High‐resolution magic‐angle‐spinning 1H NMR spectroscopy reveals different responses in choline‐containing metabolites upon gene therapy‐induced programmed cell death in rat brain glioma

Abstract

Changes in the concentrations of choline-containing metabolites (CCM) have been implicated in both cell proliferation and death processes. In this study, high-resolution magic-angle-spinning (HRMAS) 1H NMR spectroscopy was used to study metabolite changes in the CCM chemical shift region in rat glioma ex vivo during apoptosis induced by thymidine kinase-ganciclovir gene therapy. Cell density and apoptotic activity in the tumours were quantified by histological methods. HRMAS 1H NMR was able to resolve peaks from choline (Cho), glycerophosphocholine (GPC), phosphocholine (PC), taurine (Tau) and myo-inositol (myo-Ins), all of which contribute to the in vivo 1H NMR peak centred at 3.23 ppm. The early phase of apoptosis (treatment day 4), with a approximately 2.8-fold increase in the number of apoptotic nuclei (at constant cell density of 1.8 +/- 0.1 x 10(5) cells/mm3) was associated with increases in resonance intensity from GPC and PC, while Cho and Tau remained unchanged. Later stage apoptosis, accompanied by synchronous cell death (cell density declined to 0.7 +/- 0.02 x 10(5) cells/mm3), resulted in a significant decline in Tau relative to untreated tumours, while the contents of CCMs and myo-Ins detectable by 1H HRMAS were unchanged. These observations demonstrate that, while the in vivo 1H NMR peak at 3.23 ppm is indicative of cellular processes involved in apoptosis, the biochemical changes monitored by this resonance involve a number of different and chemically distinct metabolites.