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mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells.

Smalley S, Chalmers AJ, Morley SJ - Mol. Cancer (2014)

Bottom Line: MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro.We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis.Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK. s.smalley@bsms.ac.uk.

ABSTRACT

Background: Glioblastoma multiforme (GBM), the most common and most aggressive type of primary adult brain tumour, responds poorly to conventional treatment. Temozolomide (TMZ) chemotherapy remains the most commonly used treatment, despite a large proportion of tumours displaying TMZ resistance. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Tumours also often exhibit hyperactive PI3-kinase/mTOR signalling, which enables them to resynthesise proteins quickly. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity.

Methods: MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro.

Results: We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis. Western blots show that despite this, relative to loading control proteins, steady state levels of MGMT protein increased and MGMT mRNA was retained in heavy polysomes. Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels.

Conclusions: These in vitro data suggest that, counterintuitively, mTOR inhibition may not be a useful adjunct to TMZ therapy and that more investigation is needed before applying mTOR inhibitors in a clinical setting.

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TMZ combined with KU0063794 decreases MGMT protein levels compared to treatments with KU0063794 alone, but an increase in overall MGMT protein levels is still observed. T98G cells were incubated in the absence (lane 1) or presence of TMZ and KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A Proteins were visualised by Western blotting using the antiserum indicated. B. MGMT protein levels in (A) were quantified and expressed relative to the α tubulin loading control. Error bars are the SE (n = 3). Confidence limits were set: *p = <0.2, **p = <0.05 ***p = <0.005. C. Cells were incubated in the absence or presence of KU0063794, TMZ and KU0063794 or TMZ alone. Cells were incubated with [35S] methionine, as described in Materials and Methods. Incorporation of radioactive methionine into protein was determined as cpm/μg protein; results are presented as a% of methionine incorporated in to cells incubated in the absence of any treatments. Error bars are the S.D (n = 3).
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Figure 2: TMZ combined with KU0063794 decreases MGMT protein levels compared to treatments with KU0063794 alone, but an increase in overall MGMT protein levels is still observed. T98G cells were incubated in the absence (lane 1) or presence of TMZ and KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A Proteins were visualised by Western blotting using the antiserum indicated. B. MGMT protein levels in (A) were quantified and expressed relative to the α tubulin loading control. Error bars are the SE (n = 3). Confidence limits were set: *p = <0.2, **p = <0.05 ***p = <0.005. C. Cells were incubated in the absence or presence of KU0063794, TMZ and KU0063794 or TMZ alone. Cells were incubated with [35S] methionine, as described in Materials and Methods. Incorporation of radioactive methionine into protein was determined as cpm/μg protein; results are presented as a% of methionine incorporated in to cells incubated in the absence of any treatments. Error bars are the S.D (n = 3).

Mentions: To determine if combining mTOR inhibition with TMZ chemotherapy effectively reduced MGMT protein levels, we combined KU0063794 with TMZ and monitored 4E-BP1 phosphorylation and MGMT protein levels by SDS-PAGE and Western blotting, using alpha tubulin as a protein loading control (Figure 2A). 4E-BP1 is dephosphorylated in cells treated with both KU0063794 and TMZ, which is characteristic of mTORC1 inhibition. This is consistent with the finding that concomitant treatment of cells with both TMZ and KU0063794 resulted in the inhibition of p70S6K and increased phosphorylation of Akt Thr308 (Additional file 1: Figure S4), both of which indicate mTORC1 inhibition. Under these conditions, surprisingly, when protein loading is taken into consideration using alpha tubulin, levels of MGMT protein increased (Figure 2A). Quantification of these data from three separate experiments (using alpha tubulin as a loading control; error bars are SE) showed that although there was experimental variability between time-courses, there was a statistically significant change in protein levels at 48 and 72 hours (p = 0.12 and 0.13, respectively) (Figure 2B). These data suggest that increase in MGMT levels is most likely brought about by KU0063794 and overprinted the lack of MGMT protein level change seen in the TMZ treated cells alone (Figure 1). This was in spite of the fact that protein synthesis rates were inhibited to the same level with KU0063794 whether TMZ was present or not (Figure 2C). Confocal microscopy was used to determine the localisation of the MGMT protein within the cell (Additional file 1: Figure S5), but, MGMT was not differentially localised upon drug treatments, indicating that the change in MGMT protein levels was not a reflection of MGMT transport between cellular compartments.


mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells.

Smalley S, Chalmers AJ, Morley SJ - Mol. Cancer (2014)

TMZ combined with KU0063794 decreases MGMT protein levels compared to treatments with KU0063794 alone, but an increase in overall MGMT protein levels is still observed. T98G cells were incubated in the absence (lane 1) or presence of TMZ and KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A Proteins were visualised by Western blotting using the antiserum indicated. B. MGMT protein levels in (A) were quantified and expressed relative to the α tubulin loading control. Error bars are the SE (n = 3). Confidence limits were set: *p = <0.2, **p = <0.05 ***p = <0.005. C. Cells were incubated in the absence or presence of KU0063794, TMZ and KU0063794 or TMZ alone. Cells were incubated with [35S] methionine, as described in Materials and Methods. Incorporation of radioactive methionine into protein was determined as cpm/μg protein; results are presented as a% of methionine incorporated in to cells incubated in the absence of any treatments. Error bars are the S.D (n = 3).
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Figure 2: TMZ combined with KU0063794 decreases MGMT protein levels compared to treatments with KU0063794 alone, but an increase in overall MGMT protein levels is still observed. T98G cells were incubated in the absence (lane 1) or presence of TMZ and KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A Proteins were visualised by Western blotting using the antiserum indicated. B. MGMT protein levels in (A) were quantified and expressed relative to the α tubulin loading control. Error bars are the SE (n = 3). Confidence limits were set: *p = <0.2, **p = <0.05 ***p = <0.005. C. Cells were incubated in the absence or presence of KU0063794, TMZ and KU0063794 or TMZ alone. Cells were incubated with [35S] methionine, as described in Materials and Methods. Incorporation of radioactive methionine into protein was determined as cpm/μg protein; results are presented as a% of methionine incorporated in to cells incubated in the absence of any treatments. Error bars are the S.D (n = 3).
Mentions: To determine if combining mTOR inhibition with TMZ chemotherapy effectively reduced MGMT protein levels, we combined KU0063794 with TMZ and monitored 4E-BP1 phosphorylation and MGMT protein levels by SDS-PAGE and Western blotting, using alpha tubulin as a protein loading control (Figure 2A). 4E-BP1 is dephosphorylated in cells treated with both KU0063794 and TMZ, which is characteristic of mTORC1 inhibition. This is consistent with the finding that concomitant treatment of cells with both TMZ and KU0063794 resulted in the inhibition of p70S6K and increased phosphorylation of Akt Thr308 (Additional file 1: Figure S4), both of which indicate mTORC1 inhibition. Under these conditions, surprisingly, when protein loading is taken into consideration using alpha tubulin, levels of MGMT protein increased (Figure 2A). Quantification of these data from three separate experiments (using alpha tubulin as a loading control; error bars are SE) showed that although there was experimental variability between time-courses, there was a statistically significant change in protein levels at 48 and 72 hours (p = 0.12 and 0.13, respectively) (Figure 2B). These data suggest that increase in MGMT levels is most likely brought about by KU0063794 and overprinted the lack of MGMT protein level change seen in the TMZ treated cells alone (Figure 1). This was in spite of the fact that protein synthesis rates were inhibited to the same level with KU0063794 whether TMZ was present or not (Figure 2C). Confocal microscopy was used to determine the localisation of the MGMT protein within the cell (Additional file 1: Figure S5), but, MGMT was not differentially localised upon drug treatments, indicating that the change in MGMT protein levels was not a reflection of MGMT transport between cellular compartments.

Bottom Line: MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro.We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis.Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK. s.smalley@bsms.ac.uk.

ABSTRACT

Background: Glioblastoma multiforme (GBM), the most common and most aggressive type of primary adult brain tumour, responds poorly to conventional treatment. Temozolomide (TMZ) chemotherapy remains the most commonly used treatment, despite a large proportion of tumours displaying TMZ resistance. 60% of GBM tumours have unmethylated MGMT promoter regions, resulting in an overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is responsible for tumour resistance to TMZ chemotherapy. Tumours also often exhibit hyperactive PI3-kinase/mTOR signalling, which enables them to resynthesise proteins quickly. Since MGMT is a suicide protein that is degraded upon binding to and repairing TMZ-induced O6-methylguanine adducts, it has been hypothesized that inhibition of translation via the mTOR signalling pathway could generate a tumour-specific reduction in MGMT protein and increase TMZ sensitivity.

Methods: MGMT was monitored at the post-transcriptional, translational and protein levels, to determine what effect mTOR inhibition was having on MGMT protein expression in vitro.

Results: We show that inhibiting mTOR signalling is indeed associated with acute inhibition of protein synthesis. Western blots show that despite this, relative to loading control proteins, steady state levels of MGMT protein increased and MGMT mRNA was retained in heavy polysomes. Whilst TMZ treatment resulted in maintained MGMT protein levels, concomitant treatment of T98G cells with TMZ and KU0063794 resulted in increased MGMT protein levels without changes in total mRNA levels.

Conclusions: These in vitro data suggest that, counterintuitively, mTOR inhibition may not be a useful adjunct to TMZ therapy and that more investigation is needed before applying mTOR inhibitors in a clinical setting.

Show MeSH
Related in: MedlinePlus