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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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Effect of different hypoxic conditions on GAPDH mRNA expression in GaMG cells. For experimental settings refer to Fig. 2. (A) Semiquantitative RT-PCR analysis (B) densitometric evaluation of GAPDH and β-actin mRNA expression.
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Figure 4: Effect of different hypoxic conditions on GAPDH mRNA expression in GaMG cells. For experimental settings refer to Fig. 2. (A) Semiquantitative RT-PCR analysis (B) densitometric evaluation of GAPDH and β-actin mRNA expression.

Mentions: To investigate the effect of controlled hypoxic conditions on GAPDH mRNA expression, we performed in-vitro cell culture assays with 5%, 1% and 0.1% O2 with and without reoxygenation. No regulatory effect of these different oxygenation conditions on GAPDH expression was detectable by semiquantitative RT-PCR in the human glioblastoma cell lines U251, U373 and GaMG (Fig. 2A, 3A, 4A). The densitometric evaluation confirmed these results (Fig. 2B, 3B, 4B). Together, these data suggest that the known formation of hypoxic regions within gliobastoma tumor tissue is not accompanied by an upregulation of GAPDH mRNA.


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)

Effect of different hypoxic conditions on GAPDH mRNA expression in GaMG cells. For experimental settings refer to Fig. 2. (A) Semiquantitative RT-PCR analysis (B) densitometric evaluation of GAPDH and β-actin mRNA expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of different hypoxic conditions on GAPDH mRNA expression in GaMG cells. For experimental settings refer to Fig. 2. (A) Semiquantitative RT-PCR analysis (B) densitometric evaluation of GAPDH and β-actin mRNA expression.
Mentions: To investigate the effect of controlled hypoxic conditions on GAPDH mRNA expression, we performed in-vitro cell culture assays with 5%, 1% and 0.1% O2 with and without reoxygenation. No regulatory effect of these different oxygenation conditions on GAPDH expression was detectable by semiquantitative RT-PCR in the human glioblastoma cell lines U251, U373 and GaMG (Fig. 2A, 3A, 4A). The densitometric evaluation confirmed these results (Fig. 2B, 3B, 4B). Together, these data suggest that the known formation of hypoxic regions within gliobastoma tumor tissue is not accompanied by an upregulation of GAPDH mRNA.

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