Abstract
Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1H NMR visible lipids increased. HRMAS 1H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1H and 31P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo.
Highlights
Both in cell cultures in vitro [7, 8] and tumors in vivo [11, 12], Programmed cell death (PCD) has been shown to lead to accumulation of 1H NMR visible lipids
The cell density was 183 Ϯ 13, 154 Ϯ 14, 109 Ϯ 4, and 71 Ϯ 2 ϫ 103 cell/mm3 in days 2, 4, 6, and 8 of treated tumors, respectively; day 6 and 8 values were significantly less than that at day 0 (p Ͻ 0.01). These observations confirmed that gene therapy had induced PCD [14, 22], resulting in severe cell loss in the gliomas
We have used BT4C tumors undergoing ganciclovir-HSV-tk gene therapy as a model of PCD. This well documented model has characteristic metabolic changes associated with PCD, most notably a large increase in polyunsaturated fatty acids
Summary
Both in cell cultures in vitro [7, 8] and tumors in vivo [11, 12], PCD has been shown to lead to accumulation of 1H NMR visible lipids. We have monitored the non-lipid metabolite levels in rat BT4C gliomas during gene therapy-induced PCD in vivo in a quantitative manner as well as determined the metabolite concentrations in acid extracts of the tumor samples.
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