Consideration of metabolite efflux in radiolabelled choline kinetics

Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore may confound uptake of choline radiotracer [ 18F]fluoromethyl-[1,2- 2H4]-choline ([ 18 F]-D4-FCH). The aim of this study was to investigate how hypoxia affects choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [ 18 F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and efflux of [18 F]-D4-FCH and its phosphorylated product [ 18F]-D4-FCHP. Effects of hypoxia on [18 F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer, MDA-MB-231 cells. Mechanisms of radiotracer efflux were assessed by cell uptake and immunofluorescence in vitro, and examined in vivo (N=24). Mathematical modelling methodology was further developed to verify efflux hypothesis using [18 F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (N=17). We report a novel finding involving export of phosphorylated [18F]-D4-FCH, [18 F]-D4-FCHP, via HIF-1α-responsive efflux transporters including ABCB4 when HIF-1α level is augmented. This is supported by graphical analysis of human data with a compartmental model (M2T6k+k5) that accounts for efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting consideration of efflux in modelling of radiotracer dynamics

X-ray irradiation of yeast cells

Saccharomyces Cerevisiae yeast cells were irradiated using the sort X-ray laser-plasma source at Rutherford Laboratory. The aim was to produce a selective damage of enzyme metabolic activity at the wall and membrane level (responsible for fermentation) without interfering with respiration (taking place in mitochondria) and with nuclear and DNA activity. The source was calibrated by PIN diodes and X-ray spectrometers. Teflon stripes were chosen as targets for the UV laser, emitting X-rays at about 0.9 keV, characterised by a very large decay exponent in biological matter. X-ray doses to the different cell compartments were calculated following a Lambert-Bouguet-Beer law. After irradiation, the selective damage to metabolic activity at the membrane level was measured by monitoring CO2 production with pressure silicon detectors. Preliminary results gave evidence of pressure reduction for irradiated samples and non-linear response to doses. Also metabolic oscillations were evidenced in cell suspensions and it was shown that X-ray irradiation changed the oscillation frequency