Tài liệu Báo cáo khoa học: Characterization of 1H NMR detectable mobile lipids in cells from human adenocarcinomas doc

Characterization of 1 H NMR detectable mobile lipids in cells from human adenocarcinomas Anna Maria Luciani 1 , Sveva Grande 1 , Alessandra Palma 1 , Antonella Rosi 1 , Claudio Giovannini 2 , Orazio Sapora 3 , Vincenza Viti 1 and Laura Guidoni 1 1 Dipartimento di Tecnologie e Salute and INFN Gruppo Collegato Sanita ` , Istituto Superiore di Sanita ` , Rome, Italy 2 Dipartimento di Sanita ` Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanita ` , Rome, Italy 3 Dipartimento di Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanita ` , Rome, Italy Among the different molecules showing intense and narrow peaks in the 1 H magnetic resonance spectra, much attention has been devoted to the fatty acid sig- nals from mobile lipids (MLs), which are characterized by high mobility and, thus, differently from most cell lipids, are visible in high resolution magnetic resonance spectra. High-intensity MLs are often observed in pro- liferating cells and in tumour cells [1–4]. Many studies have found that the onset of apoptosis is accompanied by an increase in ML intensity [5–7], although other studies have not [8,9]. A number of studies have been performed in view of the possible use of MLs as spectroscopic markers of cell fate, although a clear explanation of their behav- iour has not yet been provided. Essentially, two differ- ent localizations have been proposed. In mammalian cells, ML resonances arise from lipids that are either present as microdomains with high mobility embedded in the plasma membrane bilayer [9] or exist in cytosolic lipid droplets, mostly consisting of triglycerides (TGs) [10,11]. Some studies have found that the concentra- tion of total cell TGs was consistent with the intensity of ML signals [11], whereas, more recently, changes in the size of lipid droplets were suggested [12]. In the present study, we examined the 1 H NMR ML signals of cultured tumour cells, specifically HeLa and Keywords cell cycle; cell metabolism; lipids; magnetic resonance spectroscopy; tumour cell lines Correspondence L. Guidoni, Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanita ` , 00161 Rome, Italy Fax: +39 06 4938 7075 Tel: +39 06 4990 2804 E-mail: guidoni@iss.it (Received 5 November 2008, revised 15 December 2008, accepted 22 December 2008) doi:10.1111/j.1742-4658.2009.06869.x Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti-cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the rela- tionship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub-G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation-induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes. Abbreviations GPC, glycerophosphorylcholine; IR, intensity ratio; ML, mobile lipid; PC, phosporylcholine; PCA, perchloric acid; PL, phospholipids; TG, triglycerides. FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1333 MCF-7 cells from human cancers. In a previous study [13], we demonstrated that these cells display intensity modulation of ML signals as a periodic event accom- panying cell growth. In a recent study [14], we also showed that these cells are differently prone to apopto- sis induced by treatment with gamma rays. We associ- ated the radiation-induced apoptosis of tumour cells with the level of reduced glutathione, as detected by 1 H NMR [14]. These cells were therefore considered for use in mag- netic resonance spectroscopy analysis for the detection of possible different trends in ML spectral features during cell growth in culture and after radiation- induced arrest in proliferation. Within this framework, a similar modulation of ML signals with growth was observed in both cell lines, whereas radiation-induced arrest in proliferation capacity resulted in a different pattern. Effects on cell cycle frequency were also observed. ML signal intensity modulation was tenta- tively related to modulation of lipid metabolism. Results Cell spectra were first examined for signal quantifica- tion after spectral assignment. Subsequently, changes in signal modulation were monitored when cell growth was arrested by means of treatment with ionizing radi- ation. Finally, a model to fit signal intensity modula- tion was proposed. Analysis of 1 H NMR spectra – spectral assignments and quantification Both cell lines displayed very similar spectral features. Besides other signals, the characteristic peaks from MLs were observed to be in agreement with the data available in the literature for cancer cells [1–4]. Under the present experimental conditions, the intense ML signals can be attributed to the fatty acid chains of neutral lipids, mostly TGs, in agreement with the data available in the literature [1,15] and on the basis of our previous observations showing that, in these cells, TG peaks are more intense in the lipid extract spectra derived from cells with high ML signals compared to spectra with low ML signals [13]. This point will be discussed further below. Figure 1 shows an example of the 1 H NMR spectra from MCF-7 cells at day 3 (Fig. 1A) and at day 6 (Fig. 1A¢) after seeding. It is worth noting that the choline-based signals at 3.2 p.p.m. display the same intensity with very different ML signals (Fig. 1A,A¢). When the ML signals were intense in the 1D spectra (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) M1 + ML1 A A′ B′ B Fig. 1. Spectra of MCF-7 cells in different proliferative conditions: 1D 1 H NMR spectra from cells at day 3 after seeding (A) and at day 6 after seeding (A¢). ML signals are at 0.89 p.p.m. (ML1), 1.28 p.p.m. (ML2) and 1.55 p.p.m. (ML3); M1 and M2 from macromolecules are also labelled. 2D 1 H NMR COSY spectra of the same cell samples at day 3 after seeding (B) and at day 6 after seeding (B¢). Cross peak A is from terminal methyl and bulk methylene coupling in ML. The reference cross peak from Lys is also labelled. Label T refers to the glycerol cross peak of triglycerides. The insert shows the glycerol cross peak region of the geminal protons of triglycerides. 1 H NMR of mobile lipids in tumour cells A. M. Luciani et al. 1334 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS (Fig. 1A), the corresponding 2D COSY spectra were also characterized by prominent cross peaks from fatty acid chains (Fig. 1B), in agreement with the data avail- able in the literature [15] and previous observations [13]. The cross peak at 4.07–4.24 p.p.m., generated by protons in the glycerol backbone of TG, was also visi- ble only in ML rich spectra (T). Details with respect to this signal are provided in Fig. 1B (insert), which shows the characteristic cross peak from the geminal protons of carbons 1 and 3 of glycerol in TG [1]. Cross peaks of lipids, including the T signal, were absent in cells characterized by low ML signals in 1D spectra (Fig. 1B¢). Very similar behaviour was observed in HeLa cells according to previously reported data [4,13]. Signal assignments in cell spectra were performed after comparison with the spectra from lipid and per- chloric acid (PCA) extracts, derived from cell samples grown and harvested under similar conditions, and with compound spectra. Assignments from the litera- ture were also taken into account [1–4,16]. Figure 2 shows typical spectra (1D and 2D COSY) from lipid and PCA extracts both relative to a MCF-7 cell sample with high MLs. It is worth noting that, in the 2D COSY spectra from extracted lipids, the glycerol geminal cross peaks of 1,3-glycerol protons of TG and of 1-glycerol protons for phospholipids (PL) are clearly separated, in agreement with the data available in the literature [1]. For peak assignments and intensity quantification, deconvolution of 1D spectra and integration of 2D cross peaks was performed as described in the Experi- mental procedures. The signal intensity refers to the peak area in the 1D spectra and to the cross peak inte- gral in the 2D spectra. Internal reference signals were used. Our study compared samples where cell volumes and cell packing change, which hinders the use of an external reference. The signal at 0.96 p.p.m., present in cells (peak M2 in Fig. 1) and PCA extract spectra, derives from poly- peptide chains and was used as the intensity reference for 1D spectra because it is indicative of cell mass. As far as the intensity reference for 2D spectra is con- cerned, the sum of peaks of Lys at 1.70–3.00 p.p.m. and Ala at 1.48–3.77 p.p.m. (Fig. 1B) was chosen as the area reference in the 2D COSY spectra. (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) (p.p.m.) A A′ B B′ Fig. 2. 1D 1 H NMR spectra of extracted lipids (A) and PCA extracts (A¢) from one representative sample of MCF-7 cells; 2D 1 H NMR spectra of extracted lipids (B) and PCA extracts (B¢) from one representative sample of MCF-7 cells. The insert shows the glycerol cross peak region of the geminal protons of TGs and PLs. A. M. Luciani et al. 1 H NMR of mobile lipids in tumour cells FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1335 For 1D spectra, deconvolution of spectra of the type shown in Fig. 1A¢, with ML signals of a very low intensity, was performed as a first step. Deconvolution of spectra of the type shown in Fig. 1A, with intense ML signals, was then performed starting from the lines and parameters previously found, by adding the signals from MLs. A typical deconvolution pattern with the used resonances is shown in Fig. 3A. The correspond- ing parameters are given in Table 1. Experiments con- ducted on different samples derived from the same culture (at least three samples) demonstrated that the SD from this procedure did not exceed 0.01 p.p.m. for chemical shifts and 10% for linewidths and intensity ratios (IRs). On the other hand, the variability of signal intensities, especially for MLs, exceeded the measurement error in spectra from samples derived from different culture, even when cells were harvested under similar growth conditions. For this reason, the spectral behaviour over time was compared among samples obtained by cells harvested at different days from the same seeding. Cholesterol peaks were present in lipid extracts at 0.70 and 1.03 p.p.m. in the 1D spectra (Fig. 2A) and with the typical cross peak at 0.87–1.50 p.p.m. in the 2D spectra (Fig. 2B). In spectra from cell samples, we could therefore assign the peak at 0.71 p.p.m. (Fig. 3A and Table 1) to the methyl group in C18 of choles- terol. This signal was present in all cell spectra, although it was broader and more intense when ML signals were present, and its IR changed from 0.07 to 0.36 as obtained by 1D spectra deconvolution. In par- allel, peaks at 1.03 and 1.50 p.p.m., as obtained from 1D spectra deconvolution (Fig. 3A and Table 1), increased, and the cross peak at 0.87–1.50 p.p.m. appeared in the cell spectra. This latter feature is evident in Fig. 1A¢,B¢. The peak at 1.03 p.p.m. could therefore be attributed to the methyl group in C19 and (p.p.m.) (p.p.m.) (p.p.m.) A B Fig. 3. Example of the deconvolution pattern of the methylene region in the 1D 1 H NMR spectra of MCF-7 cells (A); integration regions for selected cross peaks in the 2D 1 H NMR spectra of MCF-7 cells (B) (Lys1, lysine; A, bulk methylene and terminal methyl group in fatty acids; E, C4 methylene and C3 methylene in fatty acids; Lino, linolenic acid). Other ML related cross peaks are visible as peaks B and F; for assignments, see [2]. Rectangles indi- cate the area used for signal integration. Table 1. Mean values of parameters d (p.p.m.), Dm (Hz) and IR, after deconvolution (1D) and integration (2D COSY) of spectra, such as those presented in Fig. 3. Values were obtained from the spec- tra of three different samples derived from the same culture. The standard deviation was 0.01 p.p.m. for chemical shift values and 10% for linewidths and IR. Chemical shifts are referring to lactate methyl; IR are calculated with respect to M2 in 1D and to Lys1 + Ala in the 2D spectra. 1D d (p.p.m.) Dm (Hz) IR Chol 0.71 40 0.15 ML1 + M1 + Chol 0.89 24 0.16 M2 0.95 28 1.00 Chol 1.03 20 0.05 M3 1.22 46 0.14 ML2 1.28 17 3.26 ML3 1.31 23 2.70 LAC 1 1.32 8.3 0.58 LAC 2 1.34 7.1 0.67 M5 1.40 36 1.30 M6 + Chol 1.48 22 0.13 ML4 1.58 38 0.89 M7 1.70 39 0.55 Broad 2.20 870 3.10 2D d (p.p.m.) IR Lys1 1.70–3.00 1.00 Lys2 1.46–1.67 0.28 A 0.89–1.27 4.50 E 1.31–1.55 2.20 B 1.34–2.00 2.70 F 1.58–2.22 2.47 Lino 0.96–2.03 0.72 Chol 0.87–1.51 0.24 1 H NMR of mobile lipids in tumour cells A. M. Luciani et al. 1336 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS the peak at 1.50 p.p.m. to the other bulk protons of cholesterol. As far as MLs are concerned, spectra with intense ML signals could be fitted by adding a peak at 1.28 p.p.m., a second peak at 1.31 p.p.m. and a peak at 1.58 p.p.m. (Table 1 and Fig. 3A) to the spectral fitting of samples without ML signals. The presence of these peaks was paralleled by the existence of lipid cross peaks in the 2D spectra (Table 1 and Fig. 3B). The cross peak A, due to the interaction of terminal methyl group peak at 0.89 p.p.m. and the proximal methylene at 1.28 p.p.m., was used to quantify MLs because it is representative of the corresponding bulk fatty acids chains. This excludes the contribution from x-3 fatty acids, where methyl protons at 0.98 p.p.m. are coupled to the allylic methylene at 2.09 p.p.m. [16]. The signal at 0.88 p.p.m. is mostly from the terminal CH 3 of ML chains, with a minor contribution from the cholesterol C25 and C26 methyl groups, and from an unidentified macromolecule methyl group (M1). This signal is present: (a) in PCA extracts (Fig. 2A¢) and (B) in cells when ML signals are absent (Fig. 1A,B). The same considerations hold for the peaks at 1.22 and 1.40 p.p.m. (Table 1 and Figs 2 and 3). These signals most likely arise from the aggregation of large molecules because they are characterized by large linewidths (Table 1 and Figs 2 and 3). Work is in progress to clarify the nature of these structures. Signal intensities were also measured in the 2D spec- tra. A typical 2D COSY spectrum is shown in Fig. 3b, including the details of the cross peaks examined. The peaks chosen for evaluation are framed with rectangles that denote the areas used for volume integration. When very intense ML signals are present in the spec- tra, the signals related to unsaturated fatty acids also are evident, and are more intense in MCF-7 than in HeLa cell samples. Besides the cross peaks resulting from the connectivity of the vinyl protons (at 5.35 p.p.m.) to the allylic protons (at 2.05 p.p.m.) in monounsaturated chains and to the bis-allylic protons (at 2.80 p.p.m.) in polyunsaturated fatty acids (not shown), the cross peaks at 1.64–2.09 p.p.m. and 1.68– 2.24 p.p.m., attributed to arachidonic acid chains, and at 0.93–2.04 p.p.m., attributed to linolenic acid chains on the basis of a comparison with lipid extracts and from the data available in the literature, are also clearly visible in Fig. 1B,B¢. Experiments on different samples derived from the same culture (at least three samples) were also exam- ined to assess measurement errors in the 2D cross peak integration. Under these conditions, errors on cross peak volumes did not exceed 10%, whereas the vari- ability of integral values exceeded this error in the spectra from samples derived from different cultures, even when cells were harvested under similar growth conditions. For this reason, the behaviour over time of 2D COSY cell spectra were compared in samples obtained by cells harvested at different days from the same seeding. In the following, ML quantification derives from intensity measurements of the peak at 1.28 p.p.m. in 1D spectra and from the integral of cross peak A at 0.89–1.28 p.p.m Cross peaks from PL glycerols (Fig. 2B, insert) were never observed in the 2D COSY spectra of cells, even in the presence of very high ML signals in the 1D spectra. Cross peaks from glycerol protons of TG in cells were not routinely used for TG quantification because the intensities were much smal- ler and the errors were larger. ML signals were then used to monitor TG levels in cells. Biological changes and ML signal modulation with growth and in growth-arrested cells Changes in ML signals were monitored in parallel with cell growth and after cell cycle arrest due to irradiation. Cell proliferation and cell cycle Cell growth behaviour was examined in HeLa and MCF-7 cells. Cells were routinely grown as described in the Experimental procedures. Cells were sampled at different days after seeding for both NMR experiments and cell cycle measurements. Under the chosen experi- mental conditions, cells were kept in the exponential growth phase (up to 3 days from seeding). Cells were then irradiated with a single dose of 20 Gy (gamma irradiation) to provide growth arrest and cell death with different characteristics in the two cell lines, according to previous observations [14]. Figure 4 shows the cell counts as a function of time for one representative experiment in HeLa and MCF-7 cells. Compared to control samples, the differences in cell counts were larger in HeLa than in MCF-7 cells. Similar behaviour was observed in at least three independent experiments. To assess cellular transcriptional responses to radia- tion-induced DNA damage, we examined cell cycle arrest in MCF-7 and HeLa cells at 1, 2 and 3 days after treatment. Both cell lines underwent cycle arrest upon irradiation, with different characteristics. Figure 4A¢,B¢ shows the percentage of cell phases of both cell lines observed after 2 days after irradiation at 20 Gy. Although both cells were blocked in G2 ⁄ M, HeLa cells displayed a remarkable decrease in the A. M. Luciani et al. 1 H NMR of mobile lipids in tumour cells FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1337 G1 phase, whereas MCF-7 cells showed G1 block and a decreased percentage in the S phase compared to control samples. Irradiated HeLa cells showed an intense sub-G1 peak (> 20%), indicating DNA frag- mentation and the occurrence of significant apoptosis. This observation is in agreement with previous data obtained in the same cells by monitoring apoptosis as the externalization of phosphatidyserine [14]. 1 H NMR ML signals in intact cells and TG signals in lipid extracts To clarify whether intensity changes of ML signals were mainly related to changes in TG concentration, to differences in chain mobility due to structural changes, or to the different size of droplets, as recently suggested by Quintero et al. [12], we compared the behaviour of the spectra of cells and the total lipid extracted from cells. Irradiation was then used to arrest cell growth, thus providing a modification of ML signal intensity in cells. Lipids were extracted from cell samples under identical conditions. In previous experiments on these cell lines [4], we observed an intensity modulation of ML with cell growth. Consequently, the extent of the variation in intensity with irradiation was monitored after different time intervals after irradiation. Figure 5A,B shows the ML signal intensities of 1D spectra from the two cell lines run at different days. Samples of cells grown under the same conditions were irradiated and the two sets of spectra compared (control and treated samples). Similar data were obtained for 2D measurements (Fig. 5A¢,B¢), where the data are from one representa- tive experiment. Error bars indicate measurement errors. By comparing the results of at least seven inde- pendent experiments, the differences between control and irradiated samples were significant for MCF-7 cells at all time intervals examined. On the other hand, statistical significance for HeLa samples was observed at days 2 and 3 after irradiation. Figure 5C shows these differences for samples examined 2 days after irradiation. Figure 6 shows the glycerol region from two repre- sentative 1D spectra of total lipids extracted from control and irradiated HeLa (Fig. 6A,A¢) and MCF-7 (Fig. 6B,B¢) cells. A significant change in relative inten- sity of TGs (glycerol proton centred at 4.32 p.p.m.) versus PLs (glycerol proton centred at 4.42 p.p.m.) was found in irradiated samples compared to controls. Particularly, TG signals were depressed in HeLa cells (Fig. 6A,A¢), whereas an increase was evident in MCF-7 cells (Fig. 6B,B¢). Deconvolution of 1D spectra was performed to provide the relative intensities of TG versus PL, which were calculated on sn-1 and sn-3 glycerol signals centred at 4.32 p.p.m. for TG and sn-1 glycerol signals at 4.42 p.p.m. for PL (Fig. 6C). In this experiment, the calculated relative concentration of TG versus TG + PL was 15% and 12% in MCF-7 A A′ B B′ Fig. 4. Number of HeLa (A) and MCF-7 (B) cells (N) as a function of time after irradiation for both control (h) and irradiated ( ) samples. The solid black line is the fit with an exponential function. Percentage of MCF-7 (A¢) and HeLa (B¢) cells in the different cell cycle phases, measured 2 days after irradiation. One representative experiment is reported for both control and irradiated samples. 1 H NMR of mobile lipids in tumour cells A. M. Luciani et al. 1338 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS and HeLa cells, becoming 20% and 8%, respectively, after irradiation. The standard deviation in repeated calculations was 2%. The relative concentrations of TG calculated by 1 H NMR were found to be in agree- ment with that reported in previous studies [17]. Intensity measurements of 1D signals of sn-2 glycerol protons of TG at 5.29 p.p.m. and PL at 5.25 p.p.m., clearly resolved only at 700 MHz, gave similar results (not shown). This behaviour is similar to that observed for ML signals in whole cells (Fig. 5). Total extracted TG may be more abundant with respect to NMR visible TG in cells. For this reason, the spectra from extracted lipids were not used to calculate NMR visible TG in intact cells. Lipid metabolites from PCA extracts To provide further information on lipid metabolism, the PCA extracts were also analyzed. The region of choline metabolites around 3.2 p.p.m. is shown in Fig. 7 for PCA extracts of HeLa. Mean values of parameters d (p.p.m.), Dm (Hz) and IR were obtained from deconvolution of the 1D spectra (three different samples derived from the same culture) of PCA extracts and are reported in Table 2. The standard deviation was 0.005 p.p.m. for chemical shift values and 10% for linewidths and IR. Signals from the headgroups of glycerophosphoryl- choline (GPC) at 3.24 p.p.m., phosporylcholine (PC) at 3.23 p.p.m. and choline at 3.21 p.p.m. showed dif- ferent behaviour in the two cell lines after irradiation. In particular, more relevant changes in GPC ⁄ PC ratios were observed in irradiated MCF-7 cells (Fig. 7B,B¢) with respect to HeLa cells (Fig. 7A,A¢), indicating the different equilibrium of catabolism versus anabolism. Table 3 reports on the intensity of changes of the choline-based metabolites for the two cell lines after irradiation resulting from fittings of at least three different spectra. Time (day) Time (day) Time (day) Time (day) A/(Lys + Ala) c A/(Lys + Ala) i A B A′ B′ C Fig. 5. Intensity modulation of ML signals from a representative experiment (1D and 2D 1 H NMR data) for HeLa (A, A¢) and MCF-7 cells (B, B¢). Spectra were acquired at different days from seeding for both control (h) and irradiated ( ) samples (D = 20 Gy). Errors obtained from spectral fitting (1D) and integration (2D COSY) are contained within the symbols. (C) Relative IRs ML ⁄ M (control: white; irradiated: black) and A ⁄ (Lys + Ala) (controls: dotted white; irradiated: dotted black) as obtained from 1D and 2D COSY spectra of HeLa and MCF-7 cell samples. Data are the mean ± SD values of seven independent experiments. Spectra were acquired on day 5 after seed- ing and 2 days after irradiation with a single dose of 20 Gy. *P < 0.05 (t-test). A. M. Luciani et al. 1 H NMR of mobile lipids in tumour cells FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1339 Model for the ML signals To find a possible explanation for the experimental data, a model for ML intensity modulation is pro- posed. As previously reported [4,13] growth of MCF-7 and HeLa cells slows down as cells approach confluence. This finding is in agreement with the observed increase of the G1 phase in the final days in culture (Fig. 4 A¢,B¢). It is reasonable to assume that cell metabolism, including PL synthesis, slows down accordingly. On the other hand, intensities of ML (in cells) signals, mainly due to TG according to a previous study [13] as well as the present study (compare inserts in Figs 1 and 2), are characterized by a nonlinear behaviour over time in culture (Fig. 5). A mechanism that is more complicated than a simple decrease of lipid production over time must be therefore envisaged. There is a growing body of evidence indicating that lipid metabolism possesses an articulated role with respect to maintaining cell equilibrium, which takes into consideration both PL synthesis ⁄ breakdown and TG metabolism [18–20]. We may infer that there are two mechanisms inside the cell: one relative to the pro- duction of ML (and TG) with a rate constant R p and one relative to the consumption of ML (and TG) with a rate constant R c . The signal that we observe in the NMR spectra is due to the net accumulation of reserve lipids and its rate, dML ⁄ dt, is given by the difference of the rate of lipid production R p and the rate of lipid consumption R c : dML=dt ¼R p ÀR c ð1Þ We may assume that both rates R p and R c are not constant, but decrease over time in culture as a conse- quence of cell proliferation slowing down. A linear dependence of both rates can be assumed: R p ¼p 1 Àp 2 t ð2Þ R c ¼c 1 Àc 2 t ð3Þ where p 1 and c 1 are the production and consumption rates at time = 0, respectively, and p 2 and c 2 are the changes of production and consumption rates over time. By integrating Eqn (1), we obtain a second degree polynomial function for the lipid accumulation: MLðtÞ¼m 1 t 2 þm 2 tþm 3 ð4Þ where m 1 =(c 2 ) p 2 ) ⁄ 2, m 2 =(p 1 ) c 1 ) and therefore are related to the equilibrium between lipid production and consumption, and m 3 is the starting ML value. The best fit with Eqn (4) of data ML ⁄ M and A ⁄ (Lys + Ala) versus time from independent experi- ments on HeLa and MCF-7 cells gave a chi-square value that was always < 1 for both the 1D and 2D COSY experiments. Figure 5A,A¢,B,B¢ shows these fittings for a representative experiment for both cell lines. Figure 8 reports the parameters m 1 , m 2 and m 3 (mean ± SD) as obtained from fittings with Eqn (4) for the data obtained from ten samples for HeLa and ten samples for MCF-7 of cells harvested irrespective of the growth phase. (p.p.m.) (p.p.m.) (p.p.m.) A′ B′ A B C Fig. 6. Glycerol region of representative 1D 1 H NMR spectra from total lipids extracted from HeLa (A, A¢) and MCF-7 cells (B, B¢). Lower traces: control samples (A, B), upper traces: irradiated (D = 20 Gy) samples (A¢,B¢). Spectra were acquired 2 days after irradiation. Example deconvolution pattern (C) of 1D 1 H NMR spectrum for sn-2 glycerol protons of TGs (couple of doublets centered at 4.32 p.p.m., coupling 12 Hz and 4 Hz) and PLs (couple of doublets centered at 4.42 p.p.m., coupling 12 and 3 Hz). Accord- ing to this deconvolution pattern, in this spectrum, the ratio TG ⁄ (PL + TG) was 0.34. 1 H NMR of mobile lipids in tumour cells A. M. Luciani et al. 1340 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS The minimum value t m of the parabolic fitting curve, representing the time value for which production and consumption rates are equal, and the corresponding lipid value ML m are also reported. 1D and 2D data were in good agreement, although the starting intensity values and values at the minimum were different, reflecting different pools of ML inside the cells. By changing the seeding density, the minimum of the curve was shifted, but the shape of the curves did not change. It is worth noting that m 1 was always positive (i.e. c 2 was always greater than p 2 ), whereas m 2 was always negative (i.e. c 1 was always greater than p 1 ). For m 1 > 0, the parabola was concave upward. For m 2 < 0, the minimum was after time zero. (p.p.m.) (p.p.m.) (p.p.m.)(p.p.m.) A′ B′ AB C C′ Fig. 7. 1D 1 H NMR spectra of the choline- related metabolites region from PCA extracts of HeLa (A, A¢) and MCF-7 cells (B, B¢). Lower traces: control samples (A, B), upper traces (A¢,B¢): irradiated samples (D = 20 Gy). Spectra were acquired 2 days after irradiation. Deconvolution pattern of the same region (C) and of the reference signal (C¢). Table 2. Mean values of parameters d (p.p.m.), Dm (Hz) and IR after deconvolution of 1D spectra from PCA extracts of HeLa cells (Fig. 7C,C¢). Values were obtained from the spectra of three differ- ent samples derived from the same culture. The standard deviation was 0.005 p.p.m. for chemical shift values and 10% for linewidths and IR. D (p.p.m.) Dm (Hz) IR M (reference) 0.939 25.0 1.00 Choline 3.219 1.88 0.45 PC 3.226 2.20 0.70 GPC 3.236 1.65 0.53 Table 3. Mean values (three independent experiments) of IRs of the choline-related metabolites from PCA extracts of HeLa and MCF-7 cells for control and irradiated samples. The standard devia- tion was 10%. GPC PC Cho HeLacells Controlsample 0.43 0.71 0.50 Irradiated sample 0.28 0.86 0.72 MCF-7 cells Control sample 1.12 1.31 0.60 Irradiated sample 0.90 0.77 0.63 A. M. Luciani et al. 1 H NMR of mobile lipids in tumour cells FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS 1341 Although the t-test showed that differences were not significant between the two groups (HeLa and MCF- 7), m 1 values were generally higher in MCF-7 than in HeLa, thus reflecting the tendency for higher final values of ML signal intensities in MCF-7 cells (Fig. 8). Cells were then irradiated to arrest cell growth; data from irradiated cells could be still fitted through Eqn (4), as shown in Fig. 5. Figure 9 shows the parameters (mean ± SD) obtained by fitting the data from five independent experiments on HeLa cells and five inde- pendent experiments on MCF-7 cells (both irradiated and non-irradiated cells). The parameter m 1 decreased and m 2 increased after irradiation in both cell lines (Fig. 9), but only the m 2 increase in MCF-7 cells was statistically significant in independent experiments (P < 0.05; t-test). This may be due either to an increase in c 1 (consumption rate at time = 0) or a decrease in p 1 (production rate at time = 0). The relevant m 2 increase in MCF-7 cells produced the great increase of ML signals with respect to controls (Fig. 5B,B¢). In some experiments conducted on MCF-7 cells, the minimum of the parabolic curve at time > 0 was no more evident and m 2 became posi- tive. Finally, t m shifted to lower values in MCF-7 cells and the ML m value was considerably higher in irradiated MCF-7 cells compared to controls. Discussion The appearance of intense signals from bulk methylene of fatty acid chains in high resolution 1 H NMR spec- tra of cells has been studied subsequent to the first observations being made in cancer cells, lymphocytes and developing cells. On the other hand, a correlation of the intensity of these signals with metabolic parame- ters is not straightforward. Some studies noted that the signal intensity of bulk methylene is influenced by cell proliferation, as T lymphocyte activation [21] and in tumour cells by the different proliferation state [4,22]; other studies found that the onset of apoptosis correlates with the increase of lipid signals, whereas others did not [5–9]. Finally, some studies found that these signals can be affected by extreme pH conditions, which is more likely due to the effects of low pH on cell proliferation [22]. In the present study, ionizing radiation was used to affect cell growth and induce cell death in cells show- ing different attitudes with respect to undergoing radi- ation-induced apoptosis. The relevant sub-G1 peak observed in the cell cycle profile of radiation-arrested HeLa cells (Fig. 4A¢) points to significant apoptosis, in agreement with the previously observed radiation- induced apoptosis determined by phosphatidylserine 5.0 0.0 –5.0 – 10.0 5.0 0.0 –5.0 Fig. 8. Parameters m 1 , m 2 and m 3 obtained from fitting with Eqn (4) ML data, as in Fig. 5. The minimum values (t m ) of the para- bolic fitting curve and the corresponding lipid values (ML m ) are also reported. Bars represent the mean ± SD of ten indepen- dent experiments for each cell line. 5 0 –5 – 10 5 0 –5 5 0 –5 5 0 –5 – 10 Fig. 9. Parameters m 1 , m 2 and m 3 obtained from fitting with Eqn (4) ML data, as in Fig. 5, for irradiated (I) and non-irradiated (C) HeLa and MCF-7 cells. The minimum values (t m ) of the parabolic fitting curve and the corresponding lipid values (ML m ) are also reported. Bars represent the mean ± SD of five independent experiments on each cell line. *P < 0.05 (t-test). 1 H NMR of mobile lipids in tumour cells A. M. Luciani et al. 1342 FEBS Journal 276 (2009) 1333–1346 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... compilation ª 2009 FEBS 1343 1 H NMR of mobile lipids in tumour cells A M Luciani et al where the relevant increase of m2 produces the relevant intensity changes of ML ⁄ TG signals In principle, such an increase can be due to both a decrease in c1 and an increase in p1 after growth arrest Because this parameter has a tendency to become positive in MCF-7 cells, we may infer that c1 tends to become lower... Viti V & Guidoni L (2004) 1H MRS studies of signals from mobile lipids and from lipid metabolites: comparison of the behaviour in cultured tumour cells and in spheroids NMR Biomed 17, 76–91 Rosi A, Grande S, Luciani AM, Palma A, Giovannini C, Guidoni L, Sapora O & Viti V (2007) Role of glutathione in apoptosis induced by radiation as determined by 1H MR spectra of cultured tumor cells Radiat Res 167, 268–282... arrest either in the G1 phase, due to the cell checkpoint mechanism under the control of p53 protein, or in G2 After a time delay that depends on the extent of the damage, cells may enter mitosis, resulting in normal cell duplication, cell mutation or cell death Inadequate DNA repair often results in apoptosis Lack of functional p53 in HeLa cells fails to induce cell cycle arrest in G1 Cells can only... apoptosis in cancer Eur J Radiol 56, 143–153 7 Milkevitch M, Shim H, Pilatus U, Pickup S, Wehrle JP, Samid D, Poptani H, Glickson JD & Delikatny EJ (2005) Increases in NMR- visible lipid and glycerophosphocholine during phenylbutyrate-induced apoptosis in human prostate cancer cells Biochim Biophys Acta 1734, 1–12 8 Santini MT, Romano R, Rainaldi G, Ferrante A, Motta A & Indovina PL (2006) Increases in 1H- NMR. .. extracted lipids showed two separated cross peaks from PL and TG (Fig 2B, H NMR of mobile lipids in tumour cells insert) We therefore attributed ML signals to neutral lipids, mostly TG, in agreement with the data available in the literature [1,13,15] Consequently, the observed ML variations were analysed in terms of production ⁄ consumption of TG In mammalian cells, TG may be deleted to meet the needs of. .. variations in intensity of the control and irradiated samples Student’s t-test works under the assumption H NMR of mobile lipids in tumour cells of a Gaussian distribution of data, but also works remarkably well for distributions that are not accurately Gaussian [27] Variations in intensity of the examined signals are not linear with time and ⁄ or cell proliferation [13] It is therefore necessary to examine... least in this cell line The effect was less intense in HeLa cells, probably due to the tendency of the S phase to remain high in this cell line, in contrast to MCF-7 cells (Fig 4A¢,B¢) where the S phase is low after irradiation Due to a net accumulation of PL synthesis in the S phase, if a reduced S phase is paralleled by a reduction of the final step of PL, TG accumulates, as observed in MCF-7 cells. .. presaturation, with a total of 16 scans for cells and typically 1000 scans for extracts The 2D raw matrix consisted of 512 complex points along the first dimension and 128 points along the second dimension A sine function was applied in both dimensions of the time domain before Fourier transformation Integration of 1D and 2D signals was performed using 1D winnmr and 2D winnmr software (Bruker, AG, Darmstadt,... the optimized sizes of the rectangles (areas of integration) were fixed and applied to all the spectra Mean ± SD values calculated from 1D deconvolution and 2D integration of at least five spectra of samples prepared from the same cell culture were used to assess the error on the single measurement Fitting of data to obtain model parameters was performed using origin software (OriginLab Corp., Northampton,... that these NMR signals are not specifically bound to the induction of apoptosis, in agreement with recent findings [8] Second, the intensity of the ML signal, corresponding to TG levels, is increased in irradiated MCF-7 cells, which have very low S phase In mammalian cells a net accumulation of PL occurs in the S phase: a relationship between a PL synthesis block, concomitant with a reduction of S phase, . Characterization of 1 H NMR detectable mobile lipids in cells from human adenocarcinomas Anna Maria Luciani 1 , Sveva. Santini MT, Romano R, Rainaldi G, Ferrante A, Motta A & Indovina PL (2006) Increases in 1 H -NMR A. M. Luciani et al. 1 H NMR of mobile lipids in tumour