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31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells
Biochimica etBiophysicaActa
930 (1987) 179-192
179
Elsevier BBA12056
3 1 p . N M R studies of p h o s p h a t e transfer rates in T 4 7 D h u m a n breast cancer cells M. Neeman
a, E. R u s h k i n a, A . M . K a y e b a n d H . D e g a n i a
Isotope and b Hormone Research Departments, The Weizmann Institute of Science, Rehovot (Israel)
(Received 13 November 1986) (Revised manuscript received23 March 1987)
Key words: NMR, 31p.; Phosphate transfer rate; (Mammary tumor)
The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31p-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC -, yATP (0.2 raM/s), Pi -' 7ATP (0.2 m M / s ) and the conversion flATP -~ flADP (1.3 raM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.I pmol/min per mg protein) and adenylate kinase (approx. 0.4/~mol/min per mg protein) suggest that the flATP --, flADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4- 10-3 m M / s ) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.
Introduction The use of cell cultures permits the study of a homogenous cell population which can be uniformly exposed to specific nutrients, hormones and drugs. N M R spectroscopy provides a noninvasive method for characterizing the in vivo metabolism of cultured cells.
Abbreviations: PC, phosphorylcholine; PE phosphorylethanolamine; PCr, phosphocreatine; NTP, nucleoside triphosphate; GPE, glycerol phosphorylethanolamine; GPC, glycerol phosphorylcholine; SSST, steady-state saturation transfer, Correspondence: H. Degani, Isotope Department, Weizman Institute of Science, Rehovot 76100, Israel.
Monitoring of cell cultures under physiological conditions by N M R spectroscopy requires a specialized apparatus for the maintenance of viable and active cells. Initially, N M R studies of cells were performed with packed cells oxygenated by direct bubbling of oxygen, frequently at low temperature [1]. This procedure can not be used for studies over a long period of time or for monitoring metabolic parameters such as enzymatic rates. Methods that were developed later for fixing the position of the cells in the N M R tube, thus allowing perifusion with medium, permitted the maintenance of the cells' metabolic activity under steady-state conditions for relatively long periods of time [1-7]. In most of the previous N M R studies of transformed cell cultures, 3ap-NMR was used to determine the composition and concentra-
0167-4889/87/$03.50 © 1987 Elsevier SciencePublishers B.V. (Biomedical Division)
180 tion of the phosphate metabolites under various conditions [1-8]. Recently, the phosphates of MCF-7 human breast cancer cells were characterized using 31p-NMR spectroscopy [3]. Here we present 3ap-NMR studies of intact T47D human breast cancer cells maintained under physiological conditions. The emphasis in these studies was on characterizing the high-energy phosphates and on determining the kinetics of their interconversion. The kinetic measurements required development of magnetization transfer techniques which provides kinetic data for several phosphate transfer reactions within the time available for such in vivo N M R studies. 31p-NMR kinetic studies of ATP synthesis from Pi and of the creatine kinase reaction were previously employed in several systems, such as yeast cells [9-11], skeletal muscle, heart, kidney and brain [12-18]. The methods used include saturation transfer, inversion transfer and two-dimensional exchange type experiments [12-22]. H u m a n breast cancer cells differ in their metabolism and energetics from the systems mentioned above. In general, these cells resemble their cells of origin in the m a m m a r y epithelium; several fines retain receptors for steroid hormones and serve as model systems for studying hormonal responses [23,24]. Our studies were performed with cells of clone 11 from the T47D human breast cancer cell line [25,26] which contain estrogen and progesterone receptors; their growth rate is enhanced by 17/3estradiol and ifihibited by antiestrogens such as tamoxifen [27]. The experiments were complemented by biochemical studies which, with the N M R data, made it possible to characterize the energetics of this human breast cancer line. Materials
penicillin ~g/ml).
(100 u n i t s / m l )
and
mycostatin (25
Preparation of cells for NMR experiments. Approx. 5 • 10 s cells were harvested by trypsinization, when monolayers were nearly confluent. The cells were washed twice with medium and then embedded in 0.86-mm thick agarose filaments and extruded into a 10-mm N M R tube, according to the method of Foxall and Cohen [4,5]. Perifusion of the embedded cells with either R P M I or Dubelco's modified Minimal Essential Medium was initiated simultaneously with the preparation of the agarose filaments and was continued throughout the N M R measurements. The optimal rate of perifusion with medium saturated with 95% oxygen/5% CO 2 was found to be 4 m l / m i n . The medium and the N M R tube were kept at 35 ° C. The perifusion system consisted of a line which transferred the medium to the bottom of the N M R tube, and a return line placed about 10 95%02 S*/,co~
Ustd medium
and Methods
Cell line. Clone 11 of the T47D human breast cancer cell line was obtained from Y. Keydar (Tel Aviv University). The T47D fine w a s e s t a b l i s h e d from the pleural effusion of a patient with infiltrating duct carcinoma [25]. Several clones including clone 11, which showed estrogen-stimulated growth were isolated [26]. The cells were grown in monolayer culture using R P M I 1640 medium supplemented with fetal calf serum (10%), glutamine (2 mM), streptomycin (100 /Lg/ml),
Sponge fiiter~ Cells
in
agarose fitaments
Fig. 1. Scheme of cell perifusion system. Medium was maintained in a temperature-controlled vessel and was saturated with oxygen by bubbling with 95% 02/5% CO2. A peristaltic pump was used to pump the medium into the NMR tube at a rate of 4 ml/min. Overflow of medium in the NMR tube was prevented by pumping out the medium with a return line placed 10 cm above the bottom of the tube.
181 cm above the bottom (Fig. 1). During the magnetization transfer experiments the cells were perifused as described above with a phosphate-free Dulbecco's modified Minimal Essential Medium. NMR experiments. 31P-NMR spectra were recorded at 121.5 MHz by a Bruker CXP-300 spectrometer, using either 60 ° pulses and a relaxation delay of 1 s or 90 ° pulses and a relaxation delay of 7 s. The steady-state saturation transfer (SSST) experiment was performed using a selective DANTE pulse sequence [28] followed by a 90 ° observation pulse. The application of a DANTE sequence for selective saturation is possible on most commercial spectrometers and does not require any modification of instrumentation. The DANTE sequence for selective saturation included 38000 pulses of 1.3 ° with a 0.15 ms delay between them, applied at the resonance frequency of the saturated nucleus. This delay between the pulses placed the first harmonic of the DANTE sequence far outside the frequency region of the phosphates. The duration of saturation was 5.7 s. Additional relaxation delay of 1.3 s was used, following the observation pulse. The rf field was placed sequentially at the frequency of -/ATP and at three frequencies that provide control spectra for exchange from PCr, Pi and /3ATP. To minimize the error due to the long measurement time (about 5 h) 16 scans were repeatedly accumulated for each frequency (30-40 times). T1 measurements were performed using the inversion recovery method applying 180-t-90 pulses. Each measurement included 10 variable t values and a relaxation delay of 7 s. Data were recorded by accumulating sequentially 16 transients for each t value (20 times). Analysis of NMR data. An iterative nonlinear computer SAS program was employed to fit the data of the inversion recovery experiment to a two-site model (see Appendix). Ten equations (three SSST Eqns., four inversion recovery time courses and three equilibrium conditions) were used for obtaining the best fit values for ten parameters (six rate constants and four relaxation rates). A numerical FORTRAN program was used to simulate the inversion recovery experiment in a five-site exchange system (Eqn. 3 in the Appendix) using parameters obtained from the two-site model. The quality of the fit to the experimental
inversion recovery data was determined using the residual sum of squares. Scanning electron microscopy. Agarose filaments for scanning electron microscopy were prepared as for the NMR measurements. The filaments were then fixed for 45 rain in 0.1 M calcium-free phosphate buffer containing 2% gluteraldehyde. The sample was washed twice in 0.1 M phosphate buffer, dehydrated by immersing the filaments sequentially for 5 rain in 25%, 50% and 75% ethanol, then for 15 min in 100% ethanol and finally dried at the critical point of CO> The dried filaments were coated with gold and observed with a Philips analytical SEM 505/515. Assays of enzyme activities. The specific activity of adenylate kinase and creatine kinase were determined sequentially in the same cell extract, obtained by sonicating the cells in a solution containing 50 mM Tris-HC1 (pH 6.8), 5 mM magnesium acetate, 0.4 mM EDTA, 2.75 mM dithiothreitol and 250 mM sucrose. Previously described methods for measuring creatine kinase activity and adenylate kinase activity were used [29]. The assay mixture for adenylate kinase activity contained 50 mM imidazole buffer (pH 6.7), 2 mM ADP, 10 mM magnesium acetate, 20 mM D-glucose, 20 mM NAD, 5 mM EDTA, 10/~g/ml bovine serum albumin, 20 mM dithiothreitol, 2.4 units of glucose-6-phosphate dehydrogenase and 1.6 units of hexokinase . Creatine kinase activity was then measured by first adding to the above assay mixture 50 #M diadenosine-pentaphosphate (Sigma) to inhibit the activity of adenylate kinase, and then adding 25 mM PCr. The proportions of mitochondrial creatine kinase and adenylate kinase activities were assayed by measuring the activity of these enzymes in the supernatant obtained after homogenization of the cells, and in the pellet after its resuspension in buffer containing 0.5% Triton X-100 [29]. Protein was determined by the Bradford method [30], using bovine serum albumin as the protein standard. Measurement of oxygen consumption. Oxygen consumption was measured in cells embedded in agarose filaments and perifused as in the NMR experiments by placing an oxygen electrode (Orion 97-08) above the teflon retainer inside the peri-
182
fusion system (Fig. 1). The temperature was monitored continuously and maintained at 35 (+0.5) ° C. Calibration for zero oxygen was performed while perifusing degassed medium containing sodium dithionate. Calibration of 0 2 saturation was obtained with a medium gassed with 95% 02/5% CO 2. Changes in oxygen concentration due to its consumption by the cells were monitored for several hours. A 31p-NMR spectrum of a perchloric acid extract, obtained from the ceils at the end of this experiment indicated that the composition of the high-energy phosphates after perifusion was similar to that of cell extracts obtained immediately after the cells were harvested.
Fig. 2. Scanning electron micrographs of fixed, gold coated T47D cells embedded in agarose filaments.
(ex*in)
Pl
Results
~NTP+OINOP
The method of Foxall and Cohen [4] for embedding cells in agarose filaments was found suitable for studying T47D human breast cancer cells. The perifusion system used (Fig. 1) induced minimal pressure in the NMR tube making it possible to pack densely the delicate filaments and perfuse them at a rapid rate for many hours, while keeping the system intact. The stability of the cells and their metabolic state were verified by: (1) recording consecutive 3aP-NMR spectra, that showed unchanging concentrations of the phosphate metabolites; (2) measuring the oxygen consumption rate that remained unchanged, (3) taking samples frr scanning electron microscopy, which showed that the shape and the distribution of the cells did not change with time (Fig. 2). A similar examination using phase-contrast microscopy was applied to Chinese hamster lung fibroblasts embedded in agarose gel [5]. 31p-NMR spectra of T47D cells perifused with either RPMI or phosphate-free Dulbecco's modified Minimum Essential Medium, are shown in Fig. 3 (traces A and B, respectively). The signals of phosphorylethanolamine (PE), phosphorylcholine (PC), Pi, glycerol phosphorylethanolamine (GPE), glycerol phosphorylcholine (GPC), PCr, nucleoside triphosphates (NTP), primarily ATP and uridine diphosphoglucose (UDPG) were identified from their chemical shift and confirmed by adding pure compounds to an acid extract of the cells and by pH titration studies of extracts. The
G~E PC ~
yNTP÷/~NDP
B Pi (~n) I I I
I0 PPM
I
Fig. 3. 31p-NMR spectra of T47D cells (5.10 s) embedded in agarose filaments and perifused as described in the text. Each trace was obtained by accumulating 900 scans (15 rain) (60 o pulses, 1 s delay) and processed using line broadening of 15 Hz. (A) Perifusion with RPMI 1640 medium (5.6 mM Pi)- (B) Same sample perifused with phosphate-free Dulbecco's modified Minimum Essential Medium. Ex and In refer to external and internal Pi-
183
contribution of the intracellular Pi signal to the intensity of the total Pi signal was subtracted by measuring the area of the Pi signal in the same sample after an hour of perifusion with a phosphate-free Dulbecco's modified Minimum Essential Medium. The absence of Pi from the medium did not affect the concentration of the intracellular phosphates. The areas of the TATP and the flATP signals were identical within the accuracy of the measurement, resulting in an upper limit for the ADP-to-ATP ratio of 0.1. The areas of the signals of Pi and PCr relative to ATP, obtained from fully relaxed spectra (90 °, 7 s delay) are summarized in Table I. An average intracellular concentration of ATP of 4 mM was calculated using the Pi signal of the RPMI medium as a reference (5.6 mM, 90%). The magnesium-bound ATP (97%) was determined from the difference in chemical shift between the a and /3 and between the T- and /3- signals of ATP by the method of Gupta et al. [31]. Spectra recorded sequentially for several hours at constant perifusion conditions (with either RPMI 1640 or Dulbecco's modified Minimum Essential Medium) showed no significant changes in the 31p signals, indicating that the metabolism of the phosphates remained stable for that period. However, changes in the perifusion rate or in the temperature affected mostly the concentration of PCr and A T E After reducing the rate of perifusion from 4 m l / m i n to 2 m l / m i n the PCr signal disappeared and the intensity of the ATP signals decreased by about 20%. 30 min after the perifusion rate was returned to 4 m l / m i n , the intensity of the PCr and ATP signals were restored to their initial values. Raising the temperature from 25 °C to 35 °C caused a decrease of about 40% in the signal intensity of PCr (Fig. 4). This behavior lends support to the explanation by Evanochko et al. [1] that environmental factors may be responsible for reported differences in PCr concentration of cells in culture. The presence of N M R detectable concentrations of PCr in these breast cancer cells also suggests that the PCr signal detected in implanted cancers and in excised human breast tumors originate from the malignant cells and are not necessarily due to residual normal cells. The concentration of GPC and GPE relative to the ATP concentration varied considerably from
aATP ~'ATP
A
zs°c BATP
PC
II Iky° °
B
L 55"C-
I
I0 PPM
I
Fig. 4. Temperature effects on 31P-NMR spectra of T47D cells embedded in agarose filaments and perifused with phosphatefree Dulbecco's modified M i n i m u m Essential M e d i u m as described in text. (A) Cells maintained at 25 o C. (B) The same sample after raising the temperature to 35 ° C. (C) The same sample after 10 h at 35 o C.
one preparation to another (see for example Figs. 3 and 4). The factors determining the steady-state concentratio.ns of these phosphodiesters are not known [32], and further studies are now being conducted to clarify this issue. Steady-state saturation at the frequency region of the TATP and flADP signals induced a reduction (AM) in the intensity of the Pi, PCr and flATP signals (Fig. 5). The fractions of reduced intensity (AM/M) determined in five separate experiments are summarized in Table I. The apparent relaxation rates (1/~-) of Pi, PCr, /3ATP and 7ATP, and the pseudo first order rate constants for the three transfer reactions studied, were
0.36 0.54 0.18 0.41 0.40
0.33 0.14 0.37 0.43 0.41
0.18+0.014
0.18 0.18 0.20 0.16 0.20
BATP
0.31+0.10
0.39 0.45 0.27 0.26 0.17
0.36_+0.10
0.20 0.47 0.44 0.40 0.30
[PCr] [ATP]
[Pi ] a [ATP]
0.53+-0.12
0.53_+0.10 0.69_+0.16 0.37_+0.06 0.67+0.19 0.42+0.05
Pi
1/~- (s - l ) b
0.34+0.07
0.29_+0.19 0.39+0.15 0.24+0.05 0.40+_0.04 0.40+-0.03
PCr
a Intracellular concentration ratios determined from the corresponding relative signal areas. b Estimates + asymptotic standard error obtained as described in Data analysis.
0.37+0.11
PCr
Pi
AM/M
Average _+ 0.34+0.10 (S.D.)
1 2 3 4 5
Expt.
2.19+0.49
1.68+0.21 2.13+0.13 2.03+-0.15 2.00+_0.31 3.14+-0.37
yATP
KINETIC PARAMETERS OF PHOSPHATE T R A N S F E R REACTIONS IN T47D CELLS AT 35 o C
TABLE I
1.76+-0.30
1.89+_0.31 1.70_+0.21 1.575:0.12 1.38+0.20 2.26+_0.44
flATP
0.18+0.07
0.17_+0.03 0.09_+0.02 0.14+0.02 0.29_+0.08 0.21+0.02
Pi --} y-ATP
k (s-') b
0.13+_0.06
0.10+_0.07 0.21 +-0.08 0.04+-0.01 0.16+-0.017 0.16+-0.014
0.32_+0.06
0.34_+0.05 0.31 + 0.03 0.31+0.02 0.22+0.03 0.43+0.08
PCr --* "yATP flATP -~ flADP
185
SATURATION OF )-ATP PC aATP. aAOP
Gz~C
I0 PPM i
CONTROL FOR Pi
CONTF~OL FOR PCr
CONTROL FOR ~ATP
i
Fig. 5. 3 ] p - N M R saturation transfer studies of intact T47D cells. Saturation was achieved using a selective D A N T E sequence as described in Materials and Methods. Each trace was obtained by accumulating 448 transients with a delay of 7 s between them. The saturation frequencies are indicated by an arrow. Top trace: saturation of the yATP + flADP signals. Lower traces from left to right: control saturations for the Pi, PCr and flATP signals.
calculated from SSST and inversion recovery data using a two-site exchange model as described in Materials and Methods section and in the Appendix (Table I). Table I lists the best estimate _+ asymptotic S.E. of the estimate for each kinetic parameter. About twice the asymptotic standard error provides a 95% confidence interval. A numerical solution of the five-site exchange spin system equation (Eqn. 3 in the Appendix) with the parameters obtained from the two-site exchange model (see Table I), yielded either a better or about the same (two experiments) fit to the inversion recovery data, (three experiments) indicated by the residual sum of squares. This supports the above model for the exchanging spin system. No effect on the signal intensity of yATP was observed upon saturation of PCr, Pi or flATP. Estimation of the reverse pseudo first order rate constants (k_ ] --- 0.06 s- ] k_ 2 = 0.04 s- 1) from the corresponding forward constants (Table I) and the equilibrium relations (Eqn. 6 in the Appendix) indicated that they were too small relative to the yATP 7"] relaxation rate to induce any measurable change in SSST experiment. The fluxes calculated from the rate constants and the corresponding concentration of substrates (obtained from the area ratios given in Table I
and the average value of ATP concentration of 4 mM) were: I. Flux (PCr ~ y-ATP) = 0.20 + 0.11 m M / s II. Flux (Pi ~ y-ATP) = 0.20 + 0.06 m M / s III. Flux (fl-ATP ~ fl-ADP) = 1.29 + 0.27 m M / s The reverse fluxes of I and II include a flATP to flADP transfer and, therefore, contribute to the total flux of III. Since the forward and reverse fluxes were assumed to be equal (see theory and data analysis), 0.40 m M / s from I and II contribute to the total 1.29 m M / s of III. Several, enzymatic reactions could contribute to the remaining 0.89 mM/s, including the reaction catalyzed by adenylate kinase. The spec. act. of adenylate kinase, determined using a coupled assay as described in the Materials and Methods section, was 0.41 + 0.06 /~mol/ min per mg protein (n = 7), 80% of the activity was the cytosolic and 20% mitochondrial. The specific activity of creatine kinase was 0.11 + 0.03 # m o l / m i n per mg protein (n = 11), and was found exclusively in the cytosol fraction, with an isozyme distribution of 75% MM, 6% MB and 19% BB. Although the activity assays for adenylate kinase and creatine kinase are performed with excess substrates and may not necessarily represent the activity in the intact cells, it suggests that catalysis
186
6 4
z
~3Arp
TATP
~ / r = L 3 8 s "1
0'
I / r = 2 D s-'
"2 I
0
I 2
I 3
1 4
I 5
I 6
0
I
[
I
I
1
I
2
3
4
5
6
Seconds
Seconds
: 3 _o
~2
I
(3 -I -2
0
,
J,
,
5 Seconds
,
6
i
0
i
l
l
I
2
3
a
~
Seconds
Fig. 6. 3]p inversion recovery time courses of phosphates in intact T47D cells. The experimental conditions are described in Materials and Methods. The curves are the best fits (obtained as described in Data analysis) that yielded the relaxation rates given in Expt. 5 of Table I.
of the ATP to ADP flux is dominated by adenylate kinase. The rate of oxygen consumption, measured in a cell system identical to that used for the NMR studies (see Materials and Methods) was 2.4.10 -5 mmol/min. This value was calculated using a solubility of oxygen of 0.0245 ml gas/ml medium at 1 atm of gas, introducing a correction for the water vapor pressure. The total intracellular volume was 0.2 ml yielding an intracellular 0 2 flux of 2- 10-3 mM/s. The ratio between the rate of A T P synthesis from Pi (0.2 mM/s), measured by NMR, and the rate of consumption of oxygen atoms was, therefore, approx. 50. Discussion
The 31p spectra of living cells includes several signals due to exchanging phosphorus nuclei. These nuclei are either transferred from one molecule to another as in the PCr-,/ATP transfer or are exchanging via a change in their chemical environment as the flADP-~ATP exchange. The rate of these reactions can be measured by magnetization transfer techniques provided several conditions exist.
1. The signals due to the exchanging nuclei are well resolved. 2. The T1 relaxation rates of the exchanging nuclei are of the same order of magnitude as their rate of exchange. 3. The effects of intermediates (such as enzyme-substrate complexes) on the transfer of magnetization is negligible [33]. In general, the phosphate exchange reactions in vivo are coupled and require the use of a multi-site exchange model. The kinetics of such systems can be characterized by applying the method of multiple saturation transfer [18,19], provided all spin exchange processes are a priori known. The method of inversion transfer can also be applied to a multi-site exchange system [17], for the study of a specific exchange process. Using these methods requires separate experiments for determining the kinetics of each transfer and may, therefore increase the measurement time to the extent that it would become impractical for in vivo studies. A considerably faster way that can yield simultaneously estimates of the rates of several coupled reactions is provided by combining an SSST experiment to measure the transfer into common intermediate and an inversion re-
187 covery experiment. A two-site exchange model provided an adequate estimation of the relaxation rates of Pi, PCr and flATP, which are end products in the reactions studied, and for the corresponding rate constants from these substrates: k 1, k 2 and k T [18]. Since yATP participates in more than one exchange process, the two-site model provided only approximate values for its relaxation. However, a better estimated value for the ~,ATP T1 relaxation was obtained by including all the rates of exchanges in which "/ATP participates (see Appendix). It is important to note that the values calculated for the rate constants kl, k 2 and K T and the apparent Ta relaxation rates of Pi, PCr and flATP are not affected by changing slightly (30%) the estimated value of the apparent Tl relaxation rate of ~,ATP.
The kinetics of phosphate transfer reactions in T47D cells Phosphate metabolism includes enzymatic reactions that involve synthesis and breakdown of high-energy phosphates, to store and release energy, respectively. The fluxes through various phosphate-transfer reactions vary depending on the concentration of enzymes and substrates present. Biochemical assays of enzymatic activities measure initial rates at saturating substrate levels (at Vma~) and, therefore, reflect the amount of enzyme present. However, in the living cell, enzymes may be substrate limited. Only a non-invasive method which can measure the fluxes in the intact cells (such as N M R ) can determine the physiological enzymatic rates. While N M R magnetization transfer methods are suitable for such studies, they are limited in the range of rates they can determine; the rate constant (pseudo first order or first order) of a reaction has to be of the same order of magnitude as the relaxation rate of the exchanging nucleus. It is not possible to predict a priori from the specific activity of the enzyme whether a certain reaction would proceed at a rate suitable for magnetization transfer studies, for the reasons discussed above, except for in the case of a true first order exchange reaction. For instance, in T47D cells the specific activity of creatine kinase was very low (about 0.11 /~mol/ rain per mg protein) relative to that found in other organs where this reaction was studied by magne-
tization transfer: 61.5 and 8 . 4 / ~ m o l / m i n per mg protein in rat skeletal and cardiac muscle, respectively [29], and 7.4 ~tmol/min per mg protein in rat brain [29]. Thus, on the basis of this comparison and assuming that the enzyme is similar in its kinetics in all systems, one might predict that the flux through creatine kinase in T47D cells would be too small to be detected by magnetization transfer. However, upon steady state saturation of "tATP a substantial decline in the PCr signal intensity was observed, showing that in spite of the low specific activity, the pseudo first order rate constant for creatine kinase is relatively high and measurable by magnetization transfer (Table I). This could be due to the relatively high concentration of either H + or MgADP or both (see Eqn. 1 in the Appendix). Since the pH in the cells and in the organs studied previously by magnetization transfer was approximately the same (7.2-7.4), the concentration of MgADP has to be higher. This conclusion provides an independent estimate for an ADP concentration of about 5-times higher in the T47D cells than in the cardiac cells (which happened to have almost the same MM to BB isozyme ratio as the T47D cells) and close to the upper limit for ADP concentration obtained form the N M R spectrum of approx. 0.4 mM. The creatine kinase reaction is not the dominant one for ATP to ADP exchange in the T47D cells as is the case in the above mentioned organs. The flux from ADP to ATP via synthesis from Pi was the same as the creatine kinase flux (0.2 m M / s ) . The ratio between this flux and the O2 consumption rate serves to identify the energy source for ATP synthesis, namely glycolysis versus oxidative phosphorylation. A ratio close to 3 was obtained in most organs and cells studied previously by N M R thus indicating that respiration is the main source of energy there. In the T47D cells, however, the ratio was much higher (of the order of 50). Such a ratio suggests that most of the ATP synthesis is via the glycolytic pathway, despite the fact that the cells were provided with excess oxygen. Preliminary 13C-NMR studies with labelled glucose have indicated that the rate of lactate production in the T47D cells is high (about 0.18 m M / s ) and can account for the ATP synthesis measured by NMR. The high aerobic glycolysis in cancer cells was discovered in 1926 [34]. The
188 high ADP concentration in these cells, suggested from the creatine kinase kinetics, may play an important role in maintaining high rates of aerobic glycolysis, since ADP is known to be a stimulator of the regulatory enzymes of this pathway. The rate of the creatine kinase reaction and the rate of ATP synthesis from P~ contribute together about 30% of the total ATP to ADP conversion rate, determined from the /3ATP to /~ADP exchange. All kinases and ATPases contribute to this type of change in the chemical environment of the B-phosphate. It was not possible to separate the flADP signal from the 3,ATP signal in spectra of intact cells. Thus, the saturation pulse applied at the frequency range of both signals also saturated/3ADP, which in turn induced a decrease in the intensity of the/3ATP signal. It was previously suggested that such a decrease could be due to ~,ATP ~ flATP exchange via double adenylate kinase transfer [21]. This was unlikely in our system, since saturation of the/3ATP signal did not affect the ~,ATP signal. Koretsky et al. [35] have shown that in an in vitro reaction mixture of creatine kinase, the decrease in /3ATP is maximal at the /3ADP frequency, thus indicating that /3ATP-flADP exchange can cause the reduction in the/3ATP signal. In addition, we have measured similar rates for the creatine kinase reaction in solution from the decrease in either PCr or/3ATP due to the saturation of the region of the 7ATP + /3ADP signals, and from the corresponding apparent "r values obtained by inversion recovery and inversion transfer measurements. It is therefore concluded that although /3ADP can not be detected directly, the effects of its saturation can be monitored via magnetization transfer to/3ATP. The reactions responsible for most of the ATP to ADP conversion are not known. The measured specific activity of adenylate kinase was about 4-times higher than that of creatine kinase in the same assay mixture. The conditions in the assay mixture differ from those in the intact cells; however, ADP, which is usually the limiting substrate, was found to be present at a relatively high concentration, thus suggesting that adenylate kinase may be responsible for most of t h e / 3 A T P - f l A D P flux. In an isotope exchange study of adenylate kinase it was indeed shown that the A T P - A D P exchange increases as the concentration of ADP increases [36].
The energy state of the T47D cells The energy state of cells has previously been described by the 'phosphorylation potential' which is the ratio of [ATP]/([ADP]. [Pi]) a n d / o r by the 'adenylate charge' of the cell which can be calculated for any given concentrations of ATP, ADP and AMP [37]. Estimation of these parameters requires determinations of the concentrations of the adenine nucleotides. These concentrations were previously determined by assays performed on cell extracts in which some degradation of ATP during the extract preparation was inevitable. Thus, the determination of the above-mentioned parameters was subject to error. In intact T47D c~lls, the steady-state concentrations of ATP, PCr and P~ were determined from the 31P-NMR spectra. ADP could not be determined directly but its concentration was estimated to be approx. 0.4 mM (see section above). AMP could not be detected even in extracts, and therefore its concentration was negligible. Calculation of the phosphorylation potential and the adenylate energy charge in the T47D yielded an estimate of 750 M -1 and 0.95 for these parameters respectively, indicating that these cells are highly 'energized'. Another informative way to express the energy state of living cells can be provided by the turnover rate of ATP. The rate of ATP production is adjusted to the rate of ATP utilization in a dynamic steady state. The overall rate of transfer of ATP to ADP as well as the rates of the specific phosphate transfer reactions which directly related to the metabolic activity of the cells, are usually very fast and could not be determined accurately. However, 31p magnetization transfer techniques can be used to measure these transfer rates in living cells as in this study of T47D cells. In other mammalian systems studied previously by magnetization transfer techniques (brain or skeletal and cardiac muscle), the dominant reaction for ATP turnover was catalyzed by creatine kinase. In T47D cells, the creatine kinase reaction contributes about 15% of the total ATP to ADP turnover rate which is about the same as the reactions of ATP synthesis from Pi. The major part, however, of ATP to ADP conversion is contributed by other reactions of which adenylate kinase is probably the dominant one. Adenylate kinase activity was found in the mitochondrial fraction (20%) and in the cytosol.
189
Creatine kinase activity in the mitochondrial fraction was negligible; almost all the activity originated from the cytosol. This, and the fact that the rate of ATP formation from PCr and Pi were about the same, rules out a functioning of 'shuttle' mechanism model (Ref. 38 and references cited therein) for creatine kinase in the T47D cells.
Conclusions The composition and concentration of the phosphate metabolites in T47D cells indicate that these cells are highly energized. The overall rate of ATP to ADP turnover as well as the specific rates of synthesis of ATP from Pi and from PCr were measured in the intact human cell culture by a d a p t i n g 3ap magnetization transfer techniques to this system. These studies together with 02 consumption measurements and preliminary 13CN M R studies of lactate production, showed that the main source of energy is aerobic glycolysis. The results also suggested that in these cells the adenylate kinase reaction is responsible for most of the ATP to ADP conversion, and that the ADP concentration is relatively high.
Appendix Kinetic analysis in a multi-site exchange system: The exchange of 3'ATP in the T47D cells is dominated by the following reactions: I. The reaction catalyzed by creatine kinase: PCr + ADP + H + ~ ATP + Cr 2
II. The sum of reactions that form ATP from Pi:
IV. The sum of all reactions catalyzing a transfer of the y-phosphate from ATP to an acceptor molecule ( X ) to form ADP and X-P. ~T
A T P + X ~ A D P + X-P k-T
The kinetics of these transfers are characterized by the following pseudo first order rate constants: k I =,~I[ADP][H + ]
k - I =~:_l[Cr]
k2 = k2[ADP]
k-2 =~:-2
k 3 = k3 [ A D P ]
k _ 3 = k _ 3 [AMP]
(1)
All three reactions involve an actual transfer from the 3,-phosphate of ATP to the acceptor molecules, however, these reactions also include a change in the environment of the B-phosphate via flATP-to-BADP conversion. The 3,ATP-X-P exchange and the B A T P - f l A D P exchange proceed at the same rate with a pseudo first order rate constant k T = 7¢v [X]. The enzymes that catalyze the reverse direction of I, II and III as well as all other kinase s such as hexokinase , nucleoside diphosphokinase, etc., are involved in this/3-/3 conversion. Thus, kT = ~:-1 [Cr] + ~'-2 + ~:-3[ AMP] + ~ ' ( k i [ Xi])
(2)
Where i designates a reaction in which the concentration of the phosphate acceptor is [X i]. The exchanging 31p spin system in the T47D cells can therefore be described by the following scheme: kl
k2
k-1
k_ 2
PCr ~ ~,ATP ~ P~ Pi + ADP ~ ATP ~-2
kr
flADP ~ flATP k-T
III. The reaction catalyzed by adenylate kinase: 2 ADP ~ AMP+ATP 7~_3
In this spin system, the exchange-modified Bloch equation for the decay to equilibrium of the magnetization in an inversion recovery experi-
190
merit, are [39]: r M(PCr)] IM('t-ATP)|
r M(PCr) | M('~ATP)
/M(#ADP)/ LM(BATP)]
/M(BADP~ LM(BATP)
Mo(PCr)/T 1 (PCr) M o T A T P / T I (TATP) +
A =
Mo(Pi)/TI(Pi) M o f l A D P / T 1( flADP) M o ( f l A T P ) / T 1( f l A T P )
- 1/'r (PCr)
k_ 1
0
0
ka
- 1/p(yATP)
k2
k3
0
0
k_ 2
- 1/'r (Pi)
0
0
0
k_ 3
0
I/T(flADP)
kT
0
0
0
k_ T
- 1/r(flATP)
l
covery of the signals due to PCr, yATP, P, and flATP in the system studied. Introducing several approximations based on properties of the system made it possible to simulate the data using a two-site model and to obtain estimates of the kinetic parameters. A. The forward and reverse fluxes were assumed equal for all reactions, namely
0
a. k 1[PCr] = k_l [~ATPI b. k2[Pi] = k_2[TATP l (3)
Where the T values are the apparent relaxation rates defined as: ai
I(PCr)= ~-i(PCr)+kI
b. I ( T A T P ) =
T!(-/ATP) + k_ 1 + k_2 + k_ 3
c. l(pi) = ~'~1(Pi) + k2 d. I ( f l A D P ) = T ! ( f l A D P ) + k 3 +
e. I ( f l A T P ) = ~-~(flATP) + k T
k_ T
(4)
This equation (Eqn. 3) can not be solved analytically, however, it is possible to obtain best fitted values by a numerical analysis. Saturation at the frequency region of the overlapping yATP and flADP signals caused a decrease of A M in the signal intensity of Pj, PCr and flATP due to reactions I, II and III, respectively. The fractional change in the intensity ( A M / M ) is proportional to the corresponding rate constants according to:
b. A M / M ( P i ) = kE-~'(Pi)
(5)
Performing a non-selective inversion recovery experiment provided four time courses of the re-
(6)
The above equilibrium relations also hold in a closed system when the concentration of the substrates does not change with time and there is no compartmentation of substrates. These conditions were closely maintained in the cells during the kinetic measurements. (1) The cells were perifused with phosphate-free medium and could, therefore, be considered as a closed system regarding the phosphates; (2) the concentration of the substrates remained constant; (3) most of the creatine kinase activity and approx. 80% of the adenylate kinase activity was found in the cytosol. B. The intrinsic relaxation of ,/ATP was assumed to be affected by the sum of the exchange processes (III). 1T(yATP) = ~ (yATP) + k T
(7)
C. An upper limit of 0.1 was set for the A D P / A T P ratio, on the basis of the identity of the y- and flATP signal areas. In addition, the intrinsic relaxation 1 / T 1 (flADP) was assumed to be equal to 1 / T 1 (yATP) and the apparent 1/T(flADP) was therefore given by: (flADP) = - ~ (7ATP) + k T [ATPI/[ADP]
a. A M / M ( P C r ) = k~. T(PCr)
c. A M / M ( f l A T P ) = k T . ~'(flATP)
c. kT[flATP ] = k_T[flADP l
(8)
D. The inversion recovery data were analyzed assuming a two-site exchange model according to the following equation: Mi(t ) = Mi(oo ) + C 1 e (v+o + C 2 e (v-t>
(9)
191 TABLE II PARAMATERS USED FOR FIT'I'ING THE INVERSION RECOVERY DATA TO A TWO-SITE EXCHANGE MODEL The kinetic parameters for PCr, Pi AND flATP were chosen according to Eqns. 1-8. The parameters for "tATP were: kia equals k T which includes all exchange processes from yATP to all other sites; k~i includes the average contribution from the exchange with Pi and PCr. 1
I
a
--
'ra
kia kai
PCr
y-ATP
1_(yATP)
kl
y-ATP Pi+PCr
'2(1/~(Pi)+l/r(PCr)}
k w ½(kl+k2}
Pi
1 (yATP)
k 2 k_ 2
1--(BADP)
kT k_T
y-ATP
B-ATP ¢-ADP
T
T
T
k_ 1
where y± = ½{ - ( 1 / r i + 1 / % ) + { ( 1 / r i - 1/%) 2 +4(kiakai) } 1}. C 2 = { ( 1 / k a i ) ( M i ( O ) - Mi(o0))'(Y+ + 1/ri) + M ~ ( a ) - M~(0)}/{y+ - X_ } l / k a i
C 1 = Mi(0 ) - Mi(oo ) - C2
The list of parameters used for data analysis of the inversion recovery and steady state saturation experiments is given in Table II. An iterative nonlinear computer SAS program was used to fit the data to ten Equations (three SSST equations: 5a-c, four inversion recovery time courses Eqn. 9 and three equilibrium conditions Eqns. 6a-c) for obtaining the best fit values for ten parameters (six rate constants and four relaxation rates). These parameters were then used to obtain a numerical simulation of the inversion recovery data using the five-site exchange model (Eqn. 3) and to obtain the residual sum of squares from the experimental data.
Acknowledgements We are very grateful to Professor Y. Keydar for the cells and the support in the course of these studies. We also thank Professor M. Cohn and Dr. T.A. Victor for many helpful discussions and Ms.
H. Eldar for help with the computer analysis. This work was supported by the U.S. Israel Binational Science Foundation, the Israel Cancer Research Fund and the MINERVA Foundation Munich, F.R.G. References 1 Evanochko, W.T., Ng, T.C. and Glickson, J.D. (1984) Magn. Reson. Med. 1, 508-534 2 Desmoulin, F., Galons, J.P., Canioni, P., Marvaldi, J. and Cozzone, P.J. (1986) Cancer Res. 46, 3768-3774 3 Cohen, J.S., Lyon, R.C., Chen, C., Faustino, P.J., Batist, G., Shoemaker, M., Rubalcaba, E. and Cowan, K.H. (1986) Cancer Res. 46, 4087-4090 4 Foxall, D.J. and Cohen, J.S. (1983) J. Magn. Reson. 52, 346-349 5 Knop, R.H., Chert, C.W., Mitchell, J.B., Russo, A., McPherson, S. and Cohen, J.S. (1984) Biochim. Biophys. Acta 804, 275-284 6 Ugerbil, K., Guernsey, D.C., Brown, T.R., Glynn, P., Tobkes, N.J. and Edelman, I.S. (1981) Proc. Natl. Acad. Sci. USA, 78, 4843-4847 7 Karczmar, G.S., Koretsky, A.P., Sissell, M.J., Klein, M.P. and Weiner, M.W. (1983) J. Magn. Reson. 53, 123-128 8 Evanochko, W.T., Sakai, T.T., Ng, T.C., Ramakrishna, N., Kim, H.D., Zeidler, R.B., Ghanta, V.K., Brockman, R.W., Schiffer, L.M., Braunschweiger, P.G. and Glickson, J.D. (1984) Biochim. Biophys. Acta 805, 104-116 9 Alger, J.R., Den Hollander, J.A. and Shulman, R.G. (1982) Biochemistry 21, 2957-2963 10 Campbell, S.L., Jones, K.A. and Shulman, R.G. (1985) FEBS Lett. 193, 198-193 11 Brindle, K. and Kfikler, S. (1985) Biochim. Biophys. Acta 847, 285-292 12 Gadian, D.G., Radda, G.K., Brown, T.R., Chance, E.M., Dawson, M.J. and Wilkie, D.R. (1981) Biochem. J. 194, 215-228 13 Matthews, P.M., Bland, J.L., Gadian, D.G. and Radda, G.K. (1982) Biochim. Biophys. Acta 721, 312-320 14 Shoubridge, E.A., Briggs, R.W. and Radda, G.K. (1982) FEBS Lett. 140, 288-292 15 Alger, J.R. and Shulman, R.G. (1984) Q. Rev. Biophys. 17, 83-124 16 Koretsky, A.P. and Weiner, M.W. (1984) in 31p-NMR Magnetization Transfer Measurements of Phosphorous Exchange Reactions in vivo. (James, T.L. and Margulis, A.R., eds.), Biomedical Research and Education Foundation, San Francisco 17 Degani, H., Laughlin, M., Campbell, S. and Shulman, R.G. (1985) Biochemistry 24, 5510-5516 18 Ugurbil, K. (1985) J. Magn. Resort. 64, 207-219 19 Ugurbil, K., Petein, M., Maidan, R., Michurski, S. and From, A.H.L. (1986) Biochemistry 25, 100-107 20 Bittl, J.A. and Ingwall, J.S. (1985) J. Biol. Chem. 260, 3512-3517 21 Koretsky, A.P., Wang, S., Klien, M.P., James, T.L. and Weiner, M.W. (1986) Biochemistry 25, 77-84
192 22 Balaban, R.S., Kantor, H.L. and Ferretti, J.H. (1983) J. Biol. Chem. 258, 12787-12789 23 Horwitz, K.B., Zava, D.T., Thilagar, A.K., jensen, E.M. and McGuire, W.L. (1978) Cancer Res. 38, 2434-2437 24 Jakesz, R., Smith, C.A., Aitken, S., Juff, K., Schuette, W., Shackney, S. a Lippman, M. (1984) Cancer Res. 44, 619-625 25 Keydar, I., Chen, L., Karby, S., Weiss, f.R., Delarea, J. and Radu, M. (1979) Eur. J. Cancer 15, 659-669 26 Keydar, I., Ohno, T., Nayak, R., Sweet, R., SimoN, F., Weiss, F., Karby, S., Mesa-Tejada, R. and Spiegelman, S. (1984) Proc. Natl. Acad. Sci. USA 81, 4188-4192 27 Chalbos, D., Vignon, F., Keydar, I. and Rockefort, H. (1982) J. Clin. Endocrinol. Metab. 55, 26%283 28 Morris, G.A. and Freeman, R. (1978) J. Magn. Reson. 29, 433-462 29 Shatton, J.B., Morris, H.P. and Weinhouse, S. (1979) Cancer Res. 39, 492-501 30 Bradford, M.M. (1976) Analyst. Biochem. 72, 248-254
31 Gupta, R.K., Benovic, J.L. and Rose, Z.B. (1978) J. Biol. Chem. 253, 6172-6176 32 Burt, C.T. (1985) TIBS, October, 404-406 33 Brindle, K.M. and Radda, G.K. (1985) Biochim. Biophys. Acta 829, 188-201 34 Warburg, O. (1926) Uber den Stoffwechsel der Tumoren. Springer Verlag, Berlin 35 Koretsky, A.P., Basus, V.J., James, T.L., Klein, M.P. and Weiner, M.W. (1985) Magn. Reson. Med. 2, 586-594 36 Rhoads, D.G. and Lowenstein, J.M. (1968) J. Biol. Chem. 243, 3963-3972 37 Lehninger, A.L. (1979) in Biochemistry, 2nd Edn., pp. 538-539~ Worth Publishers, New York 38 Bessman, S.P. and Carpenter, C.L. (1985) Annu. Rev. Biochem. 54, 831-862 39 Leigh, J.S. and Schotland~ J. (1982) J. Magn. Reson. 51, 48-55
930 (1987) 179-192
179
Elsevier BBA12056
3 1 p . N M R studies of p h o s p h a t e transfer rates in T 4 7 D h u m a n breast cancer cells M. Neeman
a, E. R u s h k i n a, A . M . K a y e b a n d H . D e g a n i a
Isotope and b Hormone Research Departments, The Weizmann Institute of Science, Rehovot (Israel)
(Received 13 November 1986) (Revised manuscript received23 March 1987)
Key words: NMR, 31p.; Phosphate transfer rate; (Mammary tumor)
The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31p-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC -, yATP (0.2 raM/s), Pi -' 7ATP (0.2 m M / s ) and the conversion flATP -~ flADP (1.3 raM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.I pmol/min per mg protein) and adenylate kinase (approx. 0.4/~mol/min per mg protein) suggest that the flATP --, flADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4- 10-3 m M / s ) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.
Introduction The use of cell cultures permits the study of a homogenous cell population which can be uniformly exposed to specific nutrients, hormones and drugs. N M R spectroscopy provides a noninvasive method for characterizing the in vivo metabolism of cultured cells.
Abbreviations: PC, phosphorylcholine; PE phosphorylethanolamine; PCr, phosphocreatine; NTP, nucleoside triphosphate; GPE, glycerol phosphorylethanolamine; GPC, glycerol phosphorylcholine; SSST, steady-state saturation transfer, Correspondence: H. Degani, Isotope Department, Weizman Institute of Science, Rehovot 76100, Israel.
Monitoring of cell cultures under physiological conditions by N M R spectroscopy requires a specialized apparatus for the maintenance of viable and active cells. Initially, N M R studies of cells were performed with packed cells oxygenated by direct bubbling of oxygen, frequently at low temperature [1]. This procedure can not be used for studies over a long period of time or for monitoring metabolic parameters such as enzymatic rates. Methods that were developed later for fixing the position of the cells in the N M R tube, thus allowing perifusion with medium, permitted the maintenance of the cells' metabolic activity under steady-state conditions for relatively long periods of time [1-7]. In most of the previous N M R studies of transformed cell cultures, 3ap-NMR was used to determine the composition and concentra-
0167-4889/87/$03.50 © 1987 Elsevier SciencePublishers B.V. (Biomedical Division)
180 tion of the phosphate metabolites under various conditions [1-8]. Recently, the phosphates of MCF-7 human breast cancer cells were characterized using 31p-NMR spectroscopy [3]. Here we present 3ap-NMR studies of intact T47D human breast cancer cells maintained under physiological conditions. The emphasis in these studies was on characterizing the high-energy phosphates and on determining the kinetics of their interconversion. The kinetic measurements required development of magnetization transfer techniques which provides kinetic data for several phosphate transfer reactions within the time available for such in vivo N M R studies. 31p-NMR kinetic studies of ATP synthesis from Pi and of the creatine kinase reaction were previously employed in several systems, such as yeast cells [9-11], skeletal muscle, heart, kidney and brain [12-18]. The methods used include saturation transfer, inversion transfer and two-dimensional exchange type experiments [12-22]. H u m a n breast cancer cells differ in their metabolism and energetics from the systems mentioned above. In general, these cells resemble their cells of origin in the m a m m a r y epithelium; several fines retain receptors for steroid hormones and serve as model systems for studying hormonal responses [23,24]. Our studies were performed with cells of clone 11 from the T47D human breast cancer cell line [25,26] which contain estrogen and progesterone receptors; their growth rate is enhanced by 17/3estradiol and ifihibited by antiestrogens such as tamoxifen [27]. The experiments were complemented by biochemical studies which, with the N M R data, made it possible to characterize the energetics of this human breast cancer line. Materials
penicillin ~g/ml).
(100 u n i t s / m l )
and
mycostatin (25
Preparation of cells for NMR experiments. Approx. 5 • 10 s cells were harvested by trypsinization, when monolayers were nearly confluent. The cells were washed twice with medium and then embedded in 0.86-mm thick agarose filaments and extruded into a 10-mm N M R tube, according to the method of Foxall and Cohen [4,5]. Perifusion of the embedded cells with either R P M I or Dubelco's modified Minimal Essential Medium was initiated simultaneously with the preparation of the agarose filaments and was continued throughout the N M R measurements. The optimal rate of perifusion with medium saturated with 95% oxygen/5% CO 2 was found to be 4 m l / m i n . The medium and the N M R tube were kept at 35 ° C. The perifusion system consisted of a line which transferred the medium to the bottom of the N M R tube, and a return line placed about 10 95%02 S*/,co~
Ustd medium
and Methods
Cell line. Clone 11 of the T47D human breast cancer cell line was obtained from Y. Keydar (Tel Aviv University). The T47D fine w a s e s t a b l i s h e d from the pleural effusion of a patient with infiltrating duct carcinoma [25]. Several clones including clone 11, which showed estrogen-stimulated growth were isolated [26]. The cells were grown in monolayer culture using R P M I 1640 medium supplemented with fetal calf serum (10%), glutamine (2 mM), streptomycin (100 /Lg/ml),
Sponge fiiter~ Cells
in
agarose fitaments
Fig. 1. Scheme of cell perifusion system. Medium was maintained in a temperature-controlled vessel and was saturated with oxygen by bubbling with 95% 02/5% CO2. A peristaltic pump was used to pump the medium into the NMR tube at a rate of 4 ml/min. Overflow of medium in the NMR tube was prevented by pumping out the medium with a return line placed 10 cm above the bottom of the tube.
181 cm above the bottom (Fig. 1). During the magnetization transfer experiments the cells were perifused as described above with a phosphate-free Dulbecco's modified Minimal Essential Medium. NMR experiments. 31P-NMR spectra were recorded at 121.5 MHz by a Bruker CXP-300 spectrometer, using either 60 ° pulses and a relaxation delay of 1 s or 90 ° pulses and a relaxation delay of 7 s. The steady-state saturation transfer (SSST) experiment was performed using a selective DANTE pulse sequence [28] followed by a 90 ° observation pulse. The application of a DANTE sequence for selective saturation is possible on most commercial spectrometers and does not require any modification of instrumentation. The DANTE sequence for selective saturation included 38000 pulses of 1.3 ° with a 0.15 ms delay between them, applied at the resonance frequency of the saturated nucleus. This delay between the pulses placed the first harmonic of the DANTE sequence far outside the frequency region of the phosphates. The duration of saturation was 5.7 s. Additional relaxation delay of 1.3 s was used, following the observation pulse. The rf field was placed sequentially at the frequency of -/ATP and at three frequencies that provide control spectra for exchange from PCr, Pi and /3ATP. To minimize the error due to the long measurement time (about 5 h) 16 scans were repeatedly accumulated for each frequency (30-40 times). T1 measurements were performed using the inversion recovery method applying 180-t-90 pulses. Each measurement included 10 variable t values and a relaxation delay of 7 s. Data were recorded by accumulating sequentially 16 transients for each t value (20 times). Analysis of NMR data. An iterative nonlinear computer SAS program was employed to fit the data of the inversion recovery experiment to a two-site model (see Appendix). Ten equations (three SSST Eqns., four inversion recovery time courses and three equilibrium conditions) were used for obtaining the best fit values for ten parameters (six rate constants and four relaxation rates). A numerical FORTRAN program was used to simulate the inversion recovery experiment in a five-site exchange system (Eqn. 3 in the Appendix) using parameters obtained from the two-site model. The quality of the fit to the experimental
inversion recovery data was determined using the residual sum of squares. Scanning electron microscopy. Agarose filaments for scanning electron microscopy were prepared as for the NMR measurements. The filaments were then fixed for 45 rain in 0.1 M calcium-free phosphate buffer containing 2% gluteraldehyde. The sample was washed twice in 0.1 M phosphate buffer, dehydrated by immersing the filaments sequentially for 5 rain in 25%, 50% and 75% ethanol, then for 15 min in 100% ethanol and finally dried at the critical point of CO> The dried filaments were coated with gold and observed with a Philips analytical SEM 505/515. Assays of enzyme activities. The specific activity of adenylate kinase and creatine kinase were determined sequentially in the same cell extract, obtained by sonicating the cells in a solution containing 50 mM Tris-HC1 (pH 6.8), 5 mM magnesium acetate, 0.4 mM EDTA, 2.75 mM dithiothreitol and 250 mM sucrose. Previously described methods for measuring creatine kinase activity and adenylate kinase activity were used [29]. The assay mixture for adenylate kinase activity contained 50 mM imidazole buffer (pH 6.7), 2 mM ADP, 10 mM magnesium acetate, 20 mM D-glucose, 20 mM NAD, 5 mM EDTA, 10/~g/ml bovine serum albumin, 20 mM dithiothreitol, 2.4 units of glucose-6-phosphate dehydrogenase and 1.6 units of hexokinase . Creatine kinase activity was then measured by first adding to the above assay mixture 50 #M diadenosine-pentaphosphate (Sigma) to inhibit the activity of adenylate kinase, and then adding 25 mM PCr. The proportions of mitochondrial creatine kinase and adenylate kinase activities were assayed by measuring the activity of these enzymes in the supernatant obtained after homogenization of the cells, and in the pellet after its resuspension in buffer containing 0.5% Triton X-100 [29]. Protein was determined by the Bradford method [30], using bovine serum albumin as the protein standard. Measurement of oxygen consumption. Oxygen consumption was measured in cells embedded in agarose filaments and perifused as in the NMR experiments by placing an oxygen electrode (Orion 97-08) above the teflon retainer inside the peri-
182
fusion system (Fig. 1). The temperature was monitored continuously and maintained at 35 (+0.5) ° C. Calibration for zero oxygen was performed while perifusing degassed medium containing sodium dithionate. Calibration of 0 2 saturation was obtained with a medium gassed with 95% 02/5% CO 2. Changes in oxygen concentration due to its consumption by the cells were monitored for several hours. A 31p-NMR spectrum of a perchloric acid extract, obtained from the ceils at the end of this experiment indicated that the composition of the high-energy phosphates after perifusion was similar to that of cell extracts obtained immediately after the cells were harvested.
Fig. 2. Scanning electron micrographs of fixed, gold coated T47D cells embedded in agarose filaments.
(ex*in)
Pl
Results
~NTP+OINOP
The method of Foxall and Cohen [4] for embedding cells in agarose filaments was found suitable for studying T47D human breast cancer cells. The perifusion system used (Fig. 1) induced minimal pressure in the NMR tube making it possible to pack densely the delicate filaments and perfuse them at a rapid rate for many hours, while keeping the system intact. The stability of the cells and their metabolic state were verified by: (1) recording consecutive 3aP-NMR spectra, that showed unchanging concentrations of the phosphate metabolites; (2) measuring the oxygen consumption rate that remained unchanged, (3) taking samples frr scanning electron microscopy, which showed that the shape and the distribution of the cells did not change with time (Fig. 2). A similar examination using phase-contrast microscopy was applied to Chinese hamster lung fibroblasts embedded in agarose gel [5]. 31p-NMR spectra of T47D cells perifused with either RPMI or phosphate-free Dulbecco's modified Minimum Essential Medium, are shown in Fig. 3 (traces A and B, respectively). The signals of phosphorylethanolamine (PE), phosphorylcholine (PC), Pi, glycerol phosphorylethanolamine (GPE), glycerol phosphorylcholine (GPC), PCr, nucleoside triphosphates (NTP), primarily ATP and uridine diphosphoglucose (UDPG) were identified from their chemical shift and confirmed by adding pure compounds to an acid extract of the cells and by pH titration studies of extracts. The
G~E PC ~
yNTP÷/~NDP
B Pi (~n) I I I
I0 PPM
I
Fig. 3. 31p-NMR spectra of T47D cells (5.10 s) embedded in agarose filaments and perifused as described in the text. Each trace was obtained by accumulating 900 scans (15 rain) (60 o pulses, 1 s delay) and processed using line broadening of 15 Hz. (A) Perifusion with RPMI 1640 medium (5.6 mM Pi)- (B) Same sample perifused with phosphate-free Dulbecco's modified Minimum Essential Medium. Ex and In refer to external and internal Pi-
183
contribution of the intracellular Pi signal to the intensity of the total Pi signal was subtracted by measuring the area of the Pi signal in the same sample after an hour of perifusion with a phosphate-free Dulbecco's modified Minimum Essential Medium. The absence of Pi from the medium did not affect the concentration of the intracellular phosphates. The areas of the TATP and the flATP signals were identical within the accuracy of the measurement, resulting in an upper limit for the ADP-to-ATP ratio of 0.1. The areas of the signals of Pi and PCr relative to ATP, obtained from fully relaxed spectra (90 °, 7 s delay) are summarized in Table I. An average intracellular concentration of ATP of 4 mM was calculated using the Pi signal of the RPMI medium as a reference (5.6 mM, 90%). The magnesium-bound ATP (97%) was determined from the difference in chemical shift between the a and /3 and between the T- and /3- signals of ATP by the method of Gupta et al. [31]. Spectra recorded sequentially for several hours at constant perifusion conditions (with either RPMI 1640 or Dulbecco's modified Minimum Essential Medium) showed no significant changes in the 31p signals, indicating that the metabolism of the phosphates remained stable for that period. However, changes in the perifusion rate or in the temperature affected mostly the concentration of PCr and A T E After reducing the rate of perifusion from 4 m l / m i n to 2 m l / m i n the PCr signal disappeared and the intensity of the ATP signals decreased by about 20%. 30 min after the perifusion rate was returned to 4 m l / m i n , the intensity of the PCr and ATP signals were restored to their initial values. Raising the temperature from 25 °C to 35 °C caused a decrease of about 40% in the signal intensity of PCr (Fig. 4). This behavior lends support to the explanation by Evanochko et al. [1] that environmental factors may be responsible for reported differences in PCr concentration of cells in culture. The presence of N M R detectable concentrations of PCr in these breast cancer cells also suggests that the PCr signal detected in implanted cancers and in excised human breast tumors originate from the malignant cells and are not necessarily due to residual normal cells. The concentration of GPC and GPE relative to the ATP concentration varied considerably from
aATP ~'ATP
A
zs°c BATP
PC
II Iky° °
B
L 55"C-
I
I0 PPM
I
Fig. 4. Temperature effects on 31P-NMR spectra of T47D cells embedded in agarose filaments and perifused with phosphatefree Dulbecco's modified M i n i m u m Essential M e d i u m as described in text. (A) Cells maintained at 25 o C. (B) The same sample after raising the temperature to 35 ° C. (C) The same sample after 10 h at 35 o C.
one preparation to another (see for example Figs. 3 and 4). The factors determining the steady-state concentratio.ns of these phosphodiesters are not known [32], and further studies are now being conducted to clarify this issue. Steady-state saturation at the frequency region of the TATP and flADP signals induced a reduction (AM) in the intensity of the Pi, PCr and flATP signals (Fig. 5). The fractions of reduced intensity (AM/M) determined in five separate experiments are summarized in Table I. The apparent relaxation rates (1/~-) of Pi, PCr, /3ATP and 7ATP, and the pseudo first order rate constants for the three transfer reactions studied, were
0.36 0.54 0.18 0.41 0.40
0.33 0.14 0.37 0.43 0.41
0.18+0.014
0.18 0.18 0.20 0.16 0.20
BATP
0.31+0.10
0.39 0.45 0.27 0.26 0.17
0.36_+0.10
0.20 0.47 0.44 0.40 0.30
[PCr] [ATP]
[Pi ] a [ATP]
0.53+-0.12
0.53_+0.10 0.69_+0.16 0.37_+0.06 0.67+0.19 0.42+0.05
Pi
1/~- (s - l ) b
0.34+0.07
0.29_+0.19 0.39+0.15 0.24+0.05 0.40+_0.04 0.40+-0.03
PCr
a Intracellular concentration ratios determined from the corresponding relative signal areas. b Estimates + asymptotic standard error obtained as described in Data analysis.
0.37+0.11
PCr
Pi
AM/M
Average _+ 0.34+0.10 (S.D.)
1 2 3 4 5
Expt.
2.19+0.49
1.68+0.21 2.13+0.13 2.03+-0.15 2.00+_0.31 3.14+-0.37
yATP
KINETIC PARAMETERS OF PHOSPHATE T R A N S F E R REACTIONS IN T47D CELLS AT 35 o C
TABLE I
1.76+-0.30
1.89+_0.31 1.70_+0.21 1.575:0.12 1.38+0.20 2.26+_0.44
flATP
0.18+0.07
0.17_+0.03 0.09_+0.02 0.14+0.02 0.29_+0.08 0.21+0.02
Pi --} y-ATP
k (s-') b
0.13+_0.06
0.10+_0.07 0.21 +-0.08 0.04+-0.01 0.16+-0.017 0.16+-0.014
0.32_+0.06
0.34_+0.05 0.31 + 0.03 0.31+0.02 0.22+0.03 0.43+0.08
PCr --* "yATP flATP -~ flADP
185
SATURATION OF )-ATP PC aATP. aAOP
Gz~C
I0 PPM i
CONTROL FOR Pi
CONTF~OL FOR PCr
CONTROL FOR ~ATP
i
Fig. 5. 3 ] p - N M R saturation transfer studies of intact T47D cells. Saturation was achieved using a selective D A N T E sequence as described in Materials and Methods. Each trace was obtained by accumulating 448 transients with a delay of 7 s between them. The saturation frequencies are indicated by an arrow. Top trace: saturation of the yATP + flADP signals. Lower traces from left to right: control saturations for the Pi, PCr and flATP signals.
calculated from SSST and inversion recovery data using a two-site exchange model as described in Materials and Methods section and in the Appendix (Table I). Table I lists the best estimate _+ asymptotic S.E. of the estimate for each kinetic parameter. About twice the asymptotic standard error provides a 95% confidence interval. A numerical solution of the five-site exchange spin system equation (Eqn. 3 in the Appendix) with the parameters obtained from the two-site exchange model (see Table I), yielded either a better or about the same (two experiments) fit to the inversion recovery data, (three experiments) indicated by the residual sum of squares. This supports the above model for the exchanging spin system. No effect on the signal intensity of yATP was observed upon saturation of PCr, Pi or flATP. Estimation of the reverse pseudo first order rate constants (k_ ] --- 0.06 s- ] k_ 2 = 0.04 s- 1) from the corresponding forward constants (Table I) and the equilibrium relations (Eqn. 6 in the Appendix) indicated that they were too small relative to the yATP 7"] relaxation rate to induce any measurable change in SSST experiment. The fluxes calculated from the rate constants and the corresponding concentration of substrates (obtained from the area ratios given in Table I
and the average value of ATP concentration of 4 mM) were: I. Flux (PCr ~ y-ATP) = 0.20 + 0.11 m M / s II. Flux (Pi ~ y-ATP) = 0.20 + 0.06 m M / s III. Flux (fl-ATP ~ fl-ADP) = 1.29 + 0.27 m M / s The reverse fluxes of I and II include a flATP to flADP transfer and, therefore, contribute to the total flux of III. Since the forward and reverse fluxes were assumed to be equal (see theory and data analysis), 0.40 m M / s from I and II contribute to the total 1.29 m M / s of III. Several, enzymatic reactions could contribute to the remaining 0.89 mM/s, including the reaction catalyzed by adenylate kinase. The spec. act. of adenylate kinase, determined using a coupled assay as described in the Materials and Methods section, was 0.41 + 0.06 /~mol/ min per mg protein (n = 7), 80% of the activity was the cytosolic and 20% mitochondrial. The specific activity of creatine kinase was 0.11 + 0.03 # m o l / m i n per mg protein (n = 11), and was found exclusively in the cytosol fraction, with an isozyme distribution of 75% MM, 6% MB and 19% BB. Although the activity assays for adenylate kinase and creatine kinase are performed with excess substrates and may not necessarily represent the activity in the intact cells, it suggests that catalysis
186
6 4
z
~3Arp
TATP
~ / r = L 3 8 s "1
0'
I / r = 2 D s-'
"2 I
0
I 2
I 3
1 4
I 5
I 6
0
I
[
I
I
1
I
2
3
4
5
6
Seconds
Seconds
: 3 _o
~2
I
(3 -I -2
0
,
J,
,
5 Seconds
,
6
i
0
i
l
l
I
2
3
a
~
Seconds
Fig. 6. 3]p inversion recovery time courses of phosphates in intact T47D cells. The experimental conditions are described in Materials and Methods. The curves are the best fits (obtained as described in Data analysis) that yielded the relaxation rates given in Expt. 5 of Table I.
of the ATP to ADP flux is dominated by adenylate kinase. The rate of oxygen consumption, measured in a cell system identical to that used for the NMR studies (see Materials and Methods) was 2.4.10 -5 mmol/min. This value was calculated using a solubility of oxygen of 0.0245 ml gas/ml medium at 1 atm of gas, introducing a correction for the water vapor pressure. The total intracellular volume was 0.2 ml yielding an intracellular 0 2 flux of 2- 10-3 mM/s. The ratio between the rate of A T P synthesis from Pi (0.2 mM/s), measured by NMR, and the rate of consumption of oxygen atoms was, therefore, approx. 50. Discussion
The 31p spectra of living cells includes several signals due to exchanging phosphorus nuclei. These nuclei are either transferred from one molecule to another as in the PCr-,/ATP transfer or are exchanging via a change in their chemical environment as the flADP-~ATP exchange. The rate of these reactions can be measured by magnetization transfer techniques provided several conditions exist.
1. The signals due to the exchanging nuclei are well resolved. 2. The T1 relaxation rates of the exchanging nuclei are of the same order of magnitude as their rate of exchange. 3. The effects of intermediates (such as enzyme-substrate complexes) on the transfer of magnetization is negligible [33]. In general, the phosphate exchange reactions in vivo are coupled and require the use of a multi-site exchange model. The kinetics of such systems can be characterized by applying the method of multiple saturation transfer [18,19], provided all spin exchange processes are a priori known. The method of inversion transfer can also be applied to a multi-site exchange system [17], for the study of a specific exchange process. Using these methods requires separate experiments for determining the kinetics of each transfer and may, therefore increase the measurement time to the extent that it would become impractical for in vivo studies. A considerably faster way that can yield simultaneously estimates of the rates of several coupled reactions is provided by combining an SSST experiment to measure the transfer into common intermediate and an inversion re-
187 covery experiment. A two-site exchange model provided an adequate estimation of the relaxation rates of Pi, PCr and flATP, which are end products in the reactions studied, and for the corresponding rate constants from these substrates: k 1, k 2 and k T [18]. Since yATP participates in more than one exchange process, the two-site model provided only approximate values for its relaxation. However, a better estimated value for the ~,ATP T1 relaxation was obtained by including all the rates of exchanges in which "/ATP participates (see Appendix). It is important to note that the values calculated for the rate constants kl, k 2 and K T and the apparent Ta relaxation rates of Pi, PCr and flATP are not affected by changing slightly (30%) the estimated value of the apparent Tl relaxation rate of ~,ATP.
The kinetics of phosphate transfer reactions in T47D cells Phosphate metabolism includes enzymatic reactions that involve synthesis and breakdown of high-energy phosphates, to store and release energy, respectively. The fluxes through various phosphate-transfer reactions vary depending on the concentration of enzymes and substrates present. Biochemical assays of enzymatic activities measure initial rates at saturating substrate levels (at Vma~) and, therefore, reflect the amount of enzyme present. However, in the living cell, enzymes may be substrate limited. Only a non-invasive method which can measure the fluxes in the intact cells (such as N M R ) can determine the physiological enzymatic rates. While N M R magnetization transfer methods are suitable for such studies, they are limited in the range of rates they can determine; the rate constant (pseudo first order or first order) of a reaction has to be of the same order of magnitude as the relaxation rate of the exchanging nucleus. It is not possible to predict a priori from the specific activity of the enzyme whether a certain reaction would proceed at a rate suitable for magnetization transfer studies, for the reasons discussed above, except for in the case of a true first order exchange reaction. For instance, in T47D cells the specific activity of creatine kinase was very low (about 0.11 /~mol/ rain per mg protein) relative to that found in other organs where this reaction was studied by magne-
tization transfer: 61.5 and 8 . 4 / ~ m o l / m i n per mg protein in rat skeletal and cardiac muscle, respectively [29], and 7.4 ~tmol/min per mg protein in rat brain [29]. Thus, on the basis of this comparison and assuming that the enzyme is similar in its kinetics in all systems, one might predict that the flux through creatine kinase in T47D cells would be too small to be detected by magnetization transfer. However, upon steady state saturation of "tATP a substantial decline in the PCr signal intensity was observed, showing that in spite of the low specific activity, the pseudo first order rate constant for creatine kinase is relatively high and measurable by magnetization transfer (Table I). This could be due to the relatively high concentration of either H + or MgADP or both (see Eqn. 1 in the Appendix). Since the pH in the cells and in the organs studied previously by magnetization transfer was approximately the same (7.2-7.4), the concentration of MgADP has to be higher. This conclusion provides an independent estimate for an ADP concentration of about 5-times higher in the T47D cells than in the cardiac cells (which happened to have almost the same MM to BB isozyme ratio as the T47D cells) and close to the upper limit for ADP concentration obtained form the N M R spectrum of approx. 0.4 mM. The creatine kinase reaction is not the dominant one for ATP to ADP exchange in the T47D cells as is the case in the above mentioned organs. The flux from ADP to ATP via synthesis from Pi was the same as the creatine kinase flux (0.2 m M / s ) . The ratio between this flux and the O2 consumption rate serves to identify the energy source for ATP synthesis, namely glycolysis versus oxidative phosphorylation. A ratio close to 3 was obtained in most organs and cells studied previously by N M R thus indicating that respiration is the main source of energy there. In the T47D cells, however, the ratio was much higher (of the order of 50). Such a ratio suggests that most of the ATP synthesis is via the glycolytic pathway, despite the fact that the cells were provided with excess oxygen. Preliminary 13C-NMR studies with labelled glucose have indicated that the rate of lactate production in the T47D cells is high (about 0.18 m M / s ) and can account for the ATP synthesis measured by NMR. The high aerobic glycolysis in cancer cells was discovered in 1926 [34]. The
188 high ADP concentration in these cells, suggested from the creatine kinase kinetics, may play an important role in maintaining high rates of aerobic glycolysis, since ADP is known to be a stimulator of the regulatory enzymes of this pathway. The rate of the creatine kinase reaction and the rate of ATP synthesis from P~ contribute together about 30% of the total ATP to ADP conversion rate, determined from the /3ATP to /~ADP exchange. All kinases and ATPases contribute to this type of change in the chemical environment of the B-phosphate. It was not possible to separate the flADP signal from the 3,ATP signal in spectra of intact cells. Thus, the saturation pulse applied at the frequency range of both signals also saturated/3ADP, which in turn induced a decrease in the intensity of the/3ATP signal. It was previously suggested that such a decrease could be due to ~,ATP ~ flATP exchange via double adenylate kinase transfer [21]. This was unlikely in our system, since saturation of the/3ATP signal did not affect the ~,ATP signal. Koretsky et al. [35] have shown that in an in vitro reaction mixture of creatine kinase, the decrease in /3ATP is maximal at the /3ADP frequency, thus indicating that /3ATP-flADP exchange can cause the reduction in the/3ATP signal. In addition, we have measured similar rates for the creatine kinase reaction in solution from the decrease in either PCr or/3ATP due to the saturation of the region of the 7ATP + /3ADP signals, and from the corresponding apparent "r values obtained by inversion recovery and inversion transfer measurements. It is therefore concluded that although /3ADP can not be detected directly, the effects of its saturation can be monitored via magnetization transfer to/3ATP. The reactions responsible for most of the ATP to ADP conversion are not known. The measured specific activity of adenylate kinase was about 4-times higher than that of creatine kinase in the same assay mixture. The conditions in the assay mixture differ from those in the intact cells; however, ADP, which is usually the limiting substrate, was found to be present at a relatively high concentration, thus suggesting that adenylate kinase may be responsible for most of t h e / 3 A T P - f l A D P flux. In an isotope exchange study of adenylate kinase it was indeed shown that the A T P - A D P exchange increases as the concentration of ADP increases [36].
The energy state of the T47D cells The energy state of cells has previously been described by the 'phosphorylation potential' which is the ratio of [ATP]/([ADP]. [Pi]) a n d / o r by the 'adenylate charge' of the cell which can be calculated for any given concentrations of ATP, ADP and AMP [37]. Estimation of these parameters requires determinations of the concentrations of the adenine nucleotides. These concentrations were previously determined by assays performed on cell extracts in which some degradation of ATP during the extract preparation was inevitable. Thus, the determination of the above-mentioned parameters was subject to error. In intact T47D c~lls, the steady-state concentrations of ATP, PCr and P~ were determined from the 31P-NMR spectra. ADP could not be determined directly but its concentration was estimated to be approx. 0.4 mM (see section above). AMP could not be detected even in extracts, and therefore its concentration was negligible. Calculation of the phosphorylation potential and the adenylate energy charge in the T47D yielded an estimate of 750 M -1 and 0.95 for these parameters respectively, indicating that these cells are highly 'energized'. Another informative way to express the energy state of living cells can be provided by the turnover rate of ATP. The rate of ATP production is adjusted to the rate of ATP utilization in a dynamic steady state. The overall rate of transfer of ATP to ADP as well as the rates of the specific phosphate transfer reactions which directly related to the metabolic activity of the cells, are usually very fast and could not be determined accurately. However, 31p magnetization transfer techniques can be used to measure these transfer rates in living cells as in this study of T47D cells. In other mammalian systems studied previously by magnetization transfer techniques (brain or skeletal and cardiac muscle), the dominant reaction for ATP turnover was catalyzed by creatine kinase. In T47D cells, the creatine kinase reaction contributes about 15% of the total ATP to ADP turnover rate which is about the same as the reactions of ATP synthesis from Pi. The major part, however, of ATP to ADP conversion is contributed by other reactions of which adenylate kinase is probably the dominant one. Adenylate kinase activity was found in the mitochondrial fraction (20%) and in the cytosol.
189
Creatine kinase activity in the mitochondrial fraction was negligible; almost all the activity originated from the cytosol. This, and the fact that the rate of ATP formation from PCr and Pi were about the same, rules out a functioning of 'shuttle' mechanism model (Ref. 38 and references cited therein) for creatine kinase in the T47D cells.
Conclusions The composition and concentration of the phosphate metabolites in T47D cells indicate that these cells are highly energized. The overall rate of ATP to ADP turnover as well as the specific rates of synthesis of ATP from Pi and from PCr were measured in the intact human cell culture by a d a p t i n g 3ap magnetization transfer techniques to this system. These studies together with 02 consumption measurements and preliminary 13CN M R studies of lactate production, showed that the main source of energy is aerobic glycolysis. The results also suggested that in these cells the adenylate kinase reaction is responsible for most of the ATP to ADP conversion, and that the ADP concentration is relatively high.
Appendix Kinetic analysis in a multi-site exchange system: The exchange of 3'ATP in the T47D cells is dominated by the following reactions: I. The reaction catalyzed by creatine kinase: PCr + ADP + H + ~ ATP + Cr 2
II. The sum of reactions that form ATP from Pi:
IV. The sum of all reactions catalyzing a transfer of the y-phosphate from ATP to an acceptor molecule ( X ) to form ADP and X-P. ~T
A T P + X ~ A D P + X-P k-T
The kinetics of these transfers are characterized by the following pseudo first order rate constants: k I =,~I[ADP][H + ]
k - I =~:_l[Cr]
k2 = k2[ADP]
k-2 =~:-2
k 3 = k3 [ A D P ]
k _ 3 = k _ 3 [AMP]
(1)
All three reactions involve an actual transfer from the 3,-phosphate of ATP to the acceptor molecules, however, these reactions also include a change in the environment of the B-phosphate via flATP-to-BADP conversion. The 3,ATP-X-P exchange and the B A T P - f l A D P exchange proceed at the same rate with a pseudo first order rate constant k T = 7¢v [X]. The enzymes that catalyze the reverse direction of I, II and III as well as all other kinase s such as hexokinase , nucleoside diphosphokinase, etc., are involved in this/3-/3 conversion. Thus, kT = ~:-1 [Cr] + ~'-2 + ~:-3[ AMP] + ~ ' ( k i [ Xi])
(2)
Where i designates a reaction in which the concentration of the phosphate acceptor is [X i]. The exchanging 31p spin system in the T47D cells can therefore be described by the following scheme: kl
k2
k-1
k_ 2
PCr ~ ~,ATP ~ P~ Pi + ADP ~ ATP ~-2
kr
flADP ~ flATP k-T
III. The reaction catalyzed by adenylate kinase: 2 ADP ~ AMP+ATP 7~_3
In this spin system, the exchange-modified Bloch equation for the decay to equilibrium of the magnetization in an inversion recovery experi-
190
merit, are [39]: r M(PCr)] IM('t-ATP)|
r M(PCr) | M('~ATP)
/M(#ADP)/ LM(BATP)]
/M(BADP~ LM(BATP)
Mo(PCr)/T 1 (PCr) M o T A T P / T I (TATP) +
A =
Mo(Pi)/TI(Pi) M o f l A D P / T 1( flADP) M o ( f l A T P ) / T 1( f l A T P )
- 1/'r (PCr)
k_ 1
0
0
ka
- 1/p(yATP)
k2
k3
0
0
k_ 2
- 1/'r (Pi)
0
0
0
k_ 3
0
I/T(flADP)
kT
0
0
0
k_ T
- 1/r(flATP)
l
covery of the signals due to PCr, yATP, P, and flATP in the system studied. Introducing several approximations based on properties of the system made it possible to simulate the data using a two-site model and to obtain estimates of the kinetic parameters. A. The forward and reverse fluxes were assumed equal for all reactions, namely
0
a. k 1[PCr] = k_l [~ATPI b. k2[Pi] = k_2[TATP l (3)
Where the T values are the apparent relaxation rates defined as: ai
I(PCr)= ~-i(PCr)+kI
b. I ( T A T P ) =
T!(-/ATP) + k_ 1 + k_2 + k_ 3
c. l(pi) = ~'~1(Pi) + k2 d. I ( f l A D P ) = T ! ( f l A D P ) + k 3 +
e. I ( f l A T P ) = ~-~(flATP) + k T
k_ T
(4)
This equation (Eqn. 3) can not be solved analytically, however, it is possible to obtain best fitted values by a numerical analysis. Saturation at the frequency region of the overlapping yATP and flADP signals caused a decrease of A M in the signal intensity of Pj, PCr and flATP due to reactions I, II and III, respectively. The fractional change in the intensity ( A M / M ) is proportional to the corresponding rate constants according to:
b. A M / M ( P i ) = kE-~'(Pi)
(5)
Performing a non-selective inversion recovery experiment provided four time courses of the re-
(6)
The above equilibrium relations also hold in a closed system when the concentration of the substrates does not change with time and there is no compartmentation of substrates. These conditions were closely maintained in the cells during the kinetic measurements. (1) The cells were perifused with phosphate-free medium and could, therefore, be considered as a closed system regarding the phosphates; (2) the concentration of the substrates remained constant; (3) most of the creatine kinase activity and approx. 80% of the adenylate kinase activity was found in the cytosol. B. The intrinsic relaxation of ,/ATP was assumed to be affected by the sum of the exchange processes (III). 1T(yATP) = ~ (yATP) + k T
(7)
C. An upper limit of 0.1 was set for the A D P / A T P ratio, on the basis of the identity of the y- and flATP signal areas. In addition, the intrinsic relaxation 1 / T 1 (flADP) was assumed to be equal to 1 / T 1 (yATP) and the apparent 1/T(flADP) was therefore given by: (flADP) = - ~ (7ATP) + k T [ATPI/[ADP]
a. A M / M ( P C r ) = k~. T(PCr)
c. A M / M ( f l A T P ) = k T . ~'(flATP)
c. kT[flATP ] = k_T[flADP l
(8)
D. The inversion recovery data were analyzed assuming a two-site exchange model according to the following equation: Mi(t ) = Mi(oo ) + C 1 e (v+o + C 2 e (v-t>
(9)
191 TABLE II PARAMATERS USED FOR FIT'I'ING THE INVERSION RECOVERY DATA TO A TWO-SITE EXCHANGE MODEL The kinetic parameters for PCr, Pi AND flATP were chosen according to Eqns. 1-8. The parameters for "tATP were: kia equals k T which includes all exchange processes from yATP to all other sites; k~i includes the average contribution from the exchange with Pi and PCr. 1
I
a
--
'ra
kia kai
PCr
y-ATP
1_(yATP)
kl
y-ATP Pi+PCr
'2(1/~(Pi)+l/r(PCr)}
k w ½(kl+k2}
Pi
1 (yATP)
k 2 k_ 2
1--(BADP)
kT k_T
y-ATP
B-ATP ¢-ADP
T
T
T
k_ 1
where y± = ½{ - ( 1 / r i + 1 / % ) + { ( 1 / r i - 1/%) 2 +4(kiakai) } 1}. C 2 = { ( 1 / k a i ) ( M i ( O ) - Mi(o0))'(Y+ + 1/ri) + M ~ ( a ) - M~(0)}/{y+ - X_ } l / k a i
C 1 = Mi(0 ) - Mi(oo ) - C2
The list of parameters used for data analysis of the inversion recovery and steady state saturation experiments is given in Table II. An iterative nonlinear computer SAS program was used to fit the data to ten Equations (three SSST equations: 5a-c, four inversion recovery time courses Eqn. 9 and three equilibrium conditions Eqns. 6a-c) for obtaining the best fit values for ten parameters (six rate constants and four relaxation rates). These parameters were then used to obtain a numerical simulation of the inversion recovery data using the five-site exchange model (Eqn. 3) and to obtain the residual sum of squares from the experimental data.
Acknowledgements We are very grateful to Professor Y. Keydar for the cells and the support in the course of these studies. We also thank Professor M. Cohn and Dr. T.A. Victor for many helpful discussions and Ms.
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