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GAPDH is not regulated in human glioblastoma under hypoxic conditions.

Said HM, Hagemann C, Stojic J, Schoemig B, Vince GH, Flentje M, Roosen K, Vordermark D - BMC Mol. Biol. (2007)

Bottom Line: Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression.An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression.HIF-1alpha protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Würzburg, Dept. of Radiation Oncology, Germany. Said_H@klinik.uni-wuerzburg.de

ABSTRACT

Background: Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like beta-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and beta-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1alpha protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results: We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion: GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.

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Nuclear protein expression of HIF-1α in human U373, U251 and GaMG glioblastoma cells after in vitro application of different hypoxic conditions. For experimental settings refer to Fig. 2. (A) Western-blots. β-tubulin served as loading control. (B) HIF-1α protein expression strength shown as bar graphs after densitometric evaluation and normalization to the corresponding β-tubulin expression. HIF-1α was strongly expressed after 1 h at 0.1% O2, and still increased for up to 24 h hypoxia. It showed stable reduced expression after up to 48 h reoxygenation. Similar data were obtained in three independent experiments.
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Figure 9: Nuclear protein expression of HIF-1α in human U373, U251 and GaMG glioblastoma cells after in vitro application of different hypoxic conditions. For experimental settings refer to Fig. 2. (A) Western-blots. β-tubulin served as loading control. (B) HIF-1α protein expression strength shown as bar graphs after densitometric evaluation and normalization to the corresponding β-tubulin expression. HIF-1α was strongly expressed after 1 h at 0.1% O2, and still increased for up to 24 h hypoxia. It showed stable reduced expression after up to 48 h reoxygenation. Similar data were obtained in three independent experiments.

Mentions: In a representative in-vitro example in GaMG cells cultured under different oxygenation conditions, HIF-1α mRNA expression was not influenced by hypoxic conditions (Fig. 8). In contrast, HIF-1α nuclear protein clearly responded with upregulation under hypoxic and downregulation under reoxygenation or normoxic conditions in U373, U251 and GaMG (Fig. 9). HIF-1α was strongly expressed after 1 h at 0.1% O2, still increased after 24 h hypoxia and showed stable reduced expression upon reoxygenation up to 48 h (Fig. 9).


GAPDH is not regulated in human glioblastoma under hypoxic conditions.

Said HM, Hagemann C, Stojic J, Schoemig B, Vince GH, Flentje M, Roosen K, Vordermark D - BMC Mol. Biol. (2007)

Nuclear protein expression of HIF-1α in human U373, U251 and GaMG glioblastoma cells after in vitro application of different hypoxic conditions. For experimental settings refer to Fig. 2. (A) Western-blots. β-tubulin served as loading control. (B) HIF-1α protein expression strength shown as bar graphs after densitometric evaluation and normalization to the corresponding β-tubulin expression. HIF-1α was strongly expressed after 1 h at 0.1% O2, and still increased for up to 24 h hypoxia. It showed stable reduced expression after up to 48 h reoxygenation. Similar data were obtained in three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919389&req=5

Figure 9: Nuclear protein expression of HIF-1α in human U373, U251 and GaMG glioblastoma cells after in vitro application of different hypoxic conditions. For experimental settings refer to Fig. 2. (A) Western-blots. β-tubulin served as loading control. (B) HIF-1α protein expression strength shown as bar graphs after densitometric evaluation and normalization to the corresponding β-tubulin expression. HIF-1α was strongly expressed after 1 h at 0.1% O2, and still increased for up to 24 h hypoxia. It showed stable reduced expression after up to 48 h reoxygenation. Similar data were obtained in three independent experiments.
Mentions: In a representative in-vitro example in GaMG cells cultured under different oxygenation conditions, HIF-1α mRNA expression was not influenced by hypoxic conditions (Fig. 8). In contrast, HIF-1α nuclear protein clearly responded with upregulation under hypoxic and downregulation under reoxygenation or normoxic conditions in U373, U251 and GaMG (Fig. 9). HIF-1α was strongly expressed after 1 h at 0.1% O2, still increased after 24 h hypoxia and showed stable reduced expression upon reoxygenation up to 48 h (Fig. 9).

Bottom Line: Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression.An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression.HIF-1alpha protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Würzburg, Dept. of Radiation Oncology, Germany. Said_H@klinik.uni-wuerzburg.de

ABSTRACT

Background: Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions in vitro in comparison to other housekeeping genes like beta-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and beta-actin expression was comparatively examined in vivo in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1alpha protein regulation under hypoxia was also determined on mRNA level in vitro in GaMG and on protein level in U251, U373 and GaMG cells.

Results: We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied in vitro. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation in vivo.

Conclusion: GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.

Show MeSH
Related in: MedlinePlus