O6-methylguanine-DNA methyltransferase is downregulated in transformed astrocyte cells: implications for anti-glioma therapies

Abstract

Background: A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O6-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.

Results: 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.

Conclusion: 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.

Figure 1

Establishment of immortalized and transformed NHA cells. (A) Protein extracts (10 μg) from NHA cells infected with retroviral vectors expressing indicated genes (T, hTERT: S, SV40ER: R, H-RasV12; A; myrAKT) were analyzed by immunoblotting. Asterisk, active form of AKT (myrAKT) was distinguishable from wild-type form. (B) The morphologies (top panels) and anchorage-independent growth properties (bottom panels) of NHA/TS, NHA/TSR, and NHA/TSRA cells are shown.

Figure 2

Histological features of s.c. xenografts derived from the transformed NHA cells. Formalin-fixed paraffin-embedded tissue sections (5-μm-thick) of xenografts derived from NHA/TSR (left) and NHA/TSRA (right) cells were subjected to histological analyses. Low- (top) and high- (middle) magnification images (H&E staining; scale bar, 200 μm) and immunohistochemistry using an anti-Ki-67 antibody (bottom) are shown.

Figure 3

MGMT is downregulated in immortalized and transformed NHA cells. (A) The expression of MGMT mRNA in NHA (parental; lane 1), NHA/T (lane 2), NHA/TS (lane 3), NHA/TSR (lane 4), and NHA/TSRA cells (lane 5) were analyzed by semi-quantitative RT-PCR. The levels in flank xenografts derived from the NHA/TSR and NHA/TSRA cells were also tested (lanes 6 and 7). (B) Protein extracts (10 μg) from NHA (lanes 1–4) and TIG3 cells (lanes 5–7) infected with retroviral vectors expressing indicated genes (T, hTERT: S, SV40ER: R, H-RasV12; A; myrAKT) were analyzed by immunoblotting.

Figure 4

Neither treatment with 5-aza-dC/VPA nor p53 expression restores MGMT protein levels in transformed NHA cells. (A) Protein extracts (10 μg) from the transformed NHA cells treated (+) and untreated (-) with 5-aza-dC/VPA were analyzed by immunoblotting. (B) Protein extracts (10 μg) from the transformed NHA cells infected with wild type p53 (+) and control virus (-) were analyzed by immunoblotting. (C) Protein extracts (10 μg) from glioma cell lines treated (+) and untreated (-) with 5-aza-dC/VPA were analyzed by immunoblotting.

Figure 5

Expression of MGMT RNA in human glioma tissues. The mRNA levels of MGMT expression in human glioma tissues were analyzed by semi-quantitative RT-PCR.

Figure 6

VPA inhibits cell growth in transformed human astrocyte cells. (A) Dose-dependent anti-proliferative effects of VPA were shown. NHA/TSRA cells were treated with 0, 0.5 and 1.0 mM VPA for 14 days. Cell numbers were counted every other day and results were expressed in the mean of two independent experiments. (B) NHA/TSRA cells, treated (0.5 mM) and untreated (0 mM) with VPA for two weeks, were subjected to soft-agar colony formation assay. 2 × 10⁴ cells were plated, incubated at 37°C for 21 days and stained with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide. Note VPA was not included in the assay media. (C) Protein extracts (10 μg) from NHA/TSRA cells treated with VPA were analyzed by immunoblotting using indicated antibodies.