Choline kinase-alpha by regulating cell aggressiveness and drug sensitivity is a potential druggable target for ovarian cancer.

BACKGROUND: Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). METHODS: To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. RESULTS: In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by (1)H-magnetic spectroscopy analysis; (III) a 35-36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. CONCLUSION: We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

Monitoring response to cytostatic cisplatin in a HER2(+) ovary cancer model by MRI and in vitro and in vivo MR spectroscopy.

BACKGROUND: Limited knowledge is available on alterations induced by cytostatic drugs on magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters of human cancers, in absence of apoptosis or cytotoxicity. We here investigated the effects of a cytostatic cisplatin (CDDP) treatment on (1)H MRS and MRI of HER2-overexpressing epithelial ovarian cancer (EOC) cells and in vivo xenografts. METHODS: High-resolution MRS analyses were performed on in vivo passaged SKOV3.ip cells and cell/tissue extracts (16.4 or 9.4 T). In vivo MRI/MRS quantitative analyses (4.7 T) were conducted on xenografts obtained by subcutaneous implantation of SKOV3.ip cells in SCID mice. The apparent diffusion coefficient (ADC) and metabolite levels were measured. RESULTS: CDDP-induced cytostatic effects were associated with a metabolic shift of cancer cells towards accumulation of MRS-detected neutral lipids, whereas the total choline profile failed to be perturbed in both cultured cells and xenografts. In vivo MRI examinations showed delayed tumour growth in the CDDP-treated group, associated with early reduction of the ADC mean value. CONCLUSION: This study provides an integrated set of information on cancer metabolism and physiology for monitoring the response of an EOC model to a cytostatic chemotherapy, as a basis for improving the interpretation of non-invasive MR examinations of EOC patients.

1H MRS-detectable metabolic brain changes and reduced impulsive behavior in adult rats exposed to methylphenidate during adolescence.

Administration of methylphenidate (MPH, Ritalin) to children affected by attention deficit hyperactivity disorder (ADHD) is an elective therapy, which however raises concerns for public health, due to possible persistent neuro-behavioral alterations. We investigated potential long-term consequences at adulthood of MPH exposure during adolescence, by means of behavioral and brain MRS assessment in drug-free state. Wistar adolescent rats (30- to 44-day-old) were treated with MPH (0 or 2 mg/kg once/day for 14 days) and then left undisturbed until adulthood. Levels of impulsive behavior were assessed in the intolerance-to-delay task: Food-restricted rats were tested in operant chambers with two nose-poking holes, delivering one food pellet immediately, or five pellets after a delay whose length was increased over days. MPH-exposed animals showed a less marked shifting profile from the large/late to the small/soon reward, suggesting reduced basal levels of impulsivity, compared to controls. In vivo MRI-guided 1H MRS examinations at 4.7 T in anaesthetised animals revealed long-term biochemical changes in the dorsal striatum (STR), nucleus accumbens (NAcc), and prefrontal cortex (PFC) of MPH-exposed rats. Notably, total creatine and taurine, metabolites respectively involved in bioenergetics and synaptic efficiency, were up-regulated in the STR and conversely down-regulated in the NAcc of MPH-exposed rats. A strong correlation was evident between non-phosphorylated creatine in the STR and behavioral impulsivity. Moreover, unaltered total creatine and increased phospho-creatine/creatine ratio were detected in the PFC, suggesting improved cortical energetic performance. Because of this enduring rearrangement in the forebrain function, MPH-exposed animals may be more efficient when faced with delay of reinforcement. In summary, MPH exposure during adolescence produced enduring MRS-detectable biochemical modifications in brain reward-related circuits, which may account for increased self-control capacity of adult rats.

Nuclear magnetic resonance analysis of tumor necrosis factor-induced alterations of phospholipid metabolites and pH in Friend leukemia cell tumors and fibrosarcomas in mice.

The alterations induced on the pool sizes of five phospholipid metabolites, glycerol 3-phosphorycholine, glycerol 3-phosphorylethanolamine, phosphorylcholine, sn-glycerol 3-phosphate, and choline were studied by nuclear magnetic resonance (NMR) spectroscopy in murine tumors injected with recombinant murine tumor necrosis factor (TNF). Solid tumors were obtained by s.c. injection of either Friend leukemia cells (clones 3C1-8 and 745) in DBA/2 mice or murine fibrosarcoma cells (HeN4) in C3H/HeN mice. After tumor nodules had developed, TNF or bovine serum albumin was injected intratumorally. Treatment of both tumors with TNF resulted in a marked inhibition of tumor growth. 31P-NMR analyses of Friend leukemia cell tumors (and tissue extracts), 6 h after injection of TNF, showed: (a) a 1.5- to 3.5-fold decrease in the pool sizes of glycerol 3-phosphorylcholine and glycerol 3-phosphorylethanolamine; (b) a 7- to 8-fold increase of sn-glycerol 3-phosphate; (c) a 2- to 3.5-fold decrease of phosphorylcholine; (d) an alkaline shift (0.2 units) in intratumoral pH. Similar metabolic alterations occurred in TNF-treated HeN4 fibrosarcoma. 1H-NMR analyses of Friend leukemia cell tumor extracts also indicated, 6 h after tumor injection with TNF: (a) elevated choline levels (9X); (b) a 19-fold increase in the ratio [choline]/[phosporylcholine]; (c) elevated (1.4X) levels of lactic acid; and (d) a 1.6-fold decrease in the [taurine]/[glycine] ratio. The results are interpreted in the light of possible alterations in the activity of enzymes controlling the de novo biosynthesis and catabolism of phospholipids. We concluded that NMR spectroscopy can be a useful means to monitor the level of some phospholipid precursors and/or derivatives as early markers of therapeutic efficacy in intact neoplastic tissues.

31P-nuclear magnetic resonance analysis of interferon-induced alterations of phospholipid metabolites in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice.

Adult DBA/2 mice were given injections s.c. with either interferon-sensitive (745) or -resistant (3Cl-8) Friend erythroleukemia cells (FLC). After tumor nodules had developed, mouse interferon-alpha/beta was injected daily into the tumor. 31P-Nuclear magnetic resonance (NMR) spectroscopy examinations were undertaken on freshly dissected tumors at different days of treatment with either interferon or control preparations. Analysis of 745 FLC tumors in untreated mice at different days of tumor growth (day 8 to 13 after tumor implantation) showed marked increases in the levels of phosphorylcholine (PCho), glycerophosphorylethanolamine (GroPEtn) and glycerophosphorylcholine (GroPCho). In contrast high levels of PCho, GroPEtn and GroPCho were already detectable in the 3Cl-8 FLC tumors on day 8, and no significant changes were observed during subsequent tumor growth. The intracellular pH value remained practically constant in both FLC tumors. Daily intratumoral administration of either partially purified (10(7) IU/mg of protein) or highly purified (10(9) IU/mg of protein) mouse interferon-alpha/beta to both cell tumors resulted in decreases in the levels of PCho, GroPEtn and GroPCho and in increases in the intracellular pH with respect to tumors treated with control preparations or left untreated. Two days of daily treatment of mice with interferon sufficed to induce these metabolic changes which preceded the appearance of necrosis in the tumors. Treatment of FLC tumors with X-rays on day 12 of tumor growth did not result in any comparable metabolic changes 2 days after irradiation. Changes in the levels of phospholipid metabolites were not observed when 745 or 3Cl-8 cells were cultivated in the presence of interferon. As interferon induced these changes in both interferon-sensitive and -resistant tumors we conclude that interferon treatment results in host-mediated effects on the biosynthesis and/or catabolism of tumor cell phospholipids.

Proton magnetic relaxation studies of mixed phosphatidylcholine/fatty acid and mixed phosphatidylcholine bimolecular bilayers.

High resolution proton spin-lattice relaxation times (T1), spin-spin relaxation times (T2) and resonance linewidths were measured above the gel-to-liquid crystal transition temperature (Tm), in phosphatidylcholine bilayers possessing various degrees of intramolecular motional anisotrophy at the level of various alkyl chain proton groups. The experiments were designed to test the hypothesis that coupled trans-gauche isomerizations along the chains can be responsible for the anisotropic motion of phosphatidylcholine proton groups in bilayer membranes (Horwitz, A.F., Horsley, W.J. and Klein, M.O. (1972) Proc. Natl. Acad. Sci. U.S. 69,500). Systematic series of structural perturbations of the bilayer were achieved in mixed phosphatidylcholine/fatty acid and in mixed phosphatidylcholine bilayers where the degree of motional anisotrophy of the chains' proton groups was gradually reduced by progressively increasing the chain length disparity of the two components. The systematic T1 and T2 variations observed were interterpreted on the basis of the Woessner's treatment for computing the relaxation times of a spin pair reorienting randomly about an axis which, in turn, tumbles randomly (Woessner, D.E. (1962) J. Chem. Phys. 36, 1). The results confirmed in a qualitative sense the original hypothesis made by Horwitz et al. The time-averaged structural interpretations suggested by the mangetic relaxation studies are in agreement with low-angle X-ray diffraction results obtained below Tm. In addition, the T1 values evaluated at various temperatures in dipalmitoyl phosphatidylcholine vesicles incorporated with either 2H-labeled or unlabeled palmitic acid chains indicated that the average intermolecular contribution to the spin-lattice relaxation rate of the proton groups of the phosphatidylcholine chains appears comparable to the intramolecular term at temperatures moderately higher than Tm, but becomes less and less important as the temperature is further increased above the thermal transition.

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.

Phosphocholine and phosphoethanolamine during chick embryo myogenesis: a (31)P-NMR study.

Elevated contents of phosphoethanolamine (Etn-P) and/or phosphocholine (Cho-P), a common feature of most tumours with respect to normal counterparts, may also occur in non-cancerous proliferating tissues. The significance of these alterations in relation to cell proliferation, differentiation and maturation is scarcely understood. In this work, the Cho-P and Etn-P pools were measured by (31)P-NMR in extracts of chick embryo pectoral muscle at different days of development. The average concentration of these metabolites exhibited the highest values (respectively, 1.5 and 3.0 micromol/mg DNA) on days 9-11 and decreased at later stages of myogenesis. While, however, Cho-P maintained substantial levels (above 1.0 micromol/mg DNA) also during myotube formation (days 11-18) and stepwise decreased (to about 0.5 micromol/mg DNA) upon fibres' maturation, Etn-P gradually decreased between day 11 and hatching time (down to about 0.2 micromol/mg DNA). These results demonstrate that significant changes may occur in the steady-state pools of these metabolites during normal in vivo cellular development and differentiation, and are consistent with: (a) high rates of phospholipid biosynthesis reported in the literature for proliferating myoblasts; (b) sustained phosphatidylcholine synthesis maintained also during myoblast fusion; and (c) decreased requirement of phospholipid synthesis in the last phase of in ovo myofibre maturation.

The interaction of histone H3 with histone H4 and with other histones studied by 19F nuclear magnetic resonance.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells.

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.

Alterations of lipid composition in Friend leukemia cell tumors in mice treated with tumor necrosis factor-alpha.

Lipid analyses were carried out on transplantable murine Friend leukemia cell tumors, 6 h after intratumoral administration of tumor necrosis factor-alpha (TNF). The levels of the major phospholipid classes were uniformly decreased to about 70% of control values; free fatty acids were increased to about 170%; diacylglycerol was decreased to about 50% and triacylglycerol, the main lipid component, was not significantly altered. These results analysed in the light of concomitant alterations in the levels of phospholipid precursors and catabolites (determined in previous 31P NMR studies) and histological modifications demonstrated that at early stages of TNF-induced inhibition of tumor growth (a) phospholipid catabolism was significantly enhanced; (b) morphological changes were apparently correlated with alterations in the levels of phosphatidylcholine and its catabolic products.

Modulations of glycerophosphorylcholine and phosphorylcholine in Friend erythroleukemia cells upon in vitro-induced erythroid differentiation: a 31P NMR study.

A 31P NMR study has been carried out on Friend erythroleukemia cells (FLC) induced to undergo erythroid differentiation in vitro. Significant levels of glycerophosphorylcholine (GroPCho) and phosphorylcholine (P-Cho) were identified both in the untreated cells and in their PCA extracts. In FLC treated 4 days in vitro with either dimethylsulfoxide (DMSO) or hexamethylenebisacetamide (HMBA), the intracellular concentration of P-Cho was markedly increased, whereas that of GroPCho appeared to be significantly reduced. HMBA was more effective than DMSO in producing this effect. The concomitant modulations of GroPCho and P-Cho in differentiated FLC suggest the hypothesis that erythroid differentiation involves modifications of the regulatory mechanisms controlling biosynthesis and catabolism of phospholipids.

The application of nuclear magnetic resonance spectroscopy to the study of natural and model membranes.

This review article outlines some potentials and limits of the recent application of high resolution Nuclear Magnetic Resonance technique--coupled to the Fourier transformation methods--to the study of biological membranes. Molecular arrangement and dynamical structure characters can be assessed at the level of individual chemical groups in lipid bilayer regions of natural and model membranes, through the determination of physical parameters like chemical shifts, spin-lattice (T1) and spin-spin (T2) nuclear magnetic relaxation times. The results of some significant experiments carried out on single-wall lecithin vesicles as well as on intact natural membranes, are summarized and discussed. Useful information can be obtained on the lipid fatty-acid chains thermal transition, by comparing two lecithin vesicles of the same size, formed by the same host lecithin, but incorporated with different molecular components. In particular, T1 and T2 measurements, interpreted in terms of a two- (or more-) correlation time theoretical models, are able to demonstrate different degrees of motional anisotropy in bilayers formed by mixed lecithins or by mixtures of lecithin and fatty acids, possessing moderately different chain lengths [13]. Chromophore-containing molecules, like chlorophyll [12] or fluorescent probes [14] can be located, within few Angstroms, in a lipid bilayer through proton chemical shift measurements; in addition the perturbation of the lipid membrane structure, as induced by the incorporated probe, is assessed mainly in terms of the intramolecular dynamical structure of the host lecithin molecules, by means of T1 and linewidth studies. The comparison of the n.m.r. relaxation behaviour in intact membranes and in vesicles formed by their extracted lipids may, finally, provide indirect information on the lipid-protein intermolecular interactions and relative mobility, besides indicating the intramolecular mobility characters of the lipid bilayer regions of the membrane.

Intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT): a nuclear magnetic resonance study on T-lymphoblastoid cell lines with different resistance to AZT.

This paper reports the results of 31P and 1H nuclear magnetic resonance (NMR) studies on the uptake and phosphorylation of 3'-azido-3'-deoxythymidine (AZT) in the human CD4+ T-lymphoblastoid cell line CCRF-CEM (CEM-1.3) and in its AZT-resistant cell variant MT-500, isolated by prolonged culturing of CEM cells in the presence of increasing AZT concentrations. After 3 hr of incubation in the presence of 0.5 mM AZT, both AZT and its monophosphorylated form (AZT-MP) could be detected in the sensitive cell line in concentrations above the NMR detection levels. In another cell line, MOLT-4, which is less sensitive to AZT effects, the intracellular level of AZT-MP was much lower and was only slightly raised by increasing the concentration of AZT in the extracellular and intracellular compartments. In the AZT-resistant clone MT-500, characterized by a very low thymidine kinase (TK, EC 2.7.1.21) activity with respect to the parental clone, the intracellular AZT-MP concentration was below detection (<0.02 nmol/10(6) cells). Since, however, not only AZT-MP but also AZT signals failed to be detected in MT-500 extracts following cell incubation with AZT, it was concluded that a TK deficiency cannot be the exclusive mechanism of AZT resistance in these cells. The possible effects of additional mechanisms of drug resistance, such as specific AZT cell extrusion and limited permeation, are discussed, together with the new prospects offered by NMR spectroscopy to further evaluate the limiting steps for the utilization of antiretroviral nucleoside analogues.

Proton MRI of (13)C distribution by J and chemical shift editing.

The sensitivity of (13)C NMR imaging can be considerably favored by detecting the (1)H nuclei bound to (13)C nuclei via scalar J-interaction (X-filter). However, the J-editing approaches have difficulty in discriminating between compounds with similar J-constant as, for example, different glucose metabolites. In such cases, it is almost impossible to get J-edited images of a single-compound distribution, since the various molecules are distinguishable only via their chemical shift. In a recent application of J-editing to high-resolution spectroscopy, it has been shown that a more efficient chemical selectivity could be obtained by utilizing the larger chemical shift range of (13)C. This has been made by introducing frequency-selective (13)C pulses that allow a great capability of indirect chemical separation. Here a double-resonance imaging approach is proposed, based on both J-editing and (13)C chemical shift editing, which achieves a powerful chemical selectivity and is able to produce full maps of specific chemical compounds. Results are presented on a multicompartments sample containing solutions of glucose and lactic and glutamic acid in water.

13C and 31P NMR studies of glucose and 2-deoxyglucose metabolism in normal and enzyme-deficient human erythrocytes.

The flux of 13C-labeled glucose through the Embden-Meyerhof and pentose phosphate pathways was studied by 13C NMR in intact erythrocytes isolated from normal subjects or from patients suffering of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency. Similar rates of glucose catabolism and similar fluxes of the 13C-label into 2,3-bisphosphoglycerate and lactate were found, under basal conditions, in normal and in G6PD-deficient erythrocytes incubated in the presence of either [1-13C]- or D[6-13C]glucose. Exposure to oxidative stress by preincubation with tert-butylhydroperoxide induced in normal, but not in G6PD-deficient erythrocytes, a significant enhancement of glucose consumption, as well as a substantial reduction in 13C-label transfer from C1-glucose into lactate. It was also possible, by 31P NMR, to evaluate the conversion of 2-deoxyglucose to its phosphate-containing metabolites. The oxidation and subsequent decarboxylation of 2-deoxyglucose-6-phosphate was assessed in reconstituted systems and could subsequently be evidenced also in ethanolic extracts from normal (but not from G6PD-deficient) erythrocytes which had been exposed to oxidative stress. The results indicate that, in terms of glucose flux through the glycolytic pathway, there is little or no difference between normal and G6PD-deficient erythrocytes, regardless of previous exposure to oxidative stress. Faster consumption of either glucose or 2-deoxyglucose is induced, only in normal cells, by treatment with tert-butylhydroperoxide, essentially as a consequence of the activation of the pentose-phosphate pathway.

Magnetic resonance spectroscopy in cancer: phospholipid, neutral lipid and lipoprotein metabolism and function.

A Special Symposium on "Lipid Metabolism and Function in Cancer. Significance of Magnetic Resonance Spectroscopy (MRS) Measurements in Relation to Biochemical Processes and Cellular Control" was held by the EU BIOMED 1 Concerted Action for "Cancer and Brain Disease Characterization and Therapy Assessment by Quantitative MRS", within the 5th International Conference of Anticancer Research (Corfu, 18-20 October 1995). The aims of the Symposium were to discuss present knowledge and problems in the field of phospholipid, neutral lipid and lipoprotein metabolism in cancer, together with the new potential of these inter-linked areas of research arising from advanced in vitro and in vivo MRS methodologies. This guest editorial is intended to introduce the reader of the Proceedings published in this issue, into the stimulating atmosphere of the Symposium, by summarizing some of the exciting proposals, views, ideas and open questions which emerged from presentations and multi-disciplinary, plenary discussions.

Detection of neutral active phosphatidylcholine-specific phospholipase C in Friend leukemia cells before and after erythroid differentiation.

With respect to normal tissues, 31P NMR spectra of tumors usually exhibit elevated phosphomonoester (PME) and phosphodiester (PDE) signals, arising from phospholipid metabolites such as phosphocholine (PCho) and glycerophosphocholine (GroPCho) (and/or ethanolamine analogues). PME and PDE resonances may undergo significant alterations during tumor growth, at early stages of tumor response to treatment or following cell differentiation and maturation. The enzymatic mechanisms which regulate these alterations are scarcely understood. Recent studies on agonist-induced phosphatidylcholine (PC) hydrolysis by PC-specific phospholipase C (PC-plc) in cells stimulated by hormones or growth factors suggest the hypothesis that repeated transient activations of this enzyme may also contribute to the elevation of PCho levels in tumor NMR spectra. This paper reports the first direct evidence on neutral active PC-plc activity in a tumour cell system, Friend leukemia cells, either in the undifferentiated (FLC) or differentiated state (dFLC). Cell homogenates were incubated in the presence of mixed diheptanoylphosphatidylcholine/sphingomyelin unilamellar vesicles (SLUV), which were previously shown to represent a good substrate for bacterial plc. 31P NMR analyses allowed the simultaneous detection and quantification of phosphorylated metabolites produced in tumor cell homogenates by PC-plc activity, as well by enzymes active in the PC deacylation pathway. With respect to FLC, dFLC homogenates exhibited higher PC-plc activity and lower accumulation of a deacylation product, GroPCho, in agreement with the elevation in the [PCho]/[GroPCho] ratio, already reported in 31P NMR spectra of intact differentiated cells. The direct detection of PC-plc in this cell system opens novel biochemical interpretations on a series of oncological observations, such as a) transient increases in the levels of PCho and PC-derived diacylglycerols reported in immature or in transformed cells in response to agonist-receptor interactions and b) accumulation of mobile lipids in tumor cell membranes and tissues.

Differentiation of glioblastoma multiforme from astrocytomas by in vitro 1H MRS analysis of human brain tumors.

In vitro high resolution 1H NMR spectroscopy allows non-invasive metabolic evaluation of specimens derived from surgically biopsied or resected brain tumors, with the aim of identifying potential markers of different malignancy grading, and improving diagnostic and therapeutic strategies. In the present study we evaluated 36 patients affected by different brain gliomas (7 well differentiated astrocytomas, 7 anaplastic astrocytomas, 16 glioblastomas, 6 oligodendrogliomas). These analyses allowed discrimination between well differentiated and anaplastic astrocytomas (AII + AA) and glioblastoma multiforme (GM) samples on the basis of the ratio between the integrated choline-containing resonance (b"Cho") and the creatine peaks (creatine (Cr) + Phosphocreatine (PCr)). While no definite difference was found between AII and AA, significantly higher values were observed for this ratio in GM. Other signals, derived from different metabolites, such as Glycine (Gly) and N-acetyl-aspartate (NAA), may also assume relevance in differential tumor diagnosis. In this study an increased [Gly]/[Cr + PCr] ratio was observed in GM with respect to AII and AA. The NAA levels observed in our tumor specimens may be explained on the basis of tumor cell infiltration into brain adjacent tissue. Interesting, but inconclusive, are the data concerning oligodendrogliomas, which, also in well differentiated forms, exhibit increased levels of b"Cho"/(Cr + PCr) ratio. The present study confirms the role of MRS in the biochemical characterization of neoplastic brain tissue and its potential contribution to a better selection of multidisciplinary treatment strategies.