Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 22 (12), 4189-201

Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G(2)/M Phase, and Protection From Apoptosis

Affiliations

Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G(2)/M Phase, and Protection From Apoptosis

Artur Slupianek et al. Mol Cell Biol.

Abstract

Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGF beta R, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkin's lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G(2)/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G(2)/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G(2)/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

Figures

FIG. 1.
FIG. 1.
FTK-induced drug resistance. Parental BaF3 cells and cells expressing BCR/ABL-related FTKs were exposed to cisplatin or mitomycin C and plated in methylcellulose in the presence of IL-3. Cell viability was assessed after 7 days by the clonogenic assay. Results represent the mean ± standard deviation (SD) from three independent experiments.
FIG. 2.
FIG. 2.
Role of RAD51 in FTK-mediated drug resistance. (A) Expression of RAD51 in FTK-transformed cells. Total cell lysates were obtained from BaF3 cells (P) and cells transformed by the indicated FTK (BCR/ABL [B/A], TEL/ABL [T/A], TEL/JAK2 [T/J], TEL/PDGFβR [T/P], NPM/ALK [N/A], and TEL/TRKC[L] [T/T]) cultured in the presence (+) or absence (−) of IL-3. RAD51 protein expression was assessed by Western analysis. Actin was detected as a loading control. Results are representative of two experiments. (B) Role of STAT5 in FTK-stimulated transactivation of the RAD51 promoter. The luciferase assay was performed in Tkts13 cells transiently transfected with the plasmid containing the indicated FTK or the empty plasmid (control, C) along with the plasmid carrying STAT5-DNM (DNM) or empty plasmid (E) and the plasmid encoding the luciferase reporter gene driven by the RAD51 promoter. Luciferase activity is expressed in arbitrary units. (C) Role of RAD51 in FTK-mediated inhibition of drug-induced apoptosis. BaF3 cells (P) and cells stably transfected with FTKs were infected with a retroviral construct containing RAD51(AS)-IRES-GFP (black bars) or with empty IRES-GFP virus (white bars). GFP+ cells were sorted and cultured in the presence of IL-3 with 1.5 μg of cisplatin per ml or 0.06125 μg of mitomycin C per ml for 48 h. Cell viability was assessed by the trypan blue dye exclusion test (upper panels). RAD51 expression in GFP+ cells transfected with IRES-GFP (lanes 1) or RAD51(AS)-IRES-GFP (lanes 2) was determined by Western analysis (lower panel). (D) RAD51-mediated HRR in FTK-positive cells. Draa-40 recombination-reporter cells were cotransfected with plasmids encoding FTKs or empty plasmid (E), I-SceI (to induce a DSB within one of the heterozygous GFP alleles), and either an empty plasmid (white bars) or a plasmid containing RAD51(AS) cDNA (black bars). GFP+ cells were counted after 48 h by flow cytometry. RAD51 expression in cells transfected with an empty plasmid (lanes 1) or RAD51(AS) cDNA (lanes 2) was determined by Western analysis (lower panel). Results in sections B, C, and D represent the mean ± SD from three independent experiments.
FIG. 3.
FIG. 3.
Overexpression of RAD51 only partially restored therapeutic drug resistance of BCR/ABL cells treated with STI571. 32Dcl3 cells (Parental) and BCR/ABL-expressing cells were infected with a retrovirus encoding RAD51-IRES-GFP (Parental+RAD51 and BCR/ABL+RAD51 cells, respectively) or IRES-GFP (Parental and BCR/ABL cells, respectively). GFP-positive cells were isolated by fluorescence-activated cell sorting, and the ABL kinase-selective inhibitor STI571 (1 μM) was added (+) or not (−) for 24 h in the presence of IL-3. (A) Expression of RAD51 and tyrosine phosphorylation of cellular proteins were examined by immunoblotting with anti-RAD51 antibody and antiphosphotyrosine antibody, respectively. Results are representative of two independent experiments. (B) Cells were plated in methylcellulose in the presence of IL-3 with the indicated concentrations of cisplatin or mitomycin C. Colonies were counted 7 days later. Results show the percentage of clonogenic cells (mean ± SD) from three independent experiments.
FIG. 4.
FIG. 4.
Expression of Bcl-xL and induction of DNA damage-dependent G2/M delay in FTK-transformed cells. (A) Western analysis of the expression of Bcl-xL and Bcl-2 in BaF3 parental cells (Parental) and in cells transformed with the indicated FTK and cultured in the presence of IL-3. Actin was detected to show the protein load. (B) The same cells were treated with 1 μg of cisplatin per ml, and cell cycle distribution was analyzed 0, 8, 12, 16, and 24 h later by flow cytometry after staining of DNA with propidium iodide (cell cycle phases: 1, subdiploid; 2, G0/G1; 3, S; and 4, G2/M). Results represent two independent experiments.
FIG. 5.
FIG. 5.
RAD51, Bcl-xL, and G2/M delay worked in concert to induce drug resistance. (A) Expression of RAD51 and Bcl-xL (Western analysis) and (B) cell cycle (propidium iodide staining followed by flow cytometry) were examined in 32Dcl3 parental cells (parental) (lane A1, panel B1) and clones transfected with BCR/ABL (lane A2, panel B2), RAD51 (lane A3, panel B3), Bcl-xL (lane A4, panel B4), or RAD51 and Bcl-xL (lane A6, panel B6). Nocodazole was applied to induce transient G2/M arrest in parental cells (lane A5, panel B5) and in cells transfected with RAD51 (lane A7, panel B7) or RAD51 and Bcl-xL (lane A8, panel B8). (C) Cells characterized in panels A and B were plated in methylcellulose in the presence of IL-3 and cisplatin or mitomycin C. Colonies were scored after 7 days. Results represent three independent experiments (mean ± SD).
FIG. 6.
FIG. 6.
Overexpression of RAD51 and Bcl-xL and G2/M delay are exhibited by BCR/ABL-positive primary leukemia cells: role in drug resistance. (A) Downregulation of RAD51 increased sensitivity of CML-BC cells to cisplatin and mitomycin C. CML-BC patient cells were infected with RAD51(AS)-IRES-GFP virus (GFP+AS, •) or with IRES-GFP empty virus (GFP, ▪). Downregulation of RAD51 in GFP-positive cells was determined by immunofluorescence analysis visualizing the levels of endogenous RAD51 (left panel). Drug sensitivity was assessed by the trypan blue exclusion test after 48 h of exposure to the indicated concentrations of cisplatin or mitomycin C. (B) BCR/ABL enhances expression of Bcl-xL in primary bone marrow cells. Murine bone marrow cells were infected with BCR/ABL-IRES-GFP virus (GFP+BCR/ABL) or IRES-GFP (GFP) empty virus. Bcl-xL expression was examined by Western analysis in GFP-positive cells. (C) BCR/ABL causes G2/M delay essential for drug resistance. GFP+BCR/ABL-positive cells and GFP-positive control cells were treated with 0.375 μg of cisplatin per ml in the presence of IL-3, and cell cycle analysis was performed after 0, 12, 24, and 48 h (left panel). GFP+BCR/ABL cells cultured in the presence of IL-3 were left untreated (1) or treated with 2 mM caffeine (2), 0.375 μg of cisplatin per ml (3), 0.375 μg of cisplatin per ml plus 2 mM caffeine (4), 1.5 μg of cisplatin per ml (5), 1.5 μg of cisplatin per ml plus 2 mM caffeine (6), 0.1 μg of mitomycin C per ml (7), or 0.1 μg of mitomycin C per ml plus 2 mM caffeine (8). Twenty-four hours later, cells were plated in methylcellulose, and colonies were scored after 7 days (right panel). Results are presented as the percentage of colonies in experimental samples in comparison to the untreated control sample 1 (602 ± 37 colonies arose from 103 untreated GFP+BCR/ABL cells). ∗, P < 0.05 in comparison to the corresponding group not treated with caffeine. Cell cycle analysis of the selected samples (1, 3, and 4) was performed after 24 h of treatment to confirm that caffeine abolished accumulation of the cells in G2/M. Results represent two experiments.
FIG. 7.
FIG. 7.
Model of drug resistance induced by FTKs. The basal level of RAD51 in normal hematopoietic cells is regulated by the physiological signaling from the ligand (L)-receptor (R) complex. It can be further modified by DNA damage (tyrosine phosphorylation). RAD51-mediated HRR does not efficiently repair DSBs during a short G2/M phase. Remaining DSBs and other lesions trigger death pathways. This pronounced signal could not be inhibited by the basal levels of Bcl-xL resulting in the release of cytochrome c from mitochondria and activation of caspase-3. In contrast, FTK-transformed cells contain high levels of RAD51 modified by tyrosine phosphorylation. HRR is pronounced in these cells, and transient arrest in the G2/M phase provides additional time for repair. Thus, DSBs are repaired with high efficiency, and apoptotic signaling is significantly diminished. Modest apoptotic signaling in FTK-positive cells can be inhibited by elevated levels of Bcl-xL, which prevent the release of cytochrome c from mitochondria and activation of caspase-3. Because of this “antiapoptotic umbrella” provided by Bcl-xL, FTK-transformed cells can tolerate other (possibly mutagenic) DNA lesions. In addition, dysregulated HRR may result in unfaithful repair of DSBs. Taken together, FTK-positive cells can accumulate DNA lesions leading to genomic instability and malignant progression of the disease. Blue, ligand-receptor-induced events; red, FTK-induced events.

Similar articles

See all similar articles

Cited by 52 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback