Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR – ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR – ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A significant 3 – 5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR – ABL transfection induced an overexpression of DNA polymerase β, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase β in this process.
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of primitive pluripotent stem cells involving all hematopoietic lineages (Fialkow et al., 1977). The Philadelphia chromosome (Ph) is the cytogenetic hallmark of CML (Rowley, 1973). The Ph abnormality represents a reciprocal translocation, t(9;22)(q34;q11), resulting in the transfer of c-abl sequences from chromosome 9 to a site adjacent to the BCR sequences on chromosome 22 (Heisterkamp et al., 1985) which produces hybrid 190- and 210-kDa activated tyrosine kinase fusion proteins. CML almost always evolves from a chronic, relatively indolent disease to a more aggressive leukemia whose progressive stages are the accelerated phase and blast crisis characterized by increased cellular proliferation, maturation arrest, and karyotypic clonal evolution. Although activation of bcr-abl tyrosine kinase may be essential to the pathogenesis of the chronic phase, genetic alterations responsible for transition to blast crisis are unknown. It is possible that secondary genetic events may be required for this transition. Other nonrandom cytogenetic changes including duplication of Ph, +8, i(17q), +19, +21, −Y, or +Y are encountered in more than 80% of patients with CML in transition (Bernstein, 1988). In addition, frequent activation of protooncogenes and alterations in tumor-suppressor genes such as the p53 gene have been observed in approximatively 30% of patients (Ahuja et al., 1989; Feinstein et al., 1991; Kelman et al., 1989; Mashal et al., 1990). Thus, it is possible that genomic instability and spontaneous error in DNA replication may significantly contribute to the evolution of CML to blast crisis by generating the clonal heterogeneity from which subclones with an increased proliferative advantage can emerge and come to predominate in the acute leukemic phase. It has been shown that p210bcr/abl transformed cells are karyotypically unstable, develop a proliferative advantage in vitro over time, and appear to mimic the changes associated with the transition of CML to AML (Laneuville et al., 1992). In this report, we hypothesized that the BCR – ABL translocation may confer an early genomic instability at the nucleotide level by increasing the spontaneous rate of mutations. We present evidence that BCR – ABL transfected Ba/F3 cells exhibit a weak but significant mutator phenotype which is concomitant to an increase in the level and activity of DNA polymerase β, the least accurate of all the known eukaryotic DNA polymerases.
Spontaneous mutation frequencies in BCR – ABL transfected Ba/F3 cells
To test whether BCR – ABL transfection in cells could result in an increased frequency of spontaneous mutation, we measured the appearance of mutational events leading to the ouabain and 6-thioguanine (6-TG) resistant phenotypes at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci respectively. Table 1 shows the mutation frequencies which were corrected for plating efficiency. A significant threefold increase in HPRT mutant frequency was found for the p190- and p210-transfected cell lines relative to the non transfected control Ba/F3 cell line. In the case of the ouabain resistant mutants, a 4.5- and 5.4-fold increase was measured for the p190 and p210 fusions respectively. Taken together, these data showed that the activation of bcr-abl tyrosine kinase resulted in a weak but significant spontaneous mutator phenotype.
DNA polymerase β gene expression in BCR – ABL transfected Ba/F3 cells
A recent study from our laboratory demonstrated that transfection of CHO cells by a vector overexpressing the DNA Polymerase β (Pol β) resulted in a spontaneous mutator phenotype (Canitrot et al., 1998). Pol β, structurally the simplest of the five known mammalian DNA polymerases, is believed to function primarily in the repair of damaged bases (Sobol et al., 1996). It is a monomeric protein of 335 amino acids (39 kDa) that lacks exonuclease activities and appears to be the least accurate of the eukaryotic DNA polymerases in vitro (Kunkel, 1985). In vivo, it is expressed at a constant low level throughout the cell cycle (Zmudzka et al., 1988) and is inducible by some genotoxic treatments (Fornace et al., 1989). The increased mutation frequency we observed for the BCR – ABL transfected Ba/F3 cells urged us to compare the expression of Pol β between the untransfected control Ba/F3 cells and the p190- and p210-transfected Ba/F3 cells. Western blot analysis of cell extracts showed an enhanced expression of Pol β in the transfected cell lines compared to the control ones (Figure 1A). Quantitative analysis of the overexpression by PhosphoImager scanning showed a 4±0.6-fold increase for p190(5) clone derived cell line and a 2±0.3-fold increase for p210(4) clone derived cell line (Figure 1B). These two clones were used for the Pol β activity assays. In order to investigate the specificity of our observation, we analysed the level of expression of another DNA polymerase, the replicative DNA polymerase δ. We showed that it was not significantly affected in both clones (see Figure 1C). Comparable enhanced expression of Pol β was obtained when six independent transfected clones were analysed (Figure 2).
DNA polymerase β activity in BCR – ABL transfected Ba/F3 cells
To test whether the increased expression of Pol β in p190- and p210-transfected cell lines was correlated with an elevated Pol β activity, replicative cell extracts were prepared and tested for their sensitivity to the nucleotide analog ddCTP, known to strongly inhibit in vitro and in vivo the polymerization of DNA catalyzed by Pol β (Bouayadi et al., 1997; Copeland et al., 1992). In vitro, it has been demonstrated that Pol β efficiently incorporates the ddCMP chain terminator with efficiency comparable to that of dCMP when an oligonucleotide template is used (Copeland et al., 1992). A G-rich 60-mer oligonucleotide DNA template was annealed to a 5′-phosphorylated 17-mer oligonucleotide (Figure 3A) and primer extension reactions were conducted in vitro using extracts of either control or transfected cells. The newly synthetized DNA products were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. Figure 3B shows the replication of the DNA template in presence of various concentrations of ddCTP. Full-size products (60-mer) were observed after 1 h incubation at 37°C (see arrow at 60-mer in Figure 3B). Pol β activity in the replicative cell extracts was determined by monitoring the ability of the extracts to incorporate the chain terminator ddCTP, inhibiting the DNA synthesis elongation and therefore resulting in the disappearance of the full-size products. As it can be observed in Figure 3B, the increased concentrations of ddCTP induced a disappearance of the 60-mer full-size products when extracts from p190 and p210-transfected cells were used while no significant inhibition was detected for the control cell extracts (compare lane cont and lanes p190 and p210). This indicates that the Pol β activity is enhanced in the replicative extracts from the BCR – ABL transfected cells.
The evolution of CML from a chronic to an acute leukemic phase is the main determinant of patient survival. Although the molecular mechanisms underlying this progression are poorly understood, genetic instability is generally appreciated to play a central role in the pathogenesis of CML. Strong evidence supported that this phenotypic evolution is associated with the induction of genetic instability at the chromosomal level, including both numerical and structural chromosomal abnormalities in p210bcr/abl transformed cells, but these events are relatively rare occurrences in culture cells unless defects in DNA metabolism like repair, recombination, or replication, suggesting that genetic alteration at the nucleotide level may be involved (Laneuville et al., 1992). Furthermore, the secondary activation of N-ras (Ahuja et al., 1991; Collins et al., 1989; LeMaistre et al., 1989) and c-myc (McCarthy et al., 1984) genes and the functional inactivation of the anti-oncogene p53 observed frequently in patients developing blast crisis support the hypothesis of an early alteration of a gene `guardian' of the genome confering a mutator phenotype at the nucleotide level (Loeb, 1991). Finally, genomic instability of microsatellite repeats associated with a late genetic event during the evolution of CML has been demonstrated (Wada et al., 1994). For all these reasons, we explored the intriguing possibility whether p190bcr/abl and p210bcr/abl can directly interfere with these DNA transactions.
The present study shows for the first time the occurrence of a mutator phenotype at the nucleotide level in Ba/F3 cells transfected by the p190- and p210-BCR – ABL fusion proteins. Using two conventional methodologies testing the appearance of mutational events leading to a resistance phenotype, the Na-K-ATPase and the HPRT tests, we showed significant 3 – 5-fold increases of the spontaneous mutation frequency in the transfected cells.
A mutator phenotype may involve the large number of genes required to maintain the integrity of the human genome. For example, mutations in mismatch repair and nucleotide excision repair genes, as well as deficiencies that disrupt cell cycle checkpoints have been shown to predispose carriers to cancer, presumably by increasing genomic instability (Cahill et al., 1998; Wind et al., 1995). We recently identified a new category of genetic event, overexpression of Pol β, the most inaccurate of all the mammalian DNA polymerases, that increased the spontaneous mutation frequency in CHO cells, supporting the concept that cancer may involve a mutator phenotype by higher expression of an error-prone DNA polymerase (Canitrot et al., 1998). Higher expression of Pol β has been observed in many tumor cell lines (Scanlon et al., 1989), including colon and ovarian cells. Thus, we explored the possibility that the Ba/F3 cells transfected by the fusion p190 and p210 proteins, which displayed a mutator phenotype, exhibited a higher level of Pol β. We found a 2 – 4-fold increase in the amount of the enzyme and we showed that Pol β activity is also enhanced. These data suggest that loss of fidelity in DNA replication may be induced by the BCR – ABL rearrangement and may contribute to CML evolution. This inaccuracy in DNA synthesis may be one of the initial events explaining the frequent occurrence of alterations in microsatellites in the evolution of CML (Wada et al., 1994). Further work will be required to investigate the molecular links between BCR – ABL tyrosine kinase activity and expression of Pol β. For this purpose, the existence of palindromic elements in Pol β – promoter required for p21v-rasH-mediated stimulation (Kedar et al., 1990) may involve the p21 Ras proto-oncogene, which is constitutively activated in BCR – ABL cells and contribute to their transforming phenotype (Mandanas et al., 1993; Sawyers et al., 1995).
Materials and methods
Control and transfected Ba/F3 cell lines (p190 and p210 BCR – ABL) were previously described (Ahmed et al., 1998). Ba/F3 cell lines were maintained in RPMI containing 10% FCS supplemented with antibiotics in 5% CO2 at 37°C with the addition of 10% WEHI conditional medium as a source of IL-3 to the parental cell line.
Cells (3×106) were lysed in SDS containing buffer according to the protocol of Shah et al. (1995). For analysis of pol β, total cell lysates (200 μg protein) were electrophoresed in a 12% SDS – PAGE gel (to monitor pol β expression) or 7.5% SDS – PAGE (to monitor pol δ expression) and transferred to PVDF membrane (Schleicher and Schull). Blots were blocked in TBS-T (0.1% Tween) with milk (5% skimmed milk) and incubated with pol β polyclonal antibody (1/5000) (kindly provided by Dr S Wilson, Research Triangle Park, USA) or with pol δ polyclonal antibody (1/1000) (kindly provided by Pr U Hübscher, Zürich, Switzerland) followed by incubation with horseradish peroxidase conjugated anti rabbit IgG, and revealed using the ECL system (Amersham). Equal loading was determined using monoclonal antibody to actin (1/5000) (Chemicon, Euromedex, France). Different time exposures of films were scanned to avoid saturation and quantification analysis was achieved by PhosphoImager Storm-system analysis (Molecular Dynamics) using Imagequant software.
Preparation of cell extracts and DNA polymerase β activity assay
Replicative cell extracts were prepared as previously described (Hoffmann et al., 1996). Briefly, the control and BCR – ABL-transfected Ba/F3 cells were washed with ice-cold PBS, harvested by centrifugation, and the cell pellets were suspended in hypotonic buffer (10 mM Tris-HCl, pH 7.5, 10 mM KCl, 10 mM MgCl2, 1 mM DTT) containing protease inhibitors. The cells were spontaneously disrupted during incubation at 2°C. Nuclei were harvested by centrifugation and the nuclear proteins were extracted in hypotonic buffer containing 350 mM NaCl. Cytosolic and nuclear extracts proteins were precipitated by addition of ammonium sulphate and the precipitates were resuspended in dialysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM mono-K glutamic acid, and 10% glycerol) and dialyzed for 2 h at 4°C. The extracts were frozen in liquid nitrogen and stored at −70°C.
To study the pol β-dependent replication activity, a 60-mer oligonucleotide was hybridized to a 5′-32P-labeled 17-mer synthetic primer to serve as a DNA template. This template (5 ng) was replicated in vitro by the cell extracts (3 μg protein) in reactions (15 μl) containing 45 mM HEPES-KOH (pH 7.8), 7 mM MgCl2, 1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 3.4% glycerol, 65 mM potassium glutamate, 18 μg of BSA, 200 μM each dATP, dGTP, and dTTP, and various ratios of ddCTP/dCTP as indicated in the legend of Figure 3. At the end of the reaction, 5 μl of stopping buffer (90% formamide; 0.1% xylene cyanol; 0.1% bromophenol blue; 0.1 mM EDTA) were added. Samples were denatured for 10 min at 70°C and loaded onto a 15% polyacrylamide/7 M urea/30% formamide gel.
Determination of the mutation frequencies
Cells from replica cultures were counted and resuspended in RPMI supplemented with 10% FCS, 0.9% methylcellulose (Stem Cell Laboratories, TEBU, France) and 10% WEHI for the parental cell line. Cells (1×106 or 0.5×106 per 35 mm Petri dishes) were plated in selective medium containing 2 mM ouabain or 20 μM 6-TG respectively and incubated at 37°C in a humidified 5% CO2 atmosphere for 1 week in order to determine the number of the Na-K-ATPase and the HPRT mutant colonies respectively. The colonies larger than 50 cells were counted under microscope and mutant frequencies were calculated by correcting for plating efficiency.
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This work was supported by La Ligue contre le Cancer – Comité de la Haute-Garonne (grants to YC and JSH). l'Association pour la Recherche sur le Cancer (grant to JSH) and by La Ligue Nationale contre le Cancer (grant to AGT).
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Canitrot, Y., Lautier, D., Laurent, G. et al. Mutator phenotype of BCR – ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase β. Oncogene 18, 2676–2680 (1999) doi:10.1038/sj.onc.1202619
- mutator phenotype
- DNA polymerase β
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