In Vitro and in Vivo¹³C and ³¹P NMR analyses of phosphocholine metabolism in rat glioma cells

Abstract

In vivo magnetic resonance spectroscopy (MRS) has revealed that phosphomonoesters (PME) such as phosphocholine (PCho) and phosphoethanolamine (PEth) are elevated in tumors and rapidly proliferating tissues. The regulation of PME levels and their relationship to proliferation are not well known. In the present study, we investigated the regulation of PCho and PEth levels in rat glioma cells grown in vivo and in vitro using 31P and 13C MRS. However, the ability of cells to produce choline endogenously is variable. To fully understand regulation of PCho levels, it is necessary to characterize the activity of the endogenous pathway, if it exists. This was first investigated by following the metabolic fate of 13C-labeled methionine of 9L glioma tumors in vivo. Our results indicate that there is a significant amount of de novo choline synthesis in vivo. However, similar experiments performed in vitro using cells cultured in bioreactors indicated that glioma cells themselves are unable to synthesize choline de novo, suggesting that the in vivo results were due to the involvement of extra-tumoral organs, e.g., liver. Further in vitro experiments demonstrated that the uptake and phosphorylation of physiologically relevant concentrations of exogenous choline is very active in these systems. Thus, it appears that the exogenous pathway for PCho biosynthesis predominates and regulates PCho levels in glioma cells. Our results also demonstrate that PCho levels are lowest, and PEth levels are highest, in non-proliferating cells. These observations indicate that there is a decrease in the biosynthesis of PCho concomitant with a reduction in culture growth. The source of the increased PEth is, as yet, undefined.

Pediatric Brain Tumors: Magnetic Resonance Spectroscopic Imaging

“…In general, the consensus is that tissues with high proliferative potential or tissues that are oncogenically transformed are also highly cellular in the absence of compensating apoptotic mechanisms or limitations of vascular supply. Consistent with this, many studies strongly have suggested that the Cho peak detected by in vivo MRS may be elevated because the volume of interest is highly cellular (Chang et al, 1995;Miller et al, 1996b;Cheng et al, 2000b) or includes cells with high PCho which may be due to increased proliferative potential (Daly et al, 1987;Daly and Cohen, 1989;Gillies et al, 1994a;Mahmood et al, 1994;Aiken and Gillies, 1996;Aiken et al, 1996) or includes cells which are oncogenically transformed (Bhakoo et al, 1996;Aboagye and Bhujwalla, 1999;Ackerstaff et al, 2001). It was recently reported that in ex vivo high resolution MR spectra (which show much higher than the spectra obtained in vivo) of a biopsy from a cerebellar primitive neuroectodermal tumor, myo-inositol, taurine and phosphorylethanolamine contribute to the in vivo signal at 3.2 ppm, usually attributed to Cho-containing compounds (Tugnoli et al, 2001).…”

“…The Cho peak consists of water-soluble Cho-containing compounds, such as phosphocholine (PCho) and glycerophosphocholine (GPC), and free choline (Barker et al, 1994), versus membrane-bound phospatidylcholine (Miller et al, 1996a). In vivo MRS reveals that phosphomonoesters (PME), such as phosphocholine (PCho) and phosphoethanolamine (PEth), are elevated in tumors and rapidly proliferating tissues (Daly et al, 1987;Daly and Cohen, 1989;Gillies et al, 1994a). Furthermore, elevations in PCho and PEth correlate with increased cell growth or increased cell degradation, and have been shown to occur in human tumors, as well as in animal tumor models and cell lines.…”

“…For instance, actively proliferating cultures show dramatically lower PCho/PEth as compared to stationary cultures (Aiken and Gillies, 1996). Gillies et al, found that PCho levels are lowest, and PEth levels are highest in non-proliferating cells and concluded there is a decrease in the biosynthesis of PCho concomitant with a reduction in culture growth (Gillies et al, 1994b). Mahmood et al, found a strong radiation dose-dependent response in the relative PCho/PEth ratio, suggesting that changes in the PCho levels may be related to cell proliferation and/or radiation-induced membrane damage (Mahmood et al, 1994).…”

Phosphorous metabolites and steady-state energetics of transformed fibroblasts during three-dimensional growth

“…In most cases, enhanced phospholipid biosynthesis and reduced phospholipid breakdown were observed in long-term chronic situations such as tumors and high phosphomonoester (PME) levels have been hypothesized to be the best biochemical marker to distinguish some cancerous from normal tissues (16,17,43,52,55,62,65) and to monitor tumor treatment outcome (69,75). Some but not all of these observations might be due to the fact that under certain conditions PC strongly correlates with cell proliferation (4,6,21,26,62,68,72,82). Ting et al (80) compared the metabolism of different human breast cancer cells and normal mammary epithelial cells with a similar proliferative activity.…”

“…Among other things, measurement of tumor energetics by 31 P-NMR spectroscopy provides useful information about tumor oxygenation/ hypoxia and potential therapeutic resistance. However, there is some controversy about the relevance of the additional parameters that can be monitored in tumors via phosphorous NMR, e.g., phospholipid metabolism (15,16,26,43,44,62,68,79). It was previously reported that most human tumors contain high concentrations of phosphomonoesters, with the major compounds being identified as phosphorylethanolamine (PE) and phosphorylcholine (PC) (15,54,72).…”