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Keywords:

  • carmustine;
  • liver-type glutaminase (LGA);
  • O6-methylguanine-DNA methyltransferase (MGMT);
  • T98G glioblastoma cell line;
  • temozolomide

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

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.

Abbreviations used
BCNU

1,3-bis(2-chloroethyl)-1-nitrosourea, carmustine

GAC

glutaminase isoform C

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

KGA

kidney-type glutaminase

LGA

liver-type glutaminase

MGMT

O6-methylguanine-DNA methyltransferase

MSP

methylation-specific PCR

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NFκΒ

nuclear factor κΒ

PAG/GA

phosphate-activated glutaminase

PKC

protein kinase C

RYK

receptor tyrosine kinase

Sp1

specificity protein 1

TLGA

LGA-transfected cells

TMZ

temozolomide

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 3.5.1.2) 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 2.1.1.63) 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)].

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

Cell lines and culture conditions

T98G human glioblastoma cell line purchased from American Type Culture Collection was maintained in minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% antibiotics (penicillin and streptomycin). Cultures were maintained at 37°C in a humidified atmosphere with 95% air and 5% CO2. TLGA and TpcDNA cell lines were obtained by stable transfection of T98G cells with a full cDNA sequence encoding human LGA or empty pcDNA3 vector, respectively, exactly as described previously (Szeliga et al. 2009). The culture medium for the polyclonal populations of TpcDNA and TLGA cells containing the neomycin-resistance gene was supplemented with 0.5 mg/mL G418 (Sigma-Aldrich). The expression pattern of PAG/GA isoforms in all cell lines was monitored by RT-PCR as described previously (Szeliga et al. 2009).

Western blot

The cells were harvested and sonicated in radioimmunoprecipitaton assay lysis buffer supplemented with cocktails of protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 10 000 g for 10 min at 4°C, and the supernatants were collected and stored at −80°C. Protein concentration was determined using the bicinchoninic acid Protein Assay Kit (Pierce, Rockford, IL, USA). Fifty micrograms of protein was resolved on 12% polyacrylamide gels and then electrotransferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% skim milk and incubated overnight at 4°C with a human anti-MGMT antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, membranes were incubated with anti-rabbit antibody (Sigma-Aldrich) conjugated to horseradish peroxidase. Blots were developed with SuperSignal West Pico Chemiluminescence Substrate (Pierce). For loading control, membranes were stripped and reprobed with a human-specific antibody against glyceraldehyde 3-phosphate dehydrogenase (Sigma-Aldrich). Densitometric analysis was done using G:Box system and GeneTools software (Syngene, Frederick, MD, USA).

Preparation of cell extracts and MGMT activity assay

Cell extracts and MGMT activity assay were performed as described by Hermisson et al. (2006) with some modifications. Briefly, the cells were harvested and sonicated in buffer containing Tris-HCl (20 mM, pH 8.5), EDTA (1 mM), β-mercaptoethanol (1 mM), glycerol (5%), phenylmethylsulfonyl fluoride (0.1 mM). The extracts were centrifuged at 8 000 g for 10 min at 4°C; the supernatants were snap-frozen using liquid nitrogen and stored at −80°C until use. The protein concentration was determined using bicinchoninic acid Protein Assay Kit (Pierce). MGMT activity was determined using radioactive assay, in which transfer of tritium-labeled methyl group from the O6-position of guanine in the DNA to the MGMT protein in the cell extract is measured. [3H-methyl]-nitrosourea, specific activity 80 Ci/mmol, (American Radiolabeled Chemicals) was used for labeling calf thymus DNA (Sigma-Aldrich). For each assay, 100 μg of protein was incubated with [3H-methyl]-nitrosourea-labeled DNA (140 000 cpm/sample) in a buffer containing HEPES-KOH (70 mM, pH 7.8), dithiothreitol (1 mM), and EDTA (5 mM) for 90 min. The reactions were stopped by addition of 5% trichloro-acetic acid. The pellets were centrifuged at 12 000 rpm for 15 min and the remaining 3H-labeled DNA was hydrolyzed by heating in 0.1 N HCl at 70°C for 30 min. The mixtures were centrifuged again and the radioactivity of acidic supernatants was determined in a liquid scintillation counter. Data are expressed as femtomole radioactivity transferred from 3H-labeled DNA to protein/milligram of protein within the sample.

MTT test

The cells were seeded in 24-well plates and incubated in the culture medium for 24 h. Next, the cells were exposed to increasing (0–1000 μM) concentrations of TMZ (Sigma-Aldrich) or BCNU (Sigma-Aldrich) for 72 h. After this time, the medium was removed, the cells were washed with phosphate-buffered saline and incubated in the culture medium with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution at the final concentration of 0.5 mg/mL for 15 min to allow the conversion of MTT into formazan. Then the medium was replaced with dimethyl sulfoxide and absorbance was read at 570 nm using Elisa Bio-Rad Microplate Reader.

Clonogenic survival assay

Five hundred cells were seeded in six-well plates and incubated in standard medium for 24 h. Next, the cells were exposed to increasing (0–1000 μM) concentrations of TMZ (Sigma-Aldrich) or BCNU (Sigma-Aldrich) for 24 h. After this time, the cells were washed with phosphate-buffered saline and incubated with fresh medium for 14 days. Subsequently, colonies were assessed using crystal violet staining. Colonies of more than 40 cells were counted.

Growth rates

Cells were seeded in six-well plates, 1000 in each well, and allowed to grow for 24 h. The medium was then changed either into fresh standard medium (serving as control) or into the medium containing TMZ or BCNU, each at 100-μM concentration. The trypsinized cells were counted on a hemocytometer. Growth assays were determined by cell enumeration in triplicate for each cell line up to the 7th day.

Preparation of genomic DNA and methylation-specific PCR (MSP)

DNA was isolated from T98G, TpcDNA, and TLGA cells using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Promoter methylation in the CpG islands of the MGMT gene was determined by chemical modification of unmethylated, but not methylated cytosines to uracil and subsequent MSP using different primer pairs covering flanking regions of the MGMT promoter. One microgram of extracted DNA underwent bisulfite modification using a CpGenome Turbo Bisulfite Modification Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions. CpGenome Universal Methylated and Unmethylated DNAs (Merck Millipore) were used as controls. MSP was carried out on 10 ng of bisulfite-treated DNA with the most commonly used primers (Christmann et al. 2010): for the methylated reaction: 5′-TTTCGACGTTCGTAGGTTTTCGC-3′ (upper primer) and 5′-GCACTCTTCCGAAAACGAAACG-3′ (lower primer) and for the unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ (upper primer) and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′ (lower primer). In addition, quantitative analysis of the MGMT promoter methylation was performed as described by Christmann et al.(2010). In this case, the primers detecting only the methylated MGMT promoter were used: 5′- TTTCGACGTTCGTAGGTTTTCGC-3′ (upper primer) and 5′-CTCGAAACTACCACCGTCCCGA-3′ (lower primer). The methylation status of the MGMT promoter was determined in relation to the signal obtained for the β-actin promoter amplified with the primers: 5′-AGGGAGTATATAGGTTGGGGAAGTT-3′ (upper primer) and 5′-AACACACAATAACAAACACAAATTCAC-3′ (lower primer). Controls without DNA were performed for each set of PCRs. Ten microliters of each PCR product was mixed with Midori Green DNA Stain (Nippon Genetics, Dueren, Germany), separated on 10% polyacrylamide gel, and visualized under UV illumination.

Statistical analysis

All experiments described above were repeated at least four times. Results are reported as means and standard deviations. GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used to determine statistical significance and EC50 values. Statistical significance of comparisons was based on one-way anova followed by Tukey's test.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

Transfection with LGA decreases MGMT protein expression and activity

As reported earlier, LGA mRNA was detected in the T98G cells transfected with the full human LGA sequence (TLGA cells), but was below the detection level in the wild-type cells or the cells transfected with the empty vector pcDNA3 (TpcDNA cells). The LGA protein expression was likewise much higher in TLGA cells than in cells not transfected with LGA (Szeliga et al. 2009). A quantitative RT-PCR analysis carried out in the same study (Szeliga et al. 2009) revealed decreased expression of MGMT gene in TLGA cells as compared with the wild-type or TpcDNA cells. In this study we examined the expression of MGMT protein using western blot analysis. TLGA cells showed a weak immunoreactive band at the predicted size of 25 kDa on immunoblots. In contrast, both control cell lines showed a strong immunoreactive band (Fig. 1a). Densitometric analysis of band intensity revealed a 70% decrease in the expression of MGMT protein in TLGA cells as compared with the control cells (Fig. 1b). Moreover, TLGA cells exhibited a 50% decrease in the activity of MGMT protein as compared with the control cells (Fig. 1c).

image

Figure 1. Transfection with liver-type glutaminase (LGA) decreases O6-methylguanine-DNA methyltransferase (MGMT) protein expression and activity. (a) Whole-cell lysates of T98G, TpcDNA, and TLGA cells were assessed by immunoblot for MGMT protein levels. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. (b) The graph shows quantification of MGMT band intensity normalized to GAPDH. The results are mean ± SD for five protein isolations from each cell line. *< 0.001 versus T98G and TpcDNA as tested with one-way anova followed by Tukey's test. (c) MGMT activity measured in T98G, TpcDNA, and TLGA cells expressed as femtomole radioactivity transferred from 3H-labeled DNA to protein /mg of protein within the sample. The results are mean ± SD for four measurements for each cell line. *< 0.001 versus T98G and TpcDNA as tested with one-way anova followed by Tukey's test.

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Transfection with LGA sensitizes T98G cells to alkylating agents

As down-regulated MGMT activity leads to the increased cell sensitivity to the alkylating agents, we hypothesized that TLGA cells were more sensitive to these compounds as compared to the controls. To verify this hypothesis, we examined the influence of methylating temozolomide (TMZ) and chloroethylating carmustine (BCNU) on TLGA, T98G, and TpcDNA cells using two independent tests: MTT assay and clonogenic survival assay. In agreement with decreased expression and activity pf MGMT, TLGA cells displayed increased sensitivity to both compounds.

In the MTT assay, TLGA cells showed approximately 20% decrease in the amount of viable cells after treatment with 100 μM and 1000 μM TMZ as compared with the controls (Fig. 2a). The EC50 value was lowered from 698 μM for T98G cells and 746 μM for TpcDNA cells to 335 μM for TLGA cells (Fig. 2b). In the clonogenic survival assay, TLGA cells presented even more increased sensitivity to TMZ (Fig. 2c). The number of colonies formed by TLGA cells after treatment with 10 μM TMZ was 35% lower as compared with the number of colonies formed by the control cells. At higher concentrations of TMZ, the decrease in number of colonies formed by TLGA cells was even more pronounced and amounted to less than 40% of number of colonies formed by the controls. In the clonogenic survival assay, the EC50 value was decreased from 342 μM for T98G and 295 μM for TpcDNA to 33 μM for TLGA cells (Fig. 2d).

image

Figure 2. Transfection with liver-type glutaminase (LGA) sensitizes T98G cells to temozolomide (TMZ)–MTT and clonogenic survival assay. (a) The acute growth inhibition assay: T98G, TpcDNA, and TLGA cells were exposed to increasing concentrations of TMZ for 72 h and then mitochondrial activity was assessed by the MTT test. The results are mean ± SD for six independent determinations for each cell line. *< 0.01 versus T98G and TpcDNA as tested with one-way anova followed by Tukey's test. (b) EC50 values in the acute growth inhibition assay were calculated. (c) The clonogenic survival assay: T98G, TpcDNA, and TLGA cells were exposed to increasing concentrations of TMZ for 24 h and allowed to grow 2 weeks in complete medium. Colonies were assessed by crystal violet staining. The results are mean ± SD for six independent determinations for each cell line. *< 0.01 versus T98G and TpcDNA as tested with one-way anova followed by Tukey's test. (d) EC50 values in the clonogenic survival assay were calculated.

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TLGA cells showed increased sensitivity to BCNU as well. In MTT assay, viability of these cells after treatment with 1000 μM BCNU was lowered by approximately 30% with respect to the controls (Fig. 3a). The EC50 value was reduced from 465 μM for T98G and 523 μM for TpcDNA to 344 μM for TLGA cells (Fig. 3b). Again, sensitivity of TLGA cells to BCNU was more pronounced in the clonogenic survival assay. At 10 μM BCNU, number of colonies formed by TLGA cells constituted approximately 80% of colonies formed by the controls and decreased to 60% and 25% at 100 μM and 1000 μM BCNU, respectively (Fig. 3c). In this assay, EC50 was lowered from 302 μM for T98G and 401 μM for TpcDNA to 173 μM for TLGA cells (Fig. 3d).

image

Figure 3. Transfection with liver-type glutaminase (LGA) sensitizes T98G cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)–MTT and clonogenic survival assay. (a) The acute growth inhibition assay: T98G, TpcDNA, and TLGA cells were exposed to increasing concentrations of BCNU for 72 h and then mitochondrial activity was assessed by the MTT test. The results are mean ± SD for four independent determinations for each cell line. < 0.05 versus T98G, *< 0.05 versus T98G and TpcDNA as tested with one-way anova followed by Tukey's test. (b) EC50 values in the acute growth inhibition assay were calculated. (c) The clonogenic survival assay: T98G, TpcDNA, and TLGA cells were exposed to increasing concentrations of BCNU for 24 h and allowed to grow 2 weeks in complete medium. Colonies were assessed by crystal violet staining. The results are mean ± SD for seven independent determinations for each cell line. *< 0.001, < 0.05 versus T98G and TpcDNA as tested with one-way anova followed by Tukey's test. (d) EC50 values in the clonogenic survival assay were calculated.

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We have previously shown that TLGA cells grow more slowly than cells not transfected with LGA (Szeliga et al. 2009). As growth is required for TMZ and BCNU toxicity, we examined the influence of either compound on cell proliferation rate. TLGA cells not treated with TMZ or BCNU showed a decrease in growth rate as compared to non-transfected cells. Treatment of each cell line with TMZ or BCNU caused a significant reduction of cell number, with TLGA cells being more susceptible to either compound than non-transfected cells (Fig. 4).

image

Figure 4. Transfection with liver-type glutaminase (LGA) sensitizes T98G cells to temozolomide (TMZ) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)–growth curves. Twenty four hours after seeding T98G, TpcDNA, and TLGA cells were exposed to 100 μM TMZ or BCNU. The cells were counted on the 2nd, 3rd, 5th, and 7th day after seeding. The results are mean ± SD for three independent determinations for each cell line untreated or treated either with TMZ or BCNU. *< 0.05 untreated cells (each cell line) versus treated with TMZ/BCNU; #< 0.01 TLGA versus T98G and TpcDNA; TLGA treated with TMZ/BCNU versus T98G treated with TMZ/BCNU and TpcDNA treated with TMZ/BCNU as tested with one-way anova followed by Tukey's test.

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Transfection with LGA does not alter methylation status of MGMT promoter methylation site covered by most often used primers

One of the best-described mechanisms of the regulation of MGMT expression is gene silencing via promoter hypermethylation (for review see Jacinto and Esteller 2007). Therefore, we employed MSP to investigate the methylation status of MGMT promoter in T98G, TpcDNA, and TLGA cells. First, we performed qualitative analysis using the most common pairs of primers (designated as MSP-P1, see Christmann et al. 2010) covering important (most commonly used in the clinical studies) CpG islands in MGMT promoter and amplifying the methylated and unmethylated allele. T98G cells showed both the methylated and unmethylated MGMT promoter (Fig. 5a), which confirms the results obtained by other investigators (Lavon et al. 2007; Yoshino et al. 2009). TLGA cells and TpcDNA serving as a second control cell line also presented both the methylated and unmethylated MGMT promoter (Fig. 5a). Next, we performed a semiquantitative analysis of the MGMT promoter in all cell lines examined. We used a pair of primers detecting only the methylated MGMT promoter and a pair of primers covering the β-actin promoter (designated as MSP-P2, see Christmann et al. 2010). Analysis of MGMT / β-actin bands' intensity revealed no differences between the cell lines examined (Fig. 5b).

image

Figure 5. Transfection with liver-type glutaminase (LGA) does not alter methylation status of MGMT promoter. O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in T98G, TpcDNA, and TLGA cells determined by MSP assay. (a) MSP-P1 was performed using primers specific for the methylated (Meth 1) and unmethylated (Unmeth) MGMT promoter. (b) MSP-P2 was performed using primers specific for the methylated (Meth 2) MGMT promoter and β-actin (β-act) promoter. M, marker ladder; MC, control methylated DNA; UC, control unmethylated DNA; H2O, control without DNA.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

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.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References

This study was supported by the National Science Centre, grant no NN401 039238 (to MS).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. Conflict of interest
  8. References
  • Aledo J. C., Gomez-Fabre P. M., Olalla L. and Marquez J. (2000) Identification of two human glutaminase loci and tissue-specific expression of the two related genes. Mamm. Genome 11, 11071110.
  • Belanich M., Pastor M., Randall T. et al. (1996) Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival of brain tumor patients treated with carmustine. Cancer Res. 56, 783788.
  • Biswas T., Ramana C. V., Srinivasan G., Boldogh I., Hazra T. K., Chen Z., Tano K., Thompson E. B. and Mitra S. (1999) Activation of human O6-methylguanine-DNA methyltransferase gene by glucocorticoid hormone. Oncogene 18, 525532.
  • Bobola M. S., Silber J. R., Ellenbogen R. G., Geyer J. R., Blank A. and Goff R. D. (2005) O6-methylguanine-DNA methyltransferase, O6-benzylguanine, and resistance to clinical alkylators in pediatric primary brain tumor cell lines. Clin. Cancer Res. 11, 27472755.
  • Bocangel D., Sengupta S., Mitra S. and Bhakat K. K. (2009) p53-Mediated down-regulation of the human DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) via interaction with Sp1 transcription factor. Anticancer Res. 29, 37413750.
  • Boldogh I., Ramana C. V., Chen Z., Biswas T., Hazra T. K., Grösch S., Grombacher T., Mitra S. and Kaina B. (1998) Regulation of expression of the DNA repair gene O6-methylguanine-DNA methyltransferase via protein kinase C-mediated signaling. Cancer Res. 58, 39503956.
  • Brasse-Lagnel C., Lavoinne A. and Husson A. (2009) Control of mammalian gene expression by amino acids, especially glutamine. FEBS J. 276, 18261844.
  • Christmann M., Nagel G., Horn S., Krahn U., Wiewrodt D., Sommer C. and Kaina B. (2010) MGMT activity, promoter methylation and immunohistochemistry of pretreatment and recurrent malignant gliomas: a comparative study on astrocytoma and glioblastoma. Int. J. Cancer 127, 21062118.
  • Costello J. F., Futscher B. W., Kroes R. A. and Pieper R. O. (1994) Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines. Mol. Cell. Biol. 14, 65156521.
  • Curthoys N. P. and Watford M. (1995) Regulation of glutaminase activity and glutamine metabolism. Annu. Rev. Nutr. 15, 133159.
  • DeBerardinis R. J. and Cheng T. (2010) Q's next: the diverse functions of glutamine in metabolism, cell biology and cancer. Oncogene 29, 313324.
  • Elgadi K. M., Meguid R. A., Qian M., Souba W. W. and Abcouwer S. F. (1999) Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing. Physiol. Genomics 1, 5162.
  • Esteller M., Garcia-Foncillas J., Andion E., Goodman S. N., Hidalgo O. F., Vanaclocha V., Baylin S. B. and Herman J. G. (2000) Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N. Engl. J. Med. 343, 13501354.
  • Fruehauf J. P., Brem H., Brem S., Sloan A., Barger G., Huang W. and Parker R. (2006) In vitro drug response and molecular markers associated with drug resistance in malignant gliomas. Clin. Cancer Res. 12, 45234532.
  • Gomez-Fabre P. M., Aledo J. C., Del Castillo-Olivares A., Alonso F. J., Nunez De Castro I., Campos J. A. and Marquez J. (2000) Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase. Biochem. J. 345, 365375.
  • Harris L. C., Potter P. M., Tano K., Shiota S., Mitra S. and Brent T. P. (1991) Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene. Nucleic Acids Res. 19, 61636167.
  • Hegi M. E., Diserens A. C., Gorlia T. et al. (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma. N. Engl. J. Med. 352, 9971003.
  • Hermisson M., Klumpp A., Wick W., Wischhusen J., Nagel G., Roos W., Kaina B. and Weller M. (2006) O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells. J. Neurochem. 96, 766776.
  • Hu W., Zhang C., Wu R., Sun Y., Levine A. and Feng Z. (2010) Glutaminase 2, a novel p53 target gene regulating energy metabolism and antioxidant function. Proc. Natl Acad. Sci. USA 107, 74557460.
  • Jacinto F. V. and Esteller M. (2007) MGMT hypermethylation: a prognostic foe, a predictive friend. DNA Repair (Amst) 6, 11551160.
  • Jaeckle K. A., Eyre H. J., Townsend J. J., Schulman S., Knudson H. M., Belanich M., Yarosh D. B., Bearman S. I., Giroux D. J. and Schold S. C. (1998) Correlation of tumor O6 methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea: a Southwest Oncology Group study. J. Clin. Oncol. 16, 33103315.
  • Kaina B., Christmann M., Naumann S. and Roos W. P. (2007) MGMT: key node in the battle against genotoxicity, carcinogenicity an apoptosis induced by alkylating agents. DNA Repair (Amst) 6, 10791099.
  • Kanzawa T., Bedwell J., Kondo Y., Kondo S. and Germano I. M. (2003) Inhibition of DNA repair for sensitizing resistant glioma cells to temozolomide. J. Neurosurg. 99, 10471052.
  • Katoh M. and Katoh M. (2007) WNT signaling pathway and stem cell signaling network. Clin. Cancer Res. 13, 40424045.
  • Kitange G. J., Carlson B. L., Schroeder M. A., Grogan P. T., Lamont J. D., Decker P. A., Wu W., James C. D. and Sarkania J. N. (2009) Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts. Neuro Oncol. 11, 281291.
  • Kokkinakis D. M., Bocangel D. B., Schold S. C., Moschel R. C. and Pegg A. E. (2001) Thresholds of O6-alkylguanine-DNA alkyltransferase which confer significant resistance of human glial tumor xenografts to treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea or temozolomide. Clin. Cancer Res. 7, 421428.
  • Lavon I., Fuchs D., Zrihan D., Efroni G., Zelikovitch B., Fellig Y. and Siegal T. (2007) Novel mechanism whereby nuclear factor kappaB mediates DNA damage repair through regulation of O(6)-methylguanine-DNA-methyltransferase. Cancer Res. 67, 89528959.
  • Lechapt-Zalcman E., Levallet G., Dugué A. E. et al. (2012) O(6) -methylguanine-DNA methyltransferase (MGMT) promoter methylation and low MGMT-encoded protein expression as prognostic markers in glioblastoma patients treated with biodegradable carmustine wafer implants after initial surgery followed by radiotherapy with concomitant and adjuvant temozolomide. Cancer. doi: 10.1002/cncr.27441.
  • Lefranc F., Facchini V. and Kiss R. (2007) Proautophagic drugs: a novel means to combat apoptosis-resistant cancers, with a special emphasis on glioblastomas. Oncologist 12, 13951403.
  • Marquez J., de la Oliva A. R., Mates J. M., Segura J. A. and Alonso F. J. (2006) Glutaminase: a multifaceted protein not only involved in generating glutamate. Neurochem. Int. 8, 465471.
  • Van Meir E. G., Kikuchi T., Tada M., Li H., Diserens A. C., Wojcik B. E., Huang H. J., Friedmann T., de Tribolet N. and Cavenee W. K. (1994) Analysis of the p53 gene and its expression in human glioblastoma cells. Cancer Res. 54, 649652.
  • Mineura K., Izumi I., Watanabe K. and Kowada M. (1993) Influence of O6-methylguanine-DNA methyltransferase activity on chloroethylnitrosourea chemotherapy in brain tumors. Int. J. Cancer 55, 7681.
  • Nagane M., Asai A., Shibui S., Nomura K. and Kuchino Y. (1997) Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas. Neurosurgery 41, 434441.
  • Nagane M., Kobayashi K., Ohnishi A., Shimizu S. and Shiokawa Y. (2007) Prognostic significance of O6-methylguanine-DNA methyltransferase protein expression in patients with recurrent glioblastoma treated with temozolomide. Jpn. J. Clin. Oncol. 37, 897906.
  • Olalla L., Aledo J. C., Bannenberg G. and Marquez J. (2001) The C-terminus of human glutaminase L mediates association with PDZ domain-containing proteins. FEBS Lett. 488, 116122.
  • Olalla L., Gutierrez A., Campos J. A., Khan Z. U., Alonso F. J., Segura J. A., Marquez J. and Aledo J. C. (2002) Nuclear localization of L-type glutaminase in mammalian brain. J. Biol. Chem. 277, 3893938944.
  • Pegg A. E., Dolan M. E. and Moschel R. C. (1995) Structure, function and inhibition of O6-alkylguanine-DNA-alkyltransferase. Prog. Nucleic Acid Res. Mol. Biol. 51, 167223.
  • La Porta C. A. and Comolli R. (1998) PKC-dependent modulation of IkB alpha-NFkB pathway in low metastatic B16F1 murine melanoma cells and in highly metastatic BL6 cells. Anticancer Res. 18, 25912597.
  • Roos W. P., Batista L. F., Naumann S. C., Wick W., Weller M., Menck C. F. and Kaina B. (2007) Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine. Oncogene 26, 186197.
  • Szeliga M. and Obara-Michlewska M. (2009) Glutamine in neoplastic cells: focus on the expression and roles of glutaminases. Neurochem. Int. 55, 7175.
  • Szeliga M., Sidoryk M., Matyja E., Kowalczyk P. and Albrecht J. (2005) Lack of expression of the liver-type glutaminase (LGA) mRNA in human malignant gliomas. Neurosci. Lett. 374, 171173.
  • Szeliga M., Matyja E., Obara M., Grajkowska W., Czernicki T. and Albrecht J. (2008) Relative expression of mRNAS coding for glutaminase isoforms in CNS tissues and CNS tumors. Neurochem. Res. 33, 808813.
  • Szeliga M., Obara-Michlewska M., Matyja E., Łazarczyk M., Lobo C., Hilgier W., Alonso F. J., Marquez J. and Albrecht J. (2009) Transfection with liver-type glutaminase cDNA alters gene expression and reduces survival, migration and proliferation of T98G glioma cells. Glia 57, 10141023.
  • Ueda S., Mineta T., Nakahara Y., Okamoto H., Shiraishi T. and Tabuchi K. (2004) Induction of the DNA repair gene O6-methylguanine-DNA methyltransferase by dexamethasone in glioblastomas. J. Neurosurg. 101, 659663.
  • Wiewrodt D., Nagel G., Dreimüller N., Hundsberger T., Perneczky A. and Kaina B. (2008) MGMT in primary and recurrent human glioblastomas after radiation and chemotherapy and comparison with p53 status and clinical outcome. Int. J. Cancer 122, 13911399.
  • Yoshino A., Ogino A., Yachi K., Ohta T., Fukushima T., Watanabe T., Katayama Y., Okamoto Y., Narusz N. and Sano E. (2009) Effect of IFN-b on human glioma cell lines with temozolomide resistance. Int. J. Oncol. 35, 139148.