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. 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. Programmed cell death (PCD) 1The abbreviations used are: PCD, programmed cell death; HRMAS, high resolution magic angle spinning; GCV, ganciclovir; TR, repetition time; TE, echo time; STEAM, stimulated echo acquisition mode; SW, sweep width; MAS, magic angle spinning; CPMG, Carr-Purcell-Mei-boom-Gill; PCA, principle component analysis; Glx, glutamate + glutamine; CHO, choline-containing compounds.1The abbreviations used are: PCD, programmed cell death; HRMAS, high resolution magic angle spinning; GCV, ganciclovir; TR, repetition time; TE, echo time; STEAM, stimulated echo acquisition mode; SW, sweep width; MAS, magic angle spinning; CPMG, Carr-Purcell-Mei-boom-Gill; PCA, principle component analysis; Glx, glutamate + glutamine; CHO, choline-containing compounds. involves a cascade of biochemical processes in an ATP-dependent manner, and the process is associated with substantial morphological alterations in the cell interior before phagocytosis (1Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12777) Google Scholar, 2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar, 3Lipton P. Physiol. Rev. 1999; 79: 1431-1568Crossref PubMed Scopus (2571) Google Scholar). Recent evidence from cell culture studies points to a number of characteristic metabolic perturbations appearing in the early phase of PCD. These include affections of intermediary metabolism, such as accumulation of glycolytic intermediates fructose 1,6-bisphosphate, dihydroxy acetone phosphate, and glycerol-3-phosphate because of inhibition of glyceraldehyde-3-phosphate dehydrogenase (4Ronen S.M. DiStefano F. McCoy C.L. Robertson D. Smith T.A. AlSaffar N.M. Titley J. Cunningham D.C. Griffiths J.R. Leach M.O. Clarke P.A. Br. J. Cancer. 1999; 80: 1035-1041Crossref PubMed Scopus (47) Google Scholar), retention of CDP-choline (5Williams S.N. Anthony M.L. Brindle K.M. Magn. Reson. Med. 1998; 40: 411-420Crossref PubMed Scopus (68) Google Scholar) as a result of inhibition of CDP-choline:1,2-diacylglycerol choline phosphotransferase (6Anthony M.L. Zhao M. Brindle K.M. J. Biol. Chem. 1999; 274: 19686-19692Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and collapse of NAD(H) levels (5Williams S.N. Anthony M.L. Brindle K.M. Magn. Reson. Med. 1998; 40: 411-420Crossref PubMed Scopus (68) Google Scholar). The severe decline in NAD(H), leading to inhibition of glycolysis, may result from activation of poly(ADP-ribose) polymerase in apoptotic cells (5Williams S.N. Anthony M.L. Brindle K.M. Magn. Reson. Med. 1998; 40: 411-420Crossref PubMed Scopus (68) Google Scholar). Inhibition of CDP-choline:1,2-diacylglycerol choline phospotransferase leads to cessation of phosphatidylcholine biosynthesis (6Anthony M.L. Zhao M. Brindle K.M. J. Biol. Chem. 1999; 274: 19686-19692Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and it has been proposed that this might be one of the mechanisms explaining accumulation of 1H NMR lipids into cells undergoing apoptosis (7Blankenberg F.G. Storrs R.W. Naumovski L. Goralski T. Spielman D. Blood. 1996; 87: 1951-1956Crossref PubMed Google Scholar, 8Al-Saffar M.M.S. Titley J.C. Robertson D. Clarke P.A. Jackson L.E. Leach M.O. Ronen S.M. Br. J. Cancer. 2001; 86: 963-970Crossref Scopus (106) Google Scholar). Interestingly, evidence has been put forward to suggest that inhibition of phosphatidylcholine biosynthesis per se might induce PCD (9Miquel K. Pradines A. Terce F. Selmi S. Favre G. J. Biol. Chem. 1998; 273: 26179-26186Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Furthermore, cell studies have indicated altered phospholipid metabolism (10Bhakoo K.K. Bell J.D. Cell Mol. Biol. (Noisy-Le-Grand). 1997; 43: 621-629PubMed Google Scholar) and intracellular acidification in the early phase of PCD (5Williams S.N. Anthony M.L. Brindle K.M. Magn. Reson. Med. 1998; 40: 411-420Crossref PubMed Scopus (68) Google Scholar). Both in cell cultures in vitro (7Blankenberg F.G. Storrs R.W. Naumovski L. Goralski T. Spielman D. Blood. 1996; 87: 1951-1956Crossref PubMed Google Scholar, 8Al-Saffar M.M.S. Titley J.C. Robertson D. Clarke P.A. Jackson L.E. Leach M.O. Ronen S.M. Br. J. Cancer. 2001; 86: 963-970Crossref Scopus (106) Google Scholar) and tumors in vivo (11Hakumäki J.M. Poptani H. Sandmair A.-M. Ylä-Herttuala S. Kauppinen R.A. Nat. Med. 1999; 5: 1323-1327Crossref PubMed Scopus (229) Google Scholar, 12Griffin J.L. Lehtimaki K.K. Valonen P.K. Grohn O.H.J. Kettunen M.I. Yla-Herttuala S. Pitkanen A. Nicholson J.K. Kauppinen R.A. Cancer Res. 2003; 63: 3195-3201PubMed Google Scholar), PCD has been shown to lead to accumulation of 1H NMR visible lipids. However, very little is known about the behavior of low molecular weight metabolites in vivo during ongoing PCD. Because a number of chemotherapeutic drugs are known to kill cancer cells by inducing apoptosis (2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar), information on low molecular weight markers of PCD would be crucial for the assessment of treatment responses in a clinical setting by 1H NMR spectroscopy (13Nelson S.J. Vigneron D.B. Dillon W.P. NMR Biomed. 1999; 12: 123-138Crossref PubMed Scopus (139) Google Scholar). In the present study, 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. Ex vivo high resolution magic angle spinning (HRMAS) 1H NMR spectroscopy was also used to further examine the metabolite patterns in the glioma tissue. This combined NMR spectroscopic approach indicates dissimilarities in behaviors of metabolites during PCD. Animals—BT4C gliomas transfected with viral HSV-tk gene were induced by implanting 104 HSV-tk+ cells in 5 μl of Opti-MEM to a depth of 2.5 mm into the corpus callosum of female BDIX rats weighing 180-250 g, as described previously (14Poptani H. Puumalainen A.-M. Gröhn O.H.J. Loimas S. Kainulainen R. Ylä-Herttuala S. Kauppinen R.A. Cancer Gene Ther. 1998; 5: 101-109PubMed Google Scholar). Rats in the treatment group were injected with GCV (25 mg/kg, intraperitoneal, twice daily) for the duration of the study. Brains were funnel-frozen in situ as described by Ponten et al. (15Pontén U. Ratcheson R.A. Salford L.G. Siesjö B.K. J. Neurochem. 1973; 21: 1127-1138Crossref PubMed Scopus (484) Google Scholar). The brains were sectioned frozen, and tumor tissue was dissected on dry ice for ex vivo 1H NMR spectroscopy or for extraction of acid-soluble metabolites with standard perchloric acid procedure. All animal experiments were performed according to the guidelines approved by the Ethical Committee of the National Laboratory Animal Center (Kuopio, Finland). NMR—For MRI, rats (n rats and were with in and the of was monitored and to using a water was performed in a with to a was used in Rats were into a using a and and to an angle to a rat The in Scholar). for animal was were determined from repetition 2.5 echo of and of (14Poptani H. Puumalainen A.-M. Gröhn O.H.J. Loimas S. Kainulainen R. Ylä-Herttuala S. Kauppinen R.A. Cancer Gene Ther. 1998; 5: 101-109PubMed Google Scholar). were using a TE, and an M. J. Magn. Reson. with in This by the of the in a acquisition S. Magn. Reson. Med. 1995; PubMed Scopus Google Scholar). from with and were used to the stimulated echo acquisition TE, sweep 2.5 an a and water and R. Magn. Reson. Med. 1999; PubMed Scopus Google Scholar) was used for of tumor metabolites in vivo. was the tumor according to the and the the was by using the R. Magn. Reson. Med. PubMed Scopus Google Scholar). In some 1H NMR were also from the using the by as described previously M. L. J. Magn. Reson. 2001; PubMed Scopus Google Scholar) TE, SW, 2.5 Metabolite concentrations were quantified from using water peak as a reference of BT4C tumor J.M. Grohn K. Valonen P. Yla-Herttuala S. Kauppinen R.A. Cancer Gene Ther. PubMed Scopus Google Scholar), for because of water accumulation during PCD, using the J.M. Grohn K. Valonen P. Yla-Herttuala S. Kauppinen R.A. Cancer Gene Ther. PubMed Scopus Google Scholar). ex vivo 1H NMR samples of tumor tissue in were into the μl of mm acid HRMAS 1H NMR were using a with a high resolution at using a on the spectroscopy to from and TR, SW, during the of and of 5 spinning were also with a using a of echo total number of to described for the spectroscopy perchloric acid extracts were and samples in acid for and resolution 1H NMR using during SW, were at using a at The was used to 31P NMR SW, peaks as in the 1H and 31P NMR some extracts were with and In vivo were in the using in vitro with and the ex vivo in the using metabolite in the were a at ppm for a at ppm for glutamine; a at for a at for a at for a at and ppm for and a at ppm for creatine + and a at ppm for was used for of was used to the of cell and metabolite concentrations both in vivo and in of ex the non-lipid metabolite changes that PCD in the high resolution 1H NMR were using were into a using and ppm as described previously Nicholson J.K. J.C. M. J. Chem. Res. 1998; PubMed Scopus Google Scholar). in was as a to the total the and of the the was using the approach The component of the were in of to tissue and progression through PCD. This process was also for in vivo and in vitro 1H NMR group of rats (n rats and were by and with for followed by in for The brains were from the in and in for was used to the of cell damage in the as well as for quantitative cell as described previously A. Kauppinen R.A. Magn. Reson. Med. 1999; PubMed Scopus Google Scholar). The were for to apoptotic using a were in tumor tissue from high through the of tumor are shown from and rats and was that by of GCV treatment in the of tumor and this was followed by a decline in the tumor and showed substantial cell loss and and from the of the tumor with the patterns of substantial in apoptotic by The cell was in tumors. The cell was and in and of and were than that at These confirmed that gene therapy induced PCD (14Poptani H. Puumalainen A.-M. Gröhn O.H.J. Loimas S. Kainulainen R. Ylä-Herttuala S. Kauppinen R.A. Cancer Gene Ther. 1998; 5: 101-109PubMed Google Scholar, J.M. Grohn K. Valonen P. Yla-Herttuala S. Kauppinen R.A. Cancer Gene Ther. PubMed Scopus Google Scholar), in severe cell loss in the 1H NMR from BT4C in vivo are by from a of saturated lipids at ppm and a of saturated lipids at peaks from of at of at and of lipids at ppm are also 1H NMR from a tumor in vivo before well resolved peaks from + creatine at glutamate + at + glycine at taurine at choline-containing compounds with a from taurine at and + creatine at and All peaks were also visible in and 1H NMR spectroscopy was used to metabolites in and the was used to the peak from with with a with the at be that in the of the resonances were in vivo from tumors to The of both and show the changes in the metabolite peaks GCV treatment and The metabolite concentrations from are cell the concentrations of + and creatine showed a and with cell in tumors undergoing PCD, that of in the of was also was the both unsaturated by at and saturated at lipids by 1H NMR indicated in the tissue with cell as previously GCV treatment of BT4C glioma (11Hakumäki J.M. Poptani H. Sandmair A.-M. Ylä-Herttuala S. Kauppinen R.A. Nat. Med. 1999; 5: 1323-1327Crossref PubMed Scopus (229) Google Scholar, 12Griffin J.L. Lehtimaki K.K. Valonen P.K. Grohn O.H.J. Kettunen M.I. Yla-Herttuala S. Pitkanen A. Nicholson J.K. Kauppinen R.A. Cancer Res. 2003; 63: 3195-3201PubMed Google Scholar). be that the peak at ppm may have from during the from was to be small metabolite concentrations cell in BT4C metabolite concentrations per of tissue in vivo were determined from using water as an internal Metabolite concentrations were also determined from tumor in vitro by 1H NMR spectroscopy as described and Cell were determined at treatment from as described and and for in vivo peaks as + creatine Glx, CHO, ppm The and for in vitro metabolites were as Glx, choline phosphocholine glycerophosphocholine Metabolite concentrations in acid extracts of tumor are cell 1H NMR from and tumor extracts are shown The in vitro results a in concentrations of and cell and However, taurine and choline-containing compounds showed in that the during treatment from in tumors to by of However, the at and of and were from determined in the tumors. The of showed a during PCD was the in vivo observation Ex vivo HRMAS 1H NMR from and BT4C gliomas at treatment are shown in Because of the resolution with at 14.1 one from CHO, and as well as taurine was metabolite resonance and of treatment using that changes in non-lipid metabolites the progression of PCD of tumor and tissue and a associated with PCD the of the the for tumor and tissue were The of tumors resonance from lipids, and of demonstrated that was by resonances from and creatine this for from extracts of tumors and in vivo using the the was the presence of and glutamate in tissue. metabolic associated with PCD was for non-lipid metabolites component of HRMAS 1H NMR of tumors and tissue. The the of correlated in the components in the with the of the of the by components and of tumor and tissue. The metabolite that components were from the of the with or in the the than at the The the that a small to the in the tumor tissue. The of the HRMAS peaks in tumors of the treatment that were in the samples from These peaks were in and were to the nucleotides and UDP or with were also in the of tumors and the tumors uridine or 31P NMR spectroscopy was performed on the tissue extracts of the tumors and the presence of and UDP in the tumors. The assignment of the latter metabolite was confirmed by spiking experiments using both 1H and 31P The of UDP to during PCD at according to the 1H NMR resonances and from the 31P NMR of the In the present study, we have used BT4C tumors undergoing gene therapy as a of PCD. This well has characteristic metabolic changes associated with PCD, a in Furthermore, in this we have demonstrated by that of the tumors are that the of metabolic changes are from PCD M. Loimas S. P. K. R. M. R. J. Ylä-Herttuala S. Cancer Gene Ther. 1999; Scopus Google Scholar). The present results show that of 1H NMR metabolites in vivo in a during cell loss in BT4C The group of and decline in a manner the cell The group of by CHO, cell collapse of The group of 1H by both saturated and lipids, during PCD severe cell loss (11Hakumäki J.M. Poptani H. Sandmair A.-M. Ylä-Herttuala S. Kauppinen R.A. Nat. Med. 1999; 5: 1323-1327Crossref PubMed Scopus (229) Google Scholar). the behavior of 1H NMR visible lipids during PCD to of low molecular weight such as glycine and 1H NMR changes in PCD have and changes have been demonstrated both in cells in vitro (7Blankenberg F.G. Storrs R.W. Naumovski L. Goralski T. Spielman D. Blood. 1996; 87: 1951-1956Crossref PubMed Google Scholar) and tumors in vivo (11Hakumäki J.M. Poptani H. Sandmair A.-M. Ylä-Herttuala S. Kauppinen R.A. Nat. Med. 1999; 5: 1323-1327Crossref PubMed Scopus (229) Google Scholar, 12Griffin J.L. Lehtimaki K.K. Valonen P.K. Grohn O.H.J. Kettunen M.I. Yla-Herttuala S. Pitkanen A. Nicholson J.K. Kauppinen R.A. Cancer Res. 2003; 63: 3195-3201PubMed Google Scholar). The 1H NMR observation is for by accumulation of M.M.S. Titley J.C. Robertson D. Clarke P.A. Jackson L.E. Leach M.O. Ronen S.M. Br. J. Cancer. 2001; 86: 963-970Crossref Scopus (106) Google Scholar), and into the tumors leading to in intracellular and (11Hakumäki J.M. Poptani H. Sandmair A.-M. Ylä-Herttuala S. Kauppinen R.A. Nat. Med. 1999; 5: 1323-1327Crossref PubMed Scopus (229) Google Scholar). 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Reson. Med. 1998; 40: 411-420Crossref PubMed Scopus (68) Google Scholar) that this of phosphatidylcholine biosynthesis during PCD in However, we for this to the from 1H or 31P NMR of tumors. The observation of CDP-choline changes in vitro using the choline resonance at ppm in vivo is to be CDP-choline has a at with the peak in vivo and with phosphocholine in vitro in the BT4C tumor extracts and ex vivo in tumors. This resonance is with phosphocholine and and the from taurine also to this of This is using HRMAS 1H NMR spectroscopy ex vivo and indicates that previously changes of choline-containing metabolites by 1H NMR, with progression of PCD, are to be by changes in the taurine at Both in vivo and in vitro taurine concentrations, and this in CDP-choline in vivo as of the combined choline-containing an for the of resonance from choline-containing metabolites in vivo at the of PCD cell loss may be associated with a of the metabolites NMR and visible used spinning were to the of the lipids in cell J. Full Text PDF PubMed Scopus Google 1H NMR-detected are to be we have in cell membrane in with the choline-containing metabolite resonance from membrane phosphocholine to the J.L. J. Nicholson J.K. 2001; PubMed Scopus Google Scholar). is that during the of PCD, the resonance is associated with cell membrane and of choline-containing metabolites into the and This would the resonance in of cell whereas some low molecular weight metabolites in resonance HRMAS spectroscopy in conjunction with the we a doublet at ppm and resonances at and These resonances are with and resonances on uridine or The of nucleotide resonances in cell cultures has previously been both using cell extracts of and HRMAS 1H NMR spectroscopy of cells PubMed Scopus Google Scholar, J.L. J.C. Nicholson J.K. 2003; PubMed Scopus Google Scholar). Furthermore, in the latter study, a known the of nucleotides of the tumor tissue extracts with 31P NMR spectroscopy indicated that resonances were from of this nucleotide in glioma may result from metabolic with cells, because is used to in cells that such as J. Neurochem. 40: PubMed Scopus Google Scholar). the of UDP may the of nucleotides for for of Because uridine is a component of it may in the whereas the nucleotides are used to This an for the of concentrations of uridine metabolites in PubMed Scopus Google Scholar), J.L. Nicholson J.K. Chem. Res. 2001; PubMed Scopus Google Scholar), cell J.L. J.C. Nicholson J.K. 2003; PubMed Scopus Google Scholar), in this study, In the well in saturated and unsaturated during PCD, non-lipid metabolites of the progression of PCD. Cell death is by a in some with a in cell the tumors. of were in tumors the of saturated and unsaturated resonances the marker of cell death in BT4C tumors during HSV-tk gene therapy. Pitkanen for in quantitative the of and