Open AccessPhytohormone metabolism in human cells: Cytokinins are taken up and interconverted in HeLa cell culture
Author(s) -
Aoki Megan M.,
Seegobin Mark,
Kisiala Anna,
Noble Adam,
Brunetti Craig,
Emery R. J. Neil
Publication year - 2019
Publication title -
faseb bioadvances
Language(s) - English
Resource type - Journals
ISSN - 2573-9832
DOI - 10.1096/fba.2018-00032
Subject(s) - hela , biochemistry , cell culture , metabolism , biology , endogeny , cell , enzyme , cytosol , electrospray ionization , biosynthesis , microbiology and biotechnology , chemistry , mass spectrometry , genetics , chromatography
Abstract Cytokinins (CKs) encompass a group of phytohormones, known to orchestrate many critical processes in plant development. Excluding Archaea, CKs are pervasive among all kingdoms, but much less is reported about their metabolism beyond plants. Recent evidence from mammalian tissues indicates the presence of six additional CK forms beyond the previously identified, single mammalian CK, N 6 ‐isopentenyladenosine (i 6 A). There is limited understanding of CK biosynthesis pathways in mammalian systems; therefore, human cervical cancer (HeLa) cells were used to further characterize CK processing by tracking the interconversion of CKs into their various structural derivatives in mammalian cells in a time‐course study. Through high‐performance liquid chromatography‐positive electrospray ionization‐tandem mass spectrometry (HPLC‐(+ESI)‐MS/MS), we document changes in the functional profiles of endogenous CKs in a human cell line following metabolism by HeLa cell cultures. The nucleotide CK fraction (iPRP) was found exclusively within the cell pellet (0.34 pmol/10 6 cells), and the active free base (FB) form (iP) and riboside fraction (iPR) were found in greater abundance extracellularly (1.67 and 0.10 nmol/L respectively). For further confirmation, we demonstrate that HeLa cells metabolize an exogenously supplied CK, N 6 ‐benzyladenosine (BAR). In the HeLa culture supernatant, a 12‐fold decrease in BAR concentration was observed within the first 24 hours of incubation accompanied by a fivefold increase in the FB form, N 6 ‐benzyladenine (BA). These findings support the hypothesis that HeLa cells have the enzymatic pathways required for the metabolism of both endogenous and exogenous CKs.