Metabolite Changes in BT4C Rat Gliomas Undergoing Ganciclovir-Thymidine Kinase Gene Therapy-induced Programmed Cell Death as Studied by super(1)H NMR Spectroscopy in Vivo, ex Vivo, and in Vitro

• Published in: The Journal of biological chemistry 2003-11, Vol.278 (46), p.45915-45923
• Main Authors: Lehtimaeki, K K, Valonen, P K, Griffin, J L, Vaeisaenen, TH, Groehn, OHJ, Kettunen, MI, Vepsaelaeinen, J, Ylae-Herttuala, S, Nicholson, J, Kauppinen, R A
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M306209200

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 super(1)H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) super(1)H 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 super(1)H NMR visible lipids increased. HRMAS super(1)H 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 super(1)H and super(31)P 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 super(1)H 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 super(1)H 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.