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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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In-vivo protein expression of human housekeeping genes GAPDH, β-actin and γ-tubulin in biopsies of normal brain (NB), low-grade astrocytoma and glioblastoma. (A) Western-blot analysis of tissue lysates. GAPDH, β-actin and γ-tubulin are homogenously expressed by all analysed samples. (B) Expression intensities of the bands in the Western blot were densitometrically evaluated. The bar graphs show GAPDH expression after normalization to the corresponding expression of β-actin. No GAPDH upregulation in the more hypoxic glioblastoma samples in comparison to low-grade astrocytoma was detectable. (C) Densitometric evaluation of γ-tubulin expression in the human brain tumor tissues. Band intensities are shown as bar graphs. No regulatory effect of hypoxic conditions in vivo on housekeeping gene expression could be detected.
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Figure 5: In-vivo protein expression of human housekeeping genes GAPDH, β-actin and γ-tubulin in biopsies of normal brain (NB), low-grade astrocytoma and glioblastoma. (A) Western-blot analysis of tissue lysates. GAPDH, β-actin and γ-tubulin are homogenously expressed by all analysed samples. (B) Expression intensities of the bands in the Western blot were densitometrically evaluated. The bar graphs show GAPDH expression after normalization to the corresponding expression of β-actin. No GAPDH upregulation in the more hypoxic glioblastoma samples in comparison to low-grade astrocytoma was detectable. (C) Densitometric evaluation of γ-tubulin expression in the human brain tumor tissues. Band intensities are shown as bar graphs. No regulatory effect of hypoxic conditions in vivo on housekeeping gene expression could be detected.

Mentions: To exclude translational regulation of GAPDH protein expression by hypoxic conditions, Western-blot analysis was performed using lysates from the same tumor samples described above. Again, GAPDH, β-actin and as an additional housekeeping gene γ-tubulin was detected in all samples analyzed (Fig. 5A). Expression of all three proteins was very homogenously distributed among the two tumor entities, low-grade astrocytoma with known modest tumor hypoxia and glioblastoma with known severe hypoxia (densitometric analysis, Fig. 5B and 5C).


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)

In-vivo protein expression of human housekeeping genes GAPDH, β-actin and γ-tubulin in biopsies of normal brain (NB), low-grade astrocytoma and glioblastoma. (A) Western-blot analysis of tissue lysates. GAPDH, β-actin and γ-tubulin are homogenously expressed by all analysed samples. (B) Expression intensities of the bands in the Western blot were densitometrically evaluated. The bar graphs show GAPDH expression after normalization to the corresponding expression of β-actin. No GAPDH upregulation in the more hypoxic glioblastoma samples in comparison to low-grade astrocytoma was detectable. (C) Densitometric evaluation of γ-tubulin expression in the human brain tumor tissues. Band intensities are shown as bar graphs. No regulatory effect of hypoxic conditions in vivo on housekeeping gene expression could be detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: In-vivo protein expression of human housekeeping genes GAPDH, β-actin and γ-tubulin in biopsies of normal brain (NB), low-grade astrocytoma and glioblastoma. (A) Western-blot analysis of tissue lysates. GAPDH, β-actin and γ-tubulin are homogenously expressed by all analysed samples. (B) Expression intensities of the bands in the Western blot were densitometrically evaluated. The bar graphs show GAPDH expression after normalization to the corresponding expression of β-actin. No GAPDH upregulation in the more hypoxic glioblastoma samples in comparison to low-grade astrocytoma was detectable. (C) Densitometric evaluation of γ-tubulin expression in the human brain tumor tissues. Band intensities are shown as bar graphs. No regulatory effect of hypoxic conditions in vivo on housekeeping gene expression could be detected.
Mentions: To exclude translational regulation of GAPDH protein expression by hypoxic conditions, Western-blot analysis was performed using lysates from the same tumor samples described above. Again, GAPDH, β-actin and as an additional housekeeping gene γ-tubulin was detected in all samples analyzed (Fig. 5A). Expression of all three proteins was very homogenously distributed among the two tumor entities, low-grade astrocytoma with known modest tumor hypoxia and glioblastoma with known severe hypoxia (densitometric analysis, Fig. 5B and 5C).

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