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The regulation of mitochondrial DNA copy number in glioblastoma cells.

Dickinson A, Yeung KY, Donoghue J, Baker MJ, Kelly RD, McKenzie M, Johns TG, St John JC - Cell Death Differ. (2013)

Bottom Line: As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions.We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity.However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH).

View Article: PubMed Central - PubMed

Affiliation: 1] The Mitochondrial Genetics Group, Centre for Genetic Diseases, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Molecular Basis of Metabolic Disease, Division of Metabolic and Vascular Health, Warwick Medical School, The University of Warwick, Clifford Bridge Road, Coventry, CV2 2DX, UK.

ABSTRACT
As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.

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HSR-GBM1 tumor formation assay and assessment of mtDNA copy number. Tumor growth curve analysis of mtDNA depleted (mtDNA50, mtDNA20, mtDNA3 and mtDNA0.2) and non-depleted (mtDNA100) HSR-GBM1 cells (a). Kaplan–Meier survival plot for non-depleted and depleted HSR-GBM1 cells (b). Immunohistochemical labeling of proliferating cell nuclear antigen. in depleted (c) and non-depleted (d–f) HSR-GBM1 tumors. Negative control (g) and positive control (h). Quantification of proliferating cell nuclear antigen positive cells in depleted and non-depleted HSR-GBM1 tumors (i). Mean mtDNA copy number analysis of depleted and non-depleted HSR-GBM1 tumors (j). Columns represent mean values±S.E.M. *P<0.05 and **P<0.01. Scale bars=25 μm
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fig6: HSR-GBM1 tumor formation assay and assessment of mtDNA copy number. Tumor growth curve analysis of mtDNA depleted (mtDNA50, mtDNA20, mtDNA3 and mtDNA0.2) and non-depleted (mtDNA100) HSR-GBM1 cells (a). Kaplan–Meier survival plot for non-depleted and depleted HSR-GBM1 cells (b). Immunohistochemical labeling of proliferating cell nuclear antigen. in depleted (c) and non-depleted (d–f) HSR-GBM1 tumors. Negative control (g) and positive control (h). Quantification of proliferating cell nuclear antigen positive cells in depleted and non-depleted HSR-GBM1 tumors (i). Mean mtDNA copy number analysis of depleted and non-depleted HSR-GBM1 tumors (j). Columns represent mean values±S.E.M. *P<0.05 and **P<0.01. Scale bars=25 μm

Mentions: We then assessed the tumorigenic potential of cells depleted to varying levels. We depleted HSR-GBM1 cells to ∼50% (mtDNA50), 20% (mtDNA20), 3% (mtDNA3) and 0.2% (mtDNA0.2) of their original mtDNA content. These and non-depleted (mtDNA100) cells were transferred into Bagg albino (Balb/c) nude mice. During the first 40 days post-inoculation, tumors from the mtDNA100 and mtDNA50 cells developed at a faster rate than mtDNA20, mtDNA3 and mtDNA0.2 cells (Figure 6a) although this was not statistically significant. After 40 days, tumors from mtDNA50 cells grew at an accelerated rate compared with mtDNA100 tumors and this trend was maintained. Tumors from mtDNA20 cells developed slowly until day 55 and, by day 65, developed faster than mtDNA100 tumors. Tumors derived from mtDNA3 and mtDNA0.2 cells showed delayed development compared with mtDNA100 tumors (P<0.01). The frequency of tumor formation was inversely related to mtDNA depletion. MtDNA100 cells generated 11/12 tumors of which 1 regressed; 10/12 were derived from mtDNA50 cells and 2 regressed; 6/12 were generated from mtDNA20 cells, 6/12 were derived from mtDNA3 cells and 3 regressed and 2/12 were generated from mtDNA0.2 cells. Tumor formation to 500 mm3 was least in the mtDNA0.2 and greatest in the mtDNA100 cohorts (Figure 6b). These data demonstrate that increased mtDNA depletion reduces the frequency of tumor formation.


The regulation of mitochondrial DNA copy number in glioblastoma cells.

Dickinson A, Yeung KY, Donoghue J, Baker MJ, Kelly RD, McKenzie M, Johns TG, St John JC - Cell Death Differ. (2013)

HSR-GBM1 tumor formation assay and assessment of mtDNA copy number. Tumor growth curve analysis of mtDNA depleted (mtDNA50, mtDNA20, mtDNA3 and mtDNA0.2) and non-depleted (mtDNA100) HSR-GBM1 cells (a). Kaplan–Meier survival plot for non-depleted and depleted HSR-GBM1 cells (b). Immunohistochemical labeling of proliferating cell nuclear antigen. in depleted (c) and non-depleted (d–f) HSR-GBM1 tumors. Negative control (g) and positive control (h). Quantification of proliferating cell nuclear antigen positive cells in depleted and non-depleted HSR-GBM1 tumors (i). Mean mtDNA copy number analysis of depleted and non-depleted HSR-GBM1 tumors (j). Columns represent mean values±S.E.M. *P<0.05 and **P<0.01. Scale bars=25 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824586&req=5

fig6: HSR-GBM1 tumor formation assay and assessment of mtDNA copy number. Tumor growth curve analysis of mtDNA depleted (mtDNA50, mtDNA20, mtDNA3 and mtDNA0.2) and non-depleted (mtDNA100) HSR-GBM1 cells (a). Kaplan–Meier survival plot for non-depleted and depleted HSR-GBM1 cells (b). Immunohistochemical labeling of proliferating cell nuclear antigen. in depleted (c) and non-depleted (d–f) HSR-GBM1 tumors. Negative control (g) and positive control (h). Quantification of proliferating cell nuclear antigen positive cells in depleted and non-depleted HSR-GBM1 tumors (i). Mean mtDNA copy number analysis of depleted and non-depleted HSR-GBM1 tumors (j). Columns represent mean values±S.E.M. *P<0.05 and **P<0.01. Scale bars=25 μm
Mentions: We then assessed the tumorigenic potential of cells depleted to varying levels. We depleted HSR-GBM1 cells to ∼50% (mtDNA50), 20% (mtDNA20), 3% (mtDNA3) and 0.2% (mtDNA0.2) of their original mtDNA content. These and non-depleted (mtDNA100) cells were transferred into Bagg albino (Balb/c) nude mice. During the first 40 days post-inoculation, tumors from the mtDNA100 and mtDNA50 cells developed at a faster rate than mtDNA20, mtDNA3 and mtDNA0.2 cells (Figure 6a) although this was not statistically significant. After 40 days, tumors from mtDNA50 cells grew at an accelerated rate compared with mtDNA100 tumors and this trend was maintained. Tumors from mtDNA20 cells developed slowly until day 55 and, by day 65, developed faster than mtDNA100 tumors. Tumors derived from mtDNA3 and mtDNA0.2 cells showed delayed development compared with mtDNA100 tumors (P<0.01). The frequency of tumor formation was inversely related to mtDNA depletion. MtDNA100 cells generated 11/12 tumors of which 1 regressed; 10/12 were derived from mtDNA50 cells and 2 regressed; 6/12 were generated from mtDNA20 cells, 6/12 were derived from mtDNA3 cells and 3 regressed and 2/12 were generated from mtDNA0.2 cells. Tumor formation to 500 mm3 was least in the mtDNA0.2 and greatest in the mtDNA100 cohorts (Figure 6b). These data demonstrate that increased mtDNA depletion reduces the frequency of tumor formation.

Bottom Line: As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions.We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity.However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH).

View Article: PubMed Central - PubMed

Affiliation: 1] The Mitochondrial Genetics Group, Centre for Genetic Diseases, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Molecular Basis of Metabolic Disease, Division of Metabolic and Vascular Health, Warwick Medical School, The University of Warwick, Clifford Bridge Road, Coventry, CV2 2DX, UK.

ABSTRACT
As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.

Show MeSH
Related in: MedlinePlus