Phytohormone metabolism in human cells: Cytokinins are taken up and interconverted in HeLa cell culture

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, N⁶-isopentenyladenosine (i⁶A). 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/10⁶ 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, N⁶-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, N⁶-benzyladenine (BA). These findings support the hypothesis that HeLa cells have the enzymatic pathways required for the metabolism of both endogenous and exogenous CKs.

Keywords: N6‐benzyladenosine; N6‐isopentenyladenine; mammalian cells; mass spectrometry.

Figure 1

A, Observed benzyladenine metabolism demonstrating the structural derivatives (nucleotide, N6‐benzyladenine‐9‐riboside‐5′ (either mono‐, di‐, or tri‐) phosphate; riboside (BARP), benzyladenine riboside (BAR); free base (FB), and benzyladenine (BA) detected in the treated HeLa culture supernatants and the proposed enzymes responsible for the interconversions between cytokinin (CK) structural forms. This information was inferred from this study and other in vitro mammalian cell culture investigations.30, 31, 50, 51, 55 Numbers represent inferred enzymes as follows: 1. adenosine kinase (EC 2.7.1.20); 2. purine nucleotide phosphorylase (EC 2.4.2.1); 3. adenine phophoribosyltransferase (EC 2.4.2.1). The remaining arrows demonstrate alternative conversions that can occur via specific enzymes in other CK producing organisms, such as Arabidopsis.5, 6, 8, 9, 19 Bolded text refers to the CK forms that were dominant and the proposed enzyme activity that we observed in this study. B, Conversion of BAR (■) into the FB fraction, BA (●) by HeLa cells treated with 10⁻⁶ mol/L BAR throughout a 72‐h incubation period indicated by a solid line. A 10⁻⁶ mol/L BAR medium (no cell) control was incubated throughout this same period showing no breakdown of the compound over time, indicated by dashed lines and triangles (BAR, ▲; BA, ▼). The hormone concentrations were determined using high‐performance liquid chromatography‐positive electrospray ionization tandem mass spectrometry Presented values are means ± SE (n = 3). A separate ANOVA was performed for each CK form for the treated medium control and the treated cell culture supernatants with time as the dependent variable, followed by Tukey's post hoc analysis. Significant differences in CK concentration were found only in the treated cell cultures, where the same letters indicate values that are not significantly different (P < 0.05). Values are followed by letters only when significant differences were detected