mRNA exhibiting a deletion within exon 1 (Fig. 2C). We concluded that the p19Bax protein corresponded to a variant of Bax/p21Bax lacking part of the exon 1 sequence which we have called Bax (EMBL Nucleotide Sequence database accession no. AJ417988). A second ATG located at position 20 in Bax can be used as an alternative initiator methionine (18–20). Cell-free transcription/translation confirmed that the Bax sequence began at this methionine and thus lacked the first 20 amino acids of Bax (data not shown).
|
|
Mature mRNAs obtained from Bax
or Bax tumors, as well as from peritumoral (PT) and control brain tissue, were analyzed by northern blot either using exon 1 or exon 3–4 sequences as probes (Fig. 2A). In non-tumoral (NT) or PT brain tissues, the exon 3–4 probe hybridized with two major bands with a molecular weight of 1.1 and 1.6 kb, respectively; whereas the exon 1 probe hybridized with only the 1.6 kb band (Fig. 3A). In Bax tumors, the exon 1 probe labeled only the 1.6 kb RNA and did not recognize any mRNA in Bax tumors (Fig. 3A). The exon 3–4 probe hybridized with only one of the Bax encoding mRNA in each type of GBM tumor (i.e. the 1.6 kb mRNA in Bax tumors and the 1.1 kb mRNA in Bax tumors) (Fig. 3A). It should be noted that the PT tissues illustrated in Figure 3 were derived from either Bax or Bax patients. To further analyze Bax expression, we investigated its presence in a selection of normal tissues of different origins (colon, lung, spleen, thymus, leukocytes). We found that the labeling of Bax mRNAs with exon 1 or exon 3–4 probes was similar to that found in normal brain tissue although mRNAs exhibited a molecular weight inferior to that observed in brain (1.5 and 1 kb). This result suggested that Bax mRNA was also present in normal tissue outside of the central nervous system (CNS) (Fig. 3B). However, in contrast to GBM, tumoral tissues derived from colon cancer, ovary carcinoma or acute myeloid leukemia (AML) expressed both Bax and Bax transcripts, suggesting that restriction of Bax or Bax mRNA expression was specific for CNS tumors (Fig. 3B). This was confirmed by RT–PCR analysis on tumors of different origins (Table 2). It should be noted that, under our conditions, Bax appears to be encoded by the 1.5/1.6 kb mRNA in all tissues examined, contrary to what was originally reported by Oltvai et al. (14).
|
|
The regulation of the expression of Bax
is likely to be post-translationalInterestingly, we did not observe the presence of Bax
by immunoblot in tissues derived from NT brain tissue or from non-glial tumors (data not shown). This result suggested that although Bax mRNAs were present in most tissues, the corresponding protein was not found except in a small subset of glial tumors. We thus address the nature of the regulation of the expression of Bax in these tissues. As shown in Figure 4A, the in vitro translation of mRNAs purified from Bax and Bax tumors indicated that the corresponding form of Bax was synthesized in these tumors. On the other hand, both p21Bax/Bax and p19Bax/Bax were synthesized in vitro by mRNAs obtained from control tissues (brain normal or PT tissues) or non-glial tumors (Fig. 4B). The latter results suggested that p21Bax underwent a rapid post-translational degradation in non-glial tumor tissue or that factors present in tissues prevented the translation of its mRNA.
|
Analysis of the Bax gene in Bax
and Bax tumorsSouthern blots using genomic DNA digested with EcoRI and HindIII and labeled with an exon 1 or exon 3–4 probe indicated that the overall genomic structure of the Bax gene was similar in Bax
and Bax tumors (Fig. 5A). These results were in agreement with cytogenetic studies which showed no genomic or chromosomal rearrangements within the Bax gene in human GBM using an exon 1 probe (21).
|
We have analyzed the organization of the Bax gene by PCR, using several probes which encompass the whole 5'-untranslated region of Bax gene (21). As shown in Figure 5B, the Bax gene was deleted in the Bax
tumors between positions –972 (beginning of the gene) and –779. However, it should be noted that, as shown in Figure 5B, this deleted sequence did not contain any of the promoter sites proposed for Bax (12,13).
One possible explanation for the observed pattern of expression of Bax and Bax expression in glioblastoma tumors is that these two variants represented two alleles of the Bax gene (i.e. one allele ‘’ and one allele ‘’) which could be segregated in the tumors. We have examined this hypothesis by analyzing the (–972/–779) deletion observed in Bax tumors in normal tissue. As shown in Figure 5C, this deletion was not observed in normal tissue, thus suggesting that it was tumor specific rather than constitutional and ruling out the ‘alleles’ hypothesis.
Microscopic observations of both Bax and Bax GBMs indicated the presence of normal cells infiltrating the tumors. This means that some Bax should be detected in Bax tumors in PCR experiments. Indeed, the presence of a small proportion of Bax cells in Bax tumors was confirmed by PCR using internal exon 1 probes (data not shown).
Methylation of genes can be responsible for alternative transcriptions in cancers (22,23) and several CpG sites representing putative methylation sites are present in the Bax gene sequence (14,21). We used HpaII or MspI cleavage properties to determine the methylation profile of genomic DNA in Bax or Bax tumors. Similar patterns of hybridization with both exon 1 and exon 3–4 probes were found in Bax tumors but not in Bax tumors (Fig. 6A). Differences in methylation profiles between Bax and Bax tumors were confirmed using bisulfite and nested primer set PCR (24). Our results indicate that all Bax CpG sites located between the TATA box and the first ATG were methylated in Bax (Fig. 6B), the latter region corresponding to putative c-myc promoter sites (13). Quite interestingly, whereas no methylation of this gene was observed in Bax tumors, analyses of GBM which do not express Bax (Bax0 GBM) showed that all CpG islands were methylated (Fig. 6B). As a control of the methylation status of the genome in these tumors, we examined the methylation of p14ARF and p15INK4b. As illustrated in Figure 6C, p14ARF was methylated in Bax tumors but not in Bax or in Bax0 GBM. On the other hand, p15INK4b was not methylated in any of these tumors (Fig. 6C).
|
Effect of the expression of Bax
on apoptosis and tumor growthWe analyzed, in primary cultures of tumoral cells obtained from Bax
and Bax GBM (see Materials and Methods), the intracellular localization of Bax by laser confocal microscopy. As shown in Figure 7A, Bax presented a dual cytosolic/mitochondrial localization whereas Bax appeared to be essentially associated with the mitochondria. In both tumors, the antiapoptotic protein Bcl-2 appeared to be essentially mitochondrial.
|
We obtained primary cultures from Bax0 GBM and transiently transfected these cells by electroporation with Bax
or Bax transgenes at different concentrations (see Materials and Methods). After 48 h of selection with G418, immunoblot analyses showed similar levels of Bax expression with Bax or Bax transgenes (Fig. 7B). Apoptosis was then induced by a treatment with doxorubicin and quantified by measuring the release of cytosolic lactate dehydrogenase (LDH) into the culture medium or by assaying the cleaving of AC-DEVD-AMC by caspases within the cells as previously described (25). As illustrated in Figure 7B, both LDH released and cellular DEVDase activity were proportional to the quantity of Bax or Bax transgenes expressed in Bax0 GBM cells. It should be noted that very little enhanced cell death was observed upon the expression of the Bax transgenes (either Bax or Bax) in absence of a cell death inducer, suggesting an intrinsic high capacity of resistance to apoptosis in these cells. However, cells transfected with Bax were more sensitive to doxorubicin than those transfected with Bax (Fig. 7B). Linear regression plots using data presented in Figure 7B indicated that using either caspase-3-like activity or LDH activity, Bax was 3.5x more apoptogenic than Bax.
Effect of Bax on in vivo growth in athymic mice
We also analyzed the in vivo growth in the athymic nude mice using mock or Bcl-2-transfected Bax and Bax cells as xenografts. As illustrated in Figure 8, the mock transfected Bax xenografted tumors grew more steadily than the Bax counterparts. However, the expression of Bcl-2 transgene increased tumoral progression to the level of Bax (Fig. 8). It should be noted that the expression of Bcl-2 did not affect the growth of Bax tumors (Fig. 8). The latter results suggest that the slow proliferation of Bax tumors was due to the expression of Bax and not to unrelated factors co-expressed in these tumors.
|
Similar results were obtained in clonogenicity experiments performed using primary cultures obtained from either Bax
or Bax patients. Less colonies were formed with Bax than with Bax tumoral cells and this tendency was reversed in Bax cells transfected by Bcl-2 (data not shown).
Correlation between the expression of Bax and survival of GBM patients
The precedent results prompted us to determine whether the presence of Bax influenced the progression of GBM in vivo. We estimated the survival probabilities of patients bearing the or variants of Bax by the method of Kaplan–Meier. Follow-up survival data were available for 13 Bax and 42 Bax patients. As shown in Figure 9A, the survival of Bax patients was significantly increased compared to that of Bax patients (18 versus 10 months, P = 0.019). On the other hand, no difference in the survival of these patients was observed in a similar analysis using a mutation of p53 versus wild-type p53 as variable (P = 0.90) (Fig. 9B) as shown previously by others (reviewed in 26). We have recently shown that the expression of Bax was closely correlated with that of Bcl-2 in human GBM (27). The ratio of Bax to Bcl-2, in Bax or Bax tumors determined after quantification of the immunoblot was not different between Bax and Bax tumors (data not shown). These results suggest that the increased survival of Bax patients was not due to a down-regulation of Bcl-2. It should also be noted that no significant differences in sex (male:female ratio Bax, 23:19, 1.21; Bax, 7:6, 1.166), age (Bax, 55.6 ± 13.5; Bax, 54.33 ± 14.5) or Karnofski index (Bax, 78.06 ± 16.6; Bax, 86.92 ± 15.48), were found between the Bax or Bax patients. All patients were treated using similar standard treatments, a combination of surgery, chemotherapy (Temodale and Vincristine) plus radiotherapy.
|
2 = 5, P = 0.019). (B) In contrast, no influence of a mutation of p53 on patients’ survival was observed in the same population ( |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We have found, in a previous work, that the Bax:Bcl-2 ratio did not affect patients, outcome in a series of astrocytoma of high and low grades (27). However, in the present work we found that Bax status differed in GBM as although a majority of tumors expressed p21Bax (42/55), 13 tumors (24%) expressed a variant of Bax with a molecular weight of 19 kDa (Fig. 1). We have confirmed by immunoblot and RT–PCR that p19 was related to p21Bax. Interestingly, two tumors lacked completely the expression of Bax transcripts (Figs 1 and 2). We showed by SMART RT–PCR that the p19Bax corresponded to a novel variant of Bax, Bax
, resulting from a deletion in the 5' end of the Bax mRNA (Fig. 2). Bax is present in most human tissue examined (Fig. 3) and is a normal transcript of Bax in brain of 1.1 kb as confirmed by in vitro translation of mRNAs (Fig. 4). As Bax can be translated in vitro from mRNAs extracted from tissue which normally did not express this protein, we concluded that Bax was post-translationally regulated (Fig. 4). It is noteworthy that the two putative PEST sequence present in Bax are encoded by exon 2 and thus are present in the N-terminus of Bax (search performed by Pestsearch at http://www.icnet.uk/LRITu/projects/pest). The latter result suggests that proteosomal degradation (28) could regulate Bax expression in most tissues as already demonstrated for Bax in some prostate cancers (29). Several other mechanisms could intervene in the regulation of mRNA translation involving sequences located in mRNAs untranslated regions (reviewed in 30). These different hypotheses are currently under investigation in our laboratory.
Since the sequence of Bax mRNA starts within that of exon 1, it is unlikely to arise from a classical mechanism of alternative splicing. Thus, alternative transcription of Bax mRNA or changes in the sequences of the promoters of Bax in tumoral cells leading to gene silencing could be envisaged. Indeed, in support of the latter hypothesis, we found that the methylation status of the Bax gene is different in Bax and Bax tumors (Fig. 6). Methylation is usually associated with the loss of gene expression (23) and this could explain the silencing of Bax in Bax tumors (Fig. 6). Indeed, preliminary experiments suggest that treatment with the de-methylation agent 5-aza2dC triggers the transcription of Bax mRNA in Bax tumoral cells (data not shown). However, hybridization patterns of the exon 1 and exon 2–3 probes with HpaII- and MspI-digested genomic DNA suggested also a rearrangement in DNA between Bax and Bax tumors which could also be involved in the restricted expression of the variants of Bax and genomic PCR indicated that the 5' extremity of Bax gene was deleted in Bax tumors (Figs 5 and 6).
The translation of Bax mRNA starts at a methionine present at position 20 of Bax sequence and thus leads to a N-terminal truncation of the protein. The importance of the first 20 amino acids of Bax in its pro-apoptotic role has been demonstrated using genetically engineered mutants lacking the N-terminus of Bax (18). Bax exhibits a mitochondrial localization in vivo and an enhanced cytotoxicity in vitro (Fig. 7). This result is in good agreement with several recent studies which have pointed out the role of the N-terminus of Bax in its retention in the cytosol of healthy cells (18–20). The limited expression of this highly apoptogenic form of Bax in tumors suggests that either the expression of Bax is restricted to tissues that are highly resistant to apoptosis or that apoptosis sensitive malignant or normal cells which express Bax are rapidly eliminated.
Experiments with Bax or Bax xenografts indicated that Bax expression was associated by a reduced tumoral growth in vivo (Fig. 8). The fundamental role of Bax in the decrease of tumor growth is confirmed by experiments in which Bcl-2 transgene is expressed in Bax expressing cells (Fig. 8). The latter results suggest also that the improved survival of Bax patients when compared to Bax ones (Fig. 9) is directly linked to Bax function(s). In addition, the expression of Bax is apparently not associated with a late occurring event in the glioma genetic pathway or to secondary GBM as Bax-positive tumors were also found in low grade astrocytomas and oligodendrogliomas (Table 2).
Interestingly, a specific isoform of Bax which lacks the first 20 amino acids of Bax has been found expressed in ischemic tissues in rat (31). This result strongly suggests that Bax is involved in the regulation of apoptosis in the CNS under different pathological situations.
Bax inactivation due to mutations or enhanced degradation influences the response of human epithelial cancer cells to chemotherapy (32) and is an indicator of poor prognosis in several types of cancers (9,29). Our data show the first positive element to support the arguement that apoptosis is a key player in tumor progression and suggest that Bax and especially Bax could be important tools in future therapeutic strategies in human cancers.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Materials and tissue samples
Unless specified otherwise, all material used in this work was from Sigma. All culture material was from Gibco BRL.
Patients
Tumor samples were chosen from adult patients after surgical resection at the Department of Neurosurgery of the Hospital of Nantes over the years 1996–1999. Brain tumors were classified and graded according to the WHO system (15). PT tissue was obtained from normal brain tissue found at the periphery of the resected tumor or from control tissues from patients operated for other pathologies at the Department of Neurosurgery. RNAs purified from non-nervous tissues were obtained from Clontech (human panels I, II and III, K4000-1, K4000-2, K4000-3).
Patient material as well as records (diagnosis, age, sex, date of death) were used with confidentiality according to French laws and recommendations of the French National Committee of Ethic.
Methods
Western blots were performed using antibodies directed against the N-terminus of Bax (amino acids 3–16, 2 µg/ml, R&D Systems, 2282-MC), Bax exon 3 (amino acids 43–61, 2 µg/ml, Dako, A3533), C-terminus of Bax (amino acids 150–165, 2 µg/ml, Oncogene, PC66), Bcl-2 (1 µg/ml, Dako, M-0887) and actin (0.5 µg/ml, Boehringer Mannheim, 138–996). The amount of immunoreactive protein was quantified using IPLab Gel Program (Signal Analytics) after scanning with an Appligene-Oncor Imager using actin as an internal standard. p53 mutants in patient samples were analyzed by immunoblots after non-denaturing gels using antibodies raised against wild-type p53 (Oncogene Research Products Ab-2) or mutated p53 (1 µg/ml, Santa Cruz, Pab240).
RT–PCR analyses and northern blot
Total RNA from control, tumoral or peritumoral tissue was prepared using RNA Isolator (Genosys-Sigma). The reverse transcription reaction was accomplished with 1 µg total RNA and MuMLV reverse transcriptase (Promega). The PCR reaction was performed using SMART RT–PCR cDNA Synthesis kit (Clontech) and primers SMART II (Clontech) and primers listed in Table 1. For the northern blots, 3 µg poly(A)+ RNA from different glioblastoma multiforme were separated in 1.5% agarose gel then transferred to nylon membranes (GeneScreen plus Dupont) in 10x SSC by capillary flow and baked at 80°C for 2 h. mRNA was hybridized with exon 3–4 or exon 1 probes synthesized with the ‘Ready to go’ kit from Pharmacia using [35S]dATP.
Southern blots and methylation PCR
Genomic DNA was prepared from tumors using standard methods. The extracted DNA was digested with restriction enzymes EcoRI and HindIII then 10 µg digest was separated in 1.5% agarose gel and transferred to Hybond+ membranes (Amersham). DNA methylation was assayed using HpaII and MspI digestion followed by Southern blotting. DNA probes for hybridization were obtained after PCR amplification and were labeled using [35S]dATP, exon 3–4 probe was obtained after PCR amplification (PCR primers, BAX3/4S and BAX3/4AS) and exon 1 probe after amplification with BaxN and BaxASe1 (5'-GAGACCCGACG-3'). Genomic DNA fragments obtained either from Bax or Bax tumors were treated with the bisulfite modification kit from Appligene-Oncor according to the manufacturer’s instructions.
Cell culture, confocal analysis and induction of apoptosis
For cell culture, the tumors were mechanically dissociated using a Medimachine® (Dako) and 50 µm Medicons® (Dako). Cells were seeded in RPMI 1640 medium containing 10% fetal calf serum, L-glutamine, penicillin and streptomycin. One day after dissociation, cells were cultured in RPMI medium containing G5, L-glutamine, penicillin and streptomycin. Experimental data described in this study were obtained with cells obtained between the seven and 15 passages for all tumors examined (i.e. two of each Bax-deficient, Bax and Bax tumors). For confocal analysis, the tumors were mechanically dissociated and then cultured for 7 days. Bax, Bcl-2 and Mitotracker green (Molecular Probes) were analyzed by laser confocal microscopy as previously described (25). Bax-deficient cells were electroporated with Bax or Bax cDNAs and grown in the presence of G418 for 48 h prior to the induction of apoptosis with doxorubicin (2 µM) and LDH and DEVDase activities were measured as described previously (25). The clonogenicity experiments were performed on tumoral cells obtained from transfected Bax or Bax tumors grown in RPMI/0.2% agar for 8 weeks and viable cells were visualized by staining with crystal violet.
In vivo experiments
Swiss nude mice were injected subcutaneously in the flank with 106 cells obtained from Bax or Bax primary cultures transfected with either the empty plasmid (pRcCMV) or the plasmid encoding human Bcl-2 (pRcCMV-huBcl-2) and were selected as described above for Bax transfections. Tumoral progression was monitored and quantified by measuring the size of the tumors every week over a period of 25 days.
|
ACKNOWLEDGEMENTS |
|---|
We thank Drs P.Cuillière and C.Sagan (CHR Nantes) for the histopathological examination of tumors. We thank Prof. R.Breathnach (U463 INSERM, Nantes), Dr Ph.Juin (U419 INSERM, Nantes), Dr G.Kroemer (IGR/CNRS, Villejuif) and Prof. J.C.Martinou (University of Geneva) for critical readings of this manuscript. We thank our colleagues of the CHU of Nantes for the non-nervous tumoral tissues (Prof. J.L.Harousseau, Prof. C.Laboisse and Dr M.Campone) and Dr O.Delattre (Insititut Curie, Paris) for the gift of neuroblastoma cell lines. P.F.C. was supported by a fellowship from the Ligue Départementale contre le Cancer Doubs/Montbeliard. This work was supported by grants from INSERM, the Association pour la Recherche sur le Cancer and the Ligue Grand Ouest contre le Cancer.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +33 24 0084081; Fax: +33 24 0084082; Email: fval@nantes.inserm.fr
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Korsmeyer,S.J. (1999) BCL-2 gene family and the regulation of programmed cell death. Cancer Res., 59 (suppl.), 1693s–1700s.
2
Reed,J.C. (2000) Mechanisms of apoptosis. Am. J. Pathol., 157, 1415–1430.
3
Lowe,S.W. and Lin,A.W. (2000) Apoptosis and cancer. Carcinogenesis, 21, 485–495.
4
Brown,J.M. and Wouters,B.G. (1999) Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res., 59, 1391–1399.
5
Adams,J.E. and Cory,S. (1998) The Bcl-2 protein family: arbiters of cell survival. Science, 281, 1322–1326.
6 Kroemer,G. and Reed,J.C. (2000) Mitochondrial control of cell death. Nat. Med., 6, 513–519.[Web of Science][Medline]
7 Yin,C., Knudson,C., Korsmeyer,S.J. and Dyke,T. (1997) Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature, 385, 637–640.[Medline]
8 Shibata,M.A., Liu,M.L., Knudson,M.C., Shibata,E., Yoshidome,K., Bandey,T., Korsmeyer,S.J. and Green,J.E. (1999) Haploid loss of Bax leads to accelerated mammary tumor development in C3(1)/SV40-TAg transgenic mice: reduction in protective apoptotic response at the preneoplastic stage. EMBO J., 18, 2692–2701.[Web of Science][Medline]
9
Ionov,Y., Yamamoto,H., Krajewski,S., Reed,J.C. and Perucho,M. (2000) Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution. Proc. Natl Acad. Sci. USA, 97, 10872–10877.
10
Meijerink,J.P.P., Mensink,E.J.B.M., Wang,K., Sedlack,T.W., Slôetjes,A.W., deWhitte,T., Waksman,G. and Korsmeyer,S.J. (1998) Hematopoietic malignancies demonstrate loss-of-function mutations of BAX. Blood, 91, 2991–2997.
11 Abdel-Rahman,W.M., Georgiades,I.B., Curtis,L.J., Arends,M.J. and Wyllie,A.H. (1999) Role of BAX mutations in mismatch repair-deficient colorectal carcinogenesis. Oncogene, 18, 2139–2142.[Web of Science][Medline]
12 Miyashita,T. and Reed,J.C. (1995) Tumor suppressor p53 is a direct transcription activator of the human Bax gene. Cell, 80, 293–299.[Web of Science][Medline]
13
Mitchell,K.O., Ricci,M.S., Myashita,T., Dicker,D.T., Jin,Z., Reed,J.C. and El-Deiry,W.S. (2000) Bax is a transcriptional target and mediator of c-myc-induced apoptosis.Cancer Res., 60, 6318–6325.
14 Oltvai,Z.N., Milliman,C.L. and Korsmeyer,S.J. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74, 609–619.[Web of Science][Medline]
15 Kleihues,P., Soylemezoglu,F., Schauble,B., Scheithauer,B.W. and Burger,P.C. (1995) Histopathology, classification, and grading of gliomas. Glia, 3, 211–321.
16
Louis,D.N., Holland,E.C. and Cairncross,J.G. (2001) Glioma classification: a molecular reappraisal. Am. J. Pathol., 159, 779–786.
17 Wood,D.E., Thomas,A., Devi,L.A., Berman,Y., Beavis,R.C., Reed,J.C. and Newcomb,E.W. (1998) Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene, 17, 1069–1678.[Web of Science][Medline]
18
Goping,I.S., Gross,A., Lavoie,J.N., Nguyen,M., Jemmerson,R., Roth,K., Korsmeyer,S.J. and Shore,G.C. (1998) Regulated targeting of BAX to mitochondria. J. Cell Biol., 143, 207–215.
19 Temblais,K., Oliver,L., Juin,P., LeCabellec,M.T., Meflah,K. and Vallette,F.M. (1999) The C-terminus of Bax is not a membrane addressing/anchoring signal. Biochem. Biophys. Res. Commun., 260, 582–691.[Web of Science][Medline]
20 Ruffolo,S.C., Breckenridge,D.G., Nguyen,M., Goping,I.S., Goping,A., Korsmeyer,S.J., Li,H., Yuan,J. and Shore,G.C. (2001) BID-dependent and BID-independent pathways for BAX insertion into mitochondria. Cell Death Differ., 7, 1101–1108.
21 Chou,D., Miashita,T., Mohrenweiser,H.W., Ueki,K., Kastury,K., Druck,T., von Deimling,A., Huebner,K., Reed,J.C. and Louis,D.N. (1996) The BAX gene maps to the glioma candidate region at 19q13.3 but is not altered in human gliomas. Cancer Genet. Cytogenet., 88, 136–140.[Web of Science][Medline]
22
Wolffe,A.P. and Matzke,M.A. (1999) Epigenetics: regulation through repression. Science, 286, 481–486.
23 Baylin,S.B., Esteller,M., Rountree,M.R., Bachman,K.E., Schuebel,K. and Herman,J.G. (2001) Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum. Mol. Genet., 10, 387–692.
24
Herman,J.G., Graft,J.R., Myohanen,S., Nelkin,B.D. and Baylin,S.B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA, 93, 9821–9826.
25 Oliver,L., Priault,M., Tremblais,K., LeCabellec,M.-T., Meflah,K., Manon,S. and Vallette,F.M. (2000) The substitution of the C-terminus of Bax by that of Bcl-xl does not affect its subcellular localization but abrogates its pro-apoptotic properties. FEBS Lett., 487, 161–165.[Web of Science][Medline]
26 Nieder,C., Petersen,S., Petersen,C. and Thames,H.D. (2000) The challenge of p53 as prognostic and predictive factor in gliomas. Cancer Treat. Rev., 26, 67–73.
27 Martin,S., Toquet,C., Oliver,L., Cartron,P.-F., Meflah,K., Cuillère,P. and Vallette,F.M. (2001) Expression of Bcl-2, Bax and Bcl-xl in human gliomas: a re-appraisal. J. NeuroOncol., 52, 129–139.[Medline]
28
Varshavsky,A. (1996) The N-end rule: functions, mysteries, uses. Proc. Natl Acad. Sci. USA, 93, 12142–12149.
29
Li,B. and Dou,Q.P. (2000) Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc. Natl Acad. Sci. USA, 97, 3850–3855.
30
Cazzola,M. and Skoda,R.C. (2000) Translational pathophysiology: a novel mechanism of human disease. Blood, 95, 3280–3288.
31 Jin,K.L., Graham,S.H., Mao,X.O., He,X., Nagayama,T., Simon,R.P. and Greenberg,D.A. (2001) Bax kappa, a novel Bax splice variant from ischemic rat brain lacking an ART domain, promotes neuronal cell death. J. Neurochem., 77, 1508–1519.[Web of Science][Medline]
32
Zhang,L., Yu,J., Park,B.H., Kinzler,K.W. and Vogelstein,B. (2000) Role of BAX in the apoptotic response to anticancer agents. Science, 290, 989–992.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Hervouet, E. Debien, L. Campion, J. Charbord, J. Menanteau, F. M. Vallette, and P.-F. Cartron Folate Supplementation Limits the Aggressiveness of Glioma via the Remethylation of DNA Repeats Element and Genes Governing Apoptosis and Proliferation Clin. Cancer Res., May 15, 2009; 15(10): 3519 - 3529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Manero, F. Gautier, T. Gallenne, N. Cauquil, D. Gree, P.-F. Cartron, O. Geneste, R. Gree, F. M. Vallette, and P. Juin The Small Organic Compound HA14-1 Prevents Bcl-2 Interaction with Bax to Sensitize Malignant Glioma Cells to Induction of Cell Death. Cancer Res., March 1, 2006; 66(5): 2757 - 2764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Aouacheria, F. Brunet, and M. Gouy Phylogenomics of Life-Or-Death Switches in Multicellular Animals: Bcl-2, BH3-Only, and BNip Families of Apoptotic Regulators Mol. Biol. Evol., December 1, 2005; 22(12): 2395 - 2416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-F. Cartron, H. Arokium, L. Oliver, K. Meflah, S. Manon, and F. M. Vallette Distinct Domains Control the Addressing and the Insertion of Bax into Mitochondria J. Biol. Chem., March 18, 2005; 280(11): 10587 - 10598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Poncet, N. Larochette, A.-L. Pauleau, P. Boya, A.-A. Jalil, P.-F. Cartron, F. Vallette, C. Schnebelen, L. M. Bartle, A. Skaletskaya, et al. An Anti-apoptotic Viral Protein That Recruits Bax to Mitochondria J. Biol. Chem., May 21, 2004; 279(21): 22605 - 22614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-F. Cartron, P. Juin, L. Oliver, S. Martin, K. Meflah, and F. M. Vallette Nonredundant Role of Bax and Bak in Bid-Mediated Apoptosis Mol. Cell. Biol., July 1, 2003; 23(13): 4701 - 4712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Moreau, P.-F. Cartron, A. Hunt, K. Meflah, D. R. Green, G. Evan, F. M. Vallette, and P. Juin Minimal BH3 Peptides Promote Cell Death by Antagonizing Anti-apoptotic Proteins J. Biol. Chem., May 23, 2003; 278(21): 19426 - 19435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-F. Cartron, M. Priault, L. Oliver, K. Meflah, S. Manon, and F. M. Vallette The N-terminal End of Bax Contains a Mitochondrial-targeting Signal J. Biol. Chem., March 21, 2003; 278(13): 11633 - 11641. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. McIlroy, P.-F. Cartron, P. Tuffery, Y. Dudoit, A. Samri, B. Autran, F. M. Vallette, P. Debre, and I. Theodorou A triple-mutated allele of granzyme B incapable of inducing apoptosis PNAS, March 4, 2003; 100(5): 2562 - 2567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Lomonosova, T. Subramanian, and G. Chinnadurai Requirement of BAX for Efficient Adenovirus-Induced Apoptosis J. Virol., October 11, 2002; 76(22): 11283 - 11290. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||