Impact of p210 on Ultraviolet C Wavelength-induced DNA Damage and Repair

Recently, it was shown that both Bcr and Bcr-Abl can interact with xeroderma pigmentosum group B (XPB/ ERCC3), a protein implicated in DNA repair after UVinduced damage. To further analyze the effect of Bcr-Abl on the DNA damage response, we used cell lines stably transfected with the BCR-ABL gene and their parental counterparts (MBA-1 versus MO7E and Bcr-AblT1 versus 4A2 pZAP) and several assays reflecting DNA repair: the comet assay, a radioimmunoassay for cyclobutane pyrimidine dimers, and clonogenic assays. After exposure to UVC (0.5– 5.0 joules m ), the Comet assay demonstrated greater efficiency of DNA repair in the BCR-ABL-positive cells (both MBA-1 and Bcr-AblT1) when compared with their parental counterparts. Furthermore, there was less production of the UV-induced DNA adduct—cyclobutane pyrimidine dimers— as well as a more rapid rate of disappearance of these adducts and greater UV survival (clonogenic assays) in MBA-1 cells as compared with MO7E cells. Apoptosis (annexin V-FITC/ propidium iodide staining) was markedly reduced in the BCR-ABL-positive cells. These results indicate that BCRABL confers enhanced resistance to UV radiation-induced damage and increased efficiency of DNA repair and that these changes are associated with a protective antiapoptotic effect. INTRODUCTION The BCR-ABL oncogene is the molecular hallmark of Phpositive CML and results from a reciprocal translocation [t(9,22)(q34;q11)] between chromosomes 9 and 22 (1, 2). The major protein products (p190 and p210) are characterized by elevated levels of tyrosine kinase activity, which contributes to the transforming capability of BCR-ABL (3–8). There is now cogent data supporting the central role of Bcr-Abl in the pathogenesis of Ph-positive leukemias. For example, p190 or p210-bearing transgenic mice develop acute lymphocytic leukemia or a CML-like myeloproliferative disorder (9–11). In addition, therapeutic application of the BcrAbl-specific kinase inhibitor—STI 571—has yielded substantial responses in patients with Ph-positive leukemias (12, 13). The Bcr-Abl protein has been linked to a variety of cytoplasmic signaling pathways (Refs. 14–18, reviewed in Ref. 19). Interestingly, Abl resides both in the cytoplasm and the nucleus (15, 20), and Bcr—although originally localized to the cytoplasm—is now known to have a nuclear fraction as well (15, 21, 22). In the nucleus, the Bcr protein was found to associate with condensed DNA such as metaphase chromatin, as well as the highly condensed heterochromatin in interphase cells (21). Of importance in this regard, it has recently been shown (through yeast two-hybrid screening) that both Bcr and Bcr-Abl can interact with the XPB protein (23, 24). XPB is known to be critical in the DNA repair process. Indeed, patients with xeroderma pigmentosum develop serious skin damage and tumors after exposure to UV light, and protection from sunlight is critical in these individuals (reviewed in Ref. 25). Abl, through its interaction with the ATM protein product (ATM being the gene mutated in ataxia telangectasia, a disorder characterized by hypersensitivity to ionizing radiation), has also been linked to the DNA damage response process (26, 27). Further, Abl tyrosine kinase is a downstream target of phosphorylation and activation by the ATM kinase after ionizing radiation (27), and both proteins appear to mediate signals required for the assembly of Rad51 and Rad52 recombination complex (28). (Rad51 and Rad52 play a key role in DNA repair of double-strand DNA breaks that may result from drugor -irradiation-induced damage.) Attenuation of DNA repair in BCR-ABL-positive cells has been found by some investigators (24, 29, 30) but not by others (31, 32). Slupianek et al. (32) recently suggested that in response to cytotoxic drugs, Bcr-Abl actually enhances repair of double-stranded DNA breaks. To additionally assess the impact of Bcr-Abl on DNA repair, we compared UVC-induced DNA damage and repair in BCR-ABL-transfected cell lines and their parental controls using several distinct assays (33–42): (a) single-cell gel electrophoresis (Comet assay) [used to detect incision events that are part of the post-UVC DNA repair process (33)]; (b) RIA to measure specific DNA damage, i.e., CPDs (34, 35); and (c) clonogenic assays to measure cell survival (36). Our results suggest that in the presence of Bcr-Abl, UVC-induced DNA damage is decreased and repair is facilitated. Furthermore, the more efficient DNA damage response was associated with a significant reduction in UVC-induced programmed cell death. Received 11/4/02; revised 2/28/03; accepted 3/3/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Bioimmunotherapy, Box 422, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-1226; Fax: (713) 745-2374; E-mail: [email protected]. 2 The abbreviations used are: Ph, Philadelphia; CML, chronic myelogenous leukemia; XPB, xeroderma pigmentosum group B; NER, nucleotide excision repair; CPD, cyclobutane pyrimidine dimer; PI, propidium iodide; GM-CSF, granulocyte macrophage colony-stimulating factor. 3722 Vol. 9, 3722–3730, September 1, 2003 Clinical Cancer Research Research. on January 29, 2018. © 2003 American Association for Cancer clincancerres.aacrjournals.org Downloaded from MATERIALS AND METHODS Cell Lines. We used the human hematopoietic cell line, MO7E, and its stably transfected p210 derivative, MBA-1 [kindly provided by Dr. John E. Dick (University of Toronto, Toronto, Ontario, Canada) and described by Sirard et al. (37)]. We also used the human fibroblast cell line, 4A2 , and its derivatives transfected with either empty vector (pZAP) or human p210-pZAP (Bcr-AblT1) [kindly provided by Dr. Jean Y. J. Wang (University of California–San Diego, La Jolla, CA) and described by Renshaw et al. (38)]. The MBA-1 cells were maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) containing 15% FCS, whereas the growth factordependent parent cell line, MO7E, was cultured in RPMI 1640 containing 10% FCS plus 100 units/ml GM-CSF (Amgen, Thousand Oaks, CA). The 4A2 cell line and its derivatives were cultured in DMEM (BioWhittaker) containing 10% ironsupplemented FCS. All cells were grown at 37°C in a 5% CO2 incubator. UVC Irradiation. MO7E and MBA-1 cells were seeded in 6-well tissue culture plates at a concentration of 3 10 cells/well in 1.5 ml of media and irradiated with 254 nm of UV light (UVC) [UVXL-1000; Fisher Scientific, Pittsburgh, PA] ranging from 0.5 to 10.0 joules/m (Jm ). Controls were exposed to no UVC. For the comet assays (see below), the cells were allowed to incubate for different lengths of time after irradiation (15 min to 24 h) and subsequently harvested. The 4A2 derivatives were seeded 1 day before treatment under similar conditions as above. For these cells, the media was removed before UV treatment and replaced before additional incubation of the cells at 37°C. For the RIA, MO7E and MBA-1 cells were irradiated with 10 Jm 2 UVC then harvested at different time points (0–48 h) after irradiation. Single-Cell Gel Electrophoresis (Comet) Assay. The Comet assay is used to detect DNA breaks, which are reflected by the migration of DNA (the comet tail) from the nucleus toward the anode (39–41). After UVC exposure, these DNA breaks occur as part of the DNA NER process. [Generally, at low doses, UVC radiation does not induce DNA breaks as a manifestation of damage in of itself (41).] Therefore, after UVC irradiation, longer comet tails reflect more DNA repair (41). The assay was performed in accordance with previously published methods (33) and as modified by Lemay and Wood (42) using the COMET Assay Kit (Trevigen, Inc., Gaithersburg, MD). After harvesting, the cells were washed with PBS and then resuspended at a concentration of 1 10 cells/ml in PBS. The cells were combined at a ratio of 1:10 with molten, low melting point agarose kept at 37°C (Trevigen, Inc.). Sixty l of the mixture were immediately pipetted and evenly spread unto Comet slides that are specially coated with high melting point agarose, which serves as an anchor for the cells/low melting point agarose mixture (Trevigen, Inc.). The slides were kept at 4°C for 10–20 min to allow the agarose to solidify, then immersed in prechilled lysis solution (Trevigen, Inc.) for 30–60 min at 4°C. Subsequently, the slides were immersed into freshly prepared alkali solution (300 mM NaCl and 1 mM EDTA) for 20 min. The slides were transferred to an electrophoresis chamber, and fresh alkali solution was poured to sufficiently cover the slides. Electrophoresis was done at 300 mA, 20 V (constant voltage) for 30–40 min. Subsequently, the slide was rinsed briefly in Tris-EDTA (TE) solution [10 mM Tris (pH 7.5) and 1 mM EDTA] and then fixed in 100% ice-cold ethanol for 5 min. The samples were air-dried at room temperature, and the cells were viewed by staining with 40 l of Sybr green (Trevigen, Inc.) diluted 1:10,000 in TE. The cells were viewed under a fluorescence microscope (excitation/emission at 494 /521 nm; Olympus Corporation, Lake Success, NY). For quantification, we used a visual scoring system under fluorescence microscopy, which was a modification of the method of Collins et al. (43). Two hundred comets on each slide were classified by an observer blinded to the conditions on the slide. Relative tail fluorescence intensity was scored as 1 (none, 25%), 2 (low, 5–20%), 3 (medium, 20–40%), 4 (high, 40–95%), and 5 (total, 95%). Length of tail was similarly scored on a 1 to 5 scale. Fluorescence intensity score was multiplied by length of tail score, giving a final score of 1–25 for each cell. Thus, the total score for 200 comets could range from 1 (all undamaged) to 5000 (all maximally damaged). Scores on different slides were compared by the Mann-Whitney test. Scoring was performed in triplicates. FITC-conjugated Annexin V/PI Staining. To identify cells in the early and late stages of apoptosis, we used the Annexin V-FITC Apoptosis Detection Kit (BD PharMingen, San Diego, CA). Briefly, cells exposed to UV radiation were harvested, washed twice with cold PBS, then resuspended in 1 binding buffer (BD PharMingen) at a concentration of 1 10 cells/ml. One hundred l of the cell suspension were transferred to a 5-ml polypropylene tube, and 5 l each of PI (50 g/ml stock) and annexin V-FITC were added simultaneously. The cells were gently mixed and incubated at room temperature in the dark for 15 min. Three hundred l of 1 binding buffer were added to each tube, and the cells were analyzed immediately by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ). RIA. DNA phosphoproducts such as CPDs are induced by UV irradiation and can be measured by RIA (34, 35). The CPD specificity is derived from the specificity of the antiserum, which was raised against triplet-sensitized UVB-irradiated DNA (containing only CPDs). The methods used to characterize this RIA include enzymatic photoreactivation, digestion with T4 endo V, and mobility shift assays using synthetic oligonucleotides containing CPDs exclusively. For the RIA, 2–5 g of heat-denatured sample DNA were incubated with 5–10 pg of poly(dA dT) (labeled to 5 10 cpm/ g by nick translation with P-dTTP) in a total volume of 1 ml of solution [10 mM Tris (pH 7.8), 150 mM NaCl, 1 mM EDTA, and 0.15% gelatin (Sigma, St. Louis, MO)]. Antiserum was added at a dilution that yielded 30–60% binding to labeled ligand. (The antisera were raised against DNA that was dissolved in 10% acetone and irradiated with UVB light under conditions that have been shown to produce CPDs exclusively.) After incubation overnight at 4°C, the immune complex was precipitated with goat antirabbit immunoglobulin (Calbiochem, San Diego, CA) and carrier serum from nonimmunized rabbits (University of Texas M. D. Anderson Cancer Center, Science Park/Veterinary Division, Bastrop, TX). After centrifugation, the pellet was dissolved in NCS tissue solubilizer (Amersham, Arlington Heights, IL), mixed with ScintiSafe (Fisher, Pittsburgh, PA) containing 3723 Clinical Cancer Research Research. on January 29, 2018. © 2003 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 0.1% glacial acetic acid, and the P quantified by liquid scintillation spectrometry. (Under these conditions, antibody binding to an unlabeled competitor inhibits antibody binding to the radiolabeled ligand.) Sample inhibition is extrapolated through a standard (dose response) curve to determine the number of photoproducts in 10 bases. For standard, we used doublestranded salmon testes DNA (Sigma) irradiated with increasing doses of UVC radiation and heat denatured, aliquoted, and kept frozen at 20°C. Rates of photoproduct induction for the standards were previously determined using nonimmunological enzymatic and biochemical techniques and determined to be 8.1 CPDs/megabase/Jm (2). These details, as well as those concerning the specificities of the RIAs, are described by Mitchell (34, 35). Clonogenic Assays. Clonogenic survival curves were used to assess UVC radiation sensitivity. MO7E and MBA-1 cells were exposed to no radiation (control) or 0.5, 1.0, 5.0, and 10.0 Jm 2 UVC, and the clonogenic assay was performed as described previously (36). Briefly, immediately after irradiation, the cells were cultured in 0.8% methylcellulose (Fluka Chemical Corp., Ronkonkoma, NY), 10% FCS and RPMI 1640 in 1% (vol/vol) methylcellulose. [For the MO7E cells, the medium was supplemented with 250 units of GM-CSF (Amgen)]. The culture mixture was placed in 35-mm Petri dishes (Nunc, Inc., Naperville, IL) in duplicate and maintained at 37°C with 5% CO2 in air in a humidified atmosphere. Colonies were counted after 7 days using an inverted microscope. A colony was defined as a cluster of 40 cells.