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[Cancer Research 65, 6622-6630, August 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

High Mobility Group A2 Potentiates Genotoxic Stress in Part through the Modulation of Basal and DNA Damage–Dependent Phosphatidylinositol 3-Kinase–Related Protein Kinase Activation

Lee Ming Boo1, H. Helen Lin1, Vincent Chung5, Bingsen Zhou5, Stan G. Louie3, Michael A. O'Reilly6, Yun Yen1,5 and David K. Ann1,2,4

Departments of 1 Molecular Pharmacology and Toxicology, 2 Medicine, and 3 Pharmacy, and 4 Norris Cancer Center, University of Southern California, Los Angeles, California; 5 Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte, California; and 6 Department of Pediatrics, University of Rochester, Rochester, New York

Requests for reprints: David K. Ann, University of Southern California, Health Science Campus, PSC-209, 1985 Zonal Avenue, Los Angeles, CA 90033-1049. Phone: 323-442-3409; Fax: 323-224-7473; E-mail: ann{at}usc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The high mobility group A2 (HMGA2) protein belongs to the architectural transcription factor HMGA family, playing a role in chromosomal organization and transcriptional regulation. We and others have previously reported that ectopic HMGA2 expression is associated with neoplastic transformation and anchorage-independent cell proliferation. Here, we reported a correlation between increased HMGA2 expression and enhanced chemosensitivity towards topoisomerase II inhibitor, doxorubicin, in breast cancer cells. Using cells exhibiting differential HMGA2 expression and small interfering RNA technique, we showed that HMGA2 expression modulates cellular response to the genotoxicity of DNA double-strand breaks. Notably, HMGA2 enhances doxorubicin-elicited cell cycle delay in sub-G1 and G2-M and augments cell cycle dysregulation on cotreatment of doxorubicin and caffeine. We further reported that HMGA2 induces a persistent Ser139 phosphorylation of histone 2A variant X, analogous to the activation by doxorubicin-mediated genotoxic stress. Moreover, this HMGA2-dependent enhancement of cytotoxicity is further extended to other double-strand breaks elicited by cisplatin and X-ray irradiation and is not restricted to one cell type. Together, we postulated that the enhanced cytotoxicity by double-strand breaks in HMGA2-expressing cells is mediated, at least in part, through the signaling pathway of which the physiologic function is to maintain genome integrity. These findings should contribute to a greater understanding of the role of HMGA2 in promoting tumorigenesis and conveying (chemo)sensitivity towards doxorubicin and other related double-strand breaks.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
High mobility group A2 (HMGA2) protein, also known as HMGI-C, is a non-histone architectural transcription factor and belongs to HMGA family, which consists of three additional members: HMGA1a, HMGA1b, and HMGA1c (1). Expression of HMGA2 is mainly restricted to embryogenesis and becomes below detection, if not absent, in the normal adult tissues (2, 3). HMGA2 protein contains three DNA-binding domains, which have been named AT hooks, due to their ability to interact with the minor groove of AT-rich DNA sequences (4).

A strong association exists between the ectopic expression of HMGA protein and the relevant transformed phenotypes. First, HMGA2 expression is correlated with malignant phenotype of mesenchymal origin as well as epithelial origin (5–7). Second, the direct role played by these factors in tumorigenesis came from transfection of an antisense against HMGA2 in normal rat thyroid cells that prevented the neoplastic transformation induced by myeloproliferative sarcoma virus and Kristen murine sarcoma virus (8). Indeed, the transgenic mice expressing HMGA2 develop pituitary adenomas (9). Third, the expression of HMGA2 in oral squamous cell carcinoma is associated with an increased disease recurrence and metastasis, along with a reduced survival rate manifested by a facilitated epithelial-mesenchymal transition (10). In addition, HMGA2 expression in patients with breast cancer correlates with poor prognosis and metastasis (11). Despite these lines of evidence, the precise role of and the molecular events elicited by HMGA2 in tumorigenesis still need to be defined.

The phosphatidylinositol 3-kinase–related protein kinase (PIKK) family of enzymes have recently been proposed and shown to contribute as one of the major signaling pathways underlying the surveillance and maintenance of genome integrity (reviewed in refs. 12, 13). These DNA damage–activated serine/threonine protein kinases include DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated kinase (ATM), and ATM- and Rad3-related kinase (ATR; ref. 14). DNA-PK, ATM, and ATR share sequence homology and many of the same substrates. They differ with respect to the types of genotoxic stresses that induce their activation. ATM primarily responds to agents that cause DNA double-strand breaks, whereas ATR signals respond to agents that cause bulky adducts on DNA or otherwise cause stalling of replication forks and generation of ssDNA break (15, 16). The DNA-PK holoenzyme is a heterodimer of 70 and 80 kDa subunits, which bind to DNA double-strand breaks, recruiting and activating a 470 kDa catalytic subunit, DNA-PKcs. Numerous studies have shown that cells lacking DNA-PK are hypersensitive to ionizing radiation and cross-linking agents and defective in double-strand break repair (17).

Activated ATM, ATR, and DNA-PK can initiate G2 cell cycle arrest, signaling through Chk1, Chk2, or p53. In addition, they can also phosphorylate histone 2A variant X (H2AX). H2AX is randomly deposited throughout chromatin, comprising ~10% of total nucleosomal histone H2A (18). A highly conserved serine residue at position 139 of H2AX is phosphorylated by ATM, ATR, or DNA-PK in response to DNA damage (19–21). It is estimated that hundreds to thousands of H2AX molecules are phosphorylated per DNA double-stranded break (20). Phosphorylation of H2AX is thought to amplify the DNA damage signal by enhancing and stabilizing the recruitment of DNA damage sensor proteins and DNA repair proteins in response to DNA damage or replication stress (22).

Here we report that HMGA2 expression is associated with enhanced selective chemosensitivity towards topoisomerase (topo) II inhibitor, doxorubicin, in breast cancer HS578T and salivary Pa-4/HMGA2 cells. Doxorubicin is an anthracycline chemotherapeutic agent, which functions, in part, by stabilizing topo II-DNA complex and resulting in double-strand breaks on colliding with replication fork (23). We found that there is a rapid H2AX Ser139 phosphorylation in response to genotoxicity elicited by doxorubicin in HMGA2-underexpressing cells, including Pa-4, HeLa, and HCC1419 cells. However, this doxorubicin-induced H2AX modification is attenuated in their HMGA2-expressing counterparts due to the increased level of basal, HMGA2-dependent H2AX phosphorylation. Moreover, treatments with caffeine (ATM/ATR inhibitor) and NU7026 (DNA-PK inhibitor) cause different profiles of doxorubicin-elicited H2AX Ser139 phosphorylation in breast cancer cells HCC1419 and HS578T, respectively. These three observations led to a hypothesis that HMGA2 induces a persistent basal H2AX Ser139 phosphorylation and perturbs doxorubicin-elicited DNA damage checkpoint control (i.e., induction of ATM/ATR/DNA-PK–dependent H2AX phosphorylation), hence promoting enhanced chemosensitivity towards doxorubicin treatment in HMGA2-expressing cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures. Salivary Pa-4 and Pa-4/HMGA2 were grown in DMEM/F12-based medium supplemented with 2% fetal bovine serum (FBS) and incubated at 35°C, as previously described (24). HS578T cells are grown in DMEM plus 10% FBS, supplemented with 10 µg/mL insulin. HCC1419 cells are grown in RPMI supplemented with 4.5 g/L glucose, 10 mmol/L HEPES, and 1 mmol/L sodium pyruvate. The pEBS7 and pEBS7-YZ5 (YZ5) cells are generous gifts from Dr. Leroy Liu, and they are maintained in DMEM and 10% FBS with 100 µg/mL hygromycin. pEBS7 and YZ5 are stable clones derived from ATM-deficient fibroblasts (AT22IJE-T) with empty vector and Flag-tagged wild-type ATM, respectively (25). Chinese hamster ovary (CHO) cells are maintained in MEM with {alpha}-modification (Sigma, St. Louis, MO). All cells, except Pa-4 and Pa-4/HMGA2 cells, are cultured in a humidified incubator at 37°C with 5% CO2. DNA-PKcs–deficient MO59J cells (26) are maintained in DMEM plus 10% FBS. ATR-kd (dominant-negative form of ATR)–containing U2OS.GK41 cells (27) are cultured in DMEM plus 10% FBS supplemented with 400 µg/mL G418 plus 50 µg/mL hygromycin. Tetracycline-inducible expression of ATR-kd was achieved by the addition of 1 µg/mL doxycycline for 48 hours before performing assays. For assays to assess H2AX Ser139 phosphorylation, only confluent cells were used.

Reagents and antibodies. Caffeine (Sigma-Aldrich) is made into a 50 mmol/L working solution in OptiMEM-I (Invitrogen, Carlsbad, CA) and NU7026 (Calbiochem, San Diego, CA) is dissolved in DMSO into a 5 mmol/L solution and stored at –20°C. Monoclonal anti–phospho-ATM (Ser1981) and monoclonal anti–phospho-H2AX (Ser139) are obtained from Upstate Cell Signaling (Charlottesville, VA). Monoclonal anti-HA antibody is from Covance (Princeton, NJ). Monoclonal anti-actin antibody is from Chemicon (Temecula, CA). Monoclonal anti–DNA-PKcs and polyclonal anti-ATM antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-FLAG M2 antibody is from Sigma; monoclonal anti–topo II{alpha} is from BD Bioscience (San Jose, CA); and polyclonal monospecific chicken anti-HMGA2 antibody was custom-made by Aves Labs (Tigard, OR) against peptide MSARGEGAGQPSTSA at the NH2 terminus of HMGA2.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays for measurement of cell viability. Cells were seeded into 24-well plates to obtain a confluency of 35% to 50% on the day of the experiment. The cells were treated with various reagents of indicated concentration and medium was changed daily for 3 days. Twenty-four to seventy-two hours after the start of treatment (depends on the cell type), 0.2 mL of 0.1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) in OptiMEM I (Invitrogen) was added to each well and the plate was incubated at 37°C for an additional 1.5 hours. The MTT solution was then aspirated and 0.2 mL isopropanol was added to each well to dissolve the formazan crystals. Absorbance was immediately read at 540 nm in a scanning multiwell spectrophotometer. The results were depicted as percentage of cell viability, reported as the mean ± SD of three independent experiments done in triplicate.

Western blot analyses. Cells were lysed in boiling SDS loading buffer, heated for 10 minutes, and centrifuged at 13,000 rpm for 10 minutes. Supernatants were collected and the protein concentrations were determined using Bradford protein assay. Twenty to forty micrograms of protein lysates were subjected to SDS-PAGE analyses. ATM and DNA-PKcs protein extractions were done according to the instructions of the manufacturer. Eighty micrograms of proteins were loaded onto SDS-PAGE gels and the separated proteins were transferred to Immobilon membranes (Millipore, Billerica, MA) overnight at 4°C.

Flow cytometry. Cells were seeded at 50% to 80% confluency in 35 mm dishes and serum starved overnight to synchronize cell cycle. After desired treatment, cells were fixed in 70% ethanol overnight and stained with 20 µg/mL propidium iodide. Flow cytometry was done at the Norris Cancer Center Flow Cytometry Core Facility using FACSCaliber (Becton Dickinson, Franklin Lakes, NJ).

Plasmid transfection and small interfering RNA transfection. For transient transfection, cells were seeded at 90% confluency, and transfections were carried out by using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. The oligonucleotides encoding HMGA2 small interfering RNA (siRNA) were 5'-CAGCAATCTGTCGCTAAGGdTdT-3' and 5'-CCTTAGCGACAGATTGCTGdTdT-3'. The oligonucleotides encoding scramble siRNA were 5'-GAGCGATCAGATGATCCACdTdT-3' and 5'-GTGGATCATCTGATCGCTCdTdT-3'. All siRNAs were synthesized by Norris Cancer Center MicroCore Facility. Transfection of siRNA was done with GeneEraser (Stratagene, La Jolla, CA) according to the instructions of the manufacturer.

RNA extraction and reverse transcription-PCR. RNA was extracted using Trizol reagent (Invitrogen) according to the protocols of the manufacturer followed by DNA-free DNase treatment (Ambion, Austin, TX). Subsequent cDNA synthesis and PCR reactions were carried out using ThermoScript reverse transcription-PCR (RT-PCR) System (Invitrogen). The following primer pairs were used for PCR reaction: HMGA2, 5'-GTGAGCCCTCTCCTAAGAGAC-3' and 5'-CTGCAGTGTCTTCTCCCTTC-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GACCACAGTCCATGCCATCAC-3' and 5'-CATACCAGGAAATGAGCTTGAC-3'.

Ionization radiation treatment and clonogenic assay. Cells were exposed to 137Cs {gamma}-irradiation at 1.55 Gy/min and irradiated with 1 to 9 Gy, returned to tissue culture incubator for 24 hours, and harvested for clonogenic assays. Briefly, irradiated cells were resuspended in clonogenic medium consisting of {alpha}-MEM supplemented with 0.9% methylcellulose, 30% fetal bovine serum, and 50 µmol/L ß-mercaptoethanol. Cells were plated in triplicate Petri dishes at 105 cells/mL/dish and cultured in a humidified 5% CO2 incubator for 7 days. Cancer cell colonies were enumerated on a grid using an inverted phase microscope of high optical resolution.

Statistical analyses. Statistical analyses are done by using two-tailed Student's t test. P < 0.05 is denoted with * and P < 0.01 is denoted with ** where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High mobility group A2 potentiates doxorubicin-induced genotoxicity. Because previous studies by others and us have shown that ectopic expression of architectural transcription factor HMGA2 is associated with cell proliferation and neoplastic transformation (10, 28), we sought to determine whether ectopic HMGA2 expression affects cellular response towards extracellular insults. Because both doxorubicin, a topo II poison, and irinotecan, a topo I inhibitor, are known to cause genotoxic stress, we treated parental Pa-4 and HMGA2-expressing Pa-4/HMGA2 cells with increasing concentrations of doxorubicin or irinotecan, followed by MTT cell viability assays. As illustrated in Fig. 1A, both doxorubicin and irinotecan treatments induced a dose-dependent growth inhibition in these two cell types. Notably, doxorubicin treatments (5 and 7 µmol/L) elicited a more pronounced cell killing in Pa-4/HMGA2 cells than in Pa-4 cells (Fig. 1A, top), whereas there was no marked difference observed between these cells towards topo I inhibitor irinotecan (Fig. 1A, bottom).



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  		Figure 1. Chemosensitivity towards doxorubicin is enhanced by HMGA2. A, HMGA2 expression selectively augments doxorubicin-mediated growth inhibition. Pa-4 and Pa-4/HMGA2 cells were incubated with increasing concentrations of doxorubicin (top) and irinotecan (bottom), respectively. Cell survival was measured by MTT assays (*, P < 0.05). The expression of HMGA2 in Pa-4 (lane 1) and Pa-4/HMGA2 (lane 2) was confirmed by Western blot analyses (inset, bottom). B, endogenous HMGA2 level in breast cancer cells HCC1419 and HS578T. Equal amounts of total RNA were used with specific primer pairs, as indicated, in RT-PCR analyses to assay the steady-state level of endogenous HMGA2 mRNAs (left). The level of GAPDH was also analyzed as a control to validate RT-PCR reaction and quantitation of RNAs. The steady-state level of HMGA2 proteins was measured by Western blot analyses with respective antibodies, as indicated (right). C, HMGA2-expressing HS578T cells are more sensitive to doxorubicin-elicited growth inhibition. HS578T, HCC1419, and CHO cells were incubated with increasing concentrations of doxorubicin and followed by MTT assays, as described in (A). Statistical analysis was done with the HS578T and HCC1419 pair (*, P < 0.05).

 
If HMGA2 expression were mainly responsible for the enhanced chemosensitivity towards doxorubicin, one would expect that other HMGA2-overexpressing cells could exhibit a similar chemosensitivity towards doxorubicin treatment. To test this possibility, breast cancer cells, HS578T and HCC1419, with different HMGA2 expression levels, and HMGA2-deficient CHO cells7 were selected. The HS578T and HCC1419 cells are both estrogen receptor and progesterone receptor negative. RT-PCR and Western blot analyses showed that HS578T cells expressed a higher endogenous level of HMGA2 than HCC1419 cells (Fig. 1B). On doxorubicin treatment, it was clear that HMGA2-proficient HS578T cells were more susceptible to doxorubicin-elicited growth inhibition than HMGA2-underexpressing HCC1419 and HMGA2-deficient CHO cells (Fig. 1C). Thus, these results support the possibility that HMGA2 sensitizes, at least in part, cellular response towards doxorubicin-induced genotoxicity.

Doxorubicin-induced G2-M arrest and sub-G1 accumulation are augmented by high mobility group A2. The topo II inhibitor doxorubicin delays the G2-M transition (29). To explore the mechanism underlying HMGA2-dependent enhancement of doxorubicin-mediated growth inhibition, we first assayed whether the effect by HMGA2 is cell cycle dependent. Not unexpectedly, doxorubicin treatment induced 50.9% of Pa-4 and 62.6% of Pa-4/HMGA2 cells to accumulate in the G2-M phase, increasing from 17.6% and 14.6% of vehicle-treated Pa-4 and Pa-4/HMGA2 cells, respectively (Fig. 2A, b and d versus a and c; summarized in e).



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  		Figure 2. HMGA2 expression induces G2-M arrest. A, HMGA2 cooperates with doxorubicin treatment. Synchronized Pa-4 cells (a and b) and Pa-4/HMGA2 (c and d) were treated with either vehicle or 1 µmol/L doxorubicin for 24 hours and subjected to fluorescence-activated cell sorting (FACS) analyses. The populations of G1 and G2-M cells were indicated by arrows. The percentage of cells in each phase of cell cycles was summarized in (e). B, HMGA2 does not affect the steady-state level of topo II{alpha}. Equal amounts of cell lysates from vehicle-treated and doxorubicin (1 µmol/L)–treated HS578T cells were subjected to Western blot analyses with an anti–topo II{alpha} antibody, as indicated. An anti-actin antibody was used to ensure equal loading and quality of protein extracts.

 
We also quantified the apoptosis of Pa-4 and Pa-4/HMGA2 cells after doxorubicin exposure by assessing the sub-G1 cell population, a hallmark of cell apoptosis. Whereas the percentage of sub-G1 phase cells was enriched by only 2-fold in doxorubicin-treated Pa-4 cells, the percentage of Pa-4/HMGA2 cells in sub-G1 phase displayed nearly a 5-fold increase after 24 hours of doxorubicin treatment (Fig. 2A, e). Together, it is possible that HMGA2 expression, together with doxorubicin-activated checkpoint control, induces more cells undergoing G2-M accumulation and apoptosis. Alternatively, because topo II{alpha} is the key enzyme target of doxorubicin and the steady-state level of topo II is inversely correlated with chemoresistance towards doxorubicin, HMGA2 may regulate the steady-state level of topo II. To test this possibility, we examined the steady-state level of topo II{alpha} in the presence and absence of HMGA2 during the course of doxorubicin treatment. As shown in Fig. 2B, the steady-state level of topo II{alpha} remained comparable during 2.5 and 5 hours of doxorubicin treatment and was independent of HMGA2 expression in HS578T cells. Thus, we ruled out the possibility that HMGA2 governs the cellular response to doxorubicin-elicited genotoxicity by modulating the steady-state levels of topo II{alpha}.

Persistent histone 2A variant X Ser139 phosphorylation in high mobility group A2–expressing cells. Subsequently, we hypothesized that HMGA2 augments doxorubicin-elicited growth inhibition by modulating cellular responses to genotoxic stress of doxorubicin-induced DNA double-strand breaks. To test this hypothesis, we first investigated whether doxorubicin treatment induces ATM activation. As shown in Fig. 3A (top), ATM Ser1981 phosphorylation was detected between 0.5 and 2 hours of post doxorubicin treatment in ATM-proficient YZ5 cells (lanes 5 and 6), indicating that doxorubicin treatment activates ATM cascade. We next examined whether there is a correlation between ATM activation and growth inhibition on doxorubicin treatment. ATM (–/–) pEBS7 and stable ATM-transfected pEBS7 and YZ5 cells were treated with increasing concentrations of doxorubicin and subsequently analyzed by MTT assays. Although both pEBS7 and YZ5 cells exhibited doxorubicin-induced cell growth inhibition, it was noted that the ATM-negative pEBS7 cells were relatively less sensitive to doxorubicin (1 µmol/L) treatment (Fig. 3A, bottom).



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  		Figure 3. H2AX Ser139 phosphorylation persists in HMGA2-proficient cells. A, doxorubicin treatment activates ATM phosphorylation. Equal amounts of protein lysates prepared from vehicle-treated and doxorubicin-treated ATM-proficient (YZ5) and ATM-deficient (pEBS7) cells, as indicated, were subjected to Western blot analyses with an anti–phospho-ATM antibody (top). YZ5 and pEBS7 cells were treated with increasing concentrations of doxorubicin and followed by MTT cell viability assays as described in Fig. 1A (bottom). B, doxorubicin-mediated ATM activation is independent of HMGA2 expression. Pa-4 and Pa-4/HMGA2 cells were treated with 2 µmol/L doxorubicin for 0, 0.5, and 2 hours, respectively. C, expression of HMGA2 induces H2AX Ser139 phosphorylation. Pa-4 and Pa-4/HMGA2 cells were treated with either vehicle or 1 µmol/L doxorubicin for 2 hours and the total cell lysates were subjected to Western blot analyses with antibodies against phospho-H2AX, H2AX, and actin, respectively (left). The effect of HMGA2 expression on the H2AX Ser139 phosphorylation profile was determined by densitometry with signals obtained (left) and expressed as relative intensity. After normalizing with that of H2AX, the steady-state level of phospho-H2AX in vehicle-treated Pa-4 cell was set to 1. Right, value represents one of three independent experiments. D, HMGA2 induces doxorubicin sensitivity and H2AX phosphorylation in HeLa cells. Top, for doxorubicin-mediated cytotoxicity assays, HeLa and HeLa/HMGA2 cells were treated and analyzed as described in Fig. 1A (**, P < 0.01). Middle and bottom, effects of doxorubicin on topo II{alpha}, p-ATM, and p-H2AX were assessed by Western blot analyses as described in (C).

 
To ascertain whether HMGA2 affects doxorubicin-induced ATM signaling activation, phosphorylation of ATM Ser1981 and H2AX Ser139, a hallmark of double-strand breaks, was assayed in Pa-4 and Pa-4/HMGA2 cells in the presence and absence of doxorubicin. As expected, doxorubicin treatment led to rapid ATM Ser1981 (Fig. 3B) and H2AX Ser139 (Fig. 3C) phosphorylation in Pa-4 cells. A comparable activation of ATM Ser1981 phosphorylation was noted in both Pa-4 and Pa-4/HMGA2 cells (Fig. 3B). Intriguingly, a persistent high level of the basal H2AX Ser139 phosphorylation, resulting in a subsequent ablation of doxorubicin-mediated induction of H2AX phosphorylation, was observed in Pa-4/HMGA2 cells (Fig. 3C). Similar HMGA2-mediated enhancement of chemosensitivity towards doxorubicin treatment (Fig. 3D, top), as well as HMGA2-dependent basal level elevation and lack of doxorubicin-mediated induction of H2AX phosphorylation, was also recapitulated in HeLa/HMGA2 cells (Fig. 3D, bottom). Consistent with results shown in Fig. 2B, the steady-state level of topo II{alpha} was not affected by HMGA2 expression and doxorubicin treatment in HeLa and HeLa/HMGA2 cells (Fig. 3D, top middle). Intriguingly, doxorubicin treatment only induced ATM activation in HeLa cells, but not in HeLa/HMGA2 cells (Fig. 3D, bottom middle). Together, we concluded that the role of HMGA2 in governing chemosensitivity towards doxorubicin and H2AX phosphorylation profile is not unique to one cell type.

Differential involvement of ataxia telangiectasia-mutated kinase/ataxia telangiectasia-mutated and Rad3-related kinase and DNA-dependent protein kinase in inducing basal and doxorubicin-elicited histone 2A variant X phosphorylation. To investigate whether the observed different H2AX phosphorylation profiles in response to doxorubicin treatment were mediated by ATM or other related protein kinases, such as ATR and DNA-PK, caffeine (30–32) and NU7026, a novel DNA-PKcs inhibitor (33), were used to uncouple these kinases. Caffeine has been reported to inhibit ATM and ATR at an IC50 of 0.2 and 1.1 mmol/L, respectively (34). It has been previously shown that NU7026 is a highly selective inhibitor of DNA-PK, but inactive against both ATM and ATR (35). Thus, treatment of Pa-4 and Pa-4/HMGA2 cells with caffeine should inhibit both ATM and ATR, and treatment with NU7026 should inhibit only DNA-PK.

As illustrated in Fig. 4A, doxorubicin-mediated H2AX Ser139 phosphorylation in Pa-4 cells was attenuated by the pretreatment with 4 mmol/L caffeine (lane 3 versus lane 2) and 10 µmol/L NU7026 (lane 4 versus lane 2), respectively. By contrast, the same dose of caffeine and NU7026 elicited a very modest, if any, effect on doxorubicin-induced H2AX phosphorylation in Pa-4/HMGA2 cells (Fig. 4A, lanes 7 and 8 versus lane 6). Similarly, a lack of doxorubicin-induced H2AX phosphorylation was observed in HMGA2-proficient and doxorubicin-sensitive HS578T cells (Fig. 4B, top, lane 4 versus lane 1), contradictory to that detected in HMGA2-underexpressing and doxorubicin-resistant HCC1419 cells (Fig. 4B, bottom, lane 4 versus lane 1). In addition, both 4 mmol/L caffeine and 10 µmol/L NU7026 ameliorated the H2AX Ser139 phosphorylation, detected in the presence of doxorubicin treatment in doxorubicin-resistant HCC1419 cells, but not in doxorubicin-sensitive HS578T cells (Fig. 4B, lanes 5 and 6 versus lane 4). Notably, neither caffeine nor NU7026 was very effective in attenuating basal, HMGA2-associated H2AX phosphorylation in HS578T cells (Fig. 4B, top, lanes 2 and 3 versus lane 1). A similar lack of effect by caffeine and NU7026 on basal H2AX Ser139 phosphorylation was also observed in Pa-4/HMGA2 cells (data not shown). Together, we concluded that there was a persistent H2AX phosphorylation detected in HMGA2-expressing cells, such as HS578T, HeLa/HMGA2, and Pa-4/HMGA2 cells, and that the HMGA2-stimulated H2AX Ser139 phosphorylation is mediated, at least in part, in a NU7026- and caffeine-insensitive manner. We further postulated that the increased level of basal H2AX phosphorylation could prevent the emergence of the doxorubicin-exerted effect on the same event in these HMGA2-expressing cells.



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  		Figure 4. Caffeine and NU7026 fail to relieve HMGA2-elicited H2AX phosphorylation. A, caffeine and NU7026 repress doxorubicin-induced H2AX phosphorylation. Pa-4 and Pa-4/HMGA2 cells were pretreated with vehicle, caffeine (4 mmol/L), or NU7026 (10 µmol/L) for 1 hour, then followed with a combination of doxorubicin (1 µmol/L), caffeine (4 mmol/L), and NU7026 (10 µmol/L) for 2 hours. Equal amounts of prepared cell lysates were subjected to Western blot analyses with either an anti–phospho-H2AX or anti-actin antibody, as indicated. B, caffeine and NU7026 down-regulate H2AX phosphorylation in HCC1419 cells (bottom), but not in HS578T cells (top). HS578T and HCC1419 cells were pretreated and treated with a combination of doxorubicin (2 µmol/L), caffeine (4 mmol/L), and NU7026 (10 µmol/L), harvested, and analyzed as described in (A). C, caffeine treatment abolishes doxorubicin-induced G2-M accumulation. Pa-4 (a-d) and Pa-4/HMGA2 (e-h) cells were pretreated with vehicle or caffeine (4 mmol/L) for 1 hour, treated with a combination of doxorubicin (1 µmol/L) and caffeine (4 mmol/L) for 48 hours, and subjected to FACS analyses. The population of G1 and G2-M was indicated by arrows. D, HMGA2 potentiates doxorubicin/caffeine-elicited apoptosis as reflected by sub-G1 population. The populations of sub-G1 cells in Fig. 4C were normalized and expressed as fold induction compared with that of vehicle-treated Pa-4 and Pa-4/HMGA2 cells.

 
To investigate whether doxorubicin-induced G2-M delay is ATM/ATR dependent, Pa-4 and Pa-4/HMGA2 were pretreated with 4 mmol/L caffeine for 1 hour, followed by treatment with a combination of 1 µmol/L doxorubicin and 4 mmol/L caffeine for 48 hours. Whereas treatment with caffeine alone did not alter the cell cycle distribution in Pa-4 cells (Fig. 4C, b versus a), the same treatment decreased the G2-M cell population in Pa-4/HMGA2 cells (Fig. 4C, f versus e). Notably, a pronounced reduction in doxorubicin-induced G2-M delay by caffeine was observed in both Pa-4 (Fig. 4C, d versus c) and Pa-4/HMGA2 cells (Fig. 4C, h versus g), confirming that ATM and/or ATR is involved in doxorubicin-elicited G2-M delay. In addition, the treatment with caffeine alone (Fig. 4C, f) or in combination with doxorubicin (Fig. 4C, h) markedly enhanced the sub-G1 population of Pa-4/HMGA2 cells (summarized in Fig. 4D). A similar cell cycle dysregulation was also observed in HeLa/HMGA2 cells cotreated with doxorubicin and caffeine (data not shown). It was apparent that there is a potential combinatory effect between doxorubicin and caffeine to commit more HMGA2-expressing cells to apoptosis. Taken together, we concluded that HMGA2 potentiates doxorubicin-mediated cell cycle dysregulation and growth inhibition by perturbing a mechanism(s) that also governs H2AX Ser139 phosphorylation.

Role of phosphatidylinositol 3-kinase–related protein kinase and high mobility group A2 in modulating histone 2A variant X Ser139 phosphorylation. We have shown above that the capacity of both caffeine and NU7026 to repress doxorubicin-elicited H2AX Ser139 phosphorylation is inversely correlated with HMGA2 expression. To find out which known member of PIKK mediates the observed doxorubicin-induced and/or HMGA2-associated H2AX phosphorylation, several cell lines that have important characteristics desirable for our studies were used, including the aforementioned pEBS7 (ATM–) and YZ5 (ATM+) cells and DNA-PKcs–deficient MO59J cells (26), to establish H2AX phosphorylation profile on doxorubicin treatment. In addition, U2OS.GK41 cells, harboring doxycycline-inducible ATR-kd (dominant-negative ATR; ref. 27), which have been pretreated with vehicle or doxycycline (to induce ATR-kd) for 48 hours, were also subjected to 2 µmol/L doxorubicin treatment to investigate the role of ATR in mediating doxorubicin-induced H2AX phosphorylation. As illustrated in Fig. 5A, ATM-deficient pEBS7 cells displayed an increase in basal H2AX phosphorylation and a lack of doxorubicin-mediated induction (top), reflecting that observed in HMGA2-expressing Pa-4/HMGA2, HeLa/HMGA2, and HS578T cells (Figs. 3C,D and 4B). Whereas, doxorubicin treatment induced H2AX phosphorylation in both ATM-proficient YZ5 cells as well as DNA-PKcs–deficient MO59J cells (Fig. 5A, top). In addition, dominant-negative ATR-kd delayed the doxorubicin-mediated induction of H2AX phosphorylation and attenuated the level of induction in U2OS.GK41 cells (Fig. 5A, bottom, lanes 2 and 3 versus lanes 5 and 6).



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  		Figure 5. Role of PIKK and HMGA2 in H2AX Ser139 phosphorylation. A, ATM mediates doxorubicin-elicited H2AX phosphorylation. The pEBS7, YZ5, MO59J, and U2OS.GK41 cells were treated with 2 µmol/L doxorubicin for 2 hours or otherwise as indicated and subjected to Western blot analyses with either an anti–phospho-H2AX or an anti-actin antibody. To analyze the effect of ATR, doxorubicin treatment was carried out in cells that have been pretreated with vehicle or doxycycline (1 µg/mL) for 48 hours to induce ATR-kd. B, HMGA2 siRNA simultaneously represses HMGA2 expression and H2AX phosphorylation. HS578T cells were transfected with vehicle, HMGA2 siRNA (100 nmol/L, and scrambled siRNA (100 nmol/L), respectively. Whole-cell lysates were prepared posttransfection at the indicated time. Equal amounts of cell lysates were subjected to Western blot analyses with respective antibodies, as indicated. C, HMGA2 siRNA reverses doxorubicin-mediated cytotoxicity. HeLa/HMGA2 and HS578T cells were transfected with 100 nmol/L HMGA2 siRNA or scrambled siRNA for 48 hours and allowed to recover for 24 hours before reseeding in a 24-well plate. The doxorubicin treatment and MTT assays were done as described in Fig. 1A (*, P < 0.05).

 
Based on the above results, we hypothesized that HMGA2 is required for conveying (chemo)sensitivity towards doxorubicin and other agents causing double-strand breaks. We first examined the effect of increased HMGA2 expression and its reversal on the level of basal H2AX phosphorylation. To test this, we down-regulated HMGA2 expression by using RNA interference approach in HeLa/HMGA2 and HS578T cells because siRNA has been shown to be a useful tool to manipulate gene expression or determine gene function (36). As shown in Fig. 5B, treatment with HMGA2 siRNA in HS578T cells virtually blocks the observed basal H2AX Ser139 phosphorylation (lanes 1-4 versus lanes 5 and 6). Notably, a time-dependent correlation between the level of endogenous HMGA2 levels and the extent of H2AX phosphorylation was established in HS578T cells.

We next investigated whether HMGA2 silencing affected doxorubicin sensitivity in HeLa/HMGA2 and HS578T cells, respectively. We assayed the effect of HMGA2 siRNA on cell viability after doxorubicin exposure and found that HMGA2 down-regulation partially enhanced cell survival by greater than 2-fold in HMGA2 siRNA–transfected, doxorubicin (2 and 4 µmol/L)–treated HeLa/HMGA2 cells (Fig. 5C, left). The lack of such a marked effect by HMGA2 siRNA observed in doxorubicin-treated HS578T cells (Fig. 5C, right) could be due to other effects inherited from siRNA approach. For example, siRNAs have been shown to induce global signaling responses, including the induction of IFN-mediated Janus-activated kinase/signal transducer and activator of transcription pathway activation (37–40). Given that, we concluded that HMGA2 expression is capable of inducing chemosensitivity towards doxorubicin treatment.

Because double-strand breaks could arise from blocked or collapsed replication forks by cisplatin treatment (41) and are induced by X-ray irradiation, the effects of HMGA2 expression on cell survival following exposure to cisplatin, a known DNA intrastrand cross-linker and X-ray, were also assessed. We found that both HMGA2-expressing HS578T and HeLa/HMGA2 cells were more sensitive to cisplatin-elicited growth inhibition than HCC1419 and parental HeLa cells (Fig. 6A). In addition, an enhanced radiosensitivity in HeLa/HMGA2 cells compared with that in HeLa cells was observed (Fig. 6B). Taken together, we concluded that overexpression of HMGA2 in HS578T and HeLa/HMGA2 cancer cells confers an elevated basal H2AX Ser139 phosphorylation, accounting for, at least in part, the increased sensitivity towards double-strand breaks exerted by doxorubicin and cisplatin treatment as well as by X-ray irradiation.



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  		Figure 6. HMGA2 elicits sensitivity towards cisplatin and radiation. A, HMGA2 conveys chemosensitivity towards cisplatin. HeLa and HeLa/HMGA2 (left), HS578T and HCC1419 (right) were treated with increasing concentration of cisplatin and cell survival was assessed by MTT assays (*, P < 0.05). B, effect of X-ray irradiation on HeLa, HeLa/HMGA2, and HS578T cells as assessed by clonogenic assays. Statistical analysis was done with HeLa and HeLa/HMGA2 pair (*, P < 0.05; **, P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HMGA2 was originally identified in undifferentiated cells, but its expression ceases on cell differentiation (42). Consistent with its growth regulatory role is the pygmy phenotype in mice in which the HMGA2 gene is homozygously inactivated (2, 43). HMGA2 is also disrupted and aberrantly expressed in many tumor types, making this gene probably the most frequently rearranged gene in human neoplasias (reviewed in ref. 44), including breast cancer, pancreatic tumors, and oral squamous cell carcinomas (5, 10, 11, 45). A surprise role of HMGA2 in modulating the biological state of cells that could serve as an indicator for selective (chemo)sensitivity towards doxorubicin treatment and other double-strand break-eliciting agents was revealed herein.

We showed that HMGA2 expression renders cell susceptible to doxorubicin-elicited, but not irinotecan-exerted, growth inhibition and doxorubicin-triggered accumulation of cells in sub-G1 and G2-M phases. In the subcellular level, overexpression of HMGA2 leads to a persistent Ser139 phosphorylation of H2AX. Moreover, by using HMGA2 siRNA to suppress endogenous HMGA2 expression in HS578T cells, a correlation between the steady-state level of HMGA2 and the extent of basal H2AX Ser139 phosphorylation was clearly established. Importantly, HMGA2 siRNA also partially reversed the observed chemosensitivity towards doxorubicin. H2AX phosphorylation is required for the recruitment of stable formation of the NBS1, 53BP1, MDC1, and BRCA1 complex at the damaged sites (46–50). Conceivably, the HMGA2-mediated, persistent H2AX phosphorylation may represent a key step that determines ensuing events in the signal transduction pathway in response to subsequent genotoxic stress. Intriguingly, the lack of a robust doxorubicin-mediated induction of H2AX Ser139 phosphorylation correlated well with the increased chemosensitivity towards doxorubicin in HMGA2-expressing HS578T, HeLa/HMGA2, and Pa-4/HMGA2 cells. One possible explanation is that the ectopic HMGA2 expression results in an adaptive mechanism to consistently phosphorylate H2AX and to desensitize H2AX from further phosphorylation by doxorubicin-elicited genotoxicity.

We extended the HMGA2-augmented sensitivity to genotoxicity from cisplatin treatment and X-ray irradiation. Because the prompt emergence of Ser139-phosphorylated H2AX after double-strand break insults and its phosphorylation can be observed over several mega bases flanking the sites of double-strand break (51), phosphorylation of H2AX at Ser139 has been regarded as a hallmark for DNA double-strand breaks. Taking together the observations that HMGA2 facilitates cell killing by three different types of DNA-damaging agents (i.e., topo II inhibitor doxorubicin, intrastranded cross-linker cisplatin, and X-ray irradiation), we postulated that HMGA2 enhanced cell killing through its effect on double-strand break–mediated genotoxic response. Two major double-strand break repair pathways for such damages are homologous recombination and DNA nonhomologous end-joining (52, 53). Double-strand breaks also activate signaling cascades that induce cell cycle checkpoint arrest and/or apoptosis (12, 16). We postulate that HMGA2 adopts an intrinsic pathway to amplify the effects of double-strand breaks, eventually leading to tumorigenesis, and doxorubicin/cisplatin/X-ray irradiation uses this HMGA2-dependent pathway to augment its effect on inducing G2-M cell cycle arrest or cell killing. It is clear that double-strand breaks also arise endogenously during processes such as meiosis and DNA replication (54). We observed a marked basal H2AX phosphorylation in HMGA2-expressing cells, supporting our notion that HMGA2-expressing cells gradually lose their ability to activate DNA repair pathways in response to double-strand break damages, eventually crossing the threshold required for cell cycle dysregulation conferred by doxorubicin/cisplatin/X-ray irradiation treatment. Recent reports that cyclin A is a downstream target of HMGA2 (28) and that the cyclin A1/cyclin-dependent kinase 2 complex regulates double-strand break repair (55) support our theory on the role of HMGA2 in governing genotoxic responses.

We speculate that a yet to be identified PIKK-related kinase is responsible for H2AX phosphorylation in the presence of HMGA2. This notion was substantiated by the observations that HS578T cells (Fig. 4B) and Pa-4/HMGA2 cells (data not shown) exhibited a caffeine- and NU7026-insensitive basal H2AX phosphorylation and that the ability of both NU7026 and caffeine to attenuate doxorubicin-elicited H2AX Ser139 phosphorylation is inversely correlated with the levels of HMGA2 expression. Alternatively, HMGA2 may interfere with the signaling, downstream of PIKK, to activate basal H2AX phosphorylation and to retard doxorubicin-mediated activation. This possibility was supported by the observation that a similar H2AX phosphorylation profile (i.e., increased level of basal phosphorylation and lack of doxorubicin inducibility) was observed in both HMGA2-expressing cells and ATM-deficient pEBS7 cells; however, doxorubicin is unable to induce ATM Ser1981 activation in HeLa/HMGA2 cells. In addition, doxorubicin treatment resulted in H2AX Ser139 phosphorylation in DNA-PK–defective MO59J cells, but not in ATM-defective pEBS7 cells. These observations suggest that H2AX is phosphorylated by ATM/ATR on doxorubicin treatment, but becomes refractory to doxorubicin-mediated phosphorylation in the presence of HMGA2. In spite of this, the signaling by ATM/ATR for cell cycle delays seems to be activated by doxorubicin treatment in these HMGA2-expressing cells. Thus, it is likely that there is a divergence in pathways leading to H2AX phosphorylation and cell cycle dysregulation in doxorubicin-treated HMGA2-expressing cells. Conceivably, we further postulate that doxorubicin and HMGA2 use distinct pathways to phosphorylate H2AX at Ser139. Notwithstanding the uncertainty about the detailed information on this process, our results unequivocally show a crucial role of HMGA2 in modulating H2AX phosphorylation and in augmenting doxorubicin/cisplatin/irradiation-elicited cell killing. It is likely that these events are directly linked, functionally cooperating downstream with genotoxic stress. Additional studies are currently in progress for a more detailed understanding of how HMGA2 alters H2AX phosphorylation profiles and activities.

DNA-damaging agents, such as doxorubicin and cisplatin, are the mainstays of cancer therapy and have achieved impressive clinical results. However, the usage of doxorubicin and cisplatin in treating patients with cancer is often plagued by various side effects, the most serious of which is doxorubicin-related cardiotoxicity and cisplatin-related nephrotoxicity, emetogenesis, and dose-limiting neurotoxicity (reviewed in refs. 56, 57). The potential for selective use of doxorubicin or cisplatin for patients with cancer cells expressing HMGA2 may underlie future successful protocol development and marginalize its associated toxicity. Profiling of patients with distinct gene expression promises to maximize the efficacy of chemotherapy and minimize toxicity in normal cells. Our study provides the insights into HMGA2-mediated modulation of selective (chemo)-sensitivity towards topo II inhibitors, such as doxorubicin, and DNA intrastranded cross-linkers, such as cisplatin, as well as irradiation therapy. The merit of these studies is particularly underpinned by recent data supporting the value of individualized pharmacotherapy (reviewed in refs. 58, 59). To our knowledge, these findings represent the first demonstration of a selective activation of DNA damage signaling pathways by the non-histone chromosomal architectural protein HMGA2, in addition to its known growth regulatory property. Our studies significantly broaden the potential clinical application of using DNA damage agents in cancer therapy based on the HMGA2-dependent pathway to selectively enhance double-strand break–elicited cell death and avoid undesired cytotoxicity.


   Acknowledgments
 
Grant support: NIH Research grants R01 DE 10742 and DE 14183 (D.K. Ann) and R01 CA 72767 (Y. Yen).

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.

We thank Dr. Leroy Liu (University of Medicine and Dentistry of New Jersey, Newark, NJ) for providing YZ5 and pEBS7 cells; Dr. Lucio Comai (University of Southern California, Los Angeles, CA) for providing MO95J cells; Xue-Fei Cao for providing HeLa/HMGA2 cells before publication; and Hong-Tao Deng, Susan Quach, Vivian Liao, and Septima Hong for technical assistance. Critical reading of the manuscript and helpful discussion by Drs. Kwang-Jin Kim (University of Southern California, Los Angeles, CA) and Hsiu-Ming Shih (Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan) are greatly appreciated.


   Footnotes
 
7 Ann DK and Cao X-F, unpublished observation. Back

Received 1/10/05. Revised 5/ 6/05. Accepted 5/17/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reeves R. Molecular biology of HMGA proteins: hubs of nuclear function. Gene 2001;277:63–81.[CrossRef][Medline]
  2. Zhou X, Benson KF, Ashar HR, Chada K. Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C. Nature 1995;376:771–4.[CrossRef][Medline]
  3. Hirning-Folz U, Wilda M, Rippe V, Bullerdiek J, Hameister H. The expression pattern of the Hmgic gene during development. Genes Chromosomes Cancer 1998;23:350–7.[CrossRef][Medline]
  4. Reeves R, Beckerbauer L. HMGI/Y proteins: flexible regulators of transcription and chromatin structure. Biochim Biophys Acta 2001;1519:13–29.[Medline]
  5. Abe N, Watanabe T, Suzuki Y, et al. An increased high-mobility group A2 expression level is associated with malignant phenotype in pancreatic exocrine tissue. Br J Cancer 2003;89:2104–9.[CrossRef][Medline]
  6. Finelli P, Pierantoni GM, Giardino D, et al. The High Mobility Group A2 gene is amplified and overexpressed in human prolactinomas. Cancer Res 2002;62:2398–405.[Abstract/Free Full Text]
  7. Masciullo V, Baldassarre G, Pentimalli F, et al. HMGA1 protein over-expression is a frequent feature of epithelial ovarian carcinomas. Carcinogenesis 2003;24:1191–8.[Abstract/Free Full Text]
  8. Berlingieri MT, Manfioletti G, Santoro M, et al. Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells. Mol Cell Biol 1995;15:1545–53.[Abstract]
  9. Fedele M, Battista S, Kenyon L, et al. Overexpression of the HMGA2 gene in transgenic mice leads to the onset of pituitary adenomas. Oncogene 2002;21:3190–8.[CrossRef][Medline]
  10. Miyazawa J, Mitoro A, Kawashiri S, Chada KK, Imai K. Expression of mesenchyme-specific gene HMGA2 in squamous cell carcinomas of the oral cavity. Cancer Res 2004;64:2024–9.[Abstract/Free Full Text]
  11. Langelotz C, Schmid P, Jakob C, et al. Expression of high-mobility-group-protein HMGI-C mRNA in the peripheral blood is an independent poor prognostic indicator for survival in metastatic breast cancer. Br J Cancer 2003;88:1406–10.[CrossRef][Medline]
  12. Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155–68.[CrossRef][Medline]
  13. Kurz EU, Lees-Miller SP. DNA damage-induced activation of ATM and ATM-dependent signaling pathways. DNA Repair (Amst) 2004;3:889–900.
  14. Durocher D, Jackson SP. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 2001;13:225–31.[CrossRef][Medline]
  15. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001;15:2177–96.[Free Full Text]
  16. Rouse J, Jackson SP. Interfaces between the detection, signaling, and repair of DNA damage. Science 2002;297:547–51.[Abstract/Free Full Text]
  17. Burma S, Chen DJ. Role of DNA-PK in the cellular response to DNA double-strand breaks. DNA Repair (Amst) 2004;3:909–18.
  18. Pilch DR, Sedelnikova OA, Redon C, et al. Characteristics of {gamma}-H2AX foci at DNA double-strand breaks sites. Biochem Cell Biol 2003;81:123–9.[CrossRef][Medline]
  19. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001;276:42462–7.[Abstract/Free Full Text]
  20. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998;273:5858–68.[Abstract/Free Full Text]
  21. Ward IM, Chen J. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 2001;276:47759–62.[Abstract/Free Full Text]
  22. Zimmerman ES, Chen J, Andersen JL, et al. Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and {gamma}-H2AX focus formation. Mol Cell Biol 2004;24:9286–94.[Abstract/Free Full Text]
  23. Li TK, Liu LF. Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol 2001;41:53–77.[CrossRef][Medline]
  24. Li D, Lin HH, McMahon M, Ma H, Ann DK. Oncogenic raf-1 induces the expression of non-histone chromosomal architectural protein HMGI-C via a p44/p42 mitogen-activated protein kinase-dependent pathway in salivary epithelial cells. J Biol Chem 1997;272:25062–70.[Abstract/Free Full Text]
  25. Li S, Ting NS, Zheng L, et al. Functional link of BRCA1 and ataxia telangiectasia gene product in DNA damage response. Nature 2000;406:210–5.[CrossRef][Medline]
  26. Lees-Miller SP, Godbout R, Chan DW, et al. Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science 1995;267:1183–5.[Abstract/Free Full Text]
  27. Nghiem P, Park PK, Kim Y-s, Desai BN, Schreiber SL. ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint. J Biol Chem 2002;277:4428–34.[Abstract/Free Full Text]
  28. Tessari MA, Gostissa M, Altamura S, et al. Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2. Mol Cell Biol 2003;23:9104–16.[Abstract/Free Full Text]
  29. Mikhailov A, Shinohara M, Rieder CL. Topoisomerase II and histone deacetylase inhibitors delay the G2-M transition by triggering the p38 MAPK checkpoint pathway. J Cell Biol 2004;166:517–26.[Abstract/Free Full Text]
  30. Zhou BB, Chaturvedi P, Spring K, et al. Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity. J Biol Chem 2000;275:10342–8.[Abstract/Free Full Text]
  31. Sarkaria JN, Busby EC, Tibbetts RS, et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 1999;59:4375–82.[Abstract/Free Full Text]
  32. Hall-Jackson CA, Cross DA, Morrice N, Smythe C. ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK. Oncogene 1999;18:6707–13.[CrossRef][Medline]
  33. Willmore E, de Caux S, Sunter NJ, et al. A novel DNA-dependent protein kinase inhibitor, NU7026, potentiates the cytotoxicity of topoisomerase II poisons used in the treatment of leukemia. Blood 2004;103:4659–65.[Abstract/Free Full Text]
  34. Cortez D. Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases. J Biol Chem 2003;278:37139–45.[Abstract/Free Full Text]
  35. Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW. Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. Cancer Res 2003;63:6008–15.[Abstract/Free Full Text]
  36. Hannon GJ. RNA interference. Nature 2002;418:244–51.[CrossRef][Medline]
  37. Kim DH, Longo M, Han Y, et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat Biotechnol 2004;22:321–5.[CrossRef][Medline]
  38. Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 2003;5:834–9.[CrossRef][Medline]
  39. Sledz CA, Williams BR. RNA interference and double-stranded-RNA-activated pathways. Biochem Soc Trans 2004;32:952–6.[CrossRef][Medline]
  40. Hornung V, Guenthner-Biller M, Bourquin C, et al. Sequence-specific potent induction of IFN-{alpha} by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 2005;11:263–70.[CrossRef][Medline]
  41. De Silva IU, McHugh PJ, Clingen PH, Hartley JA. Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells. Mol Cell Biol 2000;20:7980–90.[Abstract/Free Full Text]
  42. Zhou X, Benson KF, Przybysz K, et al. Genomic structure and expression of the murine Hmgi-c gene. Nucleic Acids Res 1996;24:4071–7.[Abstract/Free Full Text]
  43. Anand A, Chada K. In vivo modulation of Hmgic reduces obesity. Nat Genet 2000;24:377–80.[CrossRef][Medline]
  44. Tallini G, Dal Cin P. HMGI(Y) and HMGI-C dysregulation: a common occurrence in human tumors. Adv Anat Pathol 1999;6:237–46.[Medline]
  45. Rogalla P, Drechsler K, Kazmierczak B, et al. Expression of HMGI-C, a member of the high mobility group protein family, in a subset of breast cancers: relationship to histologic grade. Mol Carcinog 1997;19:153–6.[CrossRef][Medline]
  46. Wang B, Matsuoka S, Carpenter PB, Elledge SJ. 53BP1, a mediator of the DNA damage checkpoint. Science 2002;298:1435–8.[Abstract/Free Full Text]
  47. Stewart GS, Wang B, Bignell CR, Taylor AM, Elledge SJ. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 2003;421:961–6.[CrossRef][Medline]
  48. Paull TT, Rogakou EP, Yamazaki V, et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 2000;10:886–95.[CrossRef][Medline]
  49. Celeste A, Fernandez-Capetillo O, Kruhlak MJ, et al. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 2003;5:675–9.[CrossRef][Medline]
  50. Celeste A, Petersen S, Romanienko PJ, et al. Genomic instability in mice lacking histone H2AX. Science 2002;296:922–7.[Abstract/Free Full Text]
  51. Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 1999;146:905–16.[Abstract/Free Full Text]
  52. Jeggo PA. DNA breakage and repair. Adv Genet 1998;38:185–218.[Medline]
  53. Couedel C, Mills KD, Barchi M, et al. Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev 2004;18:1293–304.[Abstract/Free Full Text]
  54. Riballo E, Kuhne M, Rief N, et al. A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to {gamma}-H2AX foci. Mol Cell 2004;16:715–24.[CrossRef][Medline]
  55. Muller-Tidow C, Ji P, Diederichs S, et al. The cyclin A1-CDK2 complex regulates DNA double-strand break repair. Mol Cell Biol 2004;24:8917–28.[Abstract/Free Full Text]
  56. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004;56:185–229.[Abstract/Free Full Text]
  57. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 2005.
  58. Ross JS, Schenkein DP, Pietrusko R, et al. Targeted therapies for cancer 2004. Am J Clin Pathol 2004;122:598–609.[CrossRef][Medline]
  59. Watters JW, McLeod HL. Cancer pharmacogenomics: current and future applications. Biochim Biophys Acta 2003;1603:99–111.[Medline]



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