Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo, ex vivo, and in vitro.
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.
Lehtimäki
Kimmo K
KK
Department of Biomedical NMR and National Bio-NMR Facility, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland.
Valonen
Piia K
PK
Griffin
Julian L
JL
Väisänen
Tuula H
TH
Gröhn
Olli H J
OH
Kettunen
Mikko I
MI
Vepsäläinen
Jouko
J
Ylä-Herttuala
Seppo
S
Nicholson
Jeremy
J
Kauppinen
Risto A
RA
D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000378:metabolism; D001932:Brain Neoplasms / Q000628:therapy; D045744:Cell Line, Tumor; D002794:Choline / Q000737:chemistry; D004058:Diffusion; D015774:Ganciclovir / Q000627:therapeutic use*; D015316:Genetic Therapy; D005910:Glioma / Q000378:metabolism / Q000628:therapy*; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D009682:Magnetic Resonance Spectroscopy; D010472:Perchlorates / Q000737:chemistry; D010767:Phosphorylcholine / Q000737:chemistry; D051381:Rats; D013654:Taurine / Q000737:chemistry; D013937:Thymidine Kinase / Q000235:genetics*
D010472:Perchlorates; D010767:Phosphorylcholine; D013654:Taurine; D013937:Thymidine Kinase; D002794:Choline; D015774:Ganciclovir