O 6 -methylguanine-DNA methyltransferase is downregulated in transformed astrocyte cells: implications for anti-glioma therapies
© Sasai et al; licensee BioMed Central Ltd. 2007
Received: 26 April 2007
Accepted: 05 June 2007
Published: 05 June 2007
A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O 6 -methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways.
We established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents.
Normal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies.
Gliomas, accounting for 30% of adult primary brain tumors, are the most common primary tumors of the central nervous system and are classified into four clinical grades, with the most aggressive and lethal tumors being grade IV glioblastoma multiforme (GBM) [1, 2]. The median survival of GBM patients is less than one year from initial diagnosis, and many of the commonly used chemotherapeutic agents have limited effects on these malignant tumors . Recently, there has been increasing hope that temozolomide, a novel alkylating agent, will prove efficacious in the treatment of human glioma [4, 5]. However, a number of studies have suggested that, in tumors, O 6 -methylguanine-DNA methyltransferase (MGMT) provides resistance to treatment with temozolomide, unless expression is lost by promoter methylation or there is direct inhibition of MGMT activity . Considering the attractive efficacy of temozolomide, one of the greatest challenges facing the field may be to identify therapeutic agents that suppress MGMT expression, as such drugs may sensitize resistant glioma cells to temozolomide. Thus, establishment of more sophisticated systems to understand the functions and regulation of MGMT are highly desired.
Normal human cells, genetically modified by retroviral-mediated gene transfer, have proven important, because such systems are useful for identifying factors directly contributing to tumorigenesis, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities [7, 8]. GBMs are believed to arise from astrocyte cells by means of stepwise accumulation of genetic abnormalities [4, 9], and immortalized normal human astrocyte (NHA) cells had been established by introducing the telomerase catalytic subunit (hTERT) in combination with human papillomavirus E6/E7 to inactivate both p53 and pRb pathways . It had been systematically demonstrated that the immortalized NHA cells, expressing activated Ras (H-RasV12) or expressing both H-RasV12 and an active form of Akt (myrAKT), formed tumors consistent with human anaplastic astrocytoma or GBM in intracranial- and flank-xenografts models [10, 11]. These studies indicate that such systems are useful for glioma research to understand direct functions and regulation of genetic elements during transformation and gliomagenesis.
Here we created similar experimental models using NHA cells by introducing the simian virus 40 early region (SV40ER) instead of human papillomavirus E6/E7. The SV40ER encodes both small-t antigen, a suppressor of protein phosphatase 2A (which is downregulated in half of human glioma)  and large-T antigen, which directly binds to and inactivates p53, as well as pRB and the closely related proteins p107 and p130 . Using such genetically modified NHA cells, we demonstrate that MGMT is downregulated during oncogene-mediated transformation of astrocyte cells. Since our results indicate that downregulation of MGMT expression was not primarily dependent upon promoter hypermethylation, inhibitors of DNA methylation or histone deacetylases (HDACs) may be used in combination with alkylating agents for improved treatment of some GBM cases. We propose that the NHA cell systems are useful for investigating the mechanisms underlying MGMT expression and for improving glioma therapies.
Summary of soft agar colony formation assay and xenograft propagation experiment: NHA and NIH3T3 cells infected with retroviral vectors expressing hTERT (T), SV40ER (S), H-RasV12 (R), myrAKT (A), and/or MGMT as well as parental NHA cells were subjected to the soft-agar colony formation and xenograft propagation assays.
Colony numbers a
% Tumor incidence (n) b
664 ± 19
736 ± 51
NHA/TSR + MGMT
649 ± 34
1440 ± 56
NIH3T3/R + MGMT
1344 ± 88
Diagnosis and p53 status of human gliomas: The index of Ki-67 staining and p53 status (mutation and immunopositivity) are listed with patient ID, age at surgery, gender (F, female; M, male), and clinical diagnosis (GBM, glioblastoma, WHO grade IV; AA, anaplastic astrocytoma, WHO grade III, AE anaplastic ependymoma, WHO grade III). Images of H&E staining and immunohistochemistry are shown [see additional file 1].
Ki-67 (%) a
p53 mutations b
p53 staining c
Much of our understanding of the molecular basis of gliomagenesis derives from the study of established cell lines that are explanted from human tumors. Such cell lines are often assumed to be representative of the original diseases, and they have been extensively employed for the identification and preclinical testing of potential therapeutic compounds. However, they harbor an unknown number of genetic lesions, which complicates the study of specific molecules and pathways. Indeed, with respect to p53-mediated MGMT regulation, there have been contradictory observations, where transient knockdown of p53 caused MGMT downregulation in SF767 glioma cells without affecting promoter methylation  but increased MGMT expression in T98 cells . Since such discrepancy may depend on unidentified mutations specific for individual cell lines, it is difficult to provide constant conclusions using tumor-derived cell lines alone. Therefore, normal human cells as well as genetically engineered mouse models will prove very useful, which allow us to generate experimental models of human cancers by direct manipulation with defined genetic elements [7, 8].
Here we established glioma models from NHA cells and demonstrated that MGMT is downregulated in the transformed astrocyte cells. Although numerous studies have proposed the strong linkage between MGMT expression and promoter hypermethylation of the gene, treatment with epigenetic compounds (5-aza-dC/VPA) was unable to increase MGMT protein levels in the transformed NHA cells or in cultured tumor cells. Some previous reports demonstrated the epigenetic regulation of MGMT expression by treating tumor cell lines with 5-aza-dC and testing the MGMT levels by RT-PCR [32, 33]. However, based on the data presented here, such limited evaluation might not be sufficient, as RT-PCR assay overestimates the MGMT restoration. Since inappropriate evaluation would mislead the development of advanced therapies, protein levels or enzyme activity of MGMT in the cells treated with such compounds should be examined and compared with those in normal cells. In fact, the use of alkylating agents in combination with HDAC inhibitors has been hampered, probably because it has been suspected that such agents would increase MGMT expression. However, we showed that two-week-treatment with VPA inhibited tumor cell growth without increasing MGMT expression, suggesting potential clinical use although further preclinical studies are required.
As the treatment with 5-aza-dC/VPA slightly increased mRNA levels of MGMT in cultured glioma cells (including U87MG, U251MG and transformed NHA cells), methylation status might be involved in MGMT regulation to some extent. However, such treatment was insufficient to restore protein expression of MGMT, suggesting that the regulation mechanisms may largely depend on other cellular factors. Although p53 does not seem to contribute directly to MGMT expression, it is possible that other transcription factors, whose expressions have been downregulated or silenced during transformation and gliomagenesis, may collaborate with HDAC inhibitors to increase MGMT protein. Interestingly, the sonic hedgehog pathway has been shown to regulate the self-renewal of CD133-positive glioblastoma cells, which were resistant to temozolomide treatment . Furthermore, some of CD133-positive glioma cultures highly expressed MGMT as well as target genes of the sonic hedgehog pathway . Thus, it is possible that the regulation of MGMT expression may be partially mediated by the sonic hedgehog pathway in some cases.
As discussed above, MGMT seems to be regulated by a number of ways in gliomas. The detailed mechanisms should be further analyzed as MGMT level is a critical determinant for efficacy of therapies with alkylating agents. Thus, identification of molecules and compounds that increase MGMT expression by screening NHA/TSR or NHA/TSRA cells with cDNA- and chemical-libraries would be very useful for the development of rational drug combination. We propose that the NHA cell system creates refined human glioma models for the systematic dissection of genetic alterations and elucidation of the complexities of the signaling pathways important for gliomagenesis. These systems also provide powerful means to find and authenticate molecules of particular promise for therapeutic targeting, and the present study provides an important proof-of-principle test for such systems.
Brain tumor specimens were obtained, after informed consent, from patients undergoing tumor resection at the Kashiwaba Neurosurgical Hospital.
NHA cells (Cambrex Bio Science, Walkersville, MD, USA) were cultured in the astrocyte growth medium (AGM; Cambrex Bio Science). All other cells including immortalized NHA cells were maintained in Dulbecco's modified eagle medium (Seikagaku Co., Tokyo, Japan), supplemented with 10% fetal calf serum, 1 mM Glutamine, 50 units/ml penicillin G and 50 μg/ml streptomycin. All cultures were incubated at 37°C under a humidified atmosphere of 95% air and 5% CO2. For the combination treatment (5-aza-dC/VPA), 5-aza-dC (1 μM; Sigma, St. Louis, MO, USA) was added for an initial incubation of 48 h, after which VPA (1 mM; Sigma) was added for an additional 24 h.
Retroviral vectors and retroviral-mediated gene transfer
A cDNA fragment encoding murine ecotropic retrovirus receptor (EcoVR) was obtained from the retroviral plasmid pCX4hyg-EcoVR , and then subcloned into pCX4redEx vector [GenBank: AB296084]. Myc-His-tagged active form of mouse AKT1 cDNA, which has N-terminal myristoylation, was isolated from the pUSEamp-myr-AKT plasmid (Upstate, Charlottesville, VA, USA) and subcloned into pCX4bleo retroviral vector [GenBank: AB086388]. Full-length cDNAs for human MGMT and p53 were generated by PCR and subcloned into pCX4bleo and pCX4gfp [GenBank: AB296083] retroviral vectors, respectively. Primer sequences used in this experiment included 5'-ATG GAC AAG GAT TGT GAA-3' and 5'-TCA GTT TCG GCC AGC AGG-3' for human MGMT and 5'-CTG AAT TCA TGG AGG AGC CGC AGT CAG-3' and 5'-CCG AAT TCA GTC TGA GTC AGG CCC TTC-3' for human p53. Other retroviral vectors and the procedure of retroviral-mediated gene transfer were described previously . The murine EcoVR was first introduced into NHA cells by using amphotropic virus, in order to make human cells susceptible to the subsequent infection with ecotropic viral vectors. Infected cell populations were selected in blasticidin S (20 μg/ml), G418 (1000 μg/ml), puromycin (500 ng/ml), or zeocine (500 μg/ml) for two weeks. In all cases, cultures arose from polyclonal expansion of infected cells.
Total RNA was isolated with the TRI Reagent (Sigma) and reverse transcribed into cDNA using the oligo-dT primer (Invitrogen, Carlsbad, CA, USA) and the Superscript II (Invitrogen). The levels of MGMT were analyzed by PCR with the KOD plus DNA polymerase (Toyobo, Tokyo, Japan) using the primers described above. PCR primers for Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were described previously .
Soft-agar colony formation assay and xenograft propagation
Soft-agar colony formation assay  and xenograft propagation  were carried out as described. Female athymic nude mice (BALB/cAJcl-nu/nu) were purchased from Clea Japan (Tokyo, Japan) and all animal procedures were carried out according to the protocol approved by the institutional Animal Care and Use Committee at Hokkaido University Graduate School of Medicine.
Histological analysis and immunohistochemistry
Formalin-fixed paraffin-embedded tissues were sectioned and stained with haematoxylin and eosin (H&E) using standard protocols. Immunohistochemistry was performed using anti-Ki-67 (MIB-1; Dako, Glostrup, Denmark) and anti-p53 (DO-7; Dako) monoclonal antibodies.
Protein determination, SDS-PAGE and immunoblotting were carried out as described previously , and reactive protein signals were visualized by chemiluminescence using the ECL reagent (Amersham, Piscataway, NJ, USA) or the SuperSignal West Femto reagent (Pierce, Rockford, IL, USA). Antibodies were obtained from the following sources: anti-SV40 large T antigen (Ab-1) and anti-SV 40 small t antigen (Ab-3) monoclonal antibodies (Oncogene Research Product, San Diego, CA, USA); anti-p53 and anti-AKT polyclonal antibodies (Cell Signaling Technology, Beverly, MA, USA); anti-RAS and anti-p27KIP1 monoclonal antibodies (Transduction Laboratories, Lexington, KY, USA); anti-dimethylated Histone-H3 (Me-H3) and anti-acetylated Histone-H3 (Ac-H3) polyclonal antibodies (Upstate); anti-MGMT (MT3.1) and anti-ACTIN monoclonal antibodies (Chemicon International, Temecula, CA, USA); an anti-p21WAF1 monoclonal antibody (Ab-1; Calbiochem, San Diego, CA, USA); an anti-hTERT (L20) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
normal human astrocyte
- hTERT (T):
human telomerase catalytic subunit
- H-RasV12 (R):
- myrAKT (A):
myristoylated form (active form) of AKT
- SV40ER (S):
simian virus 40 early region
reverse transcriptase polymerase chain reaction
We thank Jared Ordway (Orion Genomics, St. Louis, MO), Christopher Calabrese (St. Jude Children's Research Hospital, Memphis, TN) and Taiko Sukezane (KAN Research Institute, Kobe, Japan) for their helpful discussion and critical reading of this manuscript. We thank Tomoyuki Shishido (Nara Institute of Science and Technology, Ikoma, Japan) for providing the reagents and Miho Nodagashira (Hokkaido University, Sapporo, Japan) for her excellent technical assistance. This work was supported by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to ST), the Suhara Memorial Foundation (to ST), and Grant-in-Aid from Ministry of Education, Culture, Sports, Science and Technology of Japan (to ST).
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