Synthesis of dinucleoside tetraphosphates in transfected cells by a firefly luciferase reporter gene

Abstract

The firefly luciferase gene is widely used as a reporter gene and its expression is generally considered to be non-toxic. In addition to its light-producing reaction, luciferase can synthesise dinucleoside polyphosphates, intracellular signalling molecules, in vitro. Here we show that COS-7 cells transfected with a luciferase expression vector accumulate up to 0.5 mM adenine-containing dinucleoside tetraphosphates (Ap4N) during the 24 h following luciferin addition. The optimal external concentration of luciferin was 0.4-0.6 mM. In agreement with its poor ability to synthesise adenine-containing dinucleoside triphosphates in vitro, the level of these compounds did not increase after transfection. Consequently, the results of experiments involving luciferase-mediated light production by live cells should now be viewed in the light of the possible effects of an increased intracellular Ap4N concentration on the properties of the system under investigation. This observation also points to a useful non-invasive procedure for the specific enhancement of intracellular Ap4N for studies directed at understanding the functions of these compounds.

Firefly luminescence: A historical perspective and recent developments

“…The report of Ap 4 A in firefly lanterns is relevant 103 and the synthesis of this compound by Luc when used as a reporter gene was recently described. 104…”

Re-evaluation of Diadenosine Tetraphosphate (Ap4A) From a Stress Metabolite to Bona Fide Secondary Messenger

“…The observed role for Ap 4 A in the initiation phase of DNA replication may also be supported by the finding that direct exposure of T47D and MCF7 breast cancer cells to 100 µM Ap 4 A slows their proliferation rate while increased NUDT2 expression (with a resulting reduction in Ap 4 A) increases their proliferation (Oka et al, 2011). However, other studies suggest that cells can tolerate greatly increased intracellular Ap 4 A without a noticeable effect on proliferation (Murphy and McLennan, 2004;Marriott et al, 2016). Thus, the cell-free system that identified the inhibition of replication initiation by Ap 4 A may reflect a response to acute changes to Ap 4 A levels that is overcome under chronic stress conditions such as in the NUDT2 knockout KBM7 cell lines.…”

Ap4A Regulates Directional Mobility and Antigen Presentation in Dendritic Cells

“…Cross-presentation is the intracellular degradation of extrinsic antigen and its presentation by MHC-I. This important DC function activates CD8 + T cells during an immunological response to intracellular pathogens (Murphy and McLennan, 2004, Murphy et al., 2000) and is a highly energy-dependent process and requires movement of the cells into finding suitable T cells. Though identical levels of phagocytic potential, Class-II MHC display and expression of co-stimulatory molecules were seen in DCs of Nudt2 fl/fl /CD11c + mice, there was an enhancement in their immune functionality.…”