Enforced Expression of Wild-Type p53 Curtails the ... · MGMT, additional strategies are needed to ensure the success of MGMT-targeted cancer therapy. In this context, numerous stud-ies

Enforced Expression of Wild-Type p53 Curtails the Transcription ofthe O6-Methylguanine-DNA Methyltransferase Gene in HumanTumor Cells and Enhances Their Sensitivity toAlkylating Agents1

Kalkunte S. Srivenugopal,2,3 Jiang Shou,2

Srinivas R. S. Mullapudi, Frederick F. Lang, Jr.,Jasti S. Rao, and Francis Ali-OsmanDepartment of Neurosurgery, The University of Texas M. D.Anderson Cancer Center, Houston, Texas 77030

ABSTRACTWe used isogenic human tumor cell lines to investigate

the specific and direct effects of wild-type (wt) p53 on theexpression of O6-methylguanine-DNA methyltransferase(MGMT), a DNA repair protein that confers tumor resist-ance to many anticancer alkylating agents. A p53-null,MGMT-proficient lung tumor cell line (H1299) was engi-neered to express wt p53 in a tetracycline-regulated system.High levels of p53 induction achieved by tetracycline with-drawal were accompanied by G1 cell cycle arrest withoutsignificant apoptosis in this cell line. p53 accumulation re-sulted in a gradual and dramatic loss of MGMT mRNA,protein, and enzyme activity, whose levels were undetectableby day 3 of induction. The loss of MGMT protein was,however, not due to its degradation because the ubiquitin-promoted in vitro degradation of MGMT, which mediatesthe cellular disposal of the repair protein, was not altered byp53. Run-on transcription assays revealed a significant re-duction in the rate of MGMT gene transcription. The neg-ative regulation of MGMT expression by wt p53 was con-firmed in two other human isogenic cell lines, namely, theGM47.23 glioblastoma, which contains a dexamethasone-inducible wt p53, and the H460 lung cancer cell line, inwhich wt p53 had been inactivated by the human papillo-mavirus E6 protein. Furthermore, a panel of four humantumor cell lines, including gliomas with wt p53 status, dis-

played markedly lower levels of MGMT gene transcriptsthan those having p53 mutations. Induction of wt p53 inthese models led to a 3- and 2-fold increase in sensitivity to1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, re-spectively, which generate the MGMT-repairableO6-alkyladducts in DNA. These results demonstrate that p53 is anegative regulator of MGMT gene expression and can createa MGMT-depleted state in human tumors similar to thatachieved byO6-benzylguanine, a potent inhibitor of MGMTcurrently undergoing clinical trials. Thus, our study exposesan additional benefit associated withp53 gene therapy andprovides a strong biochemical rationale for combining theMGMT-directed alkylators with p53 gene transfer toachieve improved antitumor efficacy.

INTRODUCTIONThe tumor suppressor genep53 encodes a sequence-

specific transcription factor that activates a variety of cellulargenes in response to DNA damage, hypoxia, stress, and manypathological states (1, 2). This unique function bestows on p53the ability to limit cell proliferation through a transient cell cycleblock or apoptosis or senescence and also to regulate the tran-scription of genes encoding many enzymes in macromolecularmetabolism (3–5). There is growing evidence that p53 not onlyfunctions to activate the transcription of its target genes(p21waf1, GADD45, cyclin G,and so forth) but also facilitatesthe repression of specific genes, such as RNA polymerase I (6),MAP4 (7), and collagenase I (8) that lack p53 consensus bindingsites. Due to a combination of deletion and/or mutation, 50% ofall human cancers lack a wt4 p53 gene allele and thus generatea functionally defective p53 protein (9). Therefore, the alteredgene expression resulting fromp53 inactivation has significantconsequences for cell cycle regulation, cellular metabolism, andcancer therapy. The ability of wt p53 to induce apoptosis,particularly when the protein is overexpressed as in gene ther-apy, has been well documented (10). However, the impact of wtp53 or its enforced expression on the cellular genes that conferanticancer drug resistance is poorly understood. Because drugresistance is a major stumbling block to successful cancer ther-apy, such a consideration is highly important.

MGMT (EC 2.1.1.63), also referred to asO6-alkylguanine-DNA alkyltransferase, is a ubiquitous DNA repair protein in-

Received 10/31/00; revised 1/2/01; accepted 1/26/01.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported by NIH Grant CA-74321 and grants from the NationalChildhood Cancer Foundation, the Pediatric Brain Tumor Foundation ofthe United States (to K. S. S.), and the Texas Advanced ResearchProgram under Grant 003657 (to F-A. O.).2 K. S. S. and J. S. contributed equally to the work reported in this study.3 To whom requests for reprints should be addressed, at Department ofNeurosurgery, Box 64, The University of Texas M. D. Anderson CancerCenter, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone:(713) 792-3821; Fax: (713) 794-5514; E-mail: [email protected].

4 The abbreviations used are: wt, wild-type; MGMT,O6-methylguanine-DNA methyltransferase; BG,O6-benzylguanine; BCNU, 1,3-bis(2-chlo-roethyl)-1-nitrosourea; Tet, tetracycline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPV, human papillomavirus; DEX,dexamethasone.

1398Vol. 7, 1398–1409, May 2001 Clinical Cancer Research

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volved in the protection of the cellular genome from the muta-genic actions of endogenous and environmental carcinogens(11). MGMT functions by a stoichiometric and suicidal reactionmechanism in which the alkyl groups bound to theO6-positionof guanine are transferred to a cysteine in its active site, result-ing in the direct restoration of the normal base and self-inacti-vation of MGMT (12). More significantly, MGMT may preventmutations in thep53gene itself (13), as inferred by the fact thatthe alkylators scavenged by MGMT are capable of inducingp53mutations (14) and that MGMT prevents mutations in therasoncogene (15). MGMT, which is highly expressed in humancancers, is also a central determinant of tumor resistance tomany clinically used anticancer alkylating agents because themethylguanine andO6-chloroethylguanine lesions induced inDNA by methylating (temozolomide, dacarbazine, and procar-bazine) and chloroethylating [BCNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] agents, respectively, are excellentsubstrates for MGMT (12, 16). In the case of bifunctionalalkylators like BCNU, the removal of the chloroethyl adducts byMGMT prevents the production of cytotoxic DNA interstrandcross-links (16). Therefore, inhibition of MGMT by powerfulpseudosubstrates such as BG, which inactivates and causeseffective depletion of cellular MGMT, has emerged as a prom-ising strategy to enhance the cytotoxicity of alkylating agents(17). BG is currently undergoing clinical trials to enhance theefficacy of chloroethylnitrosoureas in human brain tumors andother cancers (18).

Despite these advances in the biochemical modulation ofMGMT, additional strategies are needed to ensure the success ofMGMT-targeted cancer therapy. In this context, numerous stud-ies (13, 19–24) have explored the impact of p53 on MGMTexpression, but the findings have been inconsistent. Briefly,studies in murine cells have indicated that MGMT is inducibleby ionizing radiation in a classical wtp53 gene-dependentmanner (19, 20), and p53 may up-regulate the basal expressionof MGMT (21). However, evidence points to the contrary inhuman cells, with p53 being a negative regulator of MGMTexpression (22–24). A clear elucidation of the direct and specificrole of p53 on MGMT gene expression in human cells is critical;such studies are likely to provide new directions for selectingthe most effective alkylators against tumors with wt or mutantp53s. To this end, we examined the regulation of human MGMTby p53 using isogenic p53-inducible cell lines in a nonapoptoticbackground. We also assessed the sensitivity of p531 and p532

cells to alkylating drugs, which generate MGMT-repairablelesions.

MATERIALS AND METHODSReagents. All chemicals and reagents used in this study

were obtained from Sigma Chemical, Co. (St. Louis, MO) orLife Technologies, Inc. (Gaithersburg, MD). The monoclonalantibody 3.1 to MGMT protein (25) was a kind gift from Dr.Darrel Bigner of Duke University (Durham, NC). Monoclonalantibody to p53 (Do1) and actin were purchased from ChemiconInternational (Temecula, CA). Polyclonal antibodies to the HPVE6 protein were procured from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). BCNU and temozolomide (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide) were

obtained from the M. D. Anderson hospital pharmacy and theNational Cancer Institute, respectively.

Cell Lines. All cell lines used were of human origin. Thep53-null lung cancer cell line H1299; the glioblastoma cell linesT98G, U373, and U87MG; the breast cancer cell line MCF-7;and the colon tumor cell lines HT29, HCT116, and LS174Twere purchased from the American Type Culture Collection.The glioma cell line MGR2 was established in our laboratoryusing a specimen from an untreated patient. The cells weregrown in DMEM supplemented with 10% FCS. The GM47.23glioblastoma cell line is a derivative of T98G and harborsDEX-inducible wt p53 (26); it was obtained from Dr. W. E.Mercer of Thomas Jefferson University (Philadelphia, PA). TheH460 lung cancer and H460-E6 (stable transfectant) cell lines(27) were provided by Dr. Wafik El-Deiry (University of Penn-sylvania, Philadelphia, PA). All cells were cultured in 5% CO2

in a humid atmosphere at 37°C.Establishment of a p53-inducible Cell Line (H1299-

Hp53). A two-step procedure described previously (28) wasmodified to generate a p53-inducible cell line by using a Tet-regulated vector system. Human wt p53 cDNA was cloned intothe pUHD10-3 vector (29), which contains seven repeats of theTet operator linked to a cytomegalovirus minimal promoter atthe EcoRI and BamHI sites. p53 cDNA was sequenced toconfirm the wt sequence. p53-null H1299 human lung cancer(30) cells were first transfected with pUHD15–1neo/tTA (trans-activator) DNA (29) using LipofectAMINE. The cell cultureswere split the next day and maintained in 400mg/ml G418 for2 weeks. About 30 clones from this selection were cotransfectedwith the thymidine kinase promoter hygromycin plasmid (28)and the p53-1023 vector. Next, the clones were selected againsthygromycin (150mg/ml) in the presence of Tet (1mg/ml).Clones were screened using Western blot analysis for the abilityto express the p53 gene in the absence of Tet. Three clonesexpressing high levels of p53 were selected, and a clone desig-nated H1299-Hp53 was used in this study; it was maintained ina medium containing Tet (1mg/ml).

Immunofluorescence Analysis of p53 and MGMT Ex-pression. H1299-Hp53 cells were cultured in 35-mm Petridishes in the presence or absence of Tet for 3 days. Cells werefixed with cold methanol for 10 min and washed three timeswith PBS. Next, they were permeabilized with 0.2% TritonX-100 in PBS for 5 min followed by blocking with 1% goatserum and 5% BSA for 1 h. Cells were stained with monoclonalantibodies to p53 (Do1) or MGMT at 2mg/ml for 6 h, washedtwice with PBS, and incubated with FITC-conjugated goat an-timouse IgG for 1 h. The slides were counterstained with pro-pidium iodide (0.5mg/ml) to observe DNA, washed, and cov-ered with coverslips. They were viewed under a fluorescencemicroscope (Zeiss Axioskop) for red and green fluorescence,and the images were photographed.

Cell Cycle Analysis by Flow Cytometry. The H1299-Hp53 cells cultured in the presence or absence of Tet cultureswere trypsinized, washed with PBS, and fixed in 70% ethanol at4°C. The cells were pelleted and treated with RNase A (250mg/ml) and stained with propidium iodide (50mg/ml). Cellcycle phase distribution was determined using an EPICS ProfileII flow cytometer (Coulter Electronics). Apoptosis was assessedby fragmentation of DNA after agarose gel electrophoresis.

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Quantitation of MGMT and p21waf1 mRNA Levels.Total RNA was isolated by guanidinium isothiocyanate-phenolextraction (31). RNA samples (15mg) were fractionated on 1%formaldehyde-agarose gels and transferred to nylon membranes.Loading and integrity of RNA were checked by ethidium bro-mide staining of the gel and rehybridization of the blots withGAPDH cDNA. Full-length cDNAs for humanMGMT (32) orp21waf1 were 32P-labeled by random priming and used forhybridization. The blots were washed under stringent conditionswith 0.13SSC and 0.1% SDS at 65°C and exposed to film. Theautoradiographs were quantitated by densitometry.

Isolation of Nuclei and Run-on Transcription Assays.Nuclei were isolated from H1299-Hp53 cells by cell lysis in thepresence of 0.5% NP40 and 1.5 mM Mg21; further purificationby sucrose density gradient centrifugation and run-on experi-ments were performed as described previously (33). Briefly,1.5 3 107 nuclei from p53-induced and p53-uninduced cellswere incubated in the presence of 100mCi of [a-32P]UTP for 30min, the nascent32P-labeled RNA was purified, and an equalnumber of32P counts (73 106 cpm) from different treatmentswere hybridized with nylon membrane strips on which 10mg ofplasmid DNAs bearing the cDNA inserts for MGMT, p21waf1,and actin had been immobilized. Hybridization was performedat 42°C for 72 h, and the membranes were washed with 0.13SSC and 0.1% SDS at 65°C and exposed to film. Densitometrywas used to assess the gene transcription levels.

MGMT Activity Assay. MGMT activity was measuredby the transfer of3H-labeled methyl groups from theO6-posi-tion of guanine in DNA to the MGMT protein as we describedpreviously (34). Cell extracts were prepared by sonication inMGMT assay buffer [40 mM Tris-HCl (pH 8.0), 10% glycerol,1 mM EDTA, 20mM spermidine, and 0.5 mM DTT] followed bycentrifugation at 10,0003 g for 10 min. The extracts (25–150mg of protein) were supplemented with [3H]DNA enriched forO6-methylguanine (3mg; 10,000 cpm) incubated for 30 min at37°C. The reactions were quantitated after acid hydrolysis of theDNA substrate and the collection of protein precipitates andradioactivity counting as described previously (34). The activityin the linear portion of the curve was used to calculate thespecific activity (pmol CH3 groups removed/mg protein).

Assay for Ubiquitin/ATP-dependent Degradation ofMGMT Protein. The proteolysis of14C-labeled recombinantMGMT by H1299 cell extracts was monitored by SDS-PAGE.Histidine-tagged human MGMT was cloned using the pQEvector (Qiagen) and expressed inEscherichia colias describedpreviously (35). The bacteria were cultured in M9 mediumcontaining [14C]leucine (0.5mCi/ml), and synthesis of recom-binant protein was induced with 0.5 mM isopropylb-D-thioglu-coside. The14C-labeled MGMT was purified by nickel agarosechromatography as described previously (35).

Cell extracts from p53-induced and -uninduced H1299-Hp53 cells were prepared using sonication and centrifugation ina buffer containing 40 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1mM DTT, and 10% glycerol. The extracts were supplementedwith the substrate14C-labeled MGMT protein (2mg; 5000cpm). Next, Mg21 (5 mM), ATP (0.4 mM), and ubiquitin (1mg)were added either alone or in combination with each other andincubated at 37°C for 30 min. The reactions were terminated by

the addition of SDS-PAGE sample buffer and by boiling. AfterSDS-PAGE, the gels were dried and exposed to film.

Western Blot Analysis. Whole-cell lysates were pre-pared in a solution containing 62.5 mM Tris-HCl (pH 6.8), 2%SDS, and 0.5%b-mercaptoethanol. Equal amounts of totalprotein were electrophoresed on a 10% SDS-polyacrylamide geland electrotransferred to polyvinylidene difluoride membranes.The blots were blocked with 5% BSA in PBS and probed withspecific antibodies to p53, p21waf1, MGMT, and actin. Forreprobing of the blots, the bound antibodies were removed witha solution containing 20 mM Tris-HCl (pH 6.8), 100 mM b-mercaptoethanol, and 2% SDS at 45°C, followed by blocking.The immunocomplexes were visualized by incubation with ei-ther goat antimouse or goat antirabbit secondary antibodiesfollowed by chemiluminescence.

Assay for Tumor Cell Drug Sensitivity. The capillaryclonogenic assay (36) was used to assess tumor cell survivalafter drug exposure. Briefly, the p53-induced and -uninducedH1299-Hp53 and GM47.23 cells were exposed to differentconcentrations of BCNU or temozolomide for 90 min at 37°C.The cells were trypsinized, washed, and combined with a platingmixture at 2.53 106 cells/ml in DMEM containing 20% serumand 0.2% low melting point agarose. Thirtyml of these suspen-sions were drawn into sterile glass capillary tubes, cooled, andincubated in a humidified atmosphere and 5% CO2 at 37°C.After 15 days, the agarose was flushed out of the tubes ontoglass slides, and colonies of 50mm or more in diameter werecounted using a phase-contrast microscope. The survival frac-tion at each drug dose was calculated by dividing the meannumber of colonies in the drug-treated samples by that in theuntreated controls.

RESULTSInducible Expression of wt p53 in H1299 Cells and the

Accompanying Loss of MGMT Protein. To determinewhether p53 has direct and specific effects on the expression ofthe humanMGMT gene, we used the lung cancer cell lineH1299, which lacks p53 protein expression because of homozy-gous deletion of the gene (30), and obtained stable transfectantsexpressing wt p53 under the control of a Tet-responsive pro-moter. The high levels of MGMT activity (1.4 pmol [3H]methylgroups transferred/mg protein) present in these cells allowed usto study the impact of p53 on the expression of endogenousMGMT. A p53 clone with high levels of p53 expression wasselected for this study and cultured in the presence or absence ofTet. Western blotting analysis with a p53-specific antibodyrevealed that p53 expression was completely suppressed whencells were grown in the presence of Tet, whereas its synthesiswas strongly induced by Tet withdrawal in a progressive man-ner, with maximal protein levels attained on day 3 and main-tained during the later growth period (Fig. 1A, p53). The p53protein was functionally active because its expression was ac-companied by increased production of the p21waf1 protein (Fig.1A, p21waf1). The wt configuration of the expressed p53 proteinwas also confirmed by its immunoprecipitation by monoclonalantibody Pab 1620 but not by Pab 240, which recognize the wtand mutant conformations of the p53 protein, respectively (37).

The endogenous MGMT protein was analyzed by Western

1400Repression of theMGMT Gene by p53



analysis at different time points after p53 induction (Fig. 1A,MGMT). Early periods of p53 induction (6 and 12 h) did notalter the MGMT protein levels (data not shown). However, thisrepair protein disappeared from H1299-Hp53 cells in a gradualand dramatic manner at later periods of p53 induction (Fig. 1A).The reduction of MGMT appeared to follow a biphasic kineticswith a slower phase occurring during the first 2 days and thenbecoming rapid in the later period, with the MGMT proteinlevels reaching undetectable levels on day 4 (Fig. 1B). Thepattern of MGMT disappearance showed a close correlationwith that of p53 accumulation (r5 0.93). The catalytic activityof MGMT was lost from the extracts of H1299-Hp53 cells in afashion consistent with the kinetics observed for its protein loss,with an 85% decrease on day 3 after p53 induction comparedwith the same cells grown in the presence of Tet (Fig. 1C).These results obtained in an isogenic setting demonstrate astrong reciprocal association between the p53 and MGMT pro-teins.

Immunofluorescence Localization of p53 and MGMTin H1299-Hp53 Cells. Both p53 and MGMT perform theirfunctions in the nucleus, and most previous studies have re-ported a nuclear localization for these proteins (2, 38). Toexamine the subcellular distribution of the MGMT and p53proteins in the context of their counteracting association ob-served in Fig. 1, we performed indirect immunofluorescenceanalysis of these proteins in H1299 cells with and without p53induction. Fig. 2 shows that cells grown in the presence of Tetwere completely negative for the p53 stain but showed abundantnuclear staining for MGMT. In contrast, the cells on day 3 afterTet withdrawal stained intensely for p53 in the nuclei, and therewere few cells with greatly reduced MGMT staining in the

nuclear periphery. Propidium iodide staining of DNA, whichwas used as the control in p53-induced cells, indicated thatnuclear integrity was not compromised and apoptotic cell deathdid not occur. These data confirm the results shown in Fig. 1,Aand C, and suggest a functional role for p53 in regulatingMGMT protein.

ATP-driven Ubiquitin-dependent Proteolysis of MGMTWas Not Altered by p53 Expression. We have shown thatMGMT in human cells is degraded through the ubiquitin-pro-teasome pathway (39). Therefore, we investigated the possibil-ity that p53 overexpression may up-regulate the ubiquitin pro-teolytic pathway, thereby resulting in the loss of the MGMTprotein observed in Figs. 1 and 2. Crude extracts were preparedfrom H1299-p53 cells cultured with and without Tet for 3 daysand assayed for the degradation of14C-labeled human recom-binant MGMT protein (Fig. 3). The reactions were sequentiallysupplemented with the components required for the ubiquitin-dependent protein degradation (Mg21, ATP, and ubiquitin; Ref.40), incubated, and assessed for MGMT degradation usingautoradiography after SDS-PAGE. The protein staining pattern(Fig. 3, top panel) reveals that similar protein levels were usedfrom p53-induced (top right panel) and p53-uninduced (top leftpanel) cells for the reaction. Thebottom panelof Fig. 3 showsthat the degradation of radiolabeled MGMT was most stimu-lated when Mg21, ATP, and ubiquitin, which are all required forefficient catalysis of the ubiquitin conjugation and its proteaso-mal digestion of the modified proteins (40), were present in thereaction (Lane 4); however, the rate and extent of MGMTdegradation in p53-induced and -uninduced cells was very sim-ilar (Lane 4 in the bottom panelsof Fig. 3). Other studiesshowed that the levels of proteasomea and b subunits and

Fig. 1 Time-dependent loss of the endogenous MGMT protein from H1299 cells after the induction of wt p53 in a Tet-off inducible system. TheH1299-Hp53 cells maintained in the presence of Tet (1mg/ml) were grown to early log phase (Lane 1) and then cultured in the absence of Tet forthe number of days shown.A, representative Western blot analysis of MGMT, p53, p21waf1, and actin proteins. Total cell extracts (40mg of protein)were electrophoresed and immunoblotted, and the membrane was sequentially probed with monoclonal antibodies specific for p53, MGMT, p21waf1,and actin.B, densitometric quantitation of the data shown in Fig. 1A.C, quantitation of MGMT activity. After Tet withdrawal, crude extracts fromH1299-Hp53 cultures were prepared, and the transfer of3H-labeledO6-methyl groups of guanine in DNA to the MGMT protein was measured asdescribed in “Materials and Methods.” Data are the means of five independent experiments;bars, SD.p, significant atP , 0.05.




half-life of the MGMT protein were not altered by p53 induction(data not shown). These results suggest that the loss of MGMTprotein must occur through a process other than protein degra-dation.

Transcription of the MGMT Gene Is Repressed by p53Overexpression. To identify the molecular mechanism bywhich p53 inhibits MGMT expression, Northern blot analysiswas performed at different times after p53 induction in H1299cells. The steady-state levels of 0.9-kbMGMT gene transcriptsshowed a time-dependent reduction (Fig. 4A). The kineticsrevealed a significant decrease of MGMT mRNA levels by day1 after p53 induction, followed by a gradual reduction thatreached undetectable levels by day 3. It is significant to note thatMGMT RNA and protein levels decreased only after substantialaccumulation of the wt p53 protein in H1299 cells. Rehybrid-ization of these blots showed a steady increase of p21waf1

mRNA (Fig. 4A). Whereas the up-regulation ofp21waf1 geneexpression observed in this system was expected, the simulta-neous reduction of MGMT transcripts indicates a functional rolefor p53 in MGMT gene transcription.

In rodent cells, p53 has been shown to induce theMGMTgene in response to ionizing radiation (19). Because these find-ings imply that p53 at levels attainable after DNA damage mayup-regulate theMGMT gene, we reduced the Tet concentrationto 100 ng/ml (one-tenth of the routinely used concentration) toallow the expression of very low levels of wt p53 and examined

MGMT mRNA levels. Induction of p53 at low levels and itsaccumulation for 12 h did not alter the MGMT gene transcriptlevels (data not shown), suggesting that p53 may not activate theMGMT gene in human cells.

The p53-induced loss of MGMT transcripts could occurbecause of a transcriptional blockade or a posttranscriptionaldestabilization of its mRNA. To distinguish between these tworegulatory modes, we performed run-on transcription assays innuclei from p53-induced and -uninduced H1299 cells. The re-sults of a representative run-on assay after hybridization ofequal32P counts of newly synthesized RNA are shown in Fig.4B. The band intensities in this figure reflect the relative tran-scription rates ofMGMT, p21waf1, and the housekeepingGAPDH genes. In the absence of p53 induction (Tet1), a verylow level ofMGMTgene transcription was detected, as reportedpreviously (41), which disappeared completely after 3 days ofp53 induction. In contrast,p21waf1, a gene transactivated by p53,showed significant enhancement in this assay. These data, takentogether with the Northern analysis of MGMT (Fig. 4A),strongly suggest that p53-induced down-regulation of MGMTexpression occurs largely through a transcriptional repression.

Changes in MGMT Gene Expression Occur in theAbsence of Apoptosis in p53-induced H1299 Cells.Alter-ations in cell cycle progression and the possibility of apoptosisafter p53 induction were determined by flow cytometry (Fig. 5,A and B) and DNA ladder assay (Fig. 5C). The histogram

Fig. 2 Immunofluorescence analysis of the localization and expression of p53 and MGMT proteins in H1299-Hp53 cells. Cells were grown in35-mm Petri dishes for 3 days in the presence or absence of Tet, fixed, and treated with monoclonal antibodies to MGMT or p53. Theimmunoreactivity was visualized with FITC-conjugated secondary antibodies, and the slides were counterstained with the DNA stain propidium iodide(PI) to visualize nuclei. Each pair of adjacent panels depicts the same field viewed under appropriate wavelengths. Accumulation of p53 and reductionof MGMT are evident in cells grown in the absence of Tet, as is their nuclear localization.




pattern (Fig. 5A) and cell cycle phase distribution (Fig. 5B)showed that p53 accumulation was associated with a significantdecrease of cell population in S and G2-M phases and theirmarked elevation in the G1 phase of the cell cycle. The accretionof cells with sub-G1 DNA content during the time course of ourstudies was minimal, reaching only 7% on day 3 of p53 induc-tion (Fig. 5, A and B). A lack of DNA fragmentation in p53-induced cells (Fig. 5C) further demonstrates that p53 overex-pression did not trigger apoptosis in these cells. Also, theMGMT activity in human cells does not fluctuate during cellcycle progression (42). Therefore, the changes we observed inMGMT expression seem to have occurred quite independent ofthe biochemical and molecular alterations associated with cellcycle arrest and apoptosis.

Down-Regulation of MGMT Expression by wt p53 inOther Isogenic Human Tumor Cell Lines. To substantiatethe relationship between p53 function and MGMT expression,we measured the levels of MGMT protein and its activity in twoadditional paired human cancer cell lines, namely, H460 andH460-E6, and T98G and GM27.43, in which the parent andderivative cells are isogenic and differ only with respect to p53function. One member of the pair (H460-E6 lung cancer cells)is deficient in functional p53 by virtue of stable transfection ofHPV E6,whose gene product promotes degradation of the p53protein through the ubiquitin-dependent proteolytic pathway

(43). T98G glioblastoma cells harbor an endogenous mutantp53; GM47.23 is a derivative of T98G cells carrying a DEX-inducible wt p53 in the background of its mutant counterpart(26). The top panel of Fig. 6A shows that p53-inactivatedH460-E6 cells had nearly 3-fold more MGMT activity than theirparent cell line (H460), which has wt p53. Western analysis inH460-E6 cells confirmed the expression of E6 protein, unde-tectable p53 levels, and increased MGMT protein levels (Fig.6A, bottom panel). Fig. 6Brepresents MGMT protein and ac-

Fig. 3 Lack of alterations in the Mg21-ATP-dependent proteolysis of[14C]MGMT protein by p53 induction. H1299-Hp53 cells grown in thepresence or absence of Tet were trypsinized, and cell extracts wereprepared. Extracts (50mg of protein) were supplemented with purified14C-labeled recombinant MGMT protein (2mg; 5000 cpm), and prote-olysis reactions were performed with or without the addition of Mg21 (5mM), ATP (0.4 mM), and ubiquitin (1mg), as indicated. After 30 min ofincubation at 37°C, the reaction mixtures were subjected to SDS-PAGE.The gel was stained with Coomassie Blue to observe protein loading(top panel), dried, and autoradiographed to obtain the results (bottompanel).

Fig. 4 Transcriptional suppression of theMGMT gene by p53 induc-tion in H1299-Hp53 cells.A, Northern blot analysis of MGMT andp21waf1 mRNAs in H1299-p53 cells cultured in the presence (Lane 1) orabsence of Tet (Lanes 2–6) for the number of days shown. The repre-sentative pattern was obtained by electrophoresing the total RNA (15mg) on 1% formaldehyde-agarose gels, blotting onto nylon membranes,and sequential hybridization with32P-labeled cDNA probes for MGMT,p21waf1, and GAPDH.B, nuclear run-on analysis. Nuclei were isolatedfrom H1299-Hp53 cells grown in the presence or absence of Tet for 3days,32P-labeled nascent RNA was prepared, and an equal number ofcounts were hybridized with the slot-blotted plasmid-DNAs that hadcDNA inserts for the genes.




tivity levels in T98G and GM47.23 cells with and without DEXtreatment. Induction of wt p53 in GM47.23 cells resulted in a60% decrease of MGMT activity on days 3 and 4 of DEXexposure (bottom panel). In contrast, the parent T98G cells(mutant p53) did not show alterations of MGMT activity after 3days of exposure to DEX (Fig. 6B). the levels of MGMT protein(Fig. 6B, top panel) correlated well with its activity in theseexperiments. The delayed reduction of MGMT after the induc-tion of wt p53 in this model is similar to that observed afteroverexpression of p53 in H1299 cells, thereby demonstratingthat normal p53 functions to attenuate the expression of theMGMT gene.

Induction of wt p53 Enhances Cytotoxicity Exerted byClinically Active Alkylating Agents. Because MGMT-medi-ated repair ofO6-alkylguanines prevents the formation of cyto-toxic lesions by many anticancer alkylating agents and MGMTwas down-regulated by p53, we determined the clonogenicsurvival of H1299-Hp53 and GM47.23 cells after exposure tothe clinically used BCNU and temozolomide, both of whichalkylate guanine at theO6-position (12). Induction of p53 sen-sitized the H1299 cells to BCNU and temozolomide by approx-imately 3- and 1.7-fold, respectively (Fig. 7,A and B). Theinvolvement of MGMT in the increased cytotoxicity was veri-fied by using BG, a specific inhibitor of MGMT, which is wellknown to potentiate the cytotoxicity ofO6-alkylguanine-gener-ating drugs (17). The cytotoxicity of BCNU against the p53-induced H1299 cells (in which the MGMT was depleted) was

not significantly affected by BG pretreatment (15mM for 1 h),whereas a similar treatment of H1299 cells without p53 induc-tion resulted in nearly a 2-fold increase of cytotoxicity (Fig. 7A),indicating that MGMT was a major determinant of BCNUresistance in this cell line. Induction of wt p53 in GM43.27 cells,which harbor an endogenous mutant p53, also increased BCNUsensitivity, albeit to a marginal 1.4-fold (Fig. 7C). Similarly, theH460 cells were 2-fold more sensitive to BCNU than H460-E6cells, their p53-deficient counterparts (data not shown). Thesedata demonstrate that MGMT deficiency caused by p53 over-expression increases the cytotoxicity of compounds that gener-ate MGMT-consumable DNA adducts, and such a manifestationis similar to that achieved by specific inhibitors of MGMT.

Correlation between MGMT mRNA Levels and p53Status in Human Tumor Cell Lines. To gain insight into theimpact of p53 on MGMT expression under physiological con-ditions, we chose eight human cancer cell lines [four of whichharbored wtp53,and four of which harbored mutantp53 (44)]and quantitated the MGMT transcripts. We selected many gli-oma cell lines for this study because MGMT is an importantmechanism of resistance to the alkylnitrosoureas, which remainthe drugs of choice for glioma chemotherapy (16). Twentymg oftotal RNA from each cell line were subjected to Northernanalysis of MGMT mRNA followed by the rehybridization ofthe blot with GAPDH cDNA. The results (Fig. 8) show thattumor cells with wt p53 had relatively lower levels of MGMTtranscripts compared with cells with mutant p53, indicating that

Fig. 5 G1 cell cycle arrest and lack of significant apoptosis in p53-induced H1299-Hp53 cells.A, flow cytometry profiles of the H1299-Hp53 cellsin p53-uninduced (Tet1) and -induced (Tet2) state. Percentages of cells with sub-G1 DNA content are indicated.B, the distribution of cells in differentphases of the cell cycle from the data in Fig. 5Ais shown.C, ethidium bromide-stained agarose gel after electrophoresing DNA from p53-uninducedand -induced H1299-Hp53 cells.




p53 may control the basal transcription of the MGMT gene aswell. Although such an analysis needs to be performed in a largenumber of human cell lines and primary tumors, our data doessupport the notion that p53 at physiological and supraphysi-ological levels functions to curtail MGMT transcription.

DISCUSSIONBecause the tumor suppressor p53 plays a central role in

sensing DNA damage and promoting DNA repair, the possibil-ity that MGMT may be regulated by p53 has received muchattention. Previous studies performed in murine cells (19–21)and human cells (22, 23) using different experimental ap-proaches have yielded confusing results on this issue. Our studysought to clarify the direct and specific effects of p53 on MGMTgene expression in human cells by using well-defined isogeniccell pairs. The results clearly show that wt p53 functions tocurtail the transcription of theMGMT gene and that our obser-vations may have clinical relevance. The first of these systemsin which detailed studies were performed was composed of ap53-null cell line (H1299) that was engineered to express highlevels of wt p53 protein in a Tet-off inducible system. Thenuclear accumulation of p53 was accompanied by increasedexpression of its transcriptional targets, namely, p21waf1 (Figs.1A and 4,A and B), GADD45 (data not shown), and G1 cellcycle arrest (Fig. 5B). Unlike other reports of p53 overexpres-sion that result in significant levels of cell death (2, 10) andmake it difficult to rule out the contribution of apoptotic pro-cesses to the altered gene expression, lack of apoptosis in our

p53-inducible model (Fig. 5) provided a reliable system to probethe direct effect of wt p53 on MGMT gene expression. Weobserved a delayed disappearance of the MGMT protein andactivity over a period of 3–4 days, which coincided with thesimultaneous buildup of p53 protein. The loss of MGMT wasnot due to its proteolytic degradation because the ATP-depend-ent degradation of14C-labeled MGMT (which reflects the ubiq-uitin-proteasome pathway) remained similar with and withoutp53 induction (Fig. 3). We believe that this is the first study toexplore the regulation of the ubiquitin proteolytic pathway inp53-overexpressing cells, and our data suggest that high levelsof p53 may not alter the expression of the enzymes in thispathway. A progressive decline in MGMT mRNA (Fig. 4A) anda reduced transcription rate of the MGMT gene (Fig. 4B) fol-lowed p53 protein accumulation. This mutual relationship pro-vides strong evidence for a transcriptional repression of theMGMT gene by the wt p53 protein. Down-regulation of MGMTexpression by wt p53 was also demonstrated in two otherisogenic p53 models. These included the H460 human lungcancer cell line, in which p53 function had been disrupted by theE6 papillomavirus oncoprotein (43), and the GM47.23 glioblas-toma cell line, in which the stably transfected wtp53 could beinduced by DEX (26). Both of these cell pairs have been reliablyused to probe the function of wt p53 in previous studies (27, 45).MGMT expression was 3-fold higher in H460-E6 cells than inH460 cells. MGMT protein and its DNA repair activity levelswere suppressed by 3-fold in GM47.23 cells after the inductionof wt p53. Although the extent of MGMT down-regulation was

Fig. 6 Suppression of MGMT expression by wt p53 in two pairs of isogenic human tumor cell lines.A, disruption of wt p53 function in human H460lung cancer cells by HPV E6 protein increases the MGMT protein and activity levels.Top panel, MGMT activity levels in the H460 and H460-E6cells.Bottom panel, Western blot analysis of MGMT, E6, and p53 proteins in H460 and H460-E6 cells. The immunoblot was probed with antibodiesto b-actin to assess equal protein loading.B, expression of DEX-inducible wt p53 in GM47.23 human glioblastoma cells down-regulates MGMTexpression. GM47.23 cells are derived from T98G cells after the stable transfection of a wt p53 under the control of DEX-inducible promoter (26).Top panel, the Western blot of MGMT protein after treatment of the parent T98G and GM47.23 cells with 1mM DEX for the number of days shownat thebottomof the figure.Bottom panel, changes in MGMT activity in T98G and GM47.23 cells occurring after DEX treatment. A time-dependentdecrease of MGMT protein and its activity is evident in DEX-treated GM47.23 cells, but not in DEX-treated T98G cells.




not dramatic, as was observed in the inducible model, thefindings nevertheless support the inhibitory nature of p53 onMGMT transcription.

Our studies confirm and further extend the initial observa-tions of Harriset al. (22), who showed a suppression of MGMTprotein in IMR90 human fibroblasts after infection of an adeno-viral p53construct; unlike our studies, Harriset al.(22) failed torule out the involvement of apoptotic processes in MGMTdown-regulation and did not assess its consequence for cancertherapy. However, the present study and that of Harriset al.(22)involving p53 overexpression in human cells stand in starkcontrast to a number of studies in murine cells and tissues thatreported a positive regulation of MGMT expression by p53(19–21). Thus, two studies using p53-knockout mice (19, 21)and murine cell lines transfected with wt or mutantp53 (20)concluded that the induction of theMGMT gene by ionizingradiation requires a wt p53 in conformity with the establishedrole for p53 in DNA damage response. This diametrically op-posite governance of mammalian MGMTs may relate to thespecies-specific differences in the regulation of genes by p53(46) and/or to the differential responses of the human andmurine MGMTs to DNA damage. For example, an up-regula-tion of hepatic MGMT (up to 20-fold) by whole body irradiationof rats and mice (47) and an increase of MGMT mRNA and

activity (up to 6-fold) induced by ionizing radiation in murinecell cultures (48, 49) have been known for a long time. How-ever, MGMT in human cells appears to be a constitutive enzymeand does not appear to be inducible byg-radiation (50, 51) orDNA strand-breaking drugs,5 and its levels remain unchangedduring aging stress (52). Pertinent to this discussion is theobservation that no p53 binding sites are present in the MGMTgene promoter (53), although the presence of such sequences inthe introns of this gene cannot be ruled out. Because our studiespredict a repression of the MGMT gene by increased p53 levels,a careful kinetic analysis of MGMT expression afterg-radiationof human cells is necessary to further clarify this issue.

The present study and many previous studies (20, 22, 23)agree with the conclusion that cellular accumulation of wt p53protein is likely to down-regulate MGMT expression. The du-ration and the p53 protein levels required for this down-regula-tion are unclear, but it appears thatp53 gene transfer by eithertransfection (20, 22), regulated expression (this study), or infec-tion of adenoviral constructs (22) that result in modest to highlevels of p53 expression is capable of inhibiting MGMT expres-

5 K.S. Srivenugopal, unpublished observations.

Fig. 7 wt p53-induced suppression of MGMTsensitizes human tumor cells to the alkylatingagents BCNU and temozolomide.A, clonogeniccell survival of H1299-Hp53 cells in the p53-induced (no Tet) and p53-uninduced (with Tet)states after exposure to different concentrations ofBCNU or temozolomide (B). p53 was induced for6 h before drug addition. Tet (1mg/ml) was in-cluded in other assays to prevent p53 induction.Cell survival was assessed by soft agar assaysusing glass capillary tubes as described in “Mate-rials and Methods.” In some experiments, the p53-induced and -uninduced cells were first exposed toBG (15mM for 1 h) to inactivate cellular MGMT,washed, and then treated with BCNU; these re-sults are also shown in Fig. 7A.C, survival of theGM47.23 cells after BCNU treatment. Cells in theabsence or presence of 1mM DEX were exposedto different concentrations of BCNU and sub-jected to cell survival assays. Cells were exposedto each drug concentration in triplicate, and thedata represent the means of four independent de-terminations.Bars, SD.




sion, albeit to different extents. The molecular basis of thisrepression, however, is not yet clear. TheMGMTgene promoterlacks TATA and CAAT boxes and has 10 Sp1 transcriptionfactor binding sites (53). It is well established that p53 stimu-lates the expression of its target genes that possess a p53 bindingsequence and a TATA box by direct binding to itscis-elements(1) and a specific interaction with the TATA-binding protein(54). Alternatively, p53 can repress promoters that lack itsresponse element (6–8). This repression was initially believedto be restricted to TATA box-containing promoters (55); how-ever, there is growing evidence that TATA-less promoters arealso subject to p53 repression (56), and the MGMT gene be-longs to this category. A variety of mechanisms involvingprotein-protein interactions between p53 with TATA-bindingprotein (57), p53 and the p300/CBP transcriptional coactivator/histone deacetylation machinery (58), and the SP1 transcriptionfactor (59) have been proposed to explain p53-mediated tran-scriptional repression. It is possible that the large amounts of wtp53 protein produced in H1299 cells could operate through oneor more of these mechanisms to curtail the transcription of theMGMT gene. The promoter of the MGMT gene is very CpGrich, and increased cytosine methylation of this region is awell-established mechanism for transcriptional silencing of theMGMT gene, which occurs in approximately 20% of tumor celllines and a subset of primary tumors (60). Therefore, the presentstudy and previous reports (19–24) that highlight the importantrole of p53 in MGMT gene expression add a new twist to thetranscriptional regulation of this DNA repair protein in humancancers.

Reports on the relationship betweenp53 gene defects andtherapeutic response and/or survival of patients with glioma andother cancers have been inconsistent (61). In this regard, anaccurate assessment of the p53 contribution towardMGMTgeneexpression and the consequent changes in alkylator resistanceand patient survival is likely to be difficult because of themultiple roles p53 is expected to play in inducing a cell cycleblock, repairing alkylation DNA damage by non-MGMT mech-anisms, and promoting apoptosis. Despite this difficulty, our

studies bear significant implications for making the p53 genetherapy more effective through a biochemically meaningfulstrategy. As evident from the data, cellular accretion of p53 willinduce a drastic reduction or depletion of the MGMT proteinand its activity, which in turn translates into increased cytotoxiceffects by the alkylating drugs, such as BCNU and temozo-lmide. The delayed depletion of MGMT orchestrated by p53through a transcriptional block is virtually the same end resultachieved by cellular exposure to the MGMT inhibitor BG,which is currently in Phase I clinical trials for circumventingMGMT-mediated drug resistance (17, 18). Therefore, p53 genetherapy offers a rational paradigm for combining the powerfulMGMT-targeted alkylating agents into therapeutic regimens.Such a combination will introduce lethal damage in tumor DNA(interstrand cross-links), which should augment the apoptoticfunctions mediated by p53 and result in significant potentiationof cytotoxicity. On the basis of our observations in GM47.23cells (Fig. 6C) and previous reports (3, 62), this rational ap-proach may be equally applicable for tumors that harbor a wtp53 or mutant p53. p53-induced deficiency of MGMT may alsosensitize drug-resistant tumors to radiation because a synergismbetween alkylnitrosoureas and radiation has long been recog-nized (63), and a combination of these two agents is routinelyused for brain tumor therapy. Preclinical studies addressingthese specific possibilities should yield valuable information.

In summary, we conclude that production/overproductionof wt p53 protein in human tumors curtails the transcription ofthe MGMT gene and confers a MGMT-deficient phenotype,which could be potentially exploited for improving cancer ther-apy. Our study illustrates a beneficial side effect of p53 modu-lation, and it is significant to note that genes encoding othertherapeutic targets, such as the multidrug resistance protein (64),telomerase (65), and vascular endothelial growth factor (66), arealso negatively regulated by wt p53 in a manner similar to thatof the MGMT gene.

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Correction: Article on the Enforced Expression of Wild-Type p53 and the Transcription of the

O6-Methylguanine-DNA Methyltransferase Gene

In the article on Enforced Expression of Wild-Type p53 in the May 1, 200l issue of Clinical Cancer Research , the sequence of the

author listing was incorrect. The correct order is, as follows:

Jiang Shou, Kalkunte S. Srivenugopal, Srinivas R.S. Mullapudi, Frederick F. Lang, Jr., Jasti S. Rao, and Francis Ali-Osman.

Srivenugopal KS, Shou J, Mullapudi SRS, et al. Enforced expression of wild-type p53 curtails the transcription of the O6-

methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents. Clin Cancer Res

2001;7:1398–409.

Vol. 11, 2449, March 15, 2005 Clinical Cancer Research 2449

2001;7:1398-1409. Clin Cancer Res Kalkunte S. Srivenugopal, Jiang Shou, Srinivas R. S. Mullapudi, et al. Alkylating AgentsGene in Human Tumor Cells and Enhances Their Sensitivity to

-Methylguanine-DNA Methyltransferase6OTranscription of the Enforced Expression of Wild-Type p53 Curtails the

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