Inhibition of Nuclear Factor-kb Activity by Temozolomide Involves O 6 -methylguanine–induced Inhibition of P65 Dna Binding

The alkylating agent temozolomide, commonly used in the treatment of malignant glioma, causes cellular cytotoxicity by forming O 6-methylguanine adducts. In this report, we investigated whether temozolomide alters the activity of the transcription factor nuclear factor-KB (NF-KB). Temozolomide inhibits basal and tumor necrosis factor A (TNFA)–induced NF-KB transcriptional activity without altering phosphoryla-tion or degradation of inhibitor of KB-A. Inhibition of NF-KB 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 KB sites. Consistent with inhibition of NF-KB activity, temozolomide reduces basal and TNFA-induced KB-dependent gene expression. Temozolomide also inhibits NF-KB activated by inducers other than TNFA, including lipopoly-saccharide, doxorubicin, and phorbol 12-myristate 13-acetate. The inhibitory action of temozolomide on NF-KB is observed to be maximal following pretreatment of cells with temozo-lomide for 16 h and is also seen with the S N 1-type methylating agent methylnitrosourea. The ability of temozolomide to form O 6-methylguanine adducts is important for inhibition of NF-KB as is the presence of a functioning mismatch repair system. Activation of NF-KB with TNFA 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 O 6-methylguanine adducts formed by temozolomide lead to inhibition of NF-KB activity and illustrates a link between mismatch repair processing of alkylator-induced DNA damage and cell death.


Introduction
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 O 6 -methylguanine lesions (3,4).The importance of O 6 -methylguanine in mediating cell killing was shown by studies with O 6 -alkylguanine-DNA alkyltransferase (AGT; ref. 5), a protein that specifically removes alkyl groups from the O 6 position of guanine (6).Following DNA replication, O 6 -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 O 6 -methylguanine and MMR to cell death, the cytotoxic pathways downstream of MMR processing continue to be elucidated (10)(11)(12)(13).
The transcription factor nuclear factor-nB (NF-nB) 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-nB1) and p65 (RelA; ref. 16).Each NF-nB subunit contains an NH 2 -terminal Rel homology domain (RHD) that, among other functions, is important for DNA binding and dimerization (17).In unstimulated cells, NF-nB is sequestered in the cytosol bound to inhibitor of nB (InB) proteins.Following stimulation, InBa, a well-described InB protein, is phosphorylated by the InB kinase (InK) complex and thus marked for ubiquitination and proteosomal degradation.Degradation of InBa results in the release of the NF-nB subunits which translocate into the nucleus and bind specific DNA sequences in the promoter region of NF-nB-regulated genes (18).Although NF-nB is controlled primarily by InB protein degradation, transcriptional activity can be regulated at multiple different sites (18)(19)(20).In this regard, several chemotherapeutic agents have been reported to modulate the NF-nB activation pathway (21,22), with some enhancing and others inhibiting the overall activity.
In this study, we examined the effect of temozolomide on NF-nB transcriptional activation.We find that incubation with temozolomide for >6 h results in inhibition of NF-nB by an InBaindependent 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-nB by temozolomide.
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 Ag/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 Â g for 30 min.The assay for AGT activity was done as described (5) and measured as the removal of O 6 -[ 3 H]methylguanine from a 3 H-labeled methylated DNA substrate (18 Ci/mmol) after incubation with tissue extract at 37jC for 30 min.Results were expressed as fentomoles of O 6 -methylguanine released from DNA per milligram of protein.
Luciferase assay.Cells (5 Â 10 3 ) were plated overnight and cotransfected with the NF-nB luciferase reporter immunoglobulin-nB (Ig-nB)-Luc (containing three repeats of the Ign 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-nB-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 a (TNFa) as indicated.NF-nB (or Egr1) and Renilla luciferase activities were measured with the Dual-Luciferase reporter assay system (Promega Corp.) 5 h after TNFa 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 4jC 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 Ag/mL leupeptin, 5 Ag/mL aprotinin].Following the addition of 25-AL 10% NP40, the suspension was vortexed and centrifuged at 14,500 rpm for 1 min at 4jC; the supernatant was designated as the cytoplasmic fraction.Nuclei were resuspended in 50 AL 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 Ag/mL leupeptin, 5 Ag/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-Ag nuclear protein using the Promega gel shift assay system and 32 P-labeled NF-nB [or activator protein 1 (AP-1)] consensus oligonucleotide.Nuclear extract was also preincubated with 100-fold excess unlabeled NF-nB (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-nB from InB 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-KB 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-nB Assay, Active Motif).For competition, 20 pmol of wildtype 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.
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 Ag/mL anti-NF-nB-p65 (Santa Cruz Biotechnology) overnight at 4jC.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 63Â 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.
Colony-forming assay.U87 cells were plated and allowed to attach overnight.Cells were then treated with temozolomide and TNFa 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.
Statistical analysis.Results are expressed as mean F SD. Statistical significance was taken as P < 0.05 using two-tailed Student's t test.

Results
Temozolomide inhibits NF-KB-dependent transcription.To evaluate the action of temozolomide on NF-nB, we used a NF-nBresponsive luciferase reporter (25), which is 100% inhibited by the specific NF-nB inhibitor InK inhibitor III (BMS-345541; ref. 33; data not shown).In U87 cells, both basal and TNFa-induced NF-nB activities are inhibited by temozolomide with an IC 50 of 75 Amol/L (Fig. 1A; 0.1% DMSO, vehicle, has no significant effect on NF-nB luciferase; data not shown).Cell viability assay shows that treatment with 10 ng/mL TNFa and 100 Amol/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-nB by temozolomide is maximal after 16 h (Fig. 1C).Temozolomide also inhibits TNFa-induced NF-nB activity in U251 glioma cells (data not shown).In addition, temozolomide attenuates NF-nB activated by inducers other than TNFa, 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 f100 Amol/L; ref. 34) for >6 h results in inhibition of NF-nB activity.
To examine if temozolomide inhibits NF-nB-dependent transcription in vivo, we evaluated mRNA levels of several NF-nB-regulated genes.There is an increase in COX2, InBa, and MnSOD mRNA 1 and 3 h after TNFa stimulation and of X-linked inhibitor of apoptosis (XIAP) 3 h after TNFa (Fig. 1D).Pretreatment with 100 Amol/L temozolomide inhibits both basal and TNFa-induced mRNA expression of these genes and of Bcl-X L , findings consistent with the luciferase reporter studies.
Temozolomide does not inhibit TNFA-induced IKBA degradation.The primary regulatory point of NF-nB activity is at the level of InB protein degradation.Temozolomide has minimal effect on total InBa levels after 16 h (Fig. 2A, compare lanes 1 and 5) whereas TNFa treatment results in phosphorylation and almost complete degradation of InBa by 5 min (Fig. 2A, lane 2).Pretreatment with temozolomide, at a dose and duration that inhibits NF-nB transcriptional activity, does not alter TNFainduced phosphorylation or degradation of InBa (Fig. 2A).The lack of inhibitory effect of temozolomide on TNFa-induced InBa phosphorylation was confirmed with an InK activity assay showing no inhibition of InK kinase activity by concentrations of temozolomide up to 250 Amol/L (data not shown).These data Figure 1.Temozolomide inhibits NF-nB-dependent transcription.A, U87 cells were cotransfected with NF-nB-luciferase (Ig-nB-Luc) and Renilla reniformis expression vectors.After 24 h, cells were treated with temozolomide (TMZ ; 16-h pretreatment) and/or 10 ng/mL TNFa 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-nB-Luc (white columns ) or pEGR-660 (black columns ) and Renilla .Cells were untreated or treated with 100 Amol/L temozolomide (16 h pretreatment) and/or 10 ng/mL TNFa.C, U87 cells were untreated or treated with 100 Amol/L temozolomide for the times indicated either alone or followed by TNFa, as in (A).**, P < 0.001.D, RT-PCR in U87 cells.Cells were untreated or treated with 100 Amol/L temozolomide (16-h pretreatment) and/or 10 ng/mL TNFa.Columns, fold change in RNA expression of indicated genes compared with control.Representative of at least three separate experiments showing similar fold change.
show that temozolomide does not act at the level of InBa to inhibit NF-nB activity.
Temozolomide inhibits TNFA-induced NF-KB on EMSA.We next investigated whether temozolomide alters nuclear lysate binding to a NF-nB consensus probe using EMSA.TNFa stimulation results in an increase in NF-nB binding that is maximal after 30 min (Fig. 2B).Pretreatment with temozolomide for 16 h reduces TNFa-induced NF-nB 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-nB reveals that both an anti-p65 antibody and an anti-p50 antibody shift the NF-nB band whereas a c-Rel antibody has no effect (Fig. 2C, right).The residual NF-nB 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 TNFa-induced AP-1 binding (Fig. 2D), suggesting that the inhibition of NF-nB on EMSA is not a nonspecific effect.
O 6 -Methylguanine and MMR mediate inhibition of NF-nB by temozolomide.Inhibition of NF-nB by temozolomide is seen following incubation with temozolomide for an extended period (Fig. 1C), suggesting that some metabolite or intermediate is involved.Because O 6 -methylguanine is the primary cytotoxic lesion formed by temozolomide, we evaluated the role of O 6 -methylguanine in inhibition of NF-nB using cells with different AGT activities.In U87 and U251 cells, both with low AGT activity (35,36), 100 Amol/L temozolomide results in almost 100% inhibition of TNFa-induced NF-nB (Fig. 1A, and data not shown).However, in T98G glioma cells, which have high AGT activity (36), there is minimal inhibition of NF-nB by 100 Amol/L temozolomide and only 50% inhibition by 250 Amol/L temozolomide (Fig. 3A), a finding also seen on EMSA (Fig. 3B).To confirm the role of O 6 -methylguanine, we used pharmacologic and genetic studies targeting AGT.Pretreatment of T98 cells with O 6 -benzylguanine, an irreversible AGT inhibitor (5), increases inhibition of TNFa-induced NF-nB 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 (H80 AGT ), or empty vector, pcDNA3 (H80 pcDNA3 ), and AGT activity was assessed.AGT activity is below the limit of detection and 1,358 fmol/mg of protein in H80 pcDNA3 and H80 AGT cells, respectively (Fig. 3C, inset).In parental H80 cells (no detectable AGT activity) and H80 pcDNA3 cells, 100 Amol/L temozolomide significantly inhibits TNFa-induced NF-nB reporter expression (P < 0.01; Fig. 3C).However, in H80 AGT cells, concentrations as high as 250 Amol/L temozolomide have no inhibitory effect on TNFainduced NF-nB.Furthermore, the addition of O 6 -benzylguanine enhances the inhibition of NF-nB by temozolomide in H80 AGT cells but not H80 pcDNA3 cells [Fig.3D; O 6 -benzylguanine alone has no effect on the NF-nB reporter (Fig. 3A)].
Because methylation of guanine mediates the inhibition of NF-nB by temozolomide, we investigated whether inhibition of NF-nB is a finding common to agents that alkylate guanine.Whereas the S N 1-type methylating agent methylnitrosourea dosedependently inhibits NF-nB activity (Fig. 4A), the chloroethylating agent BCNU, administered at a dose that is almost 10 times more cytotoxic than 100 Amol/L temozolomide (37,38), has no inhibitory effect on TNFa-induced NF-nB in either H80 pcDNA3 cells (Fig. 4B) or H80 AGT cells (data not shown).These data suggest that O 6 -alkylation in general does not lead to inhibition of NF-nB.
We also evaluated whether temozolomide-induced formation of N 3 -methyladenine, an adduct that reportedly inhibits NF-nB via Temozolomide inhibits TNFa-induced NF-nB on EMSA but not InBa degradation.A, U87 cells were untreated, treated with 10 ng/mL TNFa for 0 to 30 min, or pretreated with 100 Amol/L temozolomide (16 h) followed by TNFa.Western blot (WB ) with anti-InBa or anti-phospho-Ser 32 -InBa antibody was done and h-actin was used as loading control.B, U87 cells were stimulated with 10 ng/mL TNFa for 15 to 60 min with and without 100 Amol/L temozolomide (16-h pretreatment).EMSA was done with NF-nB consensus oligonucleotide (oligo ).One-hundred-fold excess cold NF-nB or AP-1 oligonucleotide was given where indicated.N.S., nonspecific band.C, left, U87 cells were pretreated with 100 Amol/L temozolomide for the times indicated followed by 10 ng/mL TNFa for 30 min.Right, U87 cells were untreated or treated with TNFa 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 Amol/L temozolomide and/or stimulated with TNFa for 30 min.EMSA was done with AP-1 consensus oligonucleotide.Representative of three separate experiments with similar results.
O 6 -Methylguanine lesions mismatch with thymine, leading to activation of MMR; we therefore evaluated whether inhibition of NF-nB transcriptional activity by temozolomide requires a functional MMR system.NF-nB 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 Amol/L temozolomide inhibits TNFa-induced NF-nB 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 Amol/L temozolomide inhibits NF-nB activity in MMR-proficient TK6 cells but concentrations as high as 100 Amol/L temozolomide have no effect on NF-nB in MMR-deficient MT1 cells (Fig. 4D).These data, considered together, indicate that it is the processing of O 6 -methylguanine adducts by the MMR system that leads to the inhibition of NF-nB.
Temozolomide inhibits p65 DNA binding.Inhibition of NF-nB on EMSA without inhibition of InBa degradation suggests that temozolomide inhibits either NF-nB nuclear translocation or DNA binding.To examine this, we first looked at the cellular distribution of NF-nB using immunofluorescence staining.p65 becomes concentrated within the nucleus following TNFa 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 TNFa stimulation, a finding temporally consistent with InBa degradation (Fig. 2A), and that pretreatment with temozolomide does not significantly alter this nuclear accumulation (Supplementary Fig. S2).
These results suggest that temozolomide inhibits NF-nB DNA binding.To examine this, nuclear extracts were isolated from TK6 cells that had been stimulated with TNFa and EMSA done following incubation with temozolomide.Temozolomide at 25 Amol/L inhibits NF-nB 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 TNFa-stimulated nuclear extracts (Supplementary Fig. S4).The presence of DNA and MMR protein, both necessary for inhibition of NF-nB (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 TNFa-stimulated nuclear extracts was done using MMR-deficient MT1 cells, showing that even doses as high as 100 Amol/L temozolomide do not inhibit NF-nB (Fig. 5B, bottom).Next, for further specificity controls, we examined the affect of temozolomide on NF-nB in cytoplasmic extracts following dissociation of NF-nB from InB proteins with DOC (27).As expected, temozolomide does not inhibit NF-nB 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-X L 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 TNFa-induced nBelement binding at 1 and 3 h (Fig. 5D, bottom).Similar inhibition of nB-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 nB-element binding but does not block temozolomide-induced inhibition (Supplementary Fig. S7).A, U87 cells were pretreated with 10, 100, and 500 nmol/L methylnitrosourea (MNU ) for 16 h and/or stimulated with TNFa as shown.B, H80 pcDNA3 cells were pretreated with BCNU for 16 h and/or stimulated with TNFa.C, HCT116 and HCT116+ch3 cells were untreated (UTC ) or pretreated with 100 Amol/L temozolomide for 16 h and/or stimulated with 10 ng/mL TNFa as indicated.*, P > 0.2.D, TK6 and MT1 cells were pretreated with 2.5, 25, or 100 Amol/L temozolomide for 16 h and/or stimulated with TNFa.Representative of three separate experiments with similar results.

Cancer Research
Cancer Res 2007; 67: (14).July 15, 2007 An important regulatory mechanism of NF-nB activity involves reversible p65 phosphorylation (19).Temozolomide does not significantly alter the TNFa-induced phosphorylation of p65 at either Ser 536 (data not shown) or Ser 276 (Supplementary Fig. S8).In addition, the antioxidants N-acetylcysteine and DTT do not significantly reverse temozolomide-induced NF-nB 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/TNFA-induced cytotoxicity.Activation of NF-nB by TNFa blocks cell killing ( 14) and temozolomide inhibits NF-nB only after pretreatment for many hours (Fig. 1C); thus, when TNFa is given at the same time as temozolomide, NF-nB remains activated.In U87 cells, there is an increase in cell viability when TNFa is administered at the same time as temozolomide compared with treatment with temozolomide alone, a finding not seen when TNFa 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-nB (Fig. 3A), there is no significant increase in viability when temozolomide and TNFa are given at the same time (Fig. 6A, right).Similar results were seen with H80 cells in that TNFa, if administered at the same time as temozolomide, leads to an increase in survival only in H80 pcDNA3 but not H80 AGT cells (data not shown).The overall cytotoxicity of temozolomide pretreatment was also examined by colony-forming assay.Administration of TNFa and temozolomide at the same time results in an increase in fractional survival compared with when TNFa is given 16 h after temozolomide (P < 0.05; Fig. 6B).These results suggest that TNFa activates a pathway, presumably NF-nB, that blocks the cytotoxicity of temozolomide and that is inhibited when cells are pretreated with temozolomide, thus facilitating cell killing.

Discussion
The data presented herein show for the first time an InBaindependent mechanism for the inhibition of NF-nB activity by S N 1-type methylating agents like temozolomide.The mechanism of inhibition involves attenuation of p65 nB-element binding.Although temozolomide reduces NF-nB binding at the Bcl-X L and COX2 promoters, it has previously been reported that, in melanoma cells, temozolomide increases expression of the NF-nB-regulated chemokine CXCL8 (43).Such differences in gene expression are consistent with promoter-driven differential regulation of subsets of NF-nB-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-nB on EMSA (39); however, in this study, only the 48-h temozolomide treatment time point was evaluated and no analysis of NF-nB-dependent transcription was done.
Temozolomide inhibits NF-nB activity through formation of O 6methylguanine 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-nB can be seen in experiments with BCNU, in that BCNU does not significantly inhibit NF-nB in either AGT-positive or AGT-negative cells (Fig. 4B, and data not shown), although AGT can efficiently repair O 6 -chloroethylguanine lesions (6).Interestingly, O 6 -methylguanine adducts have previously been reported to directly block NF-nB from binding DNA in vitro (44).However, the time course of inhibition of NF-nB is not consistent with temozolomide-induced O 6 -methylguanine adducts directly affecting nB binding.In this regard, temozolomide forms a highly reactive S N 1-methylating species that methylates DNA within 1 to 2 h (45), whereas inhibition of NF-nB by temozolomide takes many hours to develop (Fig. 1C).Furthermore, when DNA binding is assessed with TNFastimulated nuclear extracts that are treated with temozolomide after isolation of the nuclei (Fig. 5B and Supplementary Fig. S4), inhibition of NF-nB 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-nB are already present within the nuclear contents and are activated following formation of O 6 -methylguanine.
Attenuation of NF-nB DNA binding suggests that temozolomide may exert its inhibitory action via an effect on the p65 RHD.In this regard, NH 2 -terminal cleavage of p65 represents one potential mechanism by which temozolomide could block NF-nB 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 Ser 276 , an important residue in the RHD involved with NF-nB

Cancer Research
Cancer Res 2007; 67: (14).July 15, 2007 transactivation (46).In addition, treatment with reducing agents does not reverse the inhibition of NF-nB, 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-nB 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.
O 6 -Methylguanine adducts and MMR are not only necessary for the inhibition of NF-nB by temozolomide but also central to the cytotoxicity of S N 1-type methylating agents.This finding raises the question of whether temozolomide-induced cell death is mediated by temozolomide-induced inhibition of NF-nB.Although there is no prior evidence to support this, our results, and the importance of NF-nB 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-X L and XIAP (Fig. 1D), a finding similar to reports that O 6 -methylguanine leads to inhibition of Bcl-2 (10), an NF-nBresponsive gene (48), and to activation of mitochondrial apoptosis cascade proteins (12).The requirement of MMR activity for inhibition of NF-nB 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, temozolomideinduced inhibition of NF-nB is blocked when the MMR system is disabled through a defect in either MutSa (MT1 cells) or MutLa (HCT116 cells; ref. 7).This finding suggests that inhibition of NF-nB by temozolomide is not mediated by a specific MMR protein but by MMR processing of O 6 -methylguanine in general.
The role of NF-nB 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-nB 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.

Figure 2 .
Figure2.Temozolomide inhibits TNFa-induced NF-nB on EMSA but not InBa degradation.A, U87 cells were untreated, treated with 10 ng/mL TNFa for 0 to 30 min, or pretreated with 100 Amol/L temozolomide (16 h) followed by TNFa.Western blot (WB ) with anti-InBa or anti-phospho-Ser 32 -InBa antibody was done and h-actin was used as loading control.B, U87 cells were stimulated with 10 ng/mL TNFa for 15 to 60 min with and without 100 Amol/L temozolomide (16-h pretreatment).EMSA was done with NF-nB consensus oligonucleotide (oligo ).One-hundred-fold excess cold NF-nB or AP-1 oligonucleotide was given where indicated.N.S., nonspecific band.C, left, U87 cells were pretreated with 100 Amol/L temozolomide for the times indicated followed by 10 ng/mL TNFa for 30 min.Right, U87 cells were untreated or treated with TNFa 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 Amol/L temozolomide and/or stimulated with TNFa for 30 min.EMSA was done with AP-1 consensus oligonucleotide.Representative of three separate experiments with similar results.

Figure 4 .
Figure 4. NF-nB is inhibited by S N 1 methylating agents in the presence of active MMR.A to D, NF-nB-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 TNFa as shown.B, H80 pcDNA3 cells were pretreated with BCNU for 16 h and/or stimulated with TNFa.C, HCT116 and HCT116+ch3 cells were untreated (UTC ) or pretreated with 100 Amol/L temozolomide for 16 h and/or stimulated with 10 ng/mL TNFa as indicated.*, P > 0.2.D, TK6 and MT1 cells were pretreated with 2.5, 25, or 100 Amol/L temozolomide for 16 h and/or stimulated with TNFa.Representative of three separate experiments with similar results.

Figure 5 .
Figure 5. Temozolomide inhibits TNFa-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 Amol/L temozolomide (16 h) and/or with 10 ng/mL TNFa 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 TNFa and nuclear fractions isolated at 30 min.Samples were then treated with 25 Amol/L temozolomide (and/or 0.1% DMSO; TK6 cells) or with the indicated concentration of temozolomide (MT1 cells) for 16 h and NF-nB EMSA was done.Supershift with anti-p65 or p50 antibody was done as indicated.Western blot of the TNFa-stimulated nuclear samples with anti-p65 antibody is shown.C, 10 ng of purified recombinant p65 protein, either untreated or incubated with 100 Amol/L temozolomide and/or DMSO for the indicated times, were analyzed by NF-nB DNA binding assay.Wild-type (wt ) or mutated (mut ) NF-nB 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-X L (top ) and COX2 (bottom ) promoter-specific primers.U87 cells were untreated or treated with 100 Amol/L temozolomide for the times shown (left ) or treated with 10 ng/mL TNFa (1 and 3 h) F 100 Amol/L temozolomide 16 h pretreatment (right ).Immunoprecipitation (IP ) was done with the antibodies indicated.Representative of at least three separate experiments with similar results.