Abstract
BackgroundA 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.ResultsWe 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.ConclusionNormal 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.
Highlights
A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas
The simian virus 40 early region (SV40ER) encodes both small-t antigen, a suppressor of protein phosphatase 2A [12] and large-T antigen, which directly binds to and inactivates p53, as well as pRB and the closely related proteins p107 and p130 [13]. Using such genetically modified normal human astrocyte (NHA) cells, we demonstrate that methylguanine-DNA methyltransferase (MGMT) is downregulated during oncogene-mediated transformation of astrocyte cells
To create an experimental model of human gliomas, NHA cells were introduced with genes for hTERT (T), SV40ER (S), H-RasV12 (R), and myrAKT (A) (Figure 1A; NHA/TS, NHA/TSR, NHA/TSRA cells)
Summary
A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. 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. There has been increasing hope that temozolomide, a novel alkylating agent, will prove efficacious in the treatment of human glioma [4,5]. A number of studies have suggested that, in tumors, O6-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 [6]. 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. Establishment of more sophisticated systems to understand the functions and regulation of MGMT are highly desired
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