- Top of page
- Materials and methods
- Conflict of interest
O6-methylguanine-DNA methyltransferase (MGMT) is a DNA-repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth-depressing transfection of a T98G glioma cell line with liver-type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA-transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non-transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100–1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.
Glutamine (Gln) plays a crucial role in the metabolism of neoplastic cells and elevated catabolism of this amino acid is observed in a wide variety of tumors (for review see DeBerardinis and Cheng 2010). Phosphate-activated glutaminase (abbreviated as PAG or GA, further referred to as PAG/GA, EC 126.96.36.199) metabolizes Gln to glutamate (Glu) and ammonia. In mammals, there are two genes coding for this enzyme: Gls encodes the kidney-type isoform (KGA), and Gls2 encodes the liver-type isoform (LGA) (Aledo et al. 2000). KGA is expressed in all mammalian tissues except liver (Curthoys and Watford 1995). Glutaminase isoform C (GAC), an alternatively spliced variant arising from Gls gene, was found in heart, pancreas, kidneys, lungs, and breast cancer cells (Elgadi et al. 1999). LGA is expressed in liver, brain, pancreas, and breast carcinoma cells (Gomez-Fabre et al. 2000).
In the central nervous system, LGA isoform shows nuclear localization (Olalla et al. 2002). Moreover, the C terminus of LGA interacts with PDZ-containing proteins (Olalla et al. 2001). In addition, two ankyrin repeats were identified in the C terminus of LGA (Marquez et al. 2006). These observations suggest that apart from the Gln-metabolizing function, LGA may play a role in the regulation of transcription (Olalla et al. 2002; Marquez et al. 2006).
Deregulated expression and/or activity of PAG/GA isoforms is a characteristic feature of different tumors and neoplastic cell lines (Szeliga and Obara-Michlewska 2009). In glioblastomas (GBM, WHO grade IV), the most malignant brain tumors, KGA and GAC are abundant, whereas LGA transcript is hardly detectable or low (Szeliga et al. 2005). Our recent study revealed that stable transfection of a full cDNA sequence encoding human LGA to glioblastoma T98G cell line decreased cell proliferation and migration and changed the expression level of 85 genes (Szeliga et al. 2009). One of the down-regulated genes, MGMT, codes for the suicide DNA-repair protein O6-methylguanine-DNA methyltransferase (MGMT, EC 188.8.131.52) that removes alkyl groups from the O6-position of guanine in DNA to its own active center (Pegg et al. 1995). O6-alkylguanine is formed by alkylating agents used in glioma therapy and provokes cell death by activating apoptosis (Roos et al. 2007) or autophagy (Lefranc et al. 2007). Therefore, O6-alkylguanine is considered to be responsible for the anticancer effect of alkylating compounds.
Elevated level of MGMT has been linked to increased cell resistance to methylating and chloroethylating agents in different glioma models (Nagane et al. 1997; Kitange et al. 2009; Yoshino et al. 2009). Moreover, a number of studies have documented correlation between MGMT status and the therapeutic response of glioma patients treated with alkylating agents (Mineura et al. 1993; Belanich et al. 1996; Hegi et al. 2005; Wiewrodt et al. 2008). The epigenetic silencing of the MGMT gene often caused by methylation of CpG islands in the promoter region has been described by different authors (for review see Jacinto and Esteller 2007).
We hypothesized that decreased MGMT gene expression observed in LGA-transfected glioblastoma cells (further defined as TLGA cells) implies a decrease in MGMT protein expression and activity. Given the crucial role of MGMT in glioma cell death induced by treatment with alkylating agents, we further speculated that decreased MGMT activity will increase cell sensitivity to methylating temozolomide (TMZ) and chloroethylating carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)].
- Top of page
- Materials and methods
- Conflict of interest
As outlined in the introduction, there are at least three glutaminase isoforms in mammalian tissues: KGA, GAC, and LGA (Elgadi et al. 1999; Aledo et al. 2000). Cerebral tumors of different origin and WHO grade express abundance of KGA and GAC, and only small amounts, or no LGA (Szeliga et al. 2005, 2008). Our previous study revealed that stable transfection of human T98G glioblastoma cell line with the full LGA sequence decreased cell proliferation and migration, and altered the expression pattern of 85 genes (Szeliga et al. 2009). One of these genes codes for MGMT, a protein playing a pivotal function in resistance to alkylating drugs often used in glioma therapy (for review see Jacinto and Esteller 2007; Kaina et al. 2007). T98G cell line is perceived as resistant to TMZ and BCNU mainly because of the high MGMT protein level and activity (Hermisson et al. 2006; Lavon et al. 2007). Therefore, we hypothesized that cells stably transfected with LGA sequence (TLGA cells) would become more sensitive to the alkylating agents as compared with T98G cells.
The key finding of this study is that TLGA cells show decreased MGMT protein expression and activity (Fig. 1). Moreover, this down-regulation of MGMT correlated with increased sensitivity of TLGA cells to methylating TMZ (Fig. 3) and chloroethylating BCNU (Fig. 4), and – consistently with expectations – suggested a causal nexus between the two phenomena. It also corroborates earlier findings in both the experimental and clinical settings, where the level of MGMT correlated well with the resistance of glioblastoma cell lines to TMZ and BCNU (Jaeckle et al. 1998; Fruehauf et al. 2006; Hermisson et al. 2006; Nagane et al. 2007). Furthermore, depletion of MGMT activity using its competitive inhibitor O6-benzylguanine (O6-BG) has been shown to restore TMZ and BCNU cytotoxicity in glioma cell lines (Kanzawa et al. 2003; Bobola et al. 2005; Hermisson et al. 2006) and xenografts (Kokkinakis et al. 2001).
The mechanism of decreased MGMT gene and protein expression observed in response to LGA transfection remains unclear. One of the best-described routes of MGMT down-regulation is via epigenetic silencing often caused by methylation of CpG islands in the promoter region (for review see Jacinto and Esteller 2007). A large body of evidence showed that glioma cells with MGMT promoter methylation are more sensitive to TMZ (Hegi et al. 2005) and BCNU (Esteller et al. 2000; Lechapt-Zalcman et al. 2012). In our study, T98G cells showed both methylated and unmethylated DNA at the locus covered by the most common pairs of primers (Christmann et al. 2010), independent of whether they were transfected or not with LGA. The latter result largely confirms data obtained by other groups (Lavon et al. 2007; Yoshino et al. 2009; but for a discrepant view see Ueda et al. 2004). As primers used in this study do not cover all possible CpG sites of the MGMT promoter, one cannot exclude that omissions of functionally relevant CpG sites might have partly entailed the observed lack of differences between TLGA and control cell lines. Nevertheless, our results suggest that methylation-independent pathways could influence MGMT expression in TLGA cells. Other mechanisms underlying decreased MGMT expression could include one of the following: (i) transcript destabilization, (ii) histone modifications, (iii) repression of transcription factors. Possibility (iii) is currently under investigation in our laboratory. Sequences for transcription activators such as: activator protein 1, activator protein 2, Sp1 (specificity protein 1), glucocorticoid responsive element (Harris et al. 1991), and NFκΒ (nuclear factor κΒ) (Lavon et al. 2007) have been identified in the promoter region of MGMT. Except AP-2, all these proteins have been described to play a role in the activation of MGMT expression (Costello et al. 1994; Boldogh et al. 1998; Biswas et al. 1999; Lavon et al. 2007; Bocangel et al. 2009). Below, we argue that activator protein 1, NfκΒ, and Sp1 deserve consideration as being engaged in the decrease of MGMT expression in TLGA cells.
Microarray analysis revealed that TLGA cells express lower amounts of RYK (receptor tyrosine kinase) transcript as compared with the controls (Szeliga et al. 2009), which was confirmed by real-time PCR (unpublished data). RYK is suspected to play a role in the non-canonical Wnt signaling pathway that involves, among others, PKC (protein kinase C) and AP-1 (for review see Katoh and Katoh 2007). PKC has been shown to regulate MGMT expression through AP-1 activation (Boldogh et al. 1998). Therefore, it can be assumed that decreased level of RYK observed as a consequence of LGA transfection results in down-regulation of PKC and/or AP-1 function, which in turn leads to reducing MGMT expression. On the other hand, PKC may also influence the NfκΒ signaling pathway (La Porta and Comolli 1998), which suggests that diminished RYK expression could also contribute to reduction of MGMT level through this pathway. Toward the same end, LGA is a target gene for p53 (Hu et al. 2010), and over-expression of Sp1 relieves, by an as yet not established mechanism, down-regulation of MGMT mediated by p53 (Bocangel et al. 2009). It would thus appear that the intrinsic absence of functional wild-type p53 in T98G cells (Van Meir et al. 1994) could be at least partially restored by the activity of LGA.
The mechanism by which LGA over-expression is translated to down-regulation of MGMT is unknown. The fact that TLGA cells show a higher Glu content and lower Gln content than non-transfected cells (Szeliga et al. 2009) would suggest altered control of MGMT gene transcription by one, or a combination of the two amino acids. In general terms, the gene transcription regulatory role of Glu has been established beyond doubt, and recent evidence strongly implicates Gln in this role as well (Brasse-Lagnel et al. 2009). To the best of our knowledge, however, experimental evidence linking Glu- or Gln-related events to MGMT transcription is not available. One other possibility to be envisaged is related to the above discussed hypothesis of LGA acting directly as a transcription factor. Clearly, further studies are needed to resolve between the above possibilities.
In conclusion, the results of this study demonstrate that stable transfection with LGA not only inhibits the growth of the cells per se but, through down-regulation of MGMT expression and activity, sensitizes T98G glioblastoma cells to TMZ and BCNU. The results encourage further research unraveling: (i) details of the mechanism by which MGMT expression is suppressed by LGA, (ii) functional and/or therapeutic implications of changes in the expression of other genes in LGA-transfected glioma cells.