A chromatographic assay for heme oxygenase activity in cultured human cells: application to artificial heme oxygenase overexpression.
Heme oxygenase (HO) activity oxidizes heme, releasing carbon monoxide; heme iron; and biliverdin, which is converted to bilirubin by biliverdin reductase. Inducible HO-I expression is a marker of oxidative stress in mammalian cells, while noninducible HO-II contributes to basal HO activity. HO-I and HO-II activities are implicated in cellular antioxidant defense mechanisms. We describe a microassay for HO activity in cultured human cells, using high-performance liquid chromatography of biliverdin and bilirubin. The assay is sufficiently sensitive to quantify basal and inducible HO activity in various human cell types. We have established human cell lines overexpressing heme oxygenase-II activity in microsomes using a metallothionein promoter-regulated expression system. Stable transformants treated with ZnCl2 express up to ninefold induction of HO activity. We have constructed human cell lines overexpressing HO-II protein and activity (5-15-fold) in the absence of tetracycline, using the HtTA-1 cell line transfected with tetracycline-regulated expression vectors (Gossen et al., Proc. Natl. Acad. Sci. USA 89, 1992). Functional HO-II overexpressing clones will be useful in investigating anti- or pro-oxidant effects of HO activity during cellular oxidative stress.
Ryter
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Swiss Institute for Experimental Cancer Research, (ISREC), Epalinges.
Kvam
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Richman
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Hartmann
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Tyrrell
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RM
D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004318:Doxycycline / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007668:Kidney; D008668:Metallothionein / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011758:Pyrroles / Q000032:analysis; D012867:Skin; D013752:Tetracycline / Q000494:pharmacology; D045725:Tetrapyrroles; D014162:Transfection
D011758:Pyrroles; D045725:Tetrapyrroles; D008668:Metallothionein; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2; D013752:Tetracycline; D004318:Doxycycline