Abstract
Macrophage-like RAW 264 and H35 hepatoma cells grown under serum-free conditions exported putrescine and an unidentified diamine into the culture medium. Unlike putrescine, the unknown compound could be detected only extracellularly. Analyses of dansylated polyamine standards and mass spectroscopy confirmed that the unknown compound was cadaverine (1,5-diaminopentane). The cells were free of mycoplasma as evidenced by a negative result using a probe specific for prokaryotic rRNA. After prophylactic treatments with two different mycoplasmacidal agents, the cells continued to export cadaverine. Attempts to "infect" a noncadaverine-exporting cell line with culture medium and cell-free lysates proved unsuccessful, establishing that cadaverine was in fact a bona fide product of these mammalian cells. Cadaverine export by RAW 264 and H35 cells was stimulated by lipopolysaccharide and insulin, respectively. However, administration of exogenous ornithine caused cadaverine export to decrease significantly with concomitant increases in putrescine export. alpha-Difluoromethylornithine, a selective inhibitor of ornithine decarboxylase, inhibited both cadaverine and putrescine export. When cells were labeled with [3H]lysine, the great majority of the radioactivity recovered in exported polyamines was found in cadaverine. The cumulative data suggested that cadaverine formation may be caused by the action of intracellular ornithine decarboxylase upon lysine to produce cadaverine, which is then effluxed from the cell with a high degree of efficiency.
Highlights
Resistant cultured mammalian cell lines in which ornithine decarboxylase gene amplification led to markedly high constitutive levels of ornithine decarboxylase activity were shown to cell-free lysates proved unsuccessful, establishing that accumulatecadaverine [8,9,10]
The cumu- shown to occur in a variety of cultured cells following selective lative data suggested that cadaverine formation may be pressure by certain drugs or growth limitation caused by concaused by the action of intracellular ornithine decarboxylase upon lysine to produce cadaverine, which is effluxed from the cell with a high degree of efficiency
We report herethat the macrophage-like RAW 264 and rat hepatoma H35 cells selectively exported relatively large amounts of cadaverine in addition to putrescine into the culture medium
Summary
Chemicals and Supplies-Cell culture media were from Life Technologies, Inc. Dansyl chlorideand polyamine standards were obtained from Fluka. To begin a n experiment, the media were again plates were analyzed for polyamines following dansylation via HPLC, replaced with fresh serum-freeDMEM containing the compounds indi- and the eluantfsrom the reverse phase colum(n0.75-ml fractions) were cated in the figures (LPS, insulin, ornithinDe,FMO). Blank plates (no mental time courseof 24 h, in the H35cell cultures, cell numbers (and cells) with culture media were treated with [3Hlornithci3nHelloy-r protein) increased by 20% in the untreated and ornithine-treatedcul- sine and analyzed to ensure that they did not contain any tures andby 50%in cultures receiving insulinA.ll cells were grownat radioactive polyamines(from nonenzymatic decarboxylation reactions). Use of Mass Spectroscopy forIdentification of the Unknown as using 0.05% trypsin with 0.02% EDTA Both cell lines were testedfor Cadaverine-Didansylputrescine andtheunknowndansylatedpeak mycoplasma with a commercial kit capable of detecting as few as lo were isolated via reverse phase HPLC and concentrated to drynessby colony-forming unitdml ofMycoplasma hominis (Gen ProbSea, n Diego) vacuum centrifugation.
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