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[Cancer Research 64, 4131-4136, June 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Control of Radiosensitivity of F9 Mouse Teratocarcinoma Cells by Regulation of Histone H2AX Gene Expression using a Tetracycline Turn-Off System

Kayo Yoshida and Takashi Morita

Department of Molecular Genetics, Graduate School of Medicine, Osaka City University, Osaka, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mouse histone H2AX has unique COOH-terminal serine residues that are phosphorylated in response to double-strand DNA breaks introduced by ionizing radiation. This suggests that H2AX acts to maintain genomic stability. We constructed a tetracycline (tet)-directed turn-off vector and integrated it into F9 mouse teratocarcinoma cells by homologous recombination. In homozygously recombined cells, expression of the histone H2AX gene was repressed to 0.02% of the expression observed in wild-type cells by the addition of doxycycline, an analog of tet. Sensitivity of cells with repressed H2AX expression to X-irradiation was increased 1.95x, indicating that DNA repair was impaired by repression of H2AX. When we s.c. injected tet-regulated F9 cells into the flanks of mice, tumor growth was slightly suppressed by X-irradiation in H2AX-repressed tumors, whereas without X-irradiation, tumor growth did not differ by H2AX status. Thus, H2AX might be a potential molecular target for sensitizing cancer cells to radiotherapy to minimize required irradiation doses.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radiotherapy is a major modality in treatment of cancer. However, exposure of tumor cells to radiation or anticancer drugs, both of which can damage DNA, induces cellular DNA repair responses. DNA repair plays a critical role in tumor cell resistance to DNA damage despite this genotoxic stress (1) . Repair of DNA damage induced by radiation has been implicated in acquired tumor cell resistance (2) . If cells become deficient in DNA repair or their DNA repair system is inhibited, more cancer cells should be killed. This strategy should sensitize tumor cells to radiation and thus improve treatment efficacy and safety.

Repair of damaged DNA is critical for maintenance of genomic stability and cellular survival (3) . Double-strand DNA breaks (DSBs), which can be induced by ionizing radiation, alkylating agents, collapse of replication forks, or mechanical stress during chromosome segregation have serious consequences in eukaryotic cells, including mammalian cells. Repair of such DSBs can involve either of two reported pathways (3) , nonhomologous end-joining (4 , 5) or homologous recombination (6, 7, 8) . In addition to DNA repair genes (9 , 10) , chromosomal structure is now recognized to play a crucial role in DNA repair in mammals. In particular, the histone H2AX has been found to act as a sensor of DNA damage by radiation and genotoxic reagents (11, 12, 13) . H2AX, a minor component of H2A, represents 10% of total H2A proteins (14) . By Northern analysis, we have detected H2AX gene expression in the thymus, spleen, testis, and small intestine. H2AX mRNA was found to be particularly abundant in the S phase of the cell cycle (15) , and the CCAAT box and E2F elements were found to participate in regulation of expression of the H2AX gene (16 , 17) . H2AX exhibits strong homology with major H2A components in its first 120 amino acids, but it has a unique COOH-terminal region that is conserved among H2As in lower eukaryotes (15) . The COOH-terminal region contains the sequence QASQEY, a consensus site of serine (Ser) phosphorylation by phosphatidylinositol 3'-kinase. Radiation-induced DSB formation results in rapid phosphorylation of H2AX by ATM and ATR kinases, producing a modified form, {gamma}-H2AX (18) . When the DSB is repaired by nonhomologous end-joining (19) or homologous recombination, H2AX participates as a sensor of DSBs. In the yeast Saccharomyces cerevisiae, histone H2A has a COOH-terminal sequence similar to that of mammalian H2AX, and phosphorylation of the above-mentioned motif in the COOH-terminal portion of H2A is required for efficient DSB repair by nonhomologous end-joining (20) . In addition, H2AX knockout mice have been shown to be genetically unstable (21 , 22) . In a recently proposed two-stage recruitment model involving H2AX, {gamma}-H2AX is not required for initial migration of factors such as NBS1, 53BP1, and Brca1 to DSBs, but it is required for subsequent association of factors with the distal part of the chromatin region of DSBs (23, 24, 25, 26) .

Thus, phosphorylation of H2AX appears to be a sensor of damage to chromosomal DNA. Control of H2AX phosphorylation is clearly important for mammals in intercellular surveillance for DNA damage and in protecting DNA against the effects of radiation during mitosis and meiosis (27 , 28) . In the present report, we describe a mouse teratocarcinoma cell line in which the histone H2AX gene was modified to permit control by exposing cells to tetracycline (tet) or doxycycline. We demonstrated that the amount of {gamma}-H2AX correlated closely with sensitivity of tumor cells to X-irradiation and that repression of H2AX might be used to sensitize cancer cells to radiotherapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Mouse F9 teratocarcinoma cells were cultured on 0.1% gelatin-coated plastic dishes in DMEM (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) at 37°C under standard conditions of 100% relative humidity in 95% air and 5% CO2.

Targeting Vectors for Tet-Off System.
We constructed a targeting vector that regulated expression of the histone H2AX gene based on tet concentration. The histone H2AX gene promoter was about 1.2 kb and contained the CCAAT box and E2F consensus sequences. The H2AX native promoter was fused to the tet-dependent transcription activator gene (tTA) so that expression of the activator gene could be controlled in the same manner as expression of the H2AX gene. The activator bound to a tet-responsive element containing tet operator (tetO) and induced transcription from a cytomegalovirus minimal promoter (PminCMV), which led to the H2AX coding sequence. Tet or doxycycline could bind to tTA and inhibit transcriptional activation of the H2AX coding sequence. The vector, based on pBluescript, contained a cassette of mouse phosphoglycerate kinase 1 (PGK) promoter derived neomycin resistance gene flanked with loxP sequences for selection.

Generation of Recombinant F9 Cells.
Mouse teratocarcinoma F9 cells were transfected with the targeting vector by electroporation. Neomycin-resistant colonies were selected and analyzed by Southern blotting using 5' and 3' external probes. The frequency of homologous recombination was 0.6%. The neomycin resistance gene was excised from the homologous recombinant clones by transient transfection with pCre-puro plasmid DNA and 2 days of selection with puromycin. Neomycin-sensitive clones were selected and further transfected with the same targeting vector. Among neomycin-resistant clones, we used Southern analysis to isolate cells that exhibited H2AX gene recombination with the targeting vector at both alleles. The frequency of homologous recombination was 0.7%. The neomycin resistance gene again was excised by transient expression of pCre-puro.

Construction of H2AX Expression Plasmid.
A 2.9-kb SspI-BsaI fragment of mouse genomic DNA spanning the whole H2AX gene from its promoter to polyadenylation signal was isolated and inserted upstream of the loxP-flanked PGK neomycin resistance gene, which was cloned in pBluescript (referred to as pH2AX).

Protein Preparation and Western Blotting.
Cells were washed with PBS and collected with a cell scraper. Cells were lysed with SDS sample buffer containing 2% SDS, 6% 2-mercaptoethanol, and 10% glycerol, followed by boiling for 5 min and sonication for 10 s. Protein samples (2–20 µg) were separated by SDS-polyacrylamide gel (12%) electrophoresis and transferred electrophoretically to a Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). The membrane was blocked and subsequently incubated with specific primary antibodies. After being washed in 0.1 M Tris-HCl-buffered saline with 0.1% Tween 20, the membrane was allowed to react with horseradish peroxidase-linked immunoglobulin for 30 min at room temperature. For detection, we used the enhanced chemiluminescence reagent of the enhanced chemiluminescence Western blot system (Amersham Pharmacia Biotech). After draining off the detection reagents, the membrane was wrapped and exposed to Hyperfilm enhanced chemiluminescence (Amersham Pharmacia Biotech) for 1 min. Developed films were scanned and analyzed using the EDAS 290 system (Eastman Kodak Co., Rochester, NY).

Antibodies.
Polyclonal anti-H2AX antibody was raised in rabbits using a COOH-terminal synthetic peptide (TVGPKAPAVGKKASQASQEY). The antiserum was affinity purified using the COOH-terminal peptide (1:5,000 dilution). An antibody against mouse {gamma}-H2AX was prepared by injecting COOH-terminal peptide with a phosphorylated Ser139 residue into rabbits. Subsequent affinity purification and absorption of the immunized serum to nonphosphorylated COOH-terminal peptide yielded a specific antibody against {gamma}-H2AX (1:1,000 dilution). We also used rabbit anti-KU80 (1:10,000 dilution; AB1357; Chemicon), rabbit anti-RAD51 (Ab-1; PC130) antibody (1:1000; Oncogene Research Products, San Diego, CA) and anti-ß-actin (1:20,000 dilution; mAB1501; Chemicon).

Exposure of Cells to X-Rays.
F9 mouse teratocarcinoma cells were trypsinized and subjected to graded doses of X-irradiation (1.00 Gy/min; MBR-1520A; X-irradiation system; Hitachi Medico, Kamakura, Japan). After irradiation, cells were harvested and seeded in 96-well plates.

Analysis of Cell Proliferation.
Cell viability was measured in 8 samples/group using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, X-irradiated F9 mouse teratocarcinoma cells were seeded in 200 µl of medium in each well of a 96-well, flat-bottomed microtiter plate (Corning Glass Works, Corning, NY). After 65 h of incubation at 37°C, MTT labeling mixture was added, and mixtures were incubated for another 4 h. Then, 100 µl of HCl-SDS were added to the mixtures for incubation overnight at 37°C. Absorbance of the samples was measured using a microtiter plate reader with a test wavelength of 570 nm and a reference wavelength of 690 nm (Wallac 1420 ARVOsx; Perkin-Elmer, Turku, Finland). The radiation dose resulting in 50% cytotoxicity (CD50) in terms of MTT dye formation was estimated by plotting the percentage of A570 nm against the radiation dose (Gy).

Tumor Growth.
F9 cells (2 x 106) were injected s.c. into the right flank of 129Sv mice supplied with water containing doxycycline (1 mg/ml). The formula used to compute tumor volume was as follows: volume = (length) x (width)2 x 1/2.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of H2AX Tet-Off Clones from F9 Cells.
We constructed a targeting vector that regulated expression of the histone H2AX gene according to concentration of added tet (Fig. 1A)Citation . Mouse teratocarcinoma F9 cells were transfected with the vector by electroporation. Neomycin-resistant colonies were selected and analyzed by Southern blotting using 5' and 3' external probes. The genomic DNA of each clone was isolated and cut by HindIII. Southern blotting using the 5' external probe detected a 15-kb band for the wild-type locus and a 7-kb band for the tet-regulated locus. The frequency of homologous recombination was 0.6%. From the homologous recombinant clones, the neomycin resistance gene was excised by transient transfection of pCre-puro plasmid DNA and 2 days of selection with puromycin (Fig. 1B)Citation . Neomycin-sensitive clones were selected and further transfected with the same H2AX Tet-off targeting vector. Among neomycin-resistant clones, we isolated cells that exhibited the H2AX gene recombination at both alleles at a frequency of 0.7% (Fig. 1A)Citation . The neomycin resistance gene was excised again by transient expression of pCre-puro.



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  		Fig. 1. Structure of H2AX targeting vector using the tetracycline (tet)-off system. A, mouse H2AX promoter was ligated to tet-dependent transcription activator gene (tTA). Transcription of the tTA gene was regulated by the H2AX promoter. The tet-responsive element and minimal cytomegalovirus promoter were located downstream and linked to the H2AX coding sequence beginning at the initiation codon, ATG. The 5'- and 3'-flanking DNA regions (5 kb) of the H2AX gene (including the GPT and PBG genes) were used for homologous recombination. A loxP-flanked PGK promoter-driven neomycin resistance gene was inserted in the vector for positive selection. Southern analyses were performed using 5' external probes for selection of homologous recombinants, which were expected to produce a shorter fragment (7 kb) with HindIII digestion. B, procedures for isolating homozygously recombined F9 cells are shown. Homologous recombinant F9 cell clones were isolated by introduction of the targeting vector and neomycin selection, followed by Southern blotting. The neomycin resistance gene was excised by transient transfection of pCre-puro.

 
Tet-Dependent Expression of H2AX.
F9 cells with both H2AX alleles modified as tet-regulated constructs, H2AX (tet/tet), were cultured in various concentrations (0–10 ng/ml) of doxycycline, an efficient analog of tet. When the cells were cultured in <0.1 ng/ml doxycycline, the amount of H2AX was relatively constant (about 130% of that in wild-type cells). However, the addition of 1.0 ng/ml doxycycline greatly decreased the amount of H2AX protein to 0.04% of that without doxycycline. At 10 ng/ml doxycycline, only a trace amount of H2AX was detected (0.02%). The concentration of ß-actin was essentially constant at all doxycycline concentrations in both wild-type and H2AX (tet/tet) cells. These results confirmed dose-dependent suppression of H2AX protein that was specific to the H2AX gene by addition of doxycycline (Fig. 2A)Citation . Furthermore, quantitative comparison showed that expression of H2AX in F9 H2AX(tet/tet) cells in medium without doxycycline also was 130% of the wild-type amount. Because tTA was intended to be transcribed under the direction of the native H2AX promoter, tTA in turn activated the cytomegalovirus minimal promoter linked to the H2AX coding sequence; the result suggested that the promoter activity of tTA was important for target gene expression in the tet-off system, although the target gene was linked to the minimal cytomegalovirus promoter. We then examined the number of days required for repression of H2AX after addition of doxycycline. As shown in Fig. 2BCitation , repression began 1 day after the addition of doxycycline. After 4 days, H2AX was present in only a trace amount. After 5 days, we could not detect H2AX protein. On the other hand, H2AX protein synthesis recovered 2–4 days after removal of doxycycline. The growth rate of cells has been reported by Celeste et al. (21) to decrease when expression of H2AX is diminished; in our findings as well, F9 H2AX(tet/tet) cells with decreased expression of H2AX grew more slowly (70%) than F9 H2AX(tet/tet) cells without doxycycline or wild-type cells (data not shown).



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  		Fig. 2. Doxycycline-dependent repression of H2AX (tet/tet) in F9 cells. A, F9 cells with tetracycline (tet)-regulated H2AX alleles and wild-type cells were cultured with various concentrations of doxycycline (0–10 ng/ml) for 6 days. In F9 cells with H2AX (tet/tet) alleles, the amounts of H2AX protein were decreased significantly by 1–10 ng/ml doxycycline. As a control, ß-actin is also shown. B, time course of H2AX repression in F9 cells with H2AX(tet/tet) alleles after addition of doxycycline (10 ng/ml). At 5 days after doxycycline addition, H2AX was not detected in F9 H2AX(tet/tet) cells. Synthesis of H2AX resumed 8 h after removal of doxycycline and recovered to normal amounts after 3–4 days.

 
Radiosensitization of F9 Cells by Repression of H2AX Expression.
We examined the radiosensitivity of F9 cells in which H2AX gene expression was reduced by doxycycline (Fig. 3A)Citation . Cells were cultured in various concentrations of doxycycline for 6 days and exposed to X-irradiation at various doses. After X-irradiation, cells were cultured in 96-well plates with medium supplemented with 10% FBS and the indicated concentrations of doxycycline. Three days later, cytotoxicity was measured by the MTT assay. Radiosensitivity was calculated as the ratio of the MTT value of irradiated cells to that of nonirradiated cells with the same content of H2AX protein. As shown in Fig. 4ACitation , sensitization to X-irradiation was observed in cells cultured with 1–10 ng/ml doxycycline over a wide range of radiation doses (0–6.0 Gy), in comparison with low doses of doxycycline (0–0.1 ng/ml). The CD50 value, the dose required to reduce absorbance in the MTT assay to 50%, was 2.39 Gy for wild-type F9 cells, whereas the CD50 for F9 H2AX(tet/tet) cells cultured in 1–10 ng/ml doxycycline was 1.22 Gy. Because we incubated irradiated cells with doxycycline-containing medium, radiation sensitivity in this experiment depended on the amount of H2AX at the time of X-irradiation and afterward. A clonogenic assay yielded results similar to the MTT findings (data not shown). Thus, we concluded that suppression of H2AX significantly sensitized F9 cells (by 1.95x) to X-irradiation.



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  		Fig. 3. H2AX expression as a regulator of radiosensitivity in F9 cells. A, F9 cells with H2AX (tet/tet) alleles and wild-type cells cultured in the indicated concentrations of doxycycline were X-irradiated at the doses indicated (0–6.0 Gy). Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. F9 cells with H2AX (tet/tet) alleles were resistant to X-irradiation to the same extent as wild-type cells in 0–0.1 ng/ml doxycycline, whereas cells treated with 1–10 ng/ml doxycycline became 1.95x more sensitive to X-irradiation, as judged from the 50% cytotoxicity dose. B, F9 cells with tetracycline (tet)-regulated H2AX alleles were cultured in the indicated concentrations of doxycycline (0–10 ng/ml) and X-irradiated at 6.0 Gy. After 30 min, cell proteins were analyzed by Western blotting. The amounts of H2AX and {gamma}-H2AX proteins were significantly decreased by 1–10 ng/ml doxycycline. However, the levels of other repair proteins, such as RAD51and KU80, and those of ß-actin were constant at all doxycycline concentrations. C, from A and B, the amounts of H2AX and {gamma}-H2AX proteins were estimated densitometrically and plotted. Simultaneously, radiosensitivity (values of CD50) of tet-regulated F9 cells was plotted. A negative relationship was detected between the radiosensitivity of cells and the amounts of H2AX and {gamma}-H2AX cellular proteins.

 


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  		Fig. 4. Rescue of H2AX function by introduction of native H2AX gene. A, mouse H2AX gene with its promoter was cloned into a vector with a loxP-flanked neomycin resistance gene (pH2AX). The tetracycline (tet)-regulated F9 cells were transformed with the plasmid pH2AX, and neomycin-resistant stable transformants were isolated. The pH2AX transformant was found to express H2AX, even with 10 ng/ml doxycycline. B, radioresistance of H2AX(tet/tet)-repressed F9 cells was rescued by pH2AX transformation. F9 cells with H2AX (tet/tet) were sensitized to X-irradiation when they were cultured in 10 ng/ml doxycycline, but transfection with pH2AX plasmid rendered them resistant to X-irradiation to the same extent as wild-type cells.

 
We examined the amount of proteins in F9 cells X-irradiated at 6.0 Gy, using Western blotting (Fig. 3B)Citation . As indicated previously, the amount of H2AX was greatly decreased at doxycycline concentrations of 1–10 ng/ml. At 0–0.1 ng/ml doxycycline, {gamma}-H2AX could be detected, with much less detected at 1–10 ng/ml doxycycline even after X-irradiation. Amounts of other DNA repair proteins (RAD51 and KU80) expressed as percentages of ß-actin were constant, irrespective of the concentrations of doxycycline. These results indicated that radiosensitization of F9 cells resulted from decreased amounts of H2AX and {gamma}-H2AX protein. Proportional relationships between CD50 value and amounts of H2AX and {gamma}-H2AX are shown in Fig. 3CCitation . Defects of H2AX expression did not alter the amounts of KU80 or RAD51 under either nonirradiated or X-irradiated conditions.

Rescue from Radiosensitization Effect by H2AX Gene.
To rescue the phenotype of H2AX-repressed cells, we constructed a plasmid (pH2AX) composed of genomic DNA of the wild-type H2AX gene with native promoter and a loxP-flanked PGK neomycin resistance gene (Fig. 4A)Citation . F9 cells with H2AX modified by the tet-off system at both alleles were transfected with the pH2AX plasmid. Stable neomycin-resistant transformants were selected, and expression of H2AX was analyzed by Western analysis. The clone was found to express H2AX protein to almost the same extent as wild-type F9 cells (Fig. 4A)Citation , even in the presence of 10 ng/ml doxycycline. Cells were X-irradiated, and cytotoxicity was analyzed (Fig. 4B)Citation . H2AX-rescued cells became as resistant to X-irradiation as wild-type F9 cells, even when cultured in 10 ng/ml doxycycline. This result confirmed that radiosensitization of F9 cells was controlled by H2AX gene expression as opposed to other factors.

Growth Retardation of F9 Tumor by H2AX Repression.
We injected wild-type and tet-regulated F9 cells (2 x 106) s.c. in the right flank of 129Sv mice supplied with water containing doxycycline (1 mg/ml). After 24 h, mice underwent whole-body X-irradiation at a dose of 6.0 Gy, and tumor volume was measured. Ten mice were examined for each data point in Fig. 5, A and BCitation . Growth rates of wild-type and H2AX-repressed tumor cells in vivo were almost the same as when not treated with X-irradiation (Fig. 5A)Citation . However, in vivo growth of F9 cells with repressed H2AX expression was slightly retarded by X-irradiation compared with growth of irradiated wild-type F9 cells (Fig. 5B)Citation .



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  		Fig. 5. Control of tumor growth in H2AX-repressed F9 cells by X-irradiation. A, wild-type and H2AX-repressed F9 teratocarcinoma cells were injected into the flank of 129Sv mice supplied with doxycycline-containing water, and tumor volumes were measured. Growth rate did not differ significantly between wild-type and H2AX-repressed F9 cells when they were not X-irradiated. B, mice injected with wild-type and H2AX-repressed F9 cells were exposed to 6.0 Gy of X-irradiation, and sizes of resulting tumors were measured. X-irradiation slightly retarded growth of H2AX-repressed tumors.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We constructed a tet-off cassette unit from components of tTA and tet-responsive element-associated minimal cytomegalovirus promoter to control expression of the H2AX histone gene in F9 mouse teratocarcinoma cells, which represent a somatic cell line known to be capable of accepting integration of exogenous DNA by homologous recombination (29 , 30) . Using F9 cells, we isolated cell clones with both H2AX alleles modified to result in a tet-regulated form. We demonstrated that radiosensitivity of H2AX-modified cells increased according to the degree of reduction of H2AX and {gamma}-H2AX. This is consistent with a recent finding by Celeste et al. (31) that loss of one H2AX allele compromises genomic integrity (haploinsufficiency) in H2AX+/– mouse embryonic fibroblasts. The severity of DNA damage (chromosomal aberrations), rates of cellular proliferation, and radiation sensitivity are proportional to the H2AX gene dose. Bassing et al. (32) and Celeste et al. (31) demonstrated that H2AX haploinsufficiency resulted in chromosomal abnormalities in normal (untransformed) peripheral T cells. Both experiments demonstrated that biallelic expression of H2AX was required for full maintenance of genomic stability.

H2AX is presently considered to retain repair-related signaling factors in the vicinity of a DSB induced by irradiation (23) . However, the physiological functions of the H2AX-derived focus-forming complex remain unclear. F9 H2AX(tet/tet) cells can be used to analyze the roles of H2AX in signaling DNA damage to DNA repair enzymes and cell cycle checkpoint proteins.

We injected F9 teratocarcinoma cells into the flank of 129Sv mice supplied with doxycycline-containing water. Although tumor growth did not differ significantly between wild-type F9 cells and H2AX-repressed F9 cells without X-irradiation, we detected a slight growth retardation that was limited to H2AX-repressed tumor cells when mice were exposed to X-rays at 6.0 Gy. Observations that H2AX dose-dependently controlled radiosensitivity of cells and that deletion of H2AX did not itself favor tumor development in mice suggested that H2AX might be a productive target for radiotherapy sensitization in treating cancer. However, Celeste et al. (31) and Bassing et al. (32) reported that H2AX deficiency acted synergistically with p53 inactivation to promote genomic instability and tumorigenesis. Thus, when we induce radiosensitization by repressing H2AX expression, p53 in cancer cells must be activated to avoid increasing genomic instability of cancer cells, which might increase the degree of malignancy in the tumor.


   ACKNOWLEDGMENTS
 
We thank Wolfgang Hillen for tTA vector and Junji Takeda, Gen Kondoh, Kyoji Horie, and Shu-hei Yoshida for helpful advice. We also thank Megumi Ohnishi, Mizuho Miyaguni, and Riyo Hirano for technical assistance and Taeko Shimada for manuscript preparation.


   FOOTNOTES
 
Grant support: Grant-in-Aid 13218113 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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.

Requests for reprints: Takashi Morita, Department of Molecular Genetics, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. Phone: 81-6-6645-3925; Fax: 81-6-6645-3927; E-mail: tmorita{at}med.osaka-cu.ac.jp

Received 8/18/03. Revised 3/29/04. Accepted 4/11/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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