Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts

Abstract

Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM tumors, and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all, GBM tumors. In this study, the relationship between MGMT protein expression and tumor response to TMZ was evaluated in four GBM xenograft lines that had been established from patient specimens and maintained by serial subcutaneous passaging in nude mice. Three MGMT unmethylated tumors displayed elevated basal MGMT protein expression, but only two of these were resistant to TMZ therapy (tumors GBM43 and GBM44), while the other (GBM14) displayed a level of TMZ sensitivity that was similar in extent to that seen in a single MGMT hypermethylated line (GBM12). In tissue culture and animal studies, TMZ treatment resulted in robust and prolonged induction of MGMT expression in the resistant GBM43 and GBM44 xenograft lines, while MGMT induction was blunted and abbreviated in GBM14. Consistent with a functional significance of MGMT induction, treatment of GBM43 with a protracted low-dose TMZ regimen was significantly less effective than a shorter high-dose regimen, while survival for GBM14 was improved with the protracted dosing regimen. In conclusion, MGMT expression is dynamically regulated in some MGMT nonmethylated tumors, and in these tumors, protracted dosing regimens may not be effective.

Fig. 1

O⁶-Methylguanine-DNA methyltransferase (MGMT) promoter methylation and protein expression in glioblastoma multiforme (GBM) xenografts. Flank tumor specimens for the indicated xenograft lines were processed for assessment of MGMT promoter methylation by methylation-specific PCR (MS-PCR) (A) and for MGMT protein expression by Western blotting (B). For the MS-PCR reaction, universally methylated DNA and normal brain DNA were used as positive (P) and negative (N) controls, respectively. The MS-PCR amplification products were resolved by electrophoresis, and the corresponding ethidium bromide–stained gel is shown. Abbreviations: Mr, relative molecular weight markers; U, unmethylated; M, methylated. GBM12 is the only MGMT methylated tumor line.

Fig. 2

Effects of temozolomide (TMZ) therapy on survival. Mice with established orthotopic xenografts for the indicated glioblastoma multiforme (GBM) tumor lines were randomized to treatment with vehicle or 120 mg/kg TMZ daily for 5 days. Mice were euthanized once they became moribund. Arrows denote when treatment was initiated. (A) GBM12 (methylated; p < 0.0001). (B) GBM14 (unmethylated; p < 0.0001). (C) GBM43 (unmethylated; p = 0.003). (D) GBM44 (unmethylated; p = 0.81).

Fig. 3

In vitro temozolomide (TMZ) cytotoxicity. Short-term cell cultures from the indicated glioblastoma multiforme (GBM) xenograft lines were treated with a single dose of TMZ, and the effects on cell survival were evaluated 96 h later using a methylene blue staining assay. Mean ± SD for relative optical density (OD) versus TMZ concentrations are plotted from three independent experiments. Significance compared with GBM14: GBM12, p < 0.001; GBM43, p < 0.76; GBM44, p < 0.20.

Fig. 4

Short versus protracted dosing regimens. Short-term cell cultures from glioblastoma multiforme (GBM) tumors GBM14 and GBM43 were treated with three doses of 100 μM temozolomide (TMZ) delivered once every 6 h (A) or once every 24 h (B). Where indicated, cultures were also dosed once with 10 μM O⁶-benzylguanine (O6BG) concurrently with the first dose of TMZ. Survival was evaluated with a methylene blue survival assay 96 h after the last dose of TMZ. Mean ± SD for relative optical density (OD) versus TMZ concentrations are plotted from two independent experiments. Survival analyses: (A) GBM14 versus GBM43, p = 0.67; GBM14 TMZ + O6BG versus TMZ only, p = 0.005; GBM43 TMZ + O6BG versus TMZ only, p = 0.0004. (B) GBM14 versus GBM43, p = 0.001; GBM14 TMZ + O6BG versus TMZ only, p = 0.71; GBM43 TMZ + O6BG versus TMZ only, p = 0.0002.

Fig. 5

Effects of temozolomide (TMZ) treatment on O⁶-methyl-guanine-DNA methyltransferase (MGMT) expression in vitro. Short-term cell cultures for glioblastoma multiforme (GBM) tumors GBM12, -14, -43, and -44 were treated with 100 μM TMZ and harvested at the indicated time points. (A) Samples were processed for Western blotting for MGMT and then β-actin. (B) Total RNA from GBM43 was reverse transcribed, and the effect of TMZ treatment on MGMT mRNA was evaluated using real-time PCR. The data from three independent experiments performed in triplicate are presented as mean ± SD.

Fig. 6

Effects of temozolomide (TMZ) treatment on O⁶-methylguanine-DNA methyltransferase (MGMT) activity and RNA expression in vivo. Mice with established flank glioblastoma multiforme (GBM) tumors were treated with TMZ at 50 mg/kg per day × 5 days, euthanized at the indicated time points, and processed for an analysis of MGMT repair activity (A) or MGMT mRNA levels by reverse transcription PCR (B). (A) For the activity assay, the MGMT activity within tumor samples is measured by incubating cell lysates with a ³²P-labeled oligonucleotide containing an O⁶-methylguanine residue within a PstI restriction site. Oligonucleotide is then isolated, cut with PstI, and resolved by electrophoresis. MGMT activity in this assay correlates with the level of the cut oligonucleotide (lower band, arrow) and was determined as a ratio of radioactivity in the uncut and cut oligonucleotide bands as determined by film densitometry. The relative change in repair activity for each tumor sample is shown. (B) Pooled total RNA from two tumor samples at each time point was reverse transcribed, followed by real-time PCR amplification. Bars represent MGMT mRNA relative to the vehicle-treated tumors.

Fig. 7

Effects of temozolomide (TMZ) treatment schedule on survival. Mice with established orthotopic xenografts for glioblastoma multiforme (GBM) tumor lines were randomized to treatment with vehicle, 120 mg/kg TMZ × 2 days, or 50 mg/kg TMZ × 10 days. Mice were euthanized once they became moribund. Arrows denote when treatment was initiated. Both TMZ treatments significantly prolonged median survival in all GBM tumor lines: GBM14, p < 0.0001; GBM 43, p < 0.03; GBM12, p < 0.0001. For GBM14 and GBM12, 50 mg/kg × 10 days was superior to 120 mg/kg × 2 days (GBM14, p < 0.0001; GBM12, p < 0.001).