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Cancer Research 67, 6889, July 15, 2007. doi: 10.1158/0008-5472.CAN-06-4496
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Inhibition of Nuclear Factor-{kappa}B Activity by Temozolomide Involves O6-Methylguanine–Induced Inhibition of p65 DNA Binding

Bakhtiar Yamini1, Xiaohong Yu1, M. Eileen Dolan2, Michael H. Wu2, Donald W. Kufe4 and Ralph R. Weichselbaum3

1 Section of Neurosurgery, Department of Surgery, 2 Section of Hematology/Oncology, Department of Medicine, and 3 Department of Radiation and Cellular Oncology, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois and 4 Dana-Farber Cancer Institute, Department of Medical Oncology, Boston, Massachusetts

Requests for reprints: Bakhtiar Yamini, MC 4066, Section of Neurosurgery, University of Chicago Hospitals, 5841 South Maryland Avenue, Chicago, IL 60637. Phone: 773-702-2475; Fax: 773-702-5234; E-mail: byamini{at}surgery.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The alkylating agent temozolomide, commonly used in the treatment of malignant glioma, causes cellular cytotoxicity by forming O6-methylguanine adducts. In this report, we investigated whether temozolomide alters the activity of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B). Temozolomide inhibits basal and tumor necrosis factor {alpha} (TNF{alpha})–induced NF-{kappa}B transcriptional activity without altering phosphorylation or degradation of inhibitor of {kappa}B-{alpha}. Inhibition of NF-{kappa}B is secondary to attenuation of p65 DNA binding, not nuclear translocation. Inhibition of DNA binding is shown both in vitro, with gel shift studies and DNA binding assays, and in vivo at {kappa}B sites. Consistent with inhibition of NF-{kappa}B activity, temozolomide reduces basal and TNF{alpha}-induced {kappa}B-dependent gene expression. Temozolomide also inhibits NF-{kappa}B activated by inducers other than TNF{alpha}, including lipopolysaccharide, doxorubicin, and phorbol 12-myristate 13-acetate. The inhibitory action of temozolomide on NF-{kappa}B is observed to be maximal following pretreatment of cells with temozolomide for 16 h and is also seen with the SN1-type methylating agent methylnitrosourea. The ability of temozolomide to form O6-methylguanine adducts is important for inhibition of NF-{kappa}B as is the presence of a functioning mismatch repair system. Activation of NF-{kappa}B with TNF{alpha} before administration of temozolomide reduces the cytotoxicity of temozolomide, whereas 16-h pretreatment with temozolomide resensitizes cells to killing. This work shows a mechanism whereby O6-methylguanine adducts formed by temozolomide lead to inhibition of NF-{kappa}B activity and illustrates a link between mismatch repair processing of alkylator-induced DNA damage and cell death. [Cancer Res 2007;67(14):6889–98]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The monofunctional alkylating agent temozolomide is routinely used for the treatment of malignant glioma (1). Although temozolomide induces the formation of several DNA adducts (2), cytotoxicity results primarily from the formation of O6-methylguanine lesions (3, 4). The importance of O6-methylguanine in mediating cell killing was shown by studies with O6-alkylguanine-DNA alkyltransferase (AGT; ref. 5), a protein that specifically removes alkyl groups from the O6 position of guanine (6). Following DNA replication, O6-methylguanine mispairs with thymine, leading to activation of the mismatch repair (MMR) system. However, as the repair process targets the newly synthesized DNA strand (7), the methylated adduct is left intact. One theory proposes that subsequent futile cycles of repair eventually result in cell death (8), whereas a second hypothesis maintains that it is the damage repair machinery itself that directly signals apoptosis (9). Although there is extensive evidence linking O6-methylguanine and MMR to cell death, the cytotoxic pathways downstream of MMR processing continue to be elucidated (10–13).

The transcription factor nuclear factor-{kappa}B (NF-{kappa}B) plays a prominent role in resistance to cell killing (14, 15) and consists of five structurally related proteins, the most abundant form consisting of the heterodimer of p50 (NF-{kappa}B1) and p65 (RelA; ref. 16). Each NF-{kappa}B subunit contains an NH2-terminal Rel homology domain (RHD) that, among other functions, is important for DNA binding and dimerization (17). In unstimulated cells, NF-{kappa}B is sequestered in the cytosol bound to inhibitor of {kappa}B (I{kappa}B) proteins. Following stimulation, I{kappa}B{alpha}, a well-described I{kappa}B protein, is phosphorylated by the I{kappa}B kinase (I{kappa}K) complex and thus marked for ubiquitination and proteosomal degradation. Degradation of I{kappa}B{alpha} results in the release of the NF-{kappa}B subunits which translocate into the nucleus and bind specific DNA sequences in the promoter region of NF-{kappa}B–regulated genes (18). Although NF-{kappa}B is controlled primarily by I{kappa}B protein degradation, transcriptional activity can be regulated at multiple different sites (18–20). In this regard, several chemotherapeutic agents have been reported to modulate the NF-{kappa}B activation pathway (21, 22), with some enhancing and others inhibiting the overall activity.

In this study, we examined the effect of temozolomide on NF-{kappa}B transcriptional activation. We find that incubation with temozolomide for >6 h results in inhibition of NF-{kappa}B by an I{kappa}B{alpha}-independent mechanism. Temozolomide inhibits the DNA binding ability of p65 without affecting its nuclear translocation. Both low cellular AGT activity and the presence of an intact MMR system are necessary for inhibition of NF-{kappa}B by temozolomide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cells. Temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute, NIH. Methylnitrosourea, lipopolysaccharide, doxorubicin, phorbol 12-myristate 13-acetate (PMA), N-acetylcysteine, DTT, 3-aminobenzamide, and O6-benzylguanine were obtained from Sigma Chemical Co. Temozolomide and methylnitrosourea were dissolved in DMSO (final concentration <0.1%, v/v); BCNU was dissolved in 1% ethanol. Human glioblastoma cell lines U87MG and T98G and colon cancer HCT116 cells were purchased from American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37°C and 5% CO2. HCT116+ch3 cells were a kind gift from Dr. C.R. Boland (Baylor University Medical Center, Dallas, TX). TK6 and MT1 cells, a gift from Dr. Giancarlo Marra (Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland), were grown in RPMI 1640 with 10% FCS.

Stable transfection. The construct pcDNA-AGT has previously been described (23). H80 glioma cells were transfected with pcDNA-AGT or with pcDNA3 by electroporation. Briefly, cells were cultured to 40% to 60% confluence and electroporated using the Electro Square Porator T820 (Genetronics, Inc.). Parameters used for electroporation were as follows: 500 V/cm, 10 pulses of 1.0 ms each in X-VIVO 10 + 1% human serum albumin. Electroporated cells were plated, grown for 24 h, and then cultured for 12 to 14 days in the presence of 250 µg/mL geneticin. Individual colonies were grown in geneticin-containing medium for preparation of cell extract as previously described (24).

AGT activity assay. Extracts were prepared from H80 stable transfectants by homogenization in 50 mmol/L Tris (pH 7.5), 0.1 mmol/L EDTA, and 5 mmol/L DTT buffer. Samples were sonicated for 1 min and centrifuged at 14,000 x g for 30 min. The assay for AGT activity was done as described (5) and measured as the removal of O6-[3H]methylguanine from a 3H-labeled methylated DNA substrate (18 Ci/mmol) after incubation with tissue extract at 37°C for 30 min. Results were expressed as fentomoles of O6-methylguanine released from DNA per milligram of protein.

Luciferase assay. Cells (5 x 103) were plated overnight and cotransfected with the NF-{kappa}B luciferase reporter immunoglobulin-{kappa}B (Ig-{kappa}B)-Luc (containing three repeats of the Ig{kappa} light chain enhancer B site; ref. 25) or the Egr1 luciferase reporter pGL3 660 (containing the minimal Egr1 promoter; ref. 26) and the Renilla reniformis, pRL-TK, expression vectors (ratio of 10:1 Ig-{kappa}B-Luc/pRL-TK) using the SuperFectin transfection kit (Qiagen). After 24 h, cells were pretreated with chemotherapeutic agent (or 0.1% DMSO, vehicle) and then with 10 ng/mL human tumor necrosis factor {alpha} (TNF{alpha}) as indicated. NF-{kappa}B (or Egr1) and Renilla luciferase activities were measured with the Dual-Luciferase reporter assay system (Promega Corp.) 5 h after TNF{alpha} stimulation. Relative luminescence was calculated as the ratio of firefly luminescence to Renilla luminescence.

Cell fractionation and electrophoretic mobility shift assay. Cells were grown and treated as indicated. They were then pelleted by centrifugation at 1,000 rpm for 5 min at 4°C and resuspended in ice-cold buffer A [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L phenylmethysulfonylfluoride (PMSF), 1 µg/mL leupeptin, 5 µg/mL aprotinin]. Following the addition of 25-µL 10% NP40, the suspension was vortexed and centrifuged at 14,500 rpm for 1 min at 4°C; the supernatant was designated as the cytoplasmic fraction. Nuclei were resuspended in 50 µL of ice-cold buffer B [20 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 25% glycerol, 1 µg/mL leupeptin, 5 µg/mL aprotinin] and centrifuged at 14,500 rpm for 5 min. The supernatant was used as the nuclear fraction and protein concentration determined by the Bradford method. Electrophoretic mobility shift assay (EMSA) was done with 10-µg nuclear protein using the Promega gel shift assay system and 32P-labeled NF-{kappa}B [or activator protein 1 (AP-1)] consensus oligonucleotide. Nuclear extract was also preincubated with 100-fold excess unlabeled NF-{kappa}B (specific competitor) or AP-1 consensus sequence (nonspecific competitor). Supershift studies were done by preincubation with anti-p65, anti-p50, or anti–c-Rel antibody (Active Motif) for 30 min.

Dissociation of NF-{kappa}B from I{kappa}B proteins was done in cytoplasmic fractions as described (27). Five micrograms of cytosolic protein were adjusted to 0.2% sodium deoxycholate (DOC; w/v) and incubated on ice for 15 min. The solution was then adjusted to 0.2% NP40 (v/v) and then incubated for an additional 15 min on ice before being analyzed.

NF-{kappa}B DNA binding assay. Five micrograms of nuclear sample or 10 ng of purified recombinant p65 protein (Active Motif) were added to each well and binding assay done according to the manufacturer's instructions (TransAM NF-{kappa}B Assay, Active Motif). For competition, 20 pmol of wild-type or mutated consensus oligonucleotide were added before the sample. Absorbance value was read at 450 nm following primary and secondary horseradish peroxidase (HRP)–conjugated antibody administration.

Western blotting. Twenty micrograms of nuclear, cytosolic, or cellular lysate were subjected to SDS-PAGE. Following electrotransfer, Immobilon-P membranes (Millipore Corp.) were probed with primary rabbit polyclonal antibody against I{kappa}B{alpha}, phospho-Ser32-I{kappa}B{alpha}, NF-{kappa}B-p65, phospho-Ser276-p65 (Cell Signaling Technology, Inc.), MSH2 (Santa Cruz Biotechnology), p65 COOH terminus (AB1604), and monoclonal anti-AGT antibody (Chemicon) diluted 1:1,000 overnight at 4°C. Antirabbit immunoglobulin G (IgG) HRP-linked secondary antibody (Cell Signaling Technology) was used at 1:1,000 dilution. Immunoreactive bands were detected by SuperSignal ECL (Pierce) and exposed to Kodak X-Omat film.

Indirect immunofluorescence imaging. U87 cells grown on glass chamber slides were treated and then washed in PBS followed by 20-min fixation with 4% paraformaldehyde. Permeabilization was done with 1% Triton X-100 in PBS for 10 min. Fixation and permeabilization were done at room temperature. Cells were then washed, blocked with 2% goat serum, and incubated with 20 µg/mL anti–NF-{kappa}B-p65 (Santa Cruz Biotechnology) overnight at 4°C. Cells were washed before incubation with affinity-purified, FITC-conjugated goat anti-rabbit secondary antibody (1:100; Santa Cruz Biotechnology) for 45 min. The samples were counterstained with 1 mg/mL 4',6-diamidino-2-phenylindole (DAPI) solution for 15 min. Following washing, cells were mounted with Citifluor medium and imaged using a Zeiss Axioplan microscope with 63x oil immersion objective (numerical aperture, 1.4). Excitation (Ex) and emission (Em) wavelengths for FITC and DAPI were Ex 480 nm, Em 525 nm and Ex 360 nm, Em 460 nm, respectively.

3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium viability assay. The assay was done as described (28). Viability in all treatment groups was assessed 48 h following administration of temozolomide.

Colony-forming assay. U87 cells were plated and allowed to attach overnight. Cells were then treated with temozolomide and TNF{alpha} and colony formation assay was done as described (29). Surviving fraction was calculated based on the plating efficiency of untreated cells.

Real-time/quantitative reverse transcription-PCR. Total RNA was isolated from U87 cells after treatment. cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen Life Technologies) and quantitative reverse transcription-PCR (RT-PCR) was done on an ABI7700 (Applied Biosystems) using SYBR Green PCR reagents as described (30). Primers for selected genes were designed based on Unigene reference sequences with PrimerExpress software (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase was used for the internal control. Fold change of gene induction was calculated relative to untreated sample.

Chromatin immunoprecipitation assay. U87 cells, treated as indicated, were cross-linked with 1% formaldehyde and analyzed with the EZ-ChIP assay kit (Upstate Biotechnology, Inc.). For serum starvation, cells were maintained in 0% FCS for 24 h and then reconstituted with 0.5% FCS. Cross-linked protein-DNA complexes were sonicated four times for 10 s, to make DNA fragments <1,000 bp in size, and immunoprecipitated with anti-p65 antibody or negative and positive control antibodies, antimouse IgG and anti–RNA polymerase II, respectively. Chromatin-antibody complexes were eluted from Protein G agarose beads with fresh 1% SDS, 0.1 mol/L NaHCO3, and 10 mmol/L DTT. Subsequently, cross-links were reversed with 5 mol/L NaCl for 16 h at 65°C. The DNA was extracted and PCR done with the following promoter specific primers previously described: Bcl-XL, forward 5'-GCACCACCTACATTCAAATCC-3' and reverse 5'-CGATGGAGGAGGAAGCAAGC-3' (31); cyclooxygenase-2 (COX2), forward 5'-CAAGGCGATCAGTCCAGAAC-3' and reverse 5'-GGTAGGCTTTGCTGTCTGAG-3' (32).

Statistical analysis. Results are expressed as mean ± SD. Statistical significance was taken as P < 0.05 using two-tailed Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Temozolomide inhibits NF-{kappa}B–dependent transcription. To evaluate the action of temozolomide on NF-{kappa}B, we used a NF-{kappa}B–responsive luciferase reporter (25), which is 100% inhibited by the specific NF-{kappa}B inhibitor I{kappa}K inhibitor III (BMS-345541; ref. 33; data not shown). In U87 cells, both basal and TNF{alpha}-induced NF-{kappa}B activities are inhibited by temozolomide with an IC50 of 75 µmol/L (Fig. 1A ; 0.1% DMSO, vehicle, has no significant effect on NF-{kappa}B luciferase; data not shown). Cell viability assay shows that treatment with 10 ng/mL TNF{alpha} and 100 µmol/L temozolomide results in only 10% decrease in survival at the time point tested in the luciferase assay (data not shown). Temozolomide has no effect on an Egr1-promoter luciferase construct (Fig. 1B), showing that the action of temozolomide is not due to a nonspecific effect on the luciferase cDNA. Time course studies show that inhibition of NF-{kappa}B by temozolomide is maximal after 16 h (Fig. 1C). Temozolomide also inhibits TNF{alpha}-induced NF-{kappa}B activity in U251 glioma cells (data not shown). In addition, temozolomide attenuates NF-{kappa}B activated by inducers other than TNF{alpha}, including lipopolysaccharide, doxorubicin, and PMA (Supplementary Fig. S1). These results indicate that incubation of glioma cells with clinically relevant doses of temozolomide (peak patient plasma temozolomide level in clinical trials is ~100 µmol/L; ref. 34) for >6 h results in inhibition of NF-{kappa}B activity.


Figure 1
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  		Figure 1. Temozolomide inhibits NF-{kappa}B–dependent transcription. A, U87 cells were cotransfected with NF-{kappa}B-luciferase (Ig-{kappa}B-Luc) and Renilla reniformis expression vectors. After 24 h, cells were treated with temozolomide (TMZ; 16-h pretreatment) and/or 10 ng/mL TNF{alpha} for an additional 5 h. Columns, mean fold change of relative luminescence from triplicate samples (untreated sample = 1); bars, SD. *, P < 0.05. B, U87 cells were cotransfected with Ig-{kappa}B-Luc (white columns) or pEGR-660 (black columns) and Renilla. Cells were untreated or treated with 100 µmol/L temozolomide (16 h pretreatment) and/or 10 ng/mL TNF{alpha}. C, U87 cells were untreated or treated with 100 µmol/L temozolomide for the times indicated either alone or followed by TNF{alpha}, as in (A). **, P < 0.001. D, RT-PCR in U87 cells. Cells were untreated or treated with 100 µmol/L temozolomide (16-h pretreatment) and/or 10 ng/mL TNF{alpha}. Columns, fold change in RNA expression of indicated genes compared with control. Representative of at least three separate experiments showing similar fold change.

 
To examine if temozolomide inhibits NF-{kappa}B–dependent transcription in vivo, we evaluated mRNA levels of several NF-{kappa}B–regulated genes. There is an increase in COX2, I{kappa}B{alpha}, and MnSOD mRNA 1 and 3 h after TNF{alpha} stimulation and of X-linked inhibitor of apoptosis (XIAP) 3 h after TNF{alpha} (Fig. 1D). Pretreatment with 100 µmol/L temozolomide inhibits both basal and TNF{alpha}-induced mRNA expression of these genes and of Bcl-XL, findings consistent with the luciferase reporter studies.

Temozolomide does not inhibit TNF{alpha}-induced I{kappa}B{alpha} degradation. The primary regulatory point of NF-{kappa}B activity is at the level of I{kappa}B protein degradation. Temozolomide has minimal effect on total I{kappa}B{alpha} levels after 16 h (Fig. 2A, compare lanes 1 and 5 ) whereas TNF{alpha} treatment results in phosphorylation and almost complete degradation of I{kappa}B{alpha} by 5 min (Fig. 2A, lane 2). Pretreatment with temozolomide, at a dose and duration that inhibits NF-{kappa}B transcriptional activity, does not alter TNF{alpha}-induced phosphorylation or degradation of I{kappa}B{alpha} (Fig. 2A). The lack of inhibitory effect of temozolomide on TNF{alpha}-induced I{kappa}B{alpha} phosphorylation was confirmed with an I{kappa}K activity assay showing no inhibition of I{kappa}K kinase activity by concentrations of temozolomide up to 250 µmol/L (data not shown). These data show that temozolomide does not act at the level of I{kappa}B{alpha} to inhibit NF-{kappa}B activity.


Figure 2
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  		Figure 2. Temozolomide inhibits TNF{alpha}-induced NF-{kappa}B on EMSA but not I{kappa}B{alpha} degradation. A, U87 cells were untreated, treated with 10 ng/mL TNF{alpha} for 0 to 30 min, or pretreated with 100 µmol/L temozolomide (16 h) followed by TNF{alpha}. Western blot (WB) with anti-I{kappa}B{alpha} or anti–phospho-Ser32-I{kappa}B{alpha} antibody was done and ß-actin was used as loading control. B, U87 cells were stimulated with 10 ng/mL TNF{alpha} for 15 to 60 min with and without 100 µmol/L temozolomide (16-h pretreatment). EMSA was done with NF-{kappa}B consensus oligonucleotide (oligo). One-hundred-fold excess cold NF-{kappa}B or AP-1 oligonucleotide was given where indicated. N.S., nonspecific band. C, left, U87 cells were pretreated with 100 µmol/L temozolomide for the times indicated followed by 10 ng/mL TNF{alpha} for 30 min. Right, U87 cells were untreated or treated with TNF{alpha} for 30 min. Three micrograms of anti-p65, anti-p50, or anti–c-Rel antibody were administered as indicated. D, U87 cells were untreated or pretreated with 100 µmol/L temozolomide and/or stimulated with TNF{alpha} for 30 min. EMSA was done with AP-1 consensus oligonucleotide. Representative of three separate experiments with similar results.

 
Temozolomide inhibits TNF{alpha}-induced NF-{kappa}B on EMSA. We next investigated whether temozolomide alters nuclear lysate binding to a NF-{kappa}B consensus probe using EMSA. TNF{alpha} stimulation results in an increase in NF-{kappa}B binding that is maximal after 30 min (Fig. 2B). Pretreatment with temozolomide for 16 h reduces TNF{alpha}-induced NF-{kappa}B at all time points tested and, consistent with the luciferase studies, inhibition is only evident with temozolomide pretreatment for >6 h (Fig. 2C, left). Analysis of the subunit composition of the translocated NF-{kappa}B reveals that both an anti-p65 antibody and an anti-p50 antibody shift the NF-{kappa}B band whereas a c-Rel antibody has no effect (Fig. 2C, right). The residual NF-{kappa}B present following incubation with an anti-p50 antibody that is shifted with an anti-p65 antibody likely represents the p65 homodimer band. EMSA with AP-1 consensus sequence shows no inhibition by temozolomide of TNF{alpha}-induced AP-1 binding (Fig. 2D), suggesting that the inhibition of NF-{kappa}B on EMSA is not a nonspecific effect.

O6-Methylguanine and MMR mediate inhibition of NF-{kappa}B by temozolomide. Inhibition of NF-{kappa}B by temozolomide is seen following incubation with temozolomide for an extended period (Fig. 1C), suggesting that some metabolite or intermediate is involved. Because O6-methylguanine is the primary cytotoxic lesion formed by temozolomide, we evaluated the role of O6-methylguanine in inhibition of NF-{kappa}B using cells with different AGT activities. In U87 and U251 cells, both with low AGT activity (35, 36), 100 µmol/L temozolomide results in almost 100% inhibition of TNF{alpha}-induced NF-{kappa}B (Fig. 1A, and data not shown). However, in T98G glioma cells, which have high AGT activity (36), there is minimal inhibition of NF-{kappa}B by 100 µmol/L temozolomide and only 50% inhibition by 250 µmol/L temozolomide (Fig. 3A ), a finding also seen on EMSA (Fig. 3B). To confirm the role of O6-methylguanine, we used pharmacologic and genetic studies targeting AGT. Pretreatment of T98 cells with O6-benzylguanine, an irreversible AGT inhibitor (5), increases inhibition of TNF{alpha}-induced NF-{kappa}B by temozolomide in both luciferase reporter studies (Fig. 3A) and EMSA (Fig. 3B, lanes 6–9). AGT was then stably expressed in H80 glioma cells using wild-type AGT, pcDNA-AGT (H80AGT), or empty vector, pcDNA3 (H80pcDNA3), and AGT activity was assessed. AGT activity is below the limit of detection and 1,358 fmol/mg of protein in H80pcDNA3 and H80AGT cells, respectively (Fig. 3C, inset). In parental H80 cells (no detectable AGT activity) and H80pcDNA3 cells, 100 µmol/L temozolomide significantly inhibits TNF{alpha}-induced NF-{kappa}B reporter expression (P < 0.01; Fig. 3C). However, in H80AGT cells, concentrations as high as 250 µmol/L temozolomide have no inhibitory effect on TNF{alpha}-induced NF-{kappa}B. Furthermore, the addition of O6-benzylguanine enhances the inhibition of NF-{kappa}B by temozolomide in H80AGT cells but not H80pcDNA3 cells [Fig. 3D; O6-benzylguanine alone has no effect on the NF-{kappa}B reporter (Fig. 3A)].


Figure 3
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  		Figure 3. O6-Methylguanine mediates inhibition of NF-{kappa}B by temozolomide. A, C, and D, NF-{kappa}B–dependent luciferase assay. Columns, mean fold change relative to control of triplicate samples; bars, SD. A, T98 cells were treated with temozolomide for 16 h, 10 ng/mL TNF{alpha}, and/or 50 µmol/L O6-benzylguanine (BG; given 1 h before temozolomide). *, P < 0.01. B, T98 cells were treated with temozolomide for 16 h, 10 ng/mL TNF{alpha}, and/or 100 µmol/L O6-benzylguanine (1 h before temozolomide) and NF-{kappa}B EMSA done. C, H80 parental and H80pcDNA3 and H80AGT cells were treated with temozolomide for 16 h and/or TNF{alpha} as indicated. *, P < 0.01. Inset, AGT activity determined as described in Materials and Methods (activity is in fentomoles per milligram of protein). D, H80AGT and H80pcDNA3 cells were treated with 100 µmol/L temozolomide for 16 h, O6-benzylguanine (1 h before temozolomide), and 10 ng/mL TNF{alpha} as in (B). Representative of three separate experiments with similar results.

 
Because methylation of guanine mediates the inhibition of NF-{kappa}B by temozolomide, we investigated whether inhibition of NF-{kappa}B is a finding common to agents that alkylate guanine. Whereas the SN1-type methylating agent methylnitrosourea dose-dependently inhibits NF-{kappa}B activity (Fig. 4A ), the chloroethylating agent BCNU, administered at a dose that is almost 10 times more cytotoxic than 100 µmol/L temozolomide (37, 38), has no inhibitory effect on TNF{alpha}-induced NF-{kappa}B in either H80pcDNA3 cells (Fig. 4B) or H80AGT cells (data not shown). These data suggest that O6-alkylation in general does not lead to inhibition of NF-{kappa}B.


Figure 4
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  		Figure 4. NF-{kappa}B is inhibited by SN1 methylating agents in the presence of active MMR. A to D, NF-{kappa}B–dependent luciferase assay. Columns, mean fold change relative to control of triplicate samples; bars, SD. A, U87 cells were pretreated with 10, 100, and 500 nmol/L methylnitrosourea (MNU) for 16 h and/or stimulated with TNF{alpha} as shown. B, H80pcDNA3 cells were pretreated with BCNU for 16 h and/or stimulated with TNF{alpha}. C, HCT116 and HCT116+ch3 cells were untreated (UTC) or pretreated with 100 µmol/L temozolomide for 16 h and/or stimulated with 10 ng/mL TNF{alpha} as indicated. *, P > 0.2. D, TK6 and MT1 cells were pretreated with 2.5, 25, or 100 µmol/L temozolomide for 16 h and/or stimulated with TNF{alpha}. Representative of three separate experiments with similar results.

 
We also evaluated whether temozolomide-induced formation of N3-methyladenine, an adduct that reportedly inhibits NF-{kappa}B via poly(ADP-ribose) polymerase 1 (PARP-1) activation (39), plays a role in inhibition of NF-{kappa}B by temozolomide. Although temozolomide activates PARP-1 (40), inhibition of PARP-1 with 3-aminobenzamide does not reverse the inhibitory effect of temozolomide on NF-{kappa}B (data not shown).

O6-Methylguanine lesions mismatch with thymine, leading to activation of MMR; we therefore evaluated whether inhibition of NF-{kappa}B transcriptional activity by temozolomide requires a functional MMR system. NF-{kappa}B activation was studied in the MMR-deficient human colon adenocarcinoma cell line HCT116 and the paired MMR-proficient cell line HCT116+ch3, which contains one copy of chromosome 3 bearing the hMh1 gene (41). Whereas 100 µmol/L temozolomide inhibits TNF{alpha}-induced NF-{kappa}B activity in HCT116+ch3 cells, there is no significant inhibition seen in HCT116 cells (P > 0.2; Fig. 4C). To confirm the importance of MMR, we studied the paired lymphoblastoid cells, TK6 and MT1 (42). Consistently, 25 µmol/L temozolomide inhibits NF-{kappa}B activity in MMR-proficient TK6 cells but concentrations as high as 100 µmol/L temozolomide have no effect on NF-{kappa}B in MMR-deficient MT1 cells (Fig. 4D). These data, considered together, indicate that it is the processing of O6-methylguanine adducts by the MMR system that leads to the inhibition of NF-{kappa}B.

Temozolomide inhibits p65 DNA binding. Inhibition of NF-{kappa}B on EMSA without inhibition of I{kappa}B{alpha} degradation suggests that temozolomide inhibits either NF-{kappa}B nuclear translocation or DNA binding. To examine this, we first looked at the cellular distribution of NF-{kappa}B using immunofluorescence staining. p65 becomes concentrated within the nucleus following TNF{alpha} stimulation (Fig. 5A ) and pretreatment with temozolomide does not alter this distribution pattern. To confirm the immunofluorescence data, Western blotting of nuclear and cytoplasmic fractions was done showing an increase in nuclear p65 following TNF{alpha} stimulation, a finding temporally consistent with I{kappa}B{alpha} degradation (Fig. 2A), and that pretreatment with temozolomide does not significantly alter this nuclear accumulation (Supplementary Fig. S2).


Figure 5
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  		Figure 5. Temozolomide inhibits TNF{alpha}-induced p65 DNA binding, not nuclear translocation. A, indirect immunofluorescence staining. U87 cells grown on chamber slides were left untreated (C) or pretreated with 100 µmol/L temozolomide (16 h) and/or with 10 ng/mL TNF{alpha} for 30 min. After fixation, p65 was localized with anti-p65 primary antibody and FITC-conjugated secondary antibody and counterstained with DAPI (left column, DAPI; middle column, FITC; right column, merged). B, TK6 cells (left) and MT1 cells (right) were untreated or stimulated with 10 ng/mL TNF{alpha} and nuclear fractions isolated at 30 min. Samples were then treated with 25 µmol/L temozolomide (and/or 0.1% DMSO; TK6 cells) or with the indicated concentration of temozolomide (MT1 cells) for 16 h and NF-{kappa}B EMSA was done. Supershift with anti-p65 or p50 antibody was done as indicated. Western blot of the TNF{alpha}-stimulated nuclear samples with anti-p65 antibody is shown. C, 10 ng of purified recombinant p65 protein, either untreated or incubated with 100 µmol/L temozolomide and/or DMSO for the indicated times, were analyzed by NF-{kappa}B DNA binding assay. Wild-type (wt) or mutated (mut) NF-{kappa}B consensus oligonucleotide (20 pmol) was administered where indicated. Columns, mean fold change in DNA binding relative to untreated p65 (set to 1) of duplicate samples; bars, SD. Western blot with anti-p65 antibody. D, ChIP assay with Bcl-XL (top) and COX2 (bottom) promoter-specific primers. U87 cells were untreated or treated with 100 µmol/L temozolomide for the times shown (left) or treated with 10 ng/mL TNF{alpha} (1 and 3 h) ± 100 µmol/L temozolomide 16 h pretreatment (right). Immunoprecipitation (IP) was done with the antibodies indicated. Representative of at least three separate experiments with similar results.

 
These results suggest that temozolomide inhibits NF-{kappa}B DNA binding. To examine this, nuclear extracts were isolated from TK6 cells that had been stimulated with TNF{alpha} and EMSA done following incubation with temozolomide. Temozolomide at 25 µmol/L inhibits NF-{kappa}B binding to the DNA probe without altering the level p65 protein (Fig. 5B, top). Similar inhibition of DNA binding is seen in U87 cells (Supplementary Fig. S3) and, consistent with the luciferase data, inhibition is only seen following incubation with temozolomide for 16 h (Supplementary Fig. S3, right). Inhibition of DNA binding by 16-h temozolomide treatment was also confirmed by an ELISA-based DNA binding assay in TNF{alpha}-stimulated nuclear extracts (Supplementary Fig. S4). The presence of DNA and MMR protein, both necessary for inhibition of NF-{kappa}B (Fig. 4), in the nuclear fraction extracts was verified by DNA gel electrophoresis (Supplementary Fig. S5, top) and Western blotting for MSH2 (Supplementary Fig. S5, right), respectively. In addition, DNA binding assay with purified recombinant p65 shows that temozolomide does not directly affect p65 DNA binding (Fig. 5C). To verify the role of MMR in the inhibition of DNA binding, EMSA of TNF{alpha}-stimulated nuclear extracts was done using MMR-deficient MT1 cells, showing that even doses as high as 100 µmol/L temozolomide do not inhibit NF-{kappa}B (Fig. 5B, bottom). Next, for further specificity controls, we examined the affect of temozolomide on NF-{kappa}B in cytoplasmic extracts following dissociation of NF-{kappa}B from I{kappa}B proteins with DOC (27). As expected, temozolomide does not inhibit NF-{kappa}B DNA binding (as assessed by EMSA or binding assay) in the cytoplasmic environment (Supplementary Fig. S6) where there are insignificant levels of DNA or MMR proteins (Supplementary Fig. S5).

We next examined whether inhibition of DNA binding is also evident in vivo by evaluating recruitment of p65 to the promoters of the Bcl-XL and COX2 genes using chromatin immunoprecipitation (ChIP) assay. Untreated U87 cells have promoter-bound p65 at baseline, and incubation with temozolomide for 16 h inhibits DNA binding without affecting RNA polymerase II binding (Fig. 5D, top). Pretreatment with temozolomide also reduces TNF{alpha}-induced {kappa}B-element binding at 1 and 3 h (Fig. 5D, bottom). Similar inhibition of {kappa}B-element binding is seen in TK6 cells, whereas no effect of temozolomide on binding is noted in MT1 cells (data not shown). Serum starvation of U87 cells for 24 h considerably reduces basal p65 {kappa}B-element binding but does not block temozolomide-induced inhibition (Supplementary Fig. S7).

An important regulatory mechanism of NF-{kappa}B activity involves reversible p65 phosphorylation (19). Temozolomide does not significantly alter the TNF{alpha}-induced phosphorylation of p65 at either Ser536 (data not shown) or Ser276 (Supplementary Fig. S8). In addition, the antioxidants N-acetylcysteine and DTT do not significantly reverse temozolomide-induced NF-{kappa}B DNA binding or luciferase expression (Supplementary Fig. S9), suggesting that redox-sensitive residues are not involved in the inhibitory action of temozolomide.

Pretreatment with temozolomide enhances combination temozolomide/TNF{alpha}–induced cytotoxicity. Activation of NF-{kappa}B by TNF{alpha} blocks cell killing (14) and temozolomide inhibits NF-{kappa}B only after pretreatment for many hours (Fig. 1C); thus, when TNF{alpha} is given at the same time as temozolomide, NF-{kappa}B remains activated. In U87 cells, there is an increase in cell viability when TNF{alpha} is administered at the same time as temozolomide compared with treatment with temozolomide alone, a finding not seen when TNF{alpha} is given 16 h after temozolomide (i.e., temozolomide pretreatment; Fig. 6A, left ). However, in T98 cells, in which temozolomide does not as effectively inhibit NF-{kappa}B (Fig. 3A), there is no significant increase in viability when temozolomide and TNF{alpha} are given at the same time (Fig. 6A, right). Similar results were seen with H80 cells in that TNF{alpha}, if administered at the same time as temozolomide, leads to an increase in survival only in H80pcDNA3 but not H80AGT cells (data not shown). The overall cytotoxicity of temozolomide pretreatment was also examined by colony-forming assay. Administration of TNF{alpha} and temozolomide at the same time results in an increase in fractional survival compared with when TNF{alpha} is given 16 h after temozolomide (P < 0.05; Fig. 6B). These results suggest that TNF{alpha} activates a pathway, presumably NF-{kappa}B, that blocks the cytotoxicity of temozolomide and that is inhibited when cells are pretreated with temozolomide, thus facilitating cell killing.


Figure 6
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  		Figure 6. Pretreatment with temozolomide enhances combination temozolomide/TNF{alpha}–induced cytotoxicity. A, U87 (left) and T98 (right) cells were untreated or treated with 25 to 100 µmol/L temozolomide alone and/or with 10 ng/mL TNF{alpha}. For combination treatment, TNF{alpha} was given 16 h after temozolomide (white columns) or at the same time as temozolomide (black columns). Cell viability was assessed by 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay 48 h following temozolomide administration. Columns, mean viability of triplicate samples relative to the untreated cells (set to 1); bars, SD. *, P < 0.02; **, P > 0.2. B, U87 cells were treated with temozolomide and TNF{alpha} alone or in combination: temozolomide was given 16 h before TNF{alpha} (Pre) or at the same time as TNF{alpha} (Same). Colony-forming assay was done. Columns, mean surviving fraction of duplicate samples relative to untreated; bars, SD. *, P < 0.05. Representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented herein show for the first time an I{kappa}B{alpha}-independent mechanism for the inhibition of NF-{kappa}B activity by SN1-type methylating agents like temozolomide. The mechanism of inhibition involves attenuation of p65 {kappa}B-element binding. Although temozolomide reduces NF-{kappa}B binding at the Bcl-XL and COX2 promoters, it has previously been reported that, in melanoma cells, temozolomide increases expression of the NF-{kappa}B–regulated chemokine CXCL8 (43). Such differences in gene expression are consistent with promoter-driven differential regulation of subsets of NF-{kappa}B–dependent genes (20) and may be due to gene-specific variations in posttranslational p65 modification. Of note, one previous report briefly showed that temozolomide decreases NF-{kappa}B on EMSA (39); however, in this study, only the 48-h temozolomide treatment time point was evaluated and no analysis of NF-{kappa}B–dependent transcription was done.

Temozolomide inhibits NF-{kappa}B activity through formation of O6-methylguanine adducts, a finding supported by experiments incorporating overexpression and inhibition of AGT. That cellular AGT level by itself is not responsible for the changes in NF-{kappa}B can be seen in experiments with BCNU, in that BCNU does not significantly inhibit NF-{kappa}B in either AGT-positive or AGT-negative cells (Fig. 4B, and data not shown), although AGT can efficiently repair O6-chloroethylguanine lesions (6). Interestingly, O6-methylguanine adducts have previously been reported to directly block NF-{kappa}B from binding DNA in vitro (44). However, the time course of inhibition of NF-{kappa}B is not consistent with temozolomide-induced O6-methylguanine adducts directly affecting {kappa}B binding. In this regard, temozolomide forms a highly reactive SN1-methylating species that methylates DNA within 1 to 2 h (45), whereas inhibition of NF-{kappa}B by temozolomide takes many hours to develop (Fig. 1C). Furthermore, when DNA binding is assessed with TNF{alpha}-stimulated nuclear extracts that are treated with temozolomide after isolation of the nuclei (Fig. 5B and Supplementary Fig. S4), inhibition of NF-{kappa}B binding is observed despite the use of an unmethylated DNA probe. These in vitro experiments using nuclear extracts also indicate that the intermediates necessary for inhibition of NF-{kappa}B are already present within the nuclear contents and are activated following formation of O6-methylguanine.

Attenuation of NF-{kappa}B DNA binding suggests that temozolomide may exert its inhibitory action via an effect on the p65 RHD. In this regard, NH2-terminal cleavage of p65 represents one potential mechanism by which temozolomide could block NF-{kappa}B DNA binding. To investigate this, we evaluated p65 following treatment with temozolomide using an antibody that specifically recognizes the COOH terminus of the molecule. No cleavage fragment formation was seen (Supplementary Fig. S10), indicating that temozolomide does not cleave the p65 RHD to block DNA binding. We also observed no effect of temozolomide on the phospho-status of Ser276, an important residue in the RHD involved with NF-{kappa}B transactivation (46). In addition, treatment with reducing agents does not reverse the inhibition of NF-{kappa}B, suggesting that, unlike certain natural compounds (47), temozolomide does not block DNA binding via an effect on redox-sensitive residues. Several other sites in the RHD are known to be important for NF-{kappa}B DNA binding (17). Although temozolomide may target these sites, it is also possible that temozolomide acts at the COOH-terminal transactivation domain of p65, resulting in a molecular conformational change that then blocks DNA binding.

O6-Methylguanine adducts and MMR are not only necessary for the inhibition of NF-{kappa}B by temozolomide but also central to the cytotoxicity of SN1-type methylating agents. This finding raises the question of whether temozolomide-induced cell death is mediated by temozolomide-induced inhibition of NF-{kappa}B. Although there is no prior evidence to support this, our results, and the importance of NF-{kappa}B activation in promoting cell survival, are consistent with such a hypothesis. In this regard, we see that temozolomide administration results in reduced expression of the antiapoptotic genes Bcl-XL and XIAP (Fig. 1D), a finding similar to reports that O6-methylguanine leads to inhibition of Bcl-2 (10), an NF-{kappa}B–responsive gene (48), and to activation of mitochondrial apoptosis cascade proteins (12). The requirement of MMR activity for inhibition of NF-{kappa}B is also reasonable in light of the central role played by MMR in DNA damage signaling (7, 49) and illustrates a potential mechanism by which MMR can target the removal of injured cells from the circulation. Interestingly, temozolomide-induced inhibition of NF-{kappa}B is blocked when the MMR system is disabled through a defect in either MutS{alpha} (MT1 cells) or MutL{alpha} (HCT116 cells; ref. 7). This finding suggests that inhibition of NF-{kappa}B by temozolomide is not mediated by a specific MMR protein but by MMR processing of O6-methylguanine in general.

The role of NF-{kappa}B in mediating cellular proliferation, oncogenesis, and resistance to therapy (15) has made it an important target in cancer therapy. Consequently, much investigation is currently focused on developing clinically useful inhibitors of this transcription factor (50). Understanding the mechanism by which temozolomide inhibits NF-{kappa}B can significantly increase our knowledge of the mechanism underlying MMR-induced damage signaling. Furthermore, exploiting this cellular damage response can potentially improve the design of rationally based combinatorial treatment strategies that are necessary for the successful management of this devastating disease.


   Acknowledgments
 
Grant support: American Cancer Society, Illinois Division, grant #05-15 and American College of Surgeons Faculty Research Fellowship Award (B. Yamini); National Cancer Institute grants R01 CA111423 (R.R. Weichselbaum), P50 CA 097247, and R01 CA81583 (M.E. Dolan); and a gift from the Foglia Family to the Center for Radiation Therapy.

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 Fangming Tang, Tom Darga, Sato Nakamura, Jula Veerapong, and Kai Bickenbach for experimental assistance; Anning Lin for helpful comments; and Mark K. Abe for critical review of the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/ 6/06. Revised 4/10/07. Accepted 5/ 3/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–96.[Abstract/Free Full Text]
  2. Newlands ES, Stevens MF, Wedge SR, Wheelhouse RT, Brock C. Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 1997;23:35–61.[CrossRef][Medline]
  3. Tisdale MJ. Antitumor imidazotetrazines-XV. Role of guanine O6 alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem Pharmacol 1987;36:457–62.[CrossRef][Medline]
  4. Baer JC, Freeman AA, Newlands ES, Watson AJ, Rafferty JA, Margison GP. Depletion of O6-alkylguanine-DNA alkyltransferase correlates with potentiation of temozolomide and CCNU toxicity in human tumour cells. Br J Cancer 1993;67:1299–302.[Medline]
  5. Dolan ME, Moschel RC, Pegg AE. Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents. Proc Natl Acad Sci U S A 1990;87:5368–72.[Abstract/Free Full Text]
  6. Gerson SL. Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 2002;20:2388–99.[Abstract/Free Full Text]
  7. Stojic L, Brun R, Jiricny J. Mismatch repair and DNA damage signalling. DNA Repair (Amst) 2004;3:1091–101.[CrossRef][Medline]
  8. Karran P, Macpherson P, Ceccotti S, Dogliotti E, Griffin S, Bignami M. O6-Methylguanine residues elicit DNA repair synthesis by human cell extracts. J Biol Chem 1993;268:15878–86.[Abstract/Free Full Text]
  9. Fishel R. The selection for mismatch repair defects in hereditary nonpolyposis colorectal cancer: revising the mutator hypothesis. Cancer Res 2001;61:7369–74.[Free Full Text]
  10. Ochs K, Kaina B. Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent. Cancer Res 2000;60:5815–24.[Abstract/Free Full Text]
  11. Hickman MJ, Samson LD. Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc Natl Acad Sci U S A 1999;96:10764–9.[Abstract/Free Full Text]
  12. Hickman MJ, Samson LD. Apoptotic signaling in response to a single type of DNA lesion, O(6)-methylguanine. Mol Cell 2004;14:105–16.[CrossRef][Medline]
  13. Hirose Y, Berger MS, Pieper RO. p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 2001;61:1957–63.[Abstract/Free Full Text]
  14. Karin M, Lin A. NF-{kappa}B at the crossroads of life and death. Nat Immunol 2002;3:221–7.[CrossRef][Medline]
  15. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-{kappa}B. J Clin Invest 2001;107:241–6.[CrossRef][Medline]
  16. Ghosh S, May MJ, Kopp EB. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225–60.[CrossRef][Medline]
  17. Chen FE, Huang DB, Chen YQ, Ghosh G. Crystal structure of p50/p65 heterodimer of transcription factor NF-{kappa}B bound to DNA. Nature 1998;391:410–3.[CrossRef][Medline]
  18. Kucharczak J, Simmons MJ, Fan Y, Gelinas C. To be, or not to be: NF-{kappa}B is the answer—role of Rel/NF-{kappa}B in the regulation of apoptosis. Oncogene 2003;22:8961–82.[CrossRef][Medline]
  19. Vermeulen L, De Wilde G, Notebaert S, Vanden Berghe W, Haegeman G. Regulation of the transcriptional activity of the nuclear factor-{kappa}B p65 subunit. Biochem Pharmacol 2002;64:963–70.[CrossRef][Medline]
  20. Anrather J, Racchumi G, Iadecola C. cis-acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-{kappa}B. J Biol Chem 2005;280:244–52.[Abstract/Free Full Text]
  21. Nakanishi C, Toi M. Nuclear factor-{kappa}B inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer 2005;5:297–309.[CrossRef][Medline]
  22. Campbell KJ, Witty JM, Rocha S, Perkins ND. Cisplatin mimics ARF tumor suppressor regulation of RelA (p65) nuclear factor-{kappa}B transactivation. Cancer Res 2006;66:929–35.[Abstract/Free Full Text]
  23. Cai Y, Wu MH, Ludeman SM, Grdina DJ, Dolan ME. Role of O6-alkylguanine-DNA alkyltransferase in protecting against cyclophosphamide-induced toxicity and mutagenicity. Cancer Res 1999;59:3059–63.[Abstract/Free Full Text]
  24. Wu MH, Yan B, Humerickhouse R, Dolan ME. Irinotecan activation by human carboxylesterases in colorectal adenocarcinoma cells. Clin Cancer Res 2002;8:2696–700.[Abstract/Free Full Text]
  25. Kanno T, Brown K, Siebenlist U. Evidence in support of a role for human T-cell leukemia virus type I Tax in activating NF-{kappa}B via stimulation of signaling pathways. J Biol Chem 1995;270:11745–8.[Abstract/Free Full Text]
  26. Park JO, Lopez CA, Gupta VK, et al. Transcriptional control of viral gene therapy by cisplatin. J Clin Invest 2002;110:403–10.[CrossRef][Medline]
  27. Han Y, Englert JA, Delude RL, Fink MP. Ethacrynic acid inhibits multiple steps in the NF-{kappa}B signaling pathway. Shock 2005;23:45–53.[CrossRef][Medline]
  28. Yamini B, Yu X, Gillespie GY, Kufe DW, Weichselbaum RR. Transcriptional targeting of adenovirally delivered tumor necrosis factor {alpha} by temozolomide in experimental glioblastoma. Cancer Res 2004;64:6381–4.[Abstract/Free Full Text]
  29. Salloum RM, Jaskowiak NT, Mauceri HJ, et al. NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity. Cancer Res 2000;60:6958–63.[Abstract/Free Full Text]
  30. Kimchi ET, Posner MC, Park JO, et al. Progression of Barrett's metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiation. Cancer Res 2005;65:3146–54.[Abstract/Free Full Text]
  31. Marinari B, Costanzo A, Marzano V, Piccolella E, Tuosto L. CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-{kappa}B subunits on IL-8 and Bcl-xL gene promoters. Proc Natl Acad Sci U S A 2004;101:6098–103.[Abstract/Free Full Text]
  32. Miao F, Gonzalo IG, Lanting L, Natarajan R. In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions. J Biol Chem 2004;279:18091–7.[Abstract/Free Full Text]
  33. Yang J, Amiri KI, Burke JR, Schmid JA, Richmond A. BMS-345541 targets inhibitor of {kappa}B kinase and induces apoptosis in melanoma: involvement of nuclear factor {kappa}B and mitochondria pathways. Clin Cancer Res 2006;12:950–60.[Abstract/Free Full Text]
  34. Brada M, Judson I, Beale P, et al. Phase I dose-escalation and pharmacokinetic study of temozolomide (SCH 52365) for refractory or relapsing malignancies. Br J Cancer 1999;81:1022–30.[CrossRef][Medline]
  35. Dolan ME, Mitchell RB, Mummert C, Moschel RC, Pegg AE. Effect of O6-benzylguanine analogues on sensitivity of human tumor cells to the cytotoxic effects of alkylating agents. Cancer Res 1991;51:3367–72.[Abstract/Free Full Text]
  36. Hermisson M, Klumpp A, Wick W, et al. O6-Methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells. J Neurochem 2006;96:766–76.[CrossRef][Medline]
  37. Jansen M, Bardenheuer W, Sorg UR, Seeber S, Flasshove M, Moritz T. Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones. Eur J Haematol 2001;67:2–13.[CrossRef][Medline]
  38. Cai Y, Wu MH, Xu-Welliver M, Pegg AE, Ludeman SM, Dolan ME. Effect of O6-benzylguanine on alkylating agent-induced toxicity and mutagenicity. In Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases. Cancer Res 2000;60:5464–9.[Abstract/Free Full Text]
  39. Tentori L, Forini O, Fossile E, et al. N3-Methyladenine induces early poly(ADP-ribosylation), reduction of nuclear factor-{kappa}B DNA binding ability, and nuclear up-regulation of telomerase activity. Mol Pharmacol 2005;67:572–81.[Abstract/Free Full Text]
  40. Tisdale MJ. Antitumour imidazotetrazines: XI. Effect of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one [CCRG 81045; M and B 39831 NSC 362856] on poly(ADP-ribose) metabolism. Br J Cancer 1985;52:789–92.[Medline]
  41. Koi M, Umar A, Chauhan DP, et al. Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N'-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. Cancer Res 1994;54:4308–12.[Abstract/Free Full Text]
  42. Kat A, Thilly WG, Fang WH, Longley MJ, Li GM, Modrich P. An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc Natl Acad Sci U S A 1993;90:6424–8.[Abstract/Free Full Text]
  43. Amiri KI, Horton LW, LaFleur BJ, Sosman JA, Richmond A. Augmenting chemosensitivity of malignant melanoma tumors via proteasome inhibition: implication for bortezomib (VELCADE, PS-341) as a therapeutic agent for malignant melanoma. Cancer Res 2004;64:4912–8.[Abstract/Free Full Text]
  44. Bonfanti M, Broggini M, Prontera C, D'Incalci M. O6-Methylguanine inhibits the binding of transcription factors to DNA. Nucleic Acids Res 1991;19:5739–42.[Abstract/Free Full Text]
  45. Bull VL, Tisdale MJ. Antitumour imidazotetrazines: XVI. Macromolecular alkylation by 3-substituted imidazotetrazinones. Biochem Pharmacol 1987;36:3215–20.[CrossRef][Medline]
  46. Okazaki T, Sakon S, Sasazuki T, et al. Phosphorylation of serine 276 is essential for p65 NF-{kappa}B subunit-dependent cellular responses. Biochem Biophys Res Commun 2003;300:807–12.[CrossRef][Medline]
  47. Garcia-Pineres AJ, Castro V, Mora G, et al. Cysteine 38 in p65/NF-{kappa}B plays a crucial role in DNA binding inhibition by sesquiterpene lactones. J Biol Chem 2001;276:39713–20.[Abstract/Free Full Text]
  48. Tamatani M, Che YH, Matsuzaki H, et al. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF{kappa}B activation in primary hippocampal neurons. J Biol Chem 1999;274:8531–8.[Abstract/Free Full Text]
  49. Wang JY, Edelmann W. Mismatch repair proteins as sensors of alkylation DNA damage. Cancer Cell 2006;9:417–8.[CrossRef][Medline]
  50. Karin M, Yamamoto Y, Wang QM. The IKK NF-{kappa}B system: a treasure trove for drug development. Nat Rev Drug Discov 2004;3:17–26.[CrossRef][Medline]



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Correction: Article on Inhibition of NF-{kappa}B by Temozolomide
Cancer Res., February 15, 2009; 69(4): 1695 - 1695.
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B. Yamini, X. Yu, P. Pytel, N. Galanopoulos, V. Rawlani, J. Veerapong, K. Bickenbach, and R. R. Weichselbaum
Adenovirally Delivered Tumor Necrosis Factor-{alpha} Improves the Antiglioma Efficacy of Concomitant Radiation and Temozolomide Therapy
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