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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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KU0063794 inhibits protein translation via mTOR kinase inhibition in T98G cells but MGMT protein levels are increased. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A. Western blotting was carried out as described in Materials and Methods B. Aliquots of protein extract were subjected to m7GTP-Sepharose affinity chromatography as described in Materials and Methods. Western blotting was carried out. All lanes were resolved on the same gel. C. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). Proteins were visualised by Western blotting using the antiserum indicated. D. MGMT protein levels in (C) 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. E. Cells were incubated in the absence or presence of KU0063794 as indicated. Mitochondrial activity (a measure of cell viability) was assessed using an MTS assay as described in Materials and Methods and is expressed relative to untreated cells (set at 100%). Error bars are the S.D (n = 3).
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Figure 3: KU0063794 inhibits protein translation via mTOR kinase inhibition in T98G cells but MGMT protein levels are increased. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A. Western blotting was carried out as described in Materials and Methods B. Aliquots of protein extract were subjected to m7GTP-Sepharose affinity chromatography as described in Materials and Methods. Western blotting was carried out. All lanes were resolved on the same gel. C. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). Proteins were visualised by Western blotting using the antiserum indicated. D. MGMT protein levels in (C) 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. E. Cells were incubated in the absence or presence of KU0063794 as indicated. Mitochondrial activity (a measure of cell viability) was assessed using an MTS assay as described in Materials and Methods and is expressed relative to untreated cells (set at 100%). Error bars are the S.D (n = 3).

Mentions: Mutations in mTOR signalling pathways in GBM cells can result in hyperactive PI3-K/mTOR signalling, promoting cell survival, protein synthesis and cell proliferation [9,10,20]. KU0063794 directly inhibits this pathway and should therefore prevent the cells from proliferating and reduce their ability to synthesise new protein. To this end, cells were treated with KU0063794 for 72 hours and protein levels monitored by SDS-PAGE and immunoblotting. As expected, phosphorylation of 4E-BP1 on either Ser65 or Thr70, and rpS6 on Ser240/244 were abrogated within 12 hours of treatment of the cells with KU0063794 (Figure 3A lane 2 vs. lane 1), which indicates reduced translation initiation. At early time points, KU0063794 had little effect on the phosphorylation of ERK1/2, p38MAPK or eIF2α (Figure 3A, lanes 1–3), but phosphorylation was decreased at later time points (lanes 4 and 5). There was no change in the total level of these proteins during the incubation period (data not shown). In contrast, the phosphorylation of Akt on Thr308 (an indicator of Akt activity) increased with time (lanes 2–5 vs. lane 1) reflecting a previously reported feedback activation of PI3K and PDK1 following mTORC1 inhibition [13,14].


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

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

KU0063794 inhibits protein translation via mTOR kinase inhibition in T98G cells but MGMT protein levels are increased. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A. Western blotting was carried out as described in Materials and Methods B. Aliquots of protein extract were subjected to m7GTP-Sepharose affinity chromatography as described in Materials and Methods. Western blotting was carried out. All lanes were resolved on the same gel. C. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). Proteins were visualised by Western blotting using the antiserum indicated. D. MGMT protein levels in (C) 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. E. Cells were incubated in the absence or presence of KU0063794 as indicated. Mitochondrial activity (a measure of cell viability) was assessed using an MTS assay as described in Materials and Methods and is expressed relative to untreated cells (set at 100%). Error bars are the S.D (n = 3).
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Figure 3: KU0063794 inhibits protein translation via mTOR kinase inhibition in T98G cells but MGMT protein levels are increased. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). A. Western blotting was carried out as described in Materials and Methods B. Aliquots of protein extract were subjected to m7GTP-Sepharose affinity chromatography as described in Materials and Methods. Western blotting was carried out. All lanes were resolved on the same gel. C. T98G cells were incubated in the absence (lane 1) or presence of KU0063794 for 12 hours (lane 2), 24 hours (lanes 3), 48 hours (lane 4) or 72 hours (lane 5). Proteins were visualised by Western blotting using the antiserum indicated. D. MGMT protein levels in (C) 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. E. Cells were incubated in the absence or presence of KU0063794 as indicated. Mitochondrial activity (a measure of cell viability) was assessed using an MTS assay as described in Materials and Methods and is expressed relative to untreated cells (set at 100%). Error bars are the S.D (n = 3).
Mentions: Mutations in mTOR signalling pathways in GBM cells can result in hyperactive PI3-K/mTOR signalling, promoting cell survival, protein synthesis and cell proliferation [9,10,20]. KU0063794 directly inhibits this pathway and should therefore prevent the cells from proliferating and reduce their ability to synthesise new protein. To this end, cells were treated with KU0063794 for 72 hours and protein levels monitored by SDS-PAGE and immunoblotting. As expected, phosphorylation of 4E-BP1 on either Ser65 or Thr70, and rpS6 on Ser240/244 were abrogated within 12 hours of treatment of the cells with KU0063794 (Figure 3A lane 2 vs. lane 1), which indicates reduced translation initiation. At early time points, KU0063794 had little effect on the phosphorylation of ERK1/2, p38MAPK or eIF2α (Figure 3A, lanes 1–3), but phosphorylation was decreased at later time points (lanes 4 and 5). There was no change in the total level of these proteins during the incubation period (data not shown). In contrast, the phosphorylation of Akt on Thr308 (an indicator of Akt activity) increased with time (lanes 2–5 vs. lane 1) reflecting a previously reported feedback activation of PI3K and PDK1 following mTORC1 inhibition [13,14].

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