PubMed · 14233407
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W SAWICKI. 1964. [ELECTRON MICROSCOPE AUTORADIOGRAPHY].. https://pubmed.ncbi.nlm.nih.gov/14233407/
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Hypoxia stimulates a number of pathways critical to cancer cell survival, including the activation of vascular endothelial growth factor (VEGF) transcription. In normal fibroblasts, hypoxia-induced activation of the protein tyrosine kinase, Src, is required for VEGF expression. We show here in both pancreatic and prostate carcinoma cell lines cobalt chloride (used to mimic hypoxia) -induced VEGF expression requires Src activation and leads to increased steady-state levels of HIF-1alpha and increased phosphorylation of signal and transducer of transcription 3 (STAT3). STAT3 and hypoxia-inducible factor (HIF)-1alpha bind simultaneously to the VEGF promoter, where they form a molecular complex with the transcription coactivators CBP/p300 and Ref-1/APE. Expression of activated Src from an inducible promoter is sufficient to increase VEGF expression and form these STAT3/HIF-1alpha-containing promoter complexes. Inhibition of DNA binding by expression of either STAT3 or HIF-1alpha dominant negative mutants significantly reduces VEGF expression. These data suggest that the binding of both STAT3 and HIF-1alpha to the VEGF promoter is required for maximum transcription of VEGF mRNA following hypoxia.
Enzymatic synthesis of alpha-(32)P and alpha-(33)P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either gamma-(32)P adenosine triphosphate (gamma-ATP) or gamma-(32)P guanosine triphosphate (gamma-GTP). Subsequent removal of these ribonucleotides is essential for the preparation of chemically pure deoxyribonucleotides. Agarose-phenyl boronate columns, which bind specifically to cis-diol moieties, have been used for the removal of ribonucleotide contaminants. However, this involves column losses and additional radiation exposure. In the present work we describe a chemical method for the improvement of the chemical purity, based on the preferential oxidation of ribose sugars by periodate. The cis-diol moiety of ribose is specifically oxidised to the dialdehyde. The excess periodate ions were destroyed using ethylene glycol. The phosphate group was then cleaved by beta-elimination using alkali. The product was purified using anion exchange chromatography. The efficiency of the process was validated using tracer gamma-(32)P ATP and alpha-(32)P dATP. Samples at various steps were analysed by TLC, autoradiography and HPLC. During the process ATP is oxidised whereas 2'-deoxyadenosine triphosphate (dATP) remains intact. The alpha-(32)P dATP synthesized by this process was assayed for its incorporation in lambda-DNA by the random priming method and was found to be effectively incorporated. The process developed is an efficient and convenient method for the preparation of chemically pure deoxyribonucleotides.
2-Deoxyribonolactone (L) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents. It exhibits unique biological effects with respect to its proclivity to form DNA-protein cross-links and promutagenic base pairs. Recent evidence suggests that the levels of this lesion caused by oxidative stress are underestimated. We have developed a simple, selective method for detecting subpicomole amounts of L in DNA. The method takes advantage of the selective reaction of the butenolide (2) derived from beta-elimination from L with a biotinylated derivative of cysteine. This method will be useful for analyzing the levels of this oxidized abasic site in DNA.