The p210 BCR-ABL tyrosine kinase of chronic myeloid leukemia causes resistance to radio-induced apoptotic death by inhibiting the proapoptotic BAX gene

TO THE EDITOR Chronic myeloid leukemia (CML) is a myeloproliferative disorder originating from a multipotential hematopoietic progenitor and leading to the illegitimate expansion of a clonal myelopoiesis that retains a normal phenotype and normal functions (reviewed in Deininger et al1). The bcr-abl-rearranged gene is the molecular marker of the disease. It arises from the juxtaposition of most of the c-abl proto-oncogene on chromosome 9 to the amino-terminal of bcr on chromosome 22. The tyrosine kinase of the abl protein constitutively activated by the oligomerization of a coiled-coil domain at the N-terminus of bcr is the causative event of CML.2 It provides leukemic hematopoietic progenitors with a proliferative advantage over its normal counterpart by transducing the mitogenic signal(s) in the absence (or in the presence of reduced amounts) of growth factors. It also prolongs the life expectancy of clonal myeloid progenitors by inhibiting apoptosis. Multiple pathways cooperate with the latter event, including the upregulation of antiapoptotic signals (such as Bcl-2 and Bcl-xL) and the abrogation of proapoptotic proteins (such as Bad) of signals proceeding from the interferon consensus-binding protein ICSBP and from the Fas receptor/Fas ligand, and lastly of most downstream effectors of apoptosis (such as the release of cytochrome c or the activation of caspase 3).1 The loss of cell cycle regulatory control and physiological response to stress, including apoptotic death, allows clonal hematopoietic progenitors to walk further steps leading, in turn, to the fully transformed phenotype of blast crisis. The appearance of additional, nonrandom chromosomal aberrations in the accelerated phase of CML or in blast crisis points to genomic instability as one main determinant of the disease progression. Indeed, CML hematopoietic progenitors exhibit discrete defects in the DNA repair machinery, including the loss of p16, the inactivation of RB protein, the enhanced expression of beta polymerase (the least faithful mammalian DNA polymerase), the inhibited catalytic function of xeroderma pigmentosum group B protein or the downregulation of DNA-dependent protein kinase.1 Still, the gene most often implicated in CML evolution is p53: it may either undergo point mutations or be functionally inactivated by its specific competitors, such as mdm2.3 Here, we describe the effects of p210 bcr-abl tyrosine kinase on Bax, a p53-dependent proapoptotic member of the Bcl-2 family of proteins. In response to DNA damage and, in particular, to ionizing radiation (IR), it is transactivated by p53 at a 39 bp region at its promoter.4 Bax induction followed by its insertion into, and consequent permeabilization of, the outer mitochondrial membranes represents a 'point of no return' in the cell commitment to apoptotic death. To correlate the p210 bcr-abl tyrosine kinase activity with Bax proapoptotic function, we generated from the 32D murine myeloid progenitor cell line several cell clones stably transducing a temperature-sensitive (ts) mutant of bcr-abl, whose protein owns constitutive tyrosine kinase activity at the permissive temperature of 33°C, but lacks it at the nonpermissive temperature of 39°C. Here, we report the results relative to three of them, namely 2D, 5B and 7B, as representative of all. Ts p210 bcr-abl-transducing cell clones kept at either temperatures were assayed for Bax induction by a low dose (4 Gy)/low-dose rate (0.05 Gy/min) gamma irradiation between 8th and 10th week from electroporation. By that time, bcr-abl expression levels did not exhibit significant differences.5 The radio-induced DNA damage elicits pathways strictly p53-dependent to either halt the cell cycle progression or trigger the apoptotic death. P53-dependent radio responses are conditional upon the wild-type (wt) status of p53 gene. However, in spite of the wt status of p53 gene (preliminarily assayed by means of the direct analysis of the gene coding sequence from 5th to 9th exon, the region accounting for most point mutations in cancer cells and cancer cell lines), the p53 protein radioinduction was precluded by p210 bcr-abl tyrosine kinase. In fact, p53 protein levels following IR exposure increased in 32D parental cell line and in 2D, 5B and 7B cell clones kept at 39°C, but not at 33°C (data not shown). Apoptosis, the death fate of irradiated cells, was almost completely abrogated by p210 bcr-abl tyrosine kinase. As shown in Figure 1a, by the 16th h from irradiation, the percentage of apoptotic cells, assayed by cytofluorimetric analysis of Annexin V staining and propidium iodine (PI) uptake, was increased from less that 2% up to 42.92.1% in 32D parental cell line and from 2.5% up to 47.11.41% in 7B cell clone (shown as representative of the other two) kept at 39°C (P<0.001 in both cases), but remained steady in 7B cell clone kept at 33°C (4.320.78 vs 3.390.11% of untreated controls) (P<0.1). Differences in the radio-induced apoptotic death relative to temperatures are statistically significant (P<0.05 or less). In irradiated 7B cells kept at 39°C, apoptosis was accelerated as a likely consequence of higher temperature (40.12.03% were already late apoptotic). The slightly significant (P<0.05) increase of late apoptotic, Annexin V/PI-positive, cell percentage (from 0.790.05 up to 11.171.01%) in 7B cell clone at 33°C suggests that necrosis rather than apoptosis was the cause of radio-induced death. Accordingly, the characteristic ladder of 200 bp DNA fragments resolved on agarose gel, caused by the endonuclease activation and chromatin cleavage into nucleosome or polynucleosome chains, was seen in irradiated 32D parental cell line and in ts p210 bcr-abl-transducing cell clones kept at 39°C, but not at 33°C (data not shown). The results of previous studies provided evidence for the key role of Bax in the radio-induced apoptotic cell death.4 According to a proposed model, Bax induction, followed by its homo- or heterodimerization and conformational changes, leads to its repositioning at the mitochondrial membranes and to the formation of pores, the last step before the mitochondrial disruption and caspase activation. Thus, we investigated IR effects on Bax expression at transcriptional and post-transcriptional levels. For this purpose, we used competitive PCR and immunoprecipitation/immunoblotting techniques. Competitive PCR is based upon the coamplification of the sample template (target), together with increasing amounts of a DNA fragment (competitor) that shares with the target the primer recognition sites, while differing in size. The linear correlation between the intensities of the amplification product signals and the amounts of competitor molecules added to single PCR reactions was preliminarily validated.5 Thus, at the equivalence point, corresponding to a 1:1 ratio between the two signals, the amount of target equals that of the competitor. The expression of G3PDH housekeeping gene was kept as an internal control. In 32D parental cell line, the constitutive Bax levels were very low: they accounted for approximately 3.3 106 molecules/g of total RNA, 220-fold less than the G3PDH housekeeping gene. In 32D parental cell line, Bax radioinduction started by 4th h and persisted up to 24th h from irradiation (data not shown). By 16th h from irradiation, the number of Bax transcript molecules was increased 3.350.27-fold (P<0.01) (Figure 2a). Unexpectedly, bcr-abl expression was associated with a significant (P < 0.01) increase of Bax transcript molecules, regardless of the p210 tyrosine kinase activity. As shown in Figure 2a, in 2D, 5B and 7B cell clones kept at 33°C Bax expression was 2.930.31-, 3.600.33- and 3.620.28-fold higher than that of 32D parental cell line, respectively, and no significant differences relative to temperature were apparent. The findings support the presence within the bcr-abl fusion gene of domain(s) other than the one coding for the abl tyrosine kinase eventually involved in Bax transcription. Alternatively, Bax overexpression might result from the enhanced expression and transcriptional activation of c-Myc by bcr-abl through mechanisms not yet completely elucidated. The human Bax is, in fact, considered as a potentially c-Myc-responsive gene based on the presence of four E-box sequences downstream of its transcriptional start site at the 5'-untranslated region.6 Bax radioinduction was significantly affected by p210 bcr-abl tyrosine kinase. As shown in Figure 2a, Bax transcript molecule numbers remained steady in irradiated 2D, 5B and 7B cell clones kept at 33°C, but were increased of 3.600.13-, 2.720.41- and 3.980.37-fold compared to untreated controls in cell clones kept at 39°C. The differences in Bax radioinduction relative to temperatures were highly significant (P<0.001). Accordingly, Bax protein levels did not undergo significant changes in irradiated 2D, 5B and 7B cell clones kept at 33°C and were, conversely, increased in 32D parental cell line and in 2D, 5B and 7B cell clones kept at 39°C (Figure 2b). Any earlier or later Bax radioinduction in 2D, 5B and 7B cell clones kept at 33°C, eventually occurring by the 4, 8, 12 and 24 h from irradiation, were excluded by experiments not shown here. In 32D parental cell line, Bax protein induction by IR resulted from a significant (P<0.001) increase in the percentage of Bax-positive cells (from 22.303.11% of untreated control up to 85.803.94%) and a concomitant intensification of Bax positivity/single cell (data not shown). Both were lacking in ts p210 bcr-abl-transducing 32D cell clones kept at 33°C. Moreover, confocal microscope analysis of Bax immunostaining revealed the protein translocation from cytosol to mitochondrial membranes in response to the radio-induced apoptogenic stimulus in 32D parental cells, but not in 2D, 5B and 7B cell clones kept at 33°C (data not shown). Thus, our results ascribe an apoptosis-'proficient' profile to 32D cells and, more in general, to hematopoietic progenitors, and the antiapoptotic potential of p210 bcr-abl tyrosine kinase, or at least part of it, to the functional inhibition of Bax. However, Bax is neither the only nor the main signal involved in the prolonged life expectancy of CML hematopoietic progenitors. Accordingly, p210 bcr-abl expression, but not its tyrosine kinase activity, was associated with Bcl-xL overexpression (Figure 2b). Bcl-2 expression in 2D, 5B and 7B cell clones at either temperatures was, conversely, quite similar to that of 32D parental cell line (Figure 2b). The last findings disagree with previous studies carried out on different cell types. Post-transcriptional mechanism(s), eventually proceeding from the block of Bax-mediated mitochondrial contact site formation by overexpressed Bcl-xL and/or from the inhibition of Bid-dependent translocation of Bax from the cytosol to mitochondria and Bax/Bak heteromerization at the mitochondrial membranes (both resulting from a putative Bcl-2 overexpression) might concur in precluding Bax proapoptotic functions.7,8 To conclude, Bax emerges as a critical mediator of apoptotic death of hematopoietic progenitors. Its inhibition allows CML hematopoietic progenitors to escape a critical physiological response to stress and provides them with a survival advantage likely contributing to their clonal evolution towards a mutator, drug-resistant phenotype.9 Bax might therefore be considered as a sensitive prognostic indicator of the disease and a potential target of novel proapoptotic molecules. This study was supported by the University of Bologna (ex 60%), MURST (Cofin 2002), Carisbo Foundation and Forlì-Cesena AIL. MM and MB are supported by the Centro Interdipartimentale di Ricerche sul Cancro 'Giorgio Prodi'. GB is a recipient of a grant entitled to Mrs Lalla Seràgnoli.