Our previous report showed that BCR/ABL kinase elevated the amount of ROS in leukemia cells, which increased the number of DSBs (11
). Here we show that 32Dcl3 cells transformed by BCR/ABL and other FTKs such as TEL/ABL, TEL/JAK2 and TEL/PDGFβR (Supplementary Figure 1
) display higher levels of ROS than the parental cells (). More ROS were also detected in primary Lin−
CP and BC CML cells in comparison to their normal counterparts ().
FTKs enhance the production of ROS and DSBs
By comparison to their respective controls, BCR/ABL and other FTK-transformed 32Dcl3 cells as well as primary Lin−
CP and BC CML cells also showed an increase in γ-H2AX nuclear foci (), which paint DSBs () (11
). This was seen both as a significant increase in the percentage of leukemic cells containing more than 20 γ-H2AX foci () as well as in the average number of γ-H2AX foci per cell (). Based on our previous reports (11
) and this work, we postulate that FTK-mediated enhancement of ROS may be responsible for the generation of an excess of DSBs in leukemic cells.
DSBs are usually repaired by HR and NHEJ, however, a relatively rare and extremely unfaithful SSA pathway can occasionally be used (8
). To examine the potential influence of FTK expression on SSA activity, cells carrying an integrated SA-GFP reporter cassette () were generated as described in the Methods
. Cells transformed by BCR/ABL or other FTKs displayed a 6 to 16-fold enhanced SSA activity by comparison to control cells (). Imatinib completely abolished the non-mutated BCR/ABL kinase-mediated activation of SSA without exerting any effect on enhanced SSA activity stimulated by the imatinib-resistant BCR/ABL-T315I mutant kinase or on the basal SSA activity measured in parental cells. This clearly shows that the enhanced SSA activity was dependent on the functionality of the BCR/ABL kinase. The various degrees of SSA stimulation obtained by different FTKs may reflect quantitative and qualitative differences in their specific kinase activities.
PCR reactions were performed on genomic DNA from GFP− and GFP+ cells using the primers shown in . The results confirmed that functional GFP gene in GFP+ cells was recovered by SSA. As expected, PCR reactions with primers 1–3 generated ~3.5 kb and ~0.8 kb bands from GFP− and GFP+ cells, respectively, detecting intact SA-GFP reporter cassette and SSA-restored GFP gene (). Thus, SSA-mediated DSB repair resulted in genomic instability associated with a loss of ~2.7 kb chromosome fragment. In contrast, PCR reactions with primers 2–3 amplified ~0.8 kb band from GFP− cells and no band from GFP+ cells confirming that a segment containing puroR had been lost during SSA.
Imatinib-resistant BCR/ABL kinase mutants may promote malignant progression in CML patients being treated with imatinib due to the different kinase activities and transforming properties they endow on hematopoietic cells (17
). Therefore, it was of interest to use the SA-GFP reporter system in a similar fashion to compare the SSA activity in 32Dcl3 cells expressing non-mutated and mutated BCR/ABL known to confer imatinib resistance (e.g.; P-Loop Y253H and E255K, T315I, and Activation Loop H396P). Accordingly, additional lines carrying the reporter construct and these mutant forms of BCR/ABL were constructed and analyzed (). The results showed that imatinib-resistant BCR/ABL kinase mutants stimulated SSA in a similar manner as the non-mutated kinase ().
Non-mutated and imatinib-resistant BCR/ABL kinase mutants stimulate SSA in a dose-dependent manner
Enhanced levels of BCR/ABL kinase expression were noted in comparisons of the cells circulating in the blood of CML patients with BC disease as compared to CP (5
). In addition, increased levels of BCR/ABL kinase have been found in the CD34+
CML cells which contain the leukemic stem and progenitor cells responsible for propagation of the malignant clone (19
). Therefore, we also investigated the impact of the level of BCR/ABL gene expression on SSA in 32Dcl3 cells expressing high and low levels of BCR/ABL (). Interestingly, the results showed a 5-fold stimulation of SSA in cells expressing lower levels of BCR/ABL and a 15-fold stimulation of SSA activity in cells expressing higher levels of BCR/ABL (). The evidence of a BCR/ABL “dose-dependent” stimulation of SSA activity was also seen in cells expressing imatinib-resistant BCR/ABL mutants, for example Y253H and E255K ().
A current model of SSA assumes that the RAD52 protein directs the annealing of complementary single strands of DNA and ERCC1/XPF endonuclease is involved in strand-processing steps (20
). FTKs and IL-3 were independently able to stimulate the expression of RAD52 and ERCC1 in 32Dcl3 cells (data not shown). FTK-transformed cells did not manifest significant changes in expression of RAD52 and ERCC1 proteins in comparison to control cells incubated under conditions where FTK-transformed cells showed enhanced SSA (). Interestingly BCR/ABL-positive cells also displayed enhanced co-localization of these proteins in the nucleus in comparison to control cells () suggesting functional modifications of RAD52 and/or ERCC1 in the leukemic cells.
BCR/ABL facilitates RAD52-ERCC1 co-localization
In summary, we have shown that BCR/ABL, TEL/ABL, TEL/JAK2 and TEL/PDGFβR oncogenic tyrosine kinases stimulate SSA, a rare and unfaithful mechanism of DSB repair. Enhanced SSA may have a significant impact on accumulation of additional chromosomal abnormalities such as intrachromosomal deletions and translocations contributing to malignant progression and further treatment resistance of MPDs expressing FTKs (3
). This speculation is further supported by the consistent demonstration of a “dose–dependent” effect of BCR/ABL on SSA activity, given the highly elevated levels of BCR/ABL expression found in CML stem and progenitor-enriched populations even prior to the emergence of BC (19
). Taken together, these findings implicate FTKs as playing an important role in contributing to the generation of treatment resistant and progressed subclones in CML and other MPDs by induction of SSA.