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Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1.

Ameri K, Burke B, Lewis CE, Harris AL - Br. J. Cancer (2002)

Bottom Line: Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements.Mutational analysis demonstrated that the base immediately 5' to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>CACGTG.A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5' flanking bases.

View Article: PubMed Central - PubMed

Affiliation: Tumour Targeting Group, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.

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(a) Electrophoretic mobility supershift assay showing binding of HIF-1α and HIF-1β in MCF-7 extracts to the trimerised SARE probe following exposure of cells for 16 hours to various O2 tensions. Bands appear in 1, 0.5 and 0% O2. These inducible complexes were supershifted in the presence of a monoclonal antibody to HIF-1α or a polyclonal antibody to HIF-1β (C=binding of constitutive factors). (b) Electrophoretic mobility supershift assay showing the inducible HIF-1 band binding to the wt SARE or M1 trimers (the HIF-binding sequence of the latter being 5′-CACGTG-3′) in nuclear extracts of MCF-7 cells exposed for 16 h to various O2 tensions. Bands appear in 1, 0.5 and 0% O2 with both probes. These are super-shifted by addition of a monoclonal antibody to HIF-1α. More extensive binding of constitutive factors (‘C’) can be seen with M1 than with the wt SARE probe.
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fig6: (a) Electrophoretic mobility supershift assay showing binding of HIF-1α and HIF-1β in MCF-7 extracts to the trimerised SARE probe following exposure of cells for 16 hours to various O2 tensions. Bands appear in 1, 0.5 and 0% O2. These inducible complexes were supershifted in the presence of a monoclonal antibody to HIF-1α or a polyclonal antibody to HIF-1β (C=binding of constitutive factors). (b) Electrophoretic mobility supershift assay showing the inducible HIF-1 band binding to the wt SARE or M1 trimers (the HIF-binding sequence of the latter being 5′-CACGTG-3′) in nuclear extracts of MCF-7 cells exposed for 16 h to various O2 tensions. Bands appear in 1, 0.5 and 0% O2 with both probes. These are super-shifted by addition of a monoclonal antibody to HIF-1α. More extensive binding of constitutive factors (‘C’) can be seen with M1 than with the wt SARE probe.

Mentions: Immunoblot analysis of HIF-1α, HIF-1β, EPAS 1, and ATF-1 in MCF-7 cells following exposure to 0, 0.5, 1, and 21% O2 (normoxia) for 16 h. Blots were stripped and re-probed for β-actin as a loading control; no differences were observed (data not shown).


Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1.

Ameri K, Burke B, Lewis CE, Harris AL - Br. J. Cancer (2002)

(a) Electrophoretic mobility supershift assay showing binding of HIF-1α and HIF-1β in MCF-7 extracts to the trimerised SARE probe following exposure of cells for 16 hours to various O2 tensions. Bands appear in 1, 0.5 and 0% O2. These inducible complexes were supershifted in the presence of a monoclonal antibody to HIF-1α or a polyclonal antibody to HIF-1β (C=binding of constitutive factors). (b) Electrophoretic mobility supershift assay showing the inducible HIF-1 band binding to the wt SARE or M1 trimers (the HIF-binding sequence of the latter being 5′-CACGTG-3′) in nuclear extracts of MCF-7 cells exposed for 16 h to various O2 tensions. Bands appear in 1, 0.5 and 0% O2 with both probes. These are super-shifted by addition of a monoclonal antibody to HIF-1α. More extensive binding of constitutive factors (‘C’) can be seen with M1 than with the wt SARE probe.
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Related In: Results  -  Collection

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fig6: (a) Electrophoretic mobility supershift assay showing binding of HIF-1α and HIF-1β in MCF-7 extracts to the trimerised SARE probe following exposure of cells for 16 hours to various O2 tensions. Bands appear in 1, 0.5 and 0% O2. These inducible complexes were supershifted in the presence of a monoclonal antibody to HIF-1α or a polyclonal antibody to HIF-1β (C=binding of constitutive factors). (b) Electrophoretic mobility supershift assay showing the inducible HIF-1 band binding to the wt SARE or M1 trimers (the HIF-binding sequence of the latter being 5′-CACGTG-3′) in nuclear extracts of MCF-7 cells exposed for 16 h to various O2 tensions. Bands appear in 1, 0.5 and 0% O2 with both probes. These are super-shifted by addition of a monoclonal antibody to HIF-1α. More extensive binding of constitutive factors (‘C’) can be seen with M1 than with the wt SARE probe.
Mentions: Immunoblot analysis of HIF-1α, HIF-1β, EPAS 1, and ATF-1 in MCF-7 cells following exposure to 0, 0.5, 1, and 21% O2 (normoxia) for 16 h. Blots were stripped and re-probed for β-actin as a loading control; no differences were observed (data not shown).

Bottom Line: Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements.Mutational analysis demonstrated that the base immediately 5' to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>CACGTG.A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5' flanking bases.

View Article: PubMed Central - PubMed

Affiliation: Tumour Targeting Group, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.

Show MeSH
Related in: MedlinePlus