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Clinical Cancer Research Vol. 7, 1398-1409, May 2001
© 2001 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

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 Agents1

Kalkunte S. Srivenugopal2,,3, Jiang Shou2, Srinivas R. S. Mullapudi, Frederick F. Lang, Jr., Jasti S. Rao and Francis Ali-Osman

Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We used isogenic human tumor cell lines to investigate the specific and direct effects of wild-type (wt) p53 on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that confers tumor resistance to many anticancer alkylating agents. A p53-null, MGMT-proficient lung tumor cell line (H1299) was engineered to express wt p53 in a tetracycline-regulated system. High levels of p53 induction achieved by tetracycline withdrawal were accompanied by G1 cell cycle arrest without significant apoptosis in this cell line. p53 accumulation resulted in a gradual and dramatic loss of MGMT mRNA, protein, and enzyme activity, whose levels were undetectable by 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 mediates the cellular disposal of the repair protein, was not altered by p53. Run-on transcription assays revealed a significant reduction in the rate of MGMT gene transcription. The negative regulation of MGMT expression by wt p53 was confirmed in two other human isogenic cell lines, namely, the GM47.23 glioblastoma, which contains a dexamethasone-inducible wt p53, and the H460 lung cancer cell line, in which wt p53 had been inactivated by the human papillomavirus E6 protein. Furthermore, a panel of four human tumor cell lines, including gliomas with wt p53 status, displayed markedly lower levels of MGMT gene transcripts than those having p53 mutations. Induction of wt p53 in these models led to a 3- and 2-fold increase in sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, respectively, which generate the MGMT-repairable O6-alkyl adducts in DNA. These results demonstrate that p53 is a negative regulator of MGMT gene expression and can create a MGMT-depleted state in human tumors similar to that achieved by O6-benzylguanine, a potent inhibitor of MGMT currently undergoing clinical trials. Thus, our study exposes an additional benefit associated with p53 gene therapy and provides a strong biochemical rationale for combining the MGMT-directed alkylators with p53 gene transfer to achieve improved antitumor efficacy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor gene p53 encodes a sequence-specific transcription factor that activates a variety of cellular genes in response to DNA damage, hypoxia, stress, and many pathological states (1 , 2) . This unique function bestows on p53 the ability to limit cell proliferation through a transient cell cycle block or apoptosis or senescence and also to regulate the transcription of genes encoding many enzymes in macromolecular metabolism (3, 4, 5) . There is growing evidence that p53 not only functions to activate the transcription of its target genes (p21waf1, GADD45, cyclin G, and so forth) but also facilitates the repression of specific genes, such as RNA polymerase I (6) , MAP4 (7) , and collagenase I (8) that lack p53 consensus binding sites. Due to a combination of deletion and/or mutation, 50% of all human cancers lack a wt4 p53 gene allele and thus generate a functionally defective p53 protein (9) . Therefore, the altered gene expression resulting from p53 inactivation has significant consequences for cell cycle regulation, cellular metabolism, and cancer therapy. The ability of wt p53 to induce apoptosis, particularly when the protein is overexpressed as in gene therapy, has been well documented (10) . However, the impact of wt p53 or its enforced expression on the cellular genes that confer anticancer drug resistance is poorly understood. Because drug resistance is a major stumbling block to successful cancer therapy, such a consideration is highly important.

MGMT (EC 2.1.1.63), also referred to as O6-alkylguanine-DNA alkyltransferase, is a ubiquitous DNA repair protein involved in the protection of the cellular genome from the mutagenic actions of endogenous and environmental carcinogens (11) . MGMT functions by a stoichiometric and suicidal reaction mechanism in which the alkyl groups bound to the O6-position of guanine are transferred to a cysteine in its active site, resulting in the direct restoration of the normal base and self-inactivation of MGMT (12) . More significantly, MGMT may prevent mutations in the p53 gene itself (13) , as inferred by the fact that the alkylators scavenged by MGMT are capable of inducing p53 mutations (14) and that MGMT prevents mutations in the ras oncogene (15) . MGMT, which is highly expressed in human cancers, is also a central determinant of tumor resistance to many clinically used anticancer alkylating agents because the methylguanine and O6-chloroethylguanine lesions induced in DNA by methylating (temozolomide, dacarbazine, and procarbazine) and chloroethylating [BCNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] agents, respectively, are excellent substrates for MGMT (12 , 16) . In the case of bifunctional alkylators like BCNU, the removal of the chloroethyl adducts by MGMT prevents the production of cytotoxic DNA interstrand cross-links (16) . Therefore, inhibition of MGMT by powerful pseudosubstrates such as BG, which inactivates and causes effective depletion of cellular MGMT, has emerged as a promising strategy to enhance the cytotoxicity of alkylating agents (17) . BG is currently undergoing clinical trials to enhance the efficacy of chloroethylnitrosoureas in human brain tumors and other cancers (18) .

Despite these advances in the biochemical modulation of MGMT, additional strategies are needed to ensure the success of MGMT-targeted cancer therapy. In this context, numerous studies (13 , 19, 20, 21, 22, 23, 24) have explored the impact of p53 on MGMT expression, but the findings have been inconsistent. Briefly, studies in murine cells have indicated that MGMT is inducible by ionizing radiation in a classical wt p53 gene-dependent manner (19 , 20) , and p53 may up-regulate the basal expression of MGMT (21) . However, evidence points to the contrary in human cells, with p53 being a negative regulator of MGMT expression (22, 23, 24) . A clear elucidation of the direct and specific role of p53 on MGMT gene expression in human cells is critical; such studies are likely to provide new directions for selecting the most effective alkylators against tumors with wt or mutant p53s. To this end, we examined the regulation of human MGMT by p53 using isogenic p53-inducible cell lines in a nonapoptotic background. We also assessed the sensitivity of p53+ and p53- cells to alkylating drugs, which generate MGMT-repairable lesions.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
All chemicals and reagents used in this study were obtained from Sigma Chemical, Co. (St. Louis, MO) or Life Technologies, Inc. (Gaithersburg, MD). The monoclonal antibody 3.1 to MGMT protein (25) was a kind gift from Dr. Darrel Bigner of Duke University (Durham, NC). Monoclonal antibody to p53 (Do1) and actin were purchased from Chemicon International (Temecula, CA). Polyclonal antibodies to the HPV E6 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 the National Cancer Institute, respectively.

Cell Lines.
All cell lines used were of human origin. The p53-null lung cancer cell line H1299; the glioblastoma cell lines T98G, U373, and U87MG; the breast cancer cell line MCF-7; and the colon tumor cell lines HT29, HCT116, and LS174T were purchased from the American Type Culture Collection. The glioma cell line MGR2 was established in our laboratory using a specimen from an untreated patient. The cells were grown in DMEM supplemented with 10% FCS. The GM47.23 glioblastoma cell line is a derivative of T98G and harbors DEX-inducible wt p53 (26) ; it was obtained from Dr. W. E. Mercer of Thomas Jefferson University (Philadelphia, PA). The H460 lung cancer and H460-E6 (stable transfectant) cell lines (27) were provided by Dr. Wafik El-Deiry (University of Pennsylvania, 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) was modified to generate a p53-inducible cell line by using a Tet-regulated vector system. Human wt p53 cDNA was cloned into the pUHD10-3 vector (29) , which contains seven repeats of the Tet operator linked to a cytomegalovirus minimal promoter at the EcoRI and BamHI sites. p53 cDNA was sequenced to confirm the wt sequence. p53-null H1299 human lung cancer (30) cells were first transfected with pUHD15-1neo/tTA (transactivator) DNA (29) using LipofectAMINE. The cell cultures were split the next day and maintained in 400 µg/ml G418 for 2 weeks. About 30 clones from this selection were cotransfected with the thymidine kinase promoter hygromycin plasmid (28) and the p53-10-3 vector. Next, the clones were selected against hygromycin (150 µg/ml) in the presence of Tet (1 µg/ml). Clones were screened using Western blot analysis for the ability to express the p53 gene in the absence of Tet. Three clones expressing high levels of p53 were selected, and a clone designated H1299-Hp53 was used in this study; it was maintained in a medium containing Tet (1 µg/ml).

Immunofluorescence Analysis of p53 and MGMT Expression.
H1299-Hp53 cells were cultured in 35-mm Petri dishes in the presence or absence of Tet for 3 days. Cells were fixed with cold methanol for 10 min and washed three times with PBS. Next, they were permeabilized with 0.2% Triton X-100 in PBS for 5 min followed by blocking with 1% goat serum and 5% BSA for 1 h. Cells were stained with monoclonal antibodies to p53 (Do1) or MGMT at 2 µg/ml for 6 h, washed twice with PBS, and incubated with FITC-conjugated goat antimouse IgG for 1 h. The slides were counterstained with propidium iodide (0.5 µg/ml) to observe DNA, washed, and covered with coverslips. They were viewed under a fluorescence microscope (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 cultures were trypsinized, washed with PBS, and fixed in 70% ethanol at 4°C. The cells were pelleted and treated with RNase A (250 µg/ml) and stained with propidium iodide (50 µg/ml). Cell cycle phase distribution was determined using an EPICS Profile II flow cytometer (Coulter Electronics). Apoptosis was assessed by fragmentation of DNA after agarose gel electrophoresis.

Quantitation of MGMT and p21waf1 mRNA Levels.
Total RNA was isolated by guanidinium isothiocyanate-phenol extraction (31) . RNA samples (15 µg) were fractionated on 1% formaldehyde-agarose gels and transferred to nylon membranes. Loading and integrity of RNA were checked by ethidium bromide staining of the gel and rehybridization of the blots with GAPDH cDNA. Full-length cDNAs for human MGMT (32) or p21waf1 were 32P-labeled by random priming and used for hybridization. The blots were washed under stringent conditions with 0.1x SSC and 0.1% SDS at 65°C and exposed to film. The autoradiographs were quantitated by densitometry.

Isolation of Nuclei and Run-on Transcription Assays.
Nuclei were isolated from H1299-Hp53 cells by cell lysis in the presence of 0.5% NP40 and 1.5 mM Mg2+; further purification by sucrose density gradient centrifugation and run-on experiments were performed as described previously (33) . Briefly, 1.5 x 107 nuclei from p53-induced and p53-uninduced cells were incubated in the presence of 100 µCi of [{alpha}-32P]UTP for 30 min, the nascent 32P-labeled RNA was purified, and an equal number of 32P counts (7 x 106 cpm) from different treatments were hybridized with nylon membrane strips on which 10 µg of plasmid DNAs bearing the cDNA inserts for MGMT, p21waf1, and actin had been immobilized. Hybridization was performed at 42°C for 72 h, and the membranes were washed with 0.1x SSC and 0.1% SDS at 65°C and exposed to film. Densitometry was used to assess the gene transcription levels.

MGMT Activity Assay.
MGMT activity was measured by the transfer of 3H-labeled methyl groups from the O6-position of guanine in DNA to the MGMT protein as we described previously (34) . Cell extracts were prepared by sonication in MGMT assay buffer [40 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EDTA, 20 µM spermidine, and 0.5 mM DTT] followed by centrifugation at 10,000 x g for 10 min. The extracts (25–150 µg of protein) were supplemented with [3H]DNA enriched for O6-methylguanine (3 µg; 10,000 cpm) incubated for 30 min at 37°C. The reactions were quantitated after acid hydrolysis of the DNA substrate and the collection of protein precipitates and radioactivity counting as described previously (34) . The activity in the linear portion of the curve was used to calculate the specific activity (pmol CH3 groups removed/mg protein).

Assay for Ubiquitin/ATP-dependent Degradation of MGMT Protein.
The proteolysis of 14C-labeled recombinant MGMT by H1299 cell extracts was monitored by SDS-PAGE. Histidine-tagged human MGMT was cloned using the pQE vector (Qiagen) and expressed in Escherichia coli as described previously (35) . The bacteria were cultured in M9 medium containing [14C]leucine (0.5 µCi/ml), and synthesis of recombinant protein was induced with 0.5 mM isopropyl ß-D-thioglucoside. The 14C-labeled MGMT was purified by nickel agarose chromatography as described previously (35) .

Cell extracts from p53-induced and -uninduced H1299-Hp53 cells were prepared using sonication and centrifugation in a buffer containing 40 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, and 10% glycerol. The extracts were supplemented with the substrate 14C-labeled MGMT protein (2 µg; 5000 cpm). Next, Mg2+ (5 mM), ATP (0.4 mM), and ubiquitin (1 µg) were added either alone or in combination with each other and incubated at 37°C for 30 min. The reactions were terminated by the addition of SDS-PAGE sample buffer and by boiling. After SDS-PAGE, the gels were dried and exposed to film.

Western Blot Analysis.
Whole-cell lysates were prepared in a solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 0.5% ß-mercaptoethanol. Equal amounts of total protein were electrophoresed on a 10% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride membranes. The blots were blocked with 5% BSA in PBS and probed with specific antibodies to p53, p21waf1, MGMT, and actin. For reprobing of the blots, the bound antibodies were removed with a solution containing 20 mM Tris-HCl (pH 6.8), 100 mM ß-mercaptoethanol, and 2% SDS at 45°C, followed by blocking. The immunocomplexes were visualized by incubation with either goat antimouse or goat antirabbit secondary antibodies followed by chemiluminescence.

Assay for Tumor Cell Drug Sensitivity.
The capillary clonogenic assay (36) was used to assess tumor cell survival after drug exposure. Briefly, the p53-induced and -uninduced H1299-Hp53 and GM47.23 cells were exposed to different concentrations of BCNU or temozolomide for 90 min at 37°C. The cells were trypsinized, washed, and combined with a plating mixture at 2.5 x 106 cells/ml in DMEM containing 20% serum and 0.2% low melting point agarose. Thirty µl of these suspensions were drawn into sterile glass capillary tubes, cooled, and incubated in a humidified atmosphere and 5% CO2 at 37°C. After 15 days, the agarose was flushed out of the tubes onto glass slides, and colonies of 50 µm or more in diameter were counted using a phase-contrast microscope. The survival fraction at each drug dose was calculated by dividing the mean number of colonies in the drug-treated samples by that in the untreated controls.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inducible Expression of wt p53 in H1299 Cells and the Accompanying Loss of MGMT Protein.
To determine whether p53 has direct and specific effects on the expression of the human MGMT gene, we used the lung cancer cell line H1299, which lacks p53 protein expression because of homozygous deletion of the gene (30) , and obtained stable transfectants expressing wt p53 under the control of a Tet-responsive promoter. The high levels of MGMT activity (1.4 pmol [3H]methyl groups transferred/mg protein) present in these cells allowed us to study the impact of p53 on the expression of endogenous MGMT. A p53 clone with high levels of p53 expression was selected for this study and cultured in the presence or absence of Tet. Western blotting analysis with a p53-specific antibody revealed that p53 expression was completely suppressed when cells were grown in the presence of Tet, whereas its synthesis was strongly induced by Tet withdrawal in a progressive manner, with maximal protein levels attained on day 3 and maintained during the later growth period (Fig. 1ACitation , p53). The p53 protein was functionally active because its expression was accompanied by increased production of the p21waf1 protein (Fig. 1ACitation , p21waf1). The wt configuration of the expressed p53 protein was also confirmed by its immunoprecipitation by monoclonal antibody Pab 1620 but not by Pab 240, which recognize the wt and mutant conformations of the p53 protein, respectively (37) .



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  		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. The H1299-Hp53 cells maintained in the presence of Tet (1 µg/ml) were grown to early log phase (Lane 1) and then cultured in the absence of Tet for the number of days shown. A, representative Western blot analysis of MGMT, p53, p21waf1, and actin proteins. Total cell extracts (40 µg 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. 1ACitation . C, quantitation of MGMT activity. After Tet withdrawal, crude extracts from H1299-Hp53 cultures were prepared, and the transfer of 3H-labeled O6-methyl groups of guanine in DNA to the MGMT protein was measured as described in "Materials and Methods." Data are the means of five independent experiments; bars, SD. *, significant at P < 0.05.

 
The endogenous MGMT protein was analyzed by Western analysis at different time points after p53 induction (Fig. 1ACitation , MGMT). Early periods of p53 induction (6 and 12 h) did not alter the MGMT protein levels (data not shown). However, this repair protein disappeared from H1299-Hp53 cells in a gradual and dramatic manner at later periods of p53 induction (Fig. 1A)Citation . The reduction of MGMT appeared to follow a biphasic kinetics with a slower phase occurring during the first 2 days and then becoming rapid in the later period, with the MGMT protein levels reaching undetectable levels on day 4 (Fig. 1B)Citation . The pattern of MGMT disappearance showed a close correlation with that of p53 accumulation (r = 0.93). The catalytic activity of MGMT was lost from the extracts of H1299-Hp53 cells in a fashion consistent with the kinetics observed for its protein loss, with an 85% decrease on day 3 after p53 induction compared with the same cells grown in the presence of Tet (Fig. 1C)Citation . These results obtained in an isogenic setting demonstrate a strong reciprocal association between the p53 and MGMT proteins.

Immunofluorescence Localization of p53 and MGMT in H1299-Hp53 Cells.
Both p53 and MGMT perform their functions in the nucleus, and most previous studies have reported a nuclear localization for these proteins (2 , 38) . To examine the subcellular distribution of the MGMT and p53 proteins in the context of their counteracting association observed in Fig. 1Citation , we performed indirect immunofluorescence analysis of these proteins in H1299 cells with and without p53 induction. Fig. 2Citation shows that cells grown in the presence of Tet were completely negative for the p53 stain but showed abundant nuclear staining for MGMT. In contrast, the cells on day 3 after Tet withdrawal stained intensely for p53 in the nuclei, and there were few cells with greatly reduced MGMT staining in the nuclear periphery. Propidium iodide staining of DNA, which was used as the control in p53-induced cells, indicated that nuclear integrity was not compromised and apoptotic cell death did not occur. These data confirm the results shown in Fig. 1, A and CCitation , and suggest a functional role for p53 in regulating MGMT protein.



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  		Fig. 2. Immunofluorescence analysis of the localization and expression of p53 and MGMT proteins in H1299-Hp53 cells. Cells were grown in 35-mm Petri dishes for 3 days in the presence or absence of Tet, fixed, and treated with monoclonal antibodies to MGMT or p53. The immunoreactivity 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 reduction of MGMT are evident in cells grown in the absence of Tet, as is their nuclear localization.

 
ATP-driven Ubiquitin-dependent Proteolysis of MGMT Was Not Altered by p53 Expression.
We have shown that MGMT in human cells is degraded through the ubiquitin-proteasome pathway (39) . Therefore, we investigated the possibility that p53 overexpression may up-regulate the ubiquitin proteolytic pathway, thereby resulting in the loss of the MGMT protein observed in Figs. 1Citation and 2Citation . Crude extracts were prepared from H1299-p53 cells cultured with and without Tet for 3 days and assayed for the degradation of 14C-labeled human recombinant MGMT protein (Fig. 3)Citation . The reactions were sequentially supplemented with the components required for the ubiquitin-dependent protein degradation (Mg2+, ATP, and ubiquitin; Ref. 40 ), incubated, and assessed for MGMT degradation using autoradiography after SDS-PAGE. The protein staining pattern (Fig. 3Citation , top panel) reveals that similar protein levels were used from p53-induced (top right panel) and p53-uninduced (top left panel) cells for the reaction. The bottom panel of Fig. 3Citation shows that the degradation of radiolabeled MGMT was most stimulated when Mg2+, ATP, and ubiquitin, which are all required for efficient catalysis of the ubiquitin conjugation and its proteasomal digestion of the modified proteins (40) , were present in the reaction (Lane 4); however, the rate and extent of MGMT degradation in p53-induced and -uninduced cells was very similar (Lane 4 in the bottom panels of Fig. 3Citation ). Other studies showed that the levels of proteasome {alpha} and ß subunits and half-life of the MGMT protein were not altered by p53 induction (data not shown). These results suggest that the loss of MGMT protein must occur through a process other than protein degradation.



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  		Fig. 3. Lack of alterations in the Mg2+-ATP-dependent proteolysis of [14C]MGMT protein by p53 induction. H1299-Hp53 cells grown in the presence or absence of Tet were trypsinized, and cell extracts were prepared. Extracts (50 µg of protein) were supplemented with purified 14C-labeled recombinant MGMT protein (2 µg; 5000 cpm), and proteolysis reactions were performed with or without the addition of Mg2+ (5 mM), ATP (0.4 mM), and ubiquitin (1 µg), as indicated. After 30 min of incubation 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 (bottom panel).

 
Transcription of the MGMT Gene Is Repressed by p53 Overexpression.
To identify the molecular mechanism by which p53 inhibits MGMT expression, Northern blot analysis was performed at different times after p53 induction in H1299 cells. The steady-state levels of 0.9-kb MGMT gene transcripts showed a time-dependent reduction (Fig. 4A)Citation . The kinetics revealed a significant decrease of MGMT mRNA levels by day 1 after p53 induction, followed by a gradual reduction that reached undetectable levels by day 3. It is significant to note that MGMT RNA and protein levels decreased only after substantial accumulation of the wt p53 protein in H1299 cells. Rehybridization of these blots showed a steady increase of p21waf1 mRNA (Fig. 4A)Citation . Whereas the up-regulation of p21waf1 gene expression observed in this system was expected, the simultaneous reduction of MGMT transcripts indicates a functional role for p53 in MGMT gene transcription.



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

 
In rodent cells, p53 has been shown to induce the MGMT gene in response to ionizing radiation (19) . Because these findings imply that p53 at levels attainable after DNA damage may up-regulate the MGMT gene, we reduced the Tet concentration to 100 ng/ml (one-tenth of the routinely used concentration) to allow the expression of very low levels of wt p53 and examined MGMT mRNA levels. Induction of p53 at low levels and its accumulation for 12 h did not alter the MGMT gene transcript levels (data not shown), suggesting that p53 may not activate the MGMT gene in human cells.

The p53-induced loss of MGMT transcripts could occur because of a transcriptional blockade or a posttranscriptional destabilization of its mRNA. To distinguish between these two regulatory modes, we performed run-on transcription assays in nuclei from p53-induced and -uninduced H1299 cells. The results of a representative run-on assay after hybridization of equal 32P counts of newly synthesized RNA are shown in Fig. 4BCitation . The band intensities in this figure reflect the relative transcription rates of MGMT, p21waf1, and the housekeeping GAPDH genes. In the absence of p53 induction (Tet+), a very low level of MGMT gene transcription was detected, as reported previously (41) , which disappeared completely after 3 days of p53 induction. In contrast, p21waf1, a gene transactivated by p53, showed significant enhancement in this assay. These data, taken together with the Northern analysis of MGMT (Fig. 4A)Citation , strongly suggest that p53-induced down-regulation of MGMT expression occurs largely through a transcriptional repression.

Changes in MGMT Gene Expression Occur in the Absence of Apoptosis in p53-induced H1299 Cells.
Alterations in cell cycle progression and the possibility of apoptosis after p53 induction were determined by flow cytometry (Fig. 5, A and B)Citation and DNA ladder assay (Fig. 5C)Citation . The histogram pattern (Fig. 5A)Citation and cell cycle phase distribution (Fig. 5B)Citation showed that p53 accumulation was associated with a significant decrease of cell population in S and G2-M phases and their marked elevation in the G1 phase of the cell cycle. The accretion of cells with sub-G1 DNA content during the time course of our studies was minimal, reaching only 7% on day 3 of p53 induction (Fig. 5, A and B)Citation . A lack of DNA fragmentation in p53-induced cells (Fig. 5C)Citation further demonstrates that p53 overexpression did not trigger apoptosis in these cells. Also, the MGMT activity in human cells does not fluctuate during cell cycle progression (42) . Therefore, the changes we observed in MGMT expression seem to have occurred quite independent of the biochemical and molecular alterations associated with cell cycle arrest and apoptosis.



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  		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 cells in p53-uninduced (Tet+) and -induced (Tet-) state. Percentages of cells with sub-G1 DNA content are indicated. B, the distribution of cells in different phases of the cell cycle from the data in Fig. 5ACitation is shown. C, ethidium bromide-stained agarose gel after electrophoresing DNA from p53-uninduced and -induced H1299-Hp53 cells.

 
Down-Regulation of MGMT Expression by wt p53 in Other Isogenic Human Tumor Cell Lines.
To substantiate the relationship between p53 function and MGMT expression, we measured the levels of MGMT protein and its activity in two additional paired human cancer cell lines, namely, H460 and H460-E6, and T98G and GM27.43, in which the parent and derivative cells are isogenic and differ only with respect to p53 function. One member of the pair (H460-E6 lung cancer cells) is deficient in functional p53 by virtue of stable transfection of HPV E6, whose gene product promotes degradation of the p53 protein through the ubiquitin-dependent proteolytic pathway (43) . T98G glioblastoma cells harbor an endogenous mutant p53; 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. 6ACitation shows that p53-inactivated H460-E6 cells had nearly 3-fold more MGMT activity than their parent cell line (H460), which has wt p53. Western analysis in H460-E6 cells confirmed the expression of E6 protein, undetectable p53 levels, and increased MGMT protein levels (Fig. 6ACitation , bottom panel). Fig. 6BCitation represents MGMT protein and activity levels in T98G and GM47.23 cells with and without DEX treatment. Induction of wt p53 in GM47.23 cells resulted in a 60% decrease of MGMT activity on days 3 and 4 of DEX exposure (bottom panel). In contrast, the parent T98G cells (mutant p53) did not show alterations of MGMT activity after 3 days of exposure to DEX (Fig. 6B)Citation . the levels of MGMT protein (Fig. 6BCitation , top panel) correlated well with its activity in these experiments. The delayed reduction of MGMT after the induction of wt p53 in this model is similar to that observed after overexpression of p53 in H1299 cells, thereby demonstrating that normal p53 functions to attenuate the expression of the MGMT gene.



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  		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 H460 lung cancer cells by HPV E6 protein increases the MGMT protein and activity levels. Top panel, MGMT activity levels in the H460 and H460-E6 cells. Bottom panel, Western blot analysis of MGMT, E6, and p53 proteins in H460 and H460-E6 cells. The immunoblot was probed with antibodies to ß-actin to assess equal protein loading. B, expression of DEX-inducible wt p53 in GM47.23 human glioblastoma cells down-regulates MGMT expression. 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 1 µM DEX for the number of days shown at the bottom of the figure. Bottom panel, changes in MGMT activity in T98G and GM47.23 cells occurring after DEX treatment. A time-dependent decrease of MGMT protein and its activity is evident in DEX-treated GM47.23 cells, but not in DEX-treated T98G cells.

 
Induction of wt p53 Enhances Cytotoxicity Exerted by Clinically Active Alkylating Agents.
Because MGMT-mediated repair of O6-alkylguanines prevents the formation of cytotoxic lesions by many anticancer alkylating agents and MGMT was down-regulated by p53, we determined the clonogenic survival of H1299-Hp53 and GM47.23 cells after exposure to the clinically used BCNU and temozolomide, both of which alkylate guanine at the O6-position (12) . Induction of p53 sensitized the H1299 cells to BCNU and temozolomide by approximately 3- and 1.7-fold, respectively (Fig. 7, A and B)Citation . The involvement of MGMT in the increased cytotoxicity was verified by using BG, a specific inhibitor of MGMT, which is well known to potentiate the cytotoxicity of O6-alkylguanine-generating 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 (15 µM for 1 h), whereas a similar treatment of H1299 cells without p53 induction resulted in nearly a 2-fold increase of cytotoxicity (Fig. 7A)Citation , indicating that MGMT was a major determinant of BCNU resistance in this cell line. Induction of wt p53 in GM43.27 cells, which harbor an endogenous mutant p53, also increased BCNU sensitivity, albeit to a marginal 1.4-fold (Fig. 7C)Citation . Similarly, the H460 cells were 2-fold more sensitive to BCNU than H460-E6 cells, their p53-deficient counterparts (data not shown). These data demonstrate that MGMT deficiency caused by p53 overexpression increases the cytotoxicity of compounds that generate MGMT-consumable DNA adducts, and such a manifestation is similar to that achieved by specific inhibitors of MGMT.



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  		Fig. 7. wt p53-induced suppression of MGMT sensitizes human tumor cells to the alkylating agents BCNU and temozolomide. A, clonogenic cell survival of H1299-Hp53 cells in the p53-induced (no Tet) and p53-uninduced (with Tet) states after exposure to different concentrations of BCNU or temozolomide (B). p53 was induced for 6 h before drug addition. Tet (1 µg/ml) was included in other assays to prevent p53 induction. Cell survival was assessed by soft agar assays using glass capillary tubes as described in "Materials and Methods." In some experiments, the p53-induced and -uninduced cells were first exposed to BG (15 µM for 1 h) to inactivate cellular MGMT, washed, and then treated with BCNU; these results are also shown in Fig. 7ACitation . C, survival of the GM47.23 cells after BCNU treatment. Cells in the absence or presence of 1 µM DEX were exposed to different concentrations of BCNU and subjected to cell survival assays. Cells were exposed to each drug concentration in triplicate, and the data represent the means of four independent determinations. Bars, SD.

 
Correlation between MGMT mRNA Levels and p53 Status in Human Tumor Cell Lines.
To gain insight into the impact of p53 on MGMT expression under physiological conditions, we chose eight human cancer cell lines [four of which harbored wt p53, and four of which harbored mutant p53 (44) ] and quantitated the MGMT transcripts. We selected many glioma cell lines for this study because MGMT is an important mechanism of resistance to the alkylnitrosoureas, which remain the drugs of choice for glioma chemotherapy (16) . Twenty µg of total RNA from each cell line were subjected to Northern analysis of MGMT mRNA followed by the rehybridization of the blot with GAPDH cDNA. The results (Fig. 8)Citation show that tumor cells with wt p53 had relatively lower levels of MGMT transcripts compared with cells with mutant p53, indicating that p53 may control the basal transcription of the MGMT gene as well. Although such an analysis needs to be performed in a large number of human cell lines and primary tumors, our data does support the notion that p53 at physiological and supraphysiological levels functions to curtail MGMT transcription.



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  		Fig. 8. Correlation of MGMT mRNA levels with p53 gene status in human tumor cell lines. Total RNA was isolated from four cell lines with mutant p53 (Lanes 1–4) and four cell lines with wt p53 (Lanes 5–8). Fifteen µg of RNA from each cell line were electrophoresed on a 1% formaldehyde-agarose gel, blotted onto a nylon membrane, and hybridized with 32P-labeled cDNA for MGMT. The blots were reprobed with cDNA for GAPDH. p53 status of the cell lines was obtained from the database (44) . Lane 1, U373 glioma (codon 273 mutation); Lane 2, T98G glioblastoma (codon 237 mutation); Lane 3, HT29 colon carcinoma (codon 273 mutation); Lane 4, MGR2 glioma (codon 273 mutation); Lane 5, U87MG glioblastoma (wt p53); Lane 6, HCT116 colon carcinoma (wt p53); Lane 7, MCF-7 breast carcinoma (wt p53); Lane 8, LS174T colon carcinoma (wt p53).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because the tumor suppressor p53 plays a central role in sensing DNA damage and promoting DNA repair, the possibility that MGMT may be regulated by p53 has received much attention. Previous studies performed in murine cells (19, 20, 21) and human cells (22 , 23) using different experimental approaches have yielded confusing results on this issue. Our study sought to clarify the direct and specific effects of p53 on MGMT gene expression in human cells by using well-defined isogenic cell pairs. The results clearly show that wt p53 functions to curtail the transcription of the MGMT gene and that our observations may have clinical relevance. The first of these systems in which detailed studies were performed was composed of a p53-null cell line (H1299) that was engineered to express high levels of wt p53 protein in a Tet-off inducible system. The nuclear accumulation of p53 was accompanied by increased expression of its transcriptional targets, namely, p21waf1 (Figs. 1ACitation and 4, A and BCitation ), GADD45 (data not shown), and G1 cell cycle arrest (Fig. 5B)Citation . Unlike other reports of p53 overexpression that result in significant levels of cell death (2 , 10) and make it difficult to rule out the contribution of apoptotic processes to the altered gene expression, lack of apoptosis in our p53-inducible model (Fig. 5)Citation provided a reliable system to probe the direct effect of wt p53 on MGMT gene expression. We observed a delayed disappearance of the MGMT protein and activity over a period of 3–4 days, which coincided with the simultaneous buildup of p53 protein. The loss of MGMT was not due to its proteolytic degradation because the ATP-dependent degradation of 14C-labeled MGMT (which reflects the ubiquitin-proteasome pathway) remained similar with and without p53 induction (Fig. 3)Citation . We believe that this is the first study to explore the regulation of the ubiquitin proteolytic pathway in p53-overexpressing cells, and our data suggest that high levels of p53 may not alter the expression of the enzymes in this pathway. A progressive decline in MGMT mRNA (Fig. 4A)Citation and a reduced transcription rate of the MGMT gene (Fig. 4B)Citation followed p53 protein accumulation. This mutual relationship provides strong evidence for a transcriptional repression of the MGMT gene by the wt p53 protein. Down-regulation of MGMT expression by wt p53 was also demonstrated in two other isogenic p53 models. These included the H460 human lung cancer cell line, in which p53 function had been disrupted by the E6 papillomavirus oncoprotein (43) , and the GM47.23 glioblastoma cell line, in which the stably transfected wt p53 could be induced by DEX (26) . Both of these cell pairs have been reliably used to probe the function of wt p53 in previous studies (27 , 45) . MGMT expression was 3-fold higher in H460-E6 cells than in H460 cells. MGMT protein and its DNA repair activity levels were suppressed by 3-fold in GM47.23 cells after the induction of wt p53. Although the extent of MGMT down-regulation was not dramatic, as was observed in the inducible model, the findings nevertheless support the inhibitory nature of p53 on MGMT transcription.

Our studies confirm and further extend the initial observations of Harris et al. (22) , who showed a suppression of MGMT protein in IMR90 human fibroblasts after infection of an adenoviral p53 construct; unlike our studies, Harris et al. (22) failed to rule out the involvement of apoptotic processes in MGMT down-regulation and did not assess its consequence for cancer therapy. However, the present study and that of Harris et al. (22) involving p53 overexpression in human cells stand in stark contrast to a number of studies in murine cells and tissues that reported a positive regulation of MGMT expression by p53 (19, 20, 21) . Thus, two studies using p53-knockout mice (19 , 21) and murine cell lines transfected with wt or mutant p53 (20) concluded that the induction of the MGMT gene by ionizing radiation requires a wt p53 in conformity with the established role for p53 in DNA damage response. This diametrically opposite governance of mammalian MGMTs may relate to the species-specific differences in the regulation of genes by p53 (46) and/or to the differential responses of the human and murine MGMTs to DNA damage. For example, an up-regulation of hepatic MGMT (up to 20-fold) by whole body irradiation of rats and mice (47) and an increase of MGMT mRNA and activity (up to 6-fold) induced by ionizing radiation in murine cell cultures (48 , 49) have been known for a long time. However, MGMT in human cells appears to be a constitutive enzyme and does not appear to be inducible by {gamma}-radiation (50 , 51) or DNA strand-breaking drugs,5 and its levels remain unchanged during aging stress (52) . Pertinent to this discussion is the observation that no p53 binding sites are present in the MGMT gene promoter (53) , although the presence of such sequences in the introns of this gene cannot be ruled out. Because our studies predict a repression of the MGMT gene by increased p53 levels, a careful kinetic analysis of MGMT expression after {gamma}-radiation of 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 p53 protein is likely to down-regulate MGMT expression. The duration and the p53 protein levels required for this down-regulation are unclear, but it appears that p53 gene transfer by either transfection (20 , 22) , regulated expression (this study), or infection of adenoviral constructs (22) that result in modest to high levels of p53 expression is capable of inhibiting MGMT expression, albeit to different extents. The molecular basis of this repression, however, is not yet clear. The MGMT gene promoter lacks TATA and CAAT boxes and has 10 Sp1 transcription factor binding sites (53) . It is well established that p53 stimulates the expression of its target genes that possess a p53 binding sequence and a TATA box by direct binding to its cis-elements (1) and a specific interaction with the TATA-binding protein (54) . Alternatively, p53 can repress promoters that lack its response element (6, 7, 8) . This repression was initially believed to be restricted to TATA box-containing promoters (55) ; however, there is growing evidence that TATA-less promoters are also subject to p53 repression (56) , and the MGMT gene belongs to this category. A variety of mechanisms involving protein-protein interactions between p53 with TATA-binding protein (57) , p53 and the p300/CBP transcriptional coactivator/histone deacetylation machinery (58) , and the SP1 transcription factor (59) have been proposed to explain p53-mediated transcriptional repression. It is possible that the large amounts of wt p53 protein produced in H1299 cells could operate through one or more of these mechanisms to curtail the transcription of the MGMT gene. The promoter of the MGMT gene is very CpG rich, and increased cytosine methylation of this region is a well-established mechanism for transcriptional silencing of the MGMT gene, which occurs in approximately 20% of tumor cell lines and a subset of primary tumors (60) . Therefore, the present study and previous reports (19, 20, 21, 22, 23, 24) that highlight the important role of p53 in MGMT gene expression add a new twist to the transcriptional regulation of this DNA repair protein in human cancers.

Reports on the relationship between p53 gene defects and therapeutic response and/or survival of patients with glioma and other cancers have been inconsistent (61) . In this regard, an accurate assessment of the p53 contribution toward MGMT gene expression and the consequent changes in alkylator resistance and patient survival is likely to be difficult because of the multiple roles p53 is expected to play in inducing a cell cycle block, repairing alkylation DNA damage by non-MGMT mechanisms, and promoting apoptosis. Despite this difficulty, our studies bear significant implications for making the p53 gene therapy more effective through a biochemically meaningful strategy. As evident from the data, cellular accretion of p53 will induce a drastic reduction or depletion of the MGMT protein and its activity, which in turn translates into increased cytotoxic effects by the alkylating drugs, such as BCNU and temozolmide. The delayed depletion of MGMT orchestrated by p53 through a transcriptional block is virtually the same end result achieved by cellular exposure to the MGMT inhibitor BG, which is currently in Phase I clinical trials for circumventing MGMT-mediated drug resistance (17 , 18) . Therefore, p53 gene therapy offers a rational paradigm for combining the powerful MGMT-targeted alkylating agents into therapeutic regimens. Such a combination will introduce lethal damage in tumor DNA (interstrand cross-links), which should augment the apoptotic functions mediated by p53 and result in significant potentiation of cytotoxicity. On the basis of our observations in GM47.23 cells (Fig. 6C)Citation and previous reports (3 , 62) , this rational approach may be equally applicable for tumors that harbor a wt p53 or mutant p53. p53-induced deficiency of MGMT may also sensitize drug-resistant tumors to radiation because a synergism between alkylnitrosoureas and radiation has long been recognized (63) , and a combination of these two agents is routinely used for brain tumor therapy. Preclinical studies addressing these specific possibilities should yield valuable information.

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


   FOOTNOTES
 
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.

1 Supported by NIH Grant CA-74321 and grants from the National Childhood Cancer Foundation, the Pediatric Brain Tumor Foundation of the United States (to K. S. S.), and the Texas Advanced Research Program under Grant 003657 (to F-A. O.). Back

2 K. S. S. and J. S. contributed equally to the work reported in this study. Back

3 To whom requests for reprints should be addressed, at Department of Neurosurgery, Box 64, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: (713) 792-3821; Fax: (713) 794-5514; E-mail: ksrivenu{at}mdanderson.org Back

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

5 K.S. Srivenugopal, unpublished observations. Back

Received 10/31/00; revised 1/ 2/01; accepted 1/26/01.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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