©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
C and P NMR Investigation of Effect of 6-Aminonicotinamide on Metabolism of RIF-1 Tumor Cells in Vitro(*)

(Received for publication, October 12, 1995; and in revised form, December 7, 1995)

James C. Street (1)(§) Umar Mahmood (1) Douglas Ballon (1) (2) Alan A. Alfieri (1) (4) Jason A. Koutcher (1) (2) (3)

From the  (1)Departments of Medical Physics, (2)Radiology and (3)Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 and (4)Beth Israel Medical Center, New York, New York 10003

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The effect of 6-aminonicotinamide on the metabolism of RIF-1 tumor cells was investigated using C and P NMR spectroscopy. 6-Aminonicotinamide can be metabolized to 6-amino-NAD(P), a competitive inhibitor of NAD(P)-requiring processes. 40 µM 6-aminonicotinamide led to an inhibition of 6-phosphogluconate dehydrogenase and an accumulation of 6-phosphogluconate. A subsequent accumulation of the 6-phosphogluconate precursor 6-phosphoglucono--lactone was observed in the C NMR spectrum. These metabolites were shown to be intracellular, although a small amount of leakage of 6-phosphoglucono--lactone occurred. The intracellular concentrations of 6-phosphogluconate and 6-phosphoglucono--lactone were 1.9 ± 0.8 µmol/10^8 cells (±1 standard deviation) and 0.8 ± 0.4 µmol/10^8 cells, respectively, after 15 h. Glucose utilization and lactate production were significantly inhibited by 6-aminonicotinamide (both p < 0.05), indicating inhibition of glycolysis. P NMR data showed that phosphocreatine was significantly depleted in cells exposed to 6-aminonicotinamide (p < 0.05). Exposure of RIF-1 cells to 6-aminonicotinamide prior to 3- or 6-Gy x-irradiation induced a supra-additive cell kill, indicating that 6-aminonicotinamide is acting as a radiosensitizer. There was no effect of 6-aminonicotinamide alone or when the drug was given postradiation, suggesting that its mechanism of action may be by inhibition of radiation-induced repair.


INTRODUCTION

6AN, (^1)an analogue of niacin, is currently being evaluated in preclinical trials as part of a multidrug anti-cancer regimen(1, 2) , having undergone clinical trials as a single agent in the 1950s(3) . It has been shown to have activity as a radiosensitizer in vitro(4) and be a potentiator of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity both in vitro and in vivo(5) . 6AN can compete with niacin in the synthesis of NAD(P), being metabolized to 6ANAD or 6ANADP, which, in turn, can act as competitive inhibitors of NAD(P)-requiring processes, such as generation of ATP by oxidative phosphorylation. 6ANADP is a particularly potent inhibitor of the PPP enzyme 6PG dehydrogenase. This leads to cytotoxicity by inhibition of nucleotide synthesis(6, 7) , poly(ADP-ribose) synthesis (5) and of glycolysis(8) .

Fig. 1summarizes the major pathways of glucose metabolism in tumor cells. Tumor cells have long been known to metabolize glucose to lactate, via glycolysis, under aerobic conditions (9) . A significant portion of glucose, however, has been shown to be metabolized to lactate via PPP(10, 11) . The PPP provides NADPH for biosynthetic processes and ribose sugars for nucleotide synthesis. Glucose 6-phosphate can be metabolized to fructose 6-phosphate via phosphoglucose isomerase as part of glycolysis or enter PPP, proceeding via the enzyme glucose 6-phosphate dehydrogenase.


Figure 1: Schematic of glucose metabolism via glycolysis and the pentose phosphate pathway highlighting the metabolites and enzymes of importance in this study. The site of inhibition by 6AN, as 6ANADP, is marked by the double diagonal. 1, hexokinase; 2, G6P dehydrogenase; 3, 6-phosphoglucono--lactonase; 4, 6PG dehydrogenase; 5, phosphopentose isomerase.



C-NMR has been widely used to investigate the pathways of cellular glucose metabolism, both in vitro and in vivo as well as in humans(11, 12, 13, 14, 15, 16, 17, 18, 19) . The effect of a number of drugs and hormones on glucose and glycogen metabolism have been studied, such as vasoactive intestinal peptide(20) , rhodamine 123 (21) and lonidamine(22) . Most of these studies have been limited to observation of glucose utilization and glycogen and lactate production, although labeled alanine and glutamate have also been observed in some systems. In this study we have investigated the effect of 6AN on the glucose metabolism of RIF-1 tumor cells using C and P NMR, and observed accumulation of the PPP intermediates 6PG and 6PGL. We also investigated the potential of 6AN as a radiosensitizer.


MATERIALS AND METHODS

Cells

RIF-1 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (Intergen, Purchase, NY). 96 h before the start of perfusion, approximately 5 times 10^7 RIF-1 cells were seeded on 0.12 g of conditioned Cultispher-G collagen beads (Hyclone Laboratories, Inc., Logan, UT) in a spinner jar in 125 ml of medium. The medium for perfusion experiments was phosphate-free RPMI 1640 supplemented with 10% fetal calf serum and 1.2 mg/liter DNase I (Sigma). The total number of cells on the beads at the time of transfer from the spinner flask to the perfusion apparatus was approximately 1.7 times 10^8 cells. Cells were counted by removing 1 ml of beads and medium from the spinner flask. Following sedimentation of the beads 0.8 ml of the supernatant was removed and replaced by 0.8 ml of collagenase (10 mg/ml). This mixture was incubated at 37 °C for 30 min. With the collagen beads digested, it was possible for the released cells to be counted by eryrthrosin B dye exclusion. The total volume of the perfusion circuit, including the reservoir, was 130 ml. The head space of the medium reservoir was gassed with O(2)/CO(2) (95:5). The design of the perfusion apparatus was as described previously for agarose threads(23) . Cells were perfused on the collagen beads as a fluidized bed in a shortened screw cap NMR tube (Wilmad, Buena, NJ). The reduction in upward force as the cross-sectional area of the tube increased at the neck was sufficient to prevent flow of cells into this part of the tube; the medium flow rate was adjusted accordingly.

NMR

Two five-turn solenoid coils, wrapped around the bead-containing volume, were used to collect either P NMR spectra or C NMR spectra. Proton-decoupled P NMR spectra were obtained using a WALTZ-16 pulse-sequence on a Bruker/GE 4.7T Omega spectrometer (Bruker NMR, Fremont, CA) operating at 81.03 MHz. P NMR data were acquired with a spectral width of 10,000 Hz, a 60° pulse angle (12-µs pulse width), a relaxation delay of 2 s, 4K data points, and 4096 signal-averaged free induction decays. Under these conditions spectra were partially saturated. 135 min of data acquisition (4096 transients) was sufficient to give a good signal:noise ratio. P NMR spectra were processed with 5-Hz exponential line broadening and referenced to PCr at 0 ppm. Proton-decoupled C NMR spectra of cells were acquired at 50.34 MHz. C NMR spectra of cells were acquired with a sweep width of 20,000 Hz, a 60° flip angle (30 µs), a recycle time of 3 s, 8192 data points, and 1024 transients. Each spectrum took 55 min to acquire. C NMR spectra were processed with 2-Hz exponential line broadening and referenced to the beta-anomer of glucose at 96.0 ppm, except where stated in the figure legends. As a known amount of labeled glucose was used in each experiment, it was used as an internal concentration standard. Since two different coils were used it was necessary to retune and shim the magnet each time the observe nucleus was changed. The NMR tube containing collagen beads was immersed in water to minimize inhomogeneities due to magnetic susceptibility gradients(24) . A Helmholtz decoupling coil was built on the outside of the water bath.

High resolution proton-decoupled C and P NMR spectra of cell extracts, at approximately pH 9, were acquired on a Bruker AC 250-MHz magnet operating at 62.90 and 101.25 MHz, respectively, using a 5-mm probe. Experimental parameters for C NMR were 20,000 Hz, 64K data points, a 60° flip angle (pulse width of 3.0 µs), and a recycle time of 4.1 s. Spectra were zero-filled and processed with 1-Hz line broadening. Spectra were referenced to the beta-anomer of glucose at 96.0 ppm. Experimental parameters for P NMR included a sweep width of 15,000 Hz, 32K data points zero-filled to 64K, a 45° flip angle of 2.75-µs pulse width, and a recycle time of 5.1 s. Free induction decays were processed with 1-Hz exponential line broadening. Spectra were referenced to GPC at 0.0 ppm, as the chemical shift of this peak is almost invariant with changes in pH(25) . Dimethyl methyl phosphonate (1 mM) was used as an internal concentration standard for calculating cellular concentrations of phosphatic metabolites. It was assumed for these calculations that the density of the extracted cells was the same as that of the deuterium oxide in which the extract was reconstituted.

Assignments of peaks in P NMR spectra of PCA extracts were made by adding known compounds to the extract and noting whether they were coincident with peaks present in the extract spectrum. In some cases assignments were confirmed by pH titration.

Experimental Protocol

After the collagen beads had been transferred to the perfusion apparatus, the magnet shimmed and the coil tuned and matched, two P NMR spectra were collected. Following this initial stabilization period, the medium reservoir was replaced by 100 ml of phosphate-free perfusion medium with [1-C]glucose (180 mg) substituted for [U-C]glucose. This volume of labeled media was much greater than the volume of media already in the perfusion circuit, so labeled and unlabeled pools were able to equilibrate during shimming and tuning. The total volume of the medium reservoir and the perfusion circuit was 130 ml. Sixteen sequential C NMR spectra were then acquired over the next 15 h. C label was then washed out of the perfusion apparatus by perfusing with two aliquots of unlabeled media, each for 15 min. A P NMR spectrum was collected before once more changing over to media containing [1-C]glucose (180 mg) and a final concentration of 40 µM 6AN. Sixteen C NMR spectra were acquired as before. Following wash-out of the label and 6AN, further C NMR spectra were acquired. It was noted that the peaks arising due to the C-1 carbons of glucose were reduced to natural abundance levels, indicating efficient removal of label and drug. Finally a P NMR spectrum was acquired prior to harvesting the cells for PCA extraction. C and P NMR data were collected from a total of three separate perfusion experiments in this manner. P NMR spectra were obtained, in these experiments, immediately before and after each period of perfusion in the presence of [1-C]glucose, with C NMR data acquired at all other times.

In addition to these experiments, three further experiments were carried out in which RIF-1 cells were perfused, both with and without 6AN, in the absence of labeled glucose. The same protocol, as described above, was followed for experiments in which P NMR spectra were acquired throughout. Unlabeled glucose was used in these experiments. Initially eight P NMR spectra were acquired prior to addition of 6AN, after which a further six spectra were acquired. Following wash-out of the drug, six more P NMR spectra were acquired. In three control experiments, in which drug was not added after 22 h, fresh perfusate was exchanged for the old at this point, and perfusion continued for an additional 22 h, for a total of 44 h.

Perchloric Acid Extracts

Chelex-100 was obtained from Sigma. All other reagents were of analytical grade. PCA extracts of excised tumors were made using slight modifications of previously described methods(26) . PCA extracts of cells on beads were made by washing sedimented beads directly into liquid nitrogen with a small volume of medium. Briefly, the cells and beads were ground to a powder in a liquid nitrogen-cooled mortar. Approximately 3 volumes of 10% PCA was added to the powdered cells. This mixture was ground further until cells and PCA were well mixed. The mixture was then transferred to a polypropylene centrifuge tube and thawed and immediately refrozen three times. Following this freezing and thawing cycle the precipitate was removed by centrifugation for 5 min. The supernatant was removed and neutralized with potassium carbonate (1 N). Following adjustment to pH 10.0, the potassium perchlorate precipitate was removed by centrifugation. The supernatant was then mixed and regularly shaken, over a period of 1 h, with Chelex-100 to remove any paramagnetic ions. Chelex-100 was subsequently removed by centrifugation. The supernatant was frozen and lyophilized prior to NMR analysis. The lyophilized extract was reconstituted in 1-2 ml of deuterated water.

Medium Extracts

A crude extraction procedure, similar to that described previously(27) , was carried out to concentrate the media and remove serum protein. Briefly, an equal volume of cold acetone was added to the harvested media sample and left on ice for 20-30 min. The precipitated protein was removed by centrifugation at 1500 times g for 10 min. The supernatant was decanted from the protein fraction, and the acetone was removed from the sample under vacuum using a rotary evaporator. The temperature of the water bath used was not in excess of 37 °C. Chelex-100 (Sigma) was added to the remaining water phase and shaken periodically over a period of approximately 2 h to chelate any paramagnetic ions present in the sample. Chelex-100 was removed by centrifugation, and the resulting solution was lyophilized. The lyophilized powder was reconstituted in 2 ml of deuterated water.

Surviving Fraction Experiments

10^5 RIF-1 cells/ml were seeded in 5 ml of RPMI 1640, supplemented with 10% fetal calf serum in 25-cm^2 culture flasks and grown for 48 h, in order to establish log phase growth. After 48 h the media was changed. Different flasks of cells were harvested by trypsinization following one of eight different treatments. Control cells were harvested without any treatment. Three flasks were treated with 40 µM 6AN for 15 h as in the NMR experiments. Of these, one flask was harvested after 15 h, one was exposed to 3-Gy irradiation after 15 h of 6AN exposure, and the other was exposed to 6-Gy irradiation. Irradiation was carried out as described previously. These irradiated flasks of cells were harvested 3 h following irradiation(23) . These conditions were reversed in other experiments, with the two doses of irradiation preceding the 6AN treatment by 3 h. Also cells were exposed to 3- or 6-Gy irradiation only and harvested 3 h later. The trypsinized cells were counted and plated on a 5-cm Petri dish in 5 ml of RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, and nonessential amino acids. A number of plating densities were used to ensure colony counts were in a range likely to give accurate data. After 9 days the plates were stained for 3 h with crystal violet and rinsed, and surviving colonies were counted. All results are quoted as mean ± 1 standard deviation of triplicate determinations.

Statistics

Paired t tests were used to evaluate differences in the metabolic response of batches of cells to perfusion in the presence and absence of 6AN. Unpaired t tests were used to compare differences between cells that were exposed to 6AN after 22 h and those that were perfused for 48 h in the absence of 6AN and to compare plating efficiencies in the surviving fraction experiments. Errors are quoted as plus or minus one standard deviation. Error bars on the figures are standard errors of the mean.


RESULTS

P NMR Experiments

Fig. 2shows P NMR spectra of RIF-1 cells obtained at intervals during a perfusion experiment, following the addition of 6AN. These spectra demonstrate a decrease in PCr and an accumulation of 6PG following the addition of 6AN (40 µM) to the perfusate. It can be seen from Fig. 3that this accumulation of 6PG continued for at least 12 h after wash-out of 6AN, albeit at a reduced rate. This 6PG peak persisted for at least 36 h following wash-out of 6AN. The extracellular concentration of inorganic phosphate was constant in all of the experiments, as the same batch of serum was used throughout. Also, the extracellular volume was much larger than the intracellular volume, so changes in P(i) with varying cell number were minimal. It was possible, therefore, to normalize metabolite ratios for cell number by dividing the ratio by the initial number of cells on the beads. P NMR data were obtained from cell perfusion experiments in which cells were perfused both in the presence and absence of C label (n = 7). There was a significant increase in PCr levels over the first 22 h of perfusion in the absence of 6AN, the PCr/P(i) ratio increasing from 0.19 ± 0.04/10^8 cells to 0.36 ± 0.12/10^8 cells (p < 0.01). 20 h after administration of 6AN, however, there was a significant drop in this ratio to 0.12 ± 0.07/10^8 cells (p < 0.05). PCr/P(i) increased from 0.36 ± 0.10/10^8 cells to 0.42 ± 0.06/10^8 cells at 40 h, in cells that continued to be perfused in the absence of 6AN (n = 3). PCr/P(i) was significantly lower after 40 h of perfusion in cells exposed to 6AN than in control cells that were not treated with the drug (p < 0.001). The observed increases in NTP/P(i) were not significant. The initial increase in PCr/P(i), coupled with the disappearance of GPC after 2-4 h, indicates that the cells were well perfused and not stressed in the first 22 h prior to addition of the drug. The maintenance of NTP and PCr levels for up to 48 h in untreated cells confirm that it is valid to compare data from the same batch of cells.


Figure 2: P NMR spectra of perfused RIF-1 tumor cells on collagen beads. A, 1-5 h after the addition of 40 µM 6AN; B, 6-10 h after the addition of 6AN; C, 11-15 h after the addition of 6AN. P NMR spectral peak assignments are as follows: 1, 6PG; 2, composite peak consisting of phosphoethanolamine and NMP; 3, phosphocholine; 4, inorganic phosphate; 5, phosphodiesters; 6, PCr; 7, -NTP; 8, alpha-NTP; 9, beta-NTP.




Figure 3: A, plot showing the increase in the 6PG:P(i) ratio after the addition of 40 µM 6AN to perfusate of perfused RIF-1 tumor cells on collagen beads. The plot also follows the change in 6PG:P(i) after wash-out of 6AN from the perfusate. B, plot showing change in PCr/P(i) over time during perfusion of RIF-1 tumor cells in which media were changed after 20 h to that containing regular media or media containing 40 µM 6AN.



Identification of 6PG and 6PGL

After 4 h of perfusion of RIF-1 cells in the presence of [1-C] glucose and 6AN (40 µM) a peak began to appear at around 178.8 ppm in the C NMR spectrum (see Fig. 4A). After a further 4 h, a second peak appeared at 178.6 ppm. The downfield peak has been assigned to 6PG, by observing an increase in the peak in both the P and C NMR spectra following addition of the authentic compound. The peak assigned to 6PG in the P NMR spectrum was coresonant with added 6PG over a wide pH range, from pH 5.5 to 9.5. The second peak, observed 0.2 ppm upfield from 6PG, was observed to slowly disappear in extracts at neutral pH. The intensity of 6PG increased as that of the second resonance decreased. 6PGL, the precursor to 6PG in PPP, is known to spontaneously hydrolyze to 6PG in solution at neutral pH even in the absence of 6-phosphoglucono--lactonase(28) . This peak was therefore assigned to 6PGL, both on the basis of this evidence and the similarity of its structure to 6PG. The rate of hydrolysis of 6PGL was much slower at pH 9 than at neutral pH. 6PGL is not commercially available. Other possible glucose metabolites were ruled out on the basis of their chemical shifts. Further evidence for these assignments comes from the fact that the chemical shifts of the two compounds reported here are very similar to those reported for the nonphosphorylated forms(29) . The chemical environment of the phosphate group in these two molecules was sufficiently similar that they could not be resolved in the P NMR spectrum of extracts, even upon pH titration of their chemical shifts.


Figure 4: A, expansion of C NMR spectrum of RIF-1 tumor cells after 15 h of perfusion in the presence of 10 mM [1-C]glucose and 40 µM 6AN. B, expansion of the C NMR spectrum of RIF-1 tumor cells 2 h after wash-out of labeled glucose and 6AN. A and B were zero-filled and processed with 1-Hz exponential line broadening. C, expansion of the C NMR spectrum of PCA extract of RIF-1 tumors. The extract was made 5 h after wash-out of C label and 6AN. D, acetone extract of perfusate from RIF-1 tumor cells perfused for 15 h in the presence of 10 mM [1-C]glucose and 40 µM 6AN. The chemical shift difference between the in vitro spectra and the extract spectra is in part due to a pH difference but is also due to the lack of intracellular interactions in extracts.



C NMR Experiments

6PG and 6PGL

In each labeling experiment, RIF-1 cells were perfused for 15 h in the presence of [1-C] glucose. Following wash-out of this label with unlabeled media, cells were perfused in the presence of both [1-C] glucose and 6AN. Each experiment, therefore, acted as its own control. Fig. 4A shows a C NMR spectrum acquired after 15 h of perfusion in the presence of 1 mmol [1-C]glucose and 40 µM 6AN, and Fig. 4B shows a C NMR spectrum following wash-out of C-labeled media and 6AN, respectively. It can be seen that following wash-out of labeled media, peaks arising due to 6PG and 6PGL persisted. Peaks due to labeled glucose and lactate were reduced to natural abundance levels (not shown). 6PG and 6PGL were diminished only slightly in the hours following wash-out of the label and drug. This suggests that these metabolites are intracellular. Fig. 4C shows a PCA extract of RIF-1 cells after perfusion in the presence of [1-C]glucose and 6AN. Fig. 4D shows an acetone extract of the C-labeled perfusate. 6PGL, but not 6PG, was found to be present. The presence of 6PG and 6PGL in PCA extracts of cells and the absence of 6PG in the perfusate provide further evidence that these metabolites are intracellular. The presence of 6PGL, but not 6PG, in the perfusate indicates that this compound was more readily leaked from the cell. Cell lysis as a mechanism for leakage of 6PGL can be ruled out, as this would be expected to yield 6PG also. Neither 6PG nor 6PGL were observed in the control experiments.

The cellular concentration of 6PG calculated from the P NMR spectra of PCA extracts, made after the 6AN wash-out phase of the perfusion experiment, was 2.0 µmol/10^8 RIF-1 cells. This was as compared with 0.3 µmol/10^8 RIF-1 cells of NTP. This value for the concentration of 6PG was used to calculate the amount of unlabeled (1% natural abundance) 6PG required to cause a doubling of the 6PG peak in the C NMR spectrum of a PCA extract of cells incubated with [1-C] glucose and 6AN. Addition of a 100 times concentration of unlabeled 6PG caused an approximate doubling of the peak assigned to 6PG in the C NMR spectrum (since the natural abundance of C is approximately 1%), indicating that it was essentially 100% C-labeled. In the perfusion apparatus the cells were restricted to a volume of 6 ml inside the NMR tube. Assuming that 6PG and 6PGL are intracellular, these metabolites are therefore restricted to this volume, whereas other labeled metabolites such as glucose and lactate are present in the entire 130 ml of the perfusion apparatus. Fig. 5charts the increase in concentration of 6PG and 6PGL following administration of 6AN, as calculated from the C NMR data. The corresponding increase in 6PG/P(i) observed in the P NMR spectrum over the same period in an experiment in which C label was not utilized is shown in Fig. 3A. As expected, it can be seen that 6PG appears at approximately the same time in both experiments. The intracellular concentrations of 6PG and 6PGL after 15 h (900 min) of perfusion in the presence of 10 mM [1-C]glucose and 40 µM 6AN were 1.9 ± 0.8 µmol/10^8 RIF-1 cells and 0.8 ± 0.4 µmol/10^8 RIF-1 cells, respectively. These values are in close agreement with the value calculated from PCA extracts.


Figure 5: Plot showing accumulation of C-labeled 6PG and 6PGL as measured from C NMR spectra of RIF-1 tumor cells perfused in the presence of 10 mM [1-C] glucose and 40 µM 6AN.



Glucose and Lactate

Fig. 6shows two series of C NMR spectra. The first series in Fig. 6A shows the metabolism of glucose to lactate over a period of 15 h in RIF-1 tumor cells perfused in the presence of 10 mM [1-C]glucose. The second series in Fig. 6B shows glucose metabolism of the same cells over 15 h in the presence of 10 mM [1-C]glucose and 40 µM 6AN. It can be seen from these two series of spectra that metabolism of glucose to lactate was reduced in the presence of 6AN. Fig. 7A shows the change in glucose concentration over time in control and treated cells. After 15 h the glucose concentration was 0.32 ± 0.03 mmol/10 cells and 0.46 ± 0.05 mmol/10 cells for control and treated cells, respectively. Glucose utilization was therefore significantly inhibited in the presence of 6AN (p < 0.05). Similarly, Fig. 7B shows that production of lactate was significantly inhibited (p < 0.05), being 0.17 ± 0.02 mmol/10 cells in control and 0.07 ± 0.01 mmol/10 cells in the presence of 6AN. H NMR analysis of the methyl peak of lactate and its H-C coupled satellites, in acetone extracts of the perfusate, showed no significant change in the percentage of labeling of lactate (data not shown). The amount of label unaccounted for in the mass balance equation, i.e. lost as carbon dioxide, was 1.67 ± 0.43 mmol/10 cells in control and 0.62 ± 0.52 mmol/10 cells in the presence of 6AN. Significantly less carbon was lost as carbon dioxide in cells treated with 6AN than in control cells (p < 0.01).


Figure 6: A, series of 16 C NMR spectra of RIF-1 tumor cells perfused in the presence of 10 mM [1-C] glucose. B, series of 16 C NMR spectra of the same RIF-1 tumor cells as in A, perfused in the presence of 10 mM [1-C] glucose and 40 µM 6AN. C NMR spectral peak assignments are as follows: 1, beta-anomer of glucose; 2, alpha-anomer of glucose; 3, lactate.




Figure 7: A, plot showing utilization of [1-C] glucose, as measured by C NMR, by RIF-1 tumor cells in the presence and absence of 40 µM 6AN. B, plot showing production of [3-C] lactate, as measured by C NMR, by RIF-1 tumor cells in the presence and absence of 40 µM 6AN.



Effect of 6AN on Radiosensitivity

The surviving fraction of RIF-1 cells was estimated following exposure to 6AN and in combination with 3- or 6-Gy irradiation in order to evaluate its potential as a radiosensitizer. The plating efficiency of RIF-1 cells following the various treatments is summarized in Table 1. 6AN alone had no significant effect, but 3 and 6 Gy caused significant cell kill with respect to control (both p < 0.05). The surviving fractions, relative to control, were 20 and 15% for 3 and 6 Gy, respectively. When 6AN was administered following radiation there was no significant reduction in the plating efficiency. When irradiation followed 6AN, however, there was a further significant reduction in the surviving fraction. When 3- or 6-Gy irradiation was followed by 6AN the plating efficiency was significantly reduced compared with irradiation alone (p < 0.05 for 3 Gy, and p < 0.001 for 6 Gy), giving surviving fractions relative to control of 11 and 6%, respectively. These data indicate potentiation of radiotherapy by 6AN when this drug is administered prior to x-irradiation.




DISCUSSION

Effect of 6AN on Pentose Phosphate Pathway

6AN can compete with nicotinamide to form 6ANAD and 6ANADP, which are competitive inhibitors of NAD(P)-requiring processes(30) . The oxidative arm of PPP is an important source of ribose 5-phosphate, which is required for nucleotide synthesis and of NADPH, which is required as a reducing equivalent in many biosynthetic reactions. 6ANADP, therefore, can cause inhibition of glucose 6-phosphate dehydrogenase and 6PG dehydrogenase, the NADP-requiring steps of this pathway. The large accumulation of 6PG and the almost 3-fold decrease in the amount of C-label lost as carbon dioxide indicate a strong inhibition of 6PG dehydrogenase. There was no detectable accumulation of glucose 6-phosphate in vitro or in extracts, in agreement with Keniry et al.(31) . As discussed below, a significant increase in glucose 6-phosphate may have been below the detection limits of this technique. While some inhibition of glucose 6-phosphate dehydrogenase cannot be ruled out, the accumulation of as much as 1.9 ± 0.8 µmol of 6PG/10^8 RIF-1 cells indicates that it was not significant relative to the inhibition of 6PG dehydrogenase. The inhibitor constant of 6ANADP for 6PG dehydrogenase has been shown to be at least 1 or 2 orders of magnitude lower than that of glucose 6-phosphate dehydrogenase and other enzymes(32, 33, 34) . It is possible, therefore, that while 6PG dehydrogenase is strongly inhibited, glucose-6-phosphate dehydrogenase could be relatively unaffected.

Immersion of the NMR tube, containing the cells, and the NMR detection coil in a water bath in order to minimize field inhomogeneities due to magnetic susceptibility has enabled us to obtain resolution in the C NMR spectrum close to that of PCA extracts (see Fig. 4) (24) . This is the first report in which any accumulation of 6PGL has been noted. The large chemical shift range of C NMR has allowed us to readily resolve this metabolite from the closely related metabolite 6PG in vitro. It is possible that the structural similarity of 6PG and 6PGL precludes the distinguishing of the two using other techniques, including P NMR. 6PGL is the direct metabolic precursor of 6PG, being hydrolyzed to 6PG via the enzyme 6PGL lactonase. Brodie and Lipmann (28) noted that 6PGL was not formed when 6PG was incubated in the presence of isolated yeast 6-phosphoglucono--lactonase, indicating that the equilibrium of the enzyme lies overwhelmingly in favor of 6PG formation. This suggests, therefore, that formation of 6PGL is probably due to competitive feedback inhibition by 6PG. Further evidence for this comes from the fact that the K(m) of the enzyme for 6PGL has been found to be low, and the cellular concentration of 6PGL in various tissues was also low (10 nmol/g of tissue)(35) . 6PG and 6PGL continued to increase throughout the incubation period, indicating that any inhibition of glucose 6-phosphate dehydrogenase, whether by feedback or by 6ANADP, was necessarily limited as discussed above. It has been shown in this study that 6PG and 6PGL are intracellular, with only a small amount of leakage to the perfusate. Like many other phosphorylated intermediates, 6PG and 6PGL transport out of the cell is restricted, leading to a large accumulation. Only the cyclic 6PGL was observed in acetone extracts of the perfusate. No 6PG, which is the open chain form of the molecule, was observed. The absence of 6PG in the perfusate indicates that 6PGL was not leaked into the perfusate via cell lysis. The similarity of the structure of 6PGL to glucose (both are pyranoses in solution) suggests that it may have reached the perfusate via the glucose transporter.

Effect of 6AN on Glycolysis

The observed reduction in glucose utilization is not in itself an indication of inhibition of glycolysis, as there was a significant flux of glucose carbon into PPP, as demonstrated by the accumulation of 6PG and 6PGL. Production of labeled lactate, however, is a direct measure of the glycolytic rate. Lactate produced via PPP is not labeled, as the label at the C-1 position is lost as carbon dioxide; hence only lactate produced via glycolysis is labeled at C-1. As can be seen from Fig. 7, lactate production, as well as glucose utilization, was reduced in cells exposed to 6AN after 200 min, and almost total inhibition occurred after 400 min. The onset of this inhibition of glycolysis correlated with the start of accumulation of 6PG. After 400 min, when inhibition of glycolysis was effectively complete, the concentration of 6PG was 1.0 ± 0.6 µmol/10^8 RIF-1. 6PG has been shown to cause inhibition of glycolysis, acting as a competitive inhibitor of phosphoglucose isomerase(35) , and Kahana et al. reported that the K(i) of 6PG for phosphoglucose isomerase was 5 µM(36) . Although some inhibition of phosphofructokinase by 6PG has been observed (37) the inhibition of phosphoglucose isomerase is more than sufficient to explain the almost complete shutdown of glycolysis following accumulation of 6PG. It is possible that 6PGL is in fact a much stronger inhibitor of phosphoglucose isomerase, as its structure, in the pyranose ring form, is much more similar to that of glucose 6-phosphate than is that of the open chained 6PG.

This inhibition of both glycolysis and PPP by administration of 6AN to RIF-1 tumor cells caused a significant reduction in PCr/P(i). The onset of PCr depletion correlated with the first appearance of 6PG and the associated inhibition of glycolysis. Inhibition of these pathways of glucose metabolism prevents the cells from synthesizing NTP via glycolysis and the Krebs cycle and NADPH via PPP. Inhibition of glycolysis is especially important to tumor cells for which this pathway is a major energy source(38, 39) . A lack of NTP synthesis will lead to a depletion of NTP as it is catabolized for maintenance energy (40) . This result is analogous to a study in which ascites tumor cells were incubated with the glycolytic inhibitor 2-deoxyglucose (41) and a previous study in which a significant depletion of ATP was observed after 4 days of exposure to 6AN(42) . Although NTP/P(i) was not significantly altered 24 h after administration of 6AN, PCr/P(i) was reduced approximately 4-fold over the same period. Keniry et al. did not observe any change in NTP/P(i) or PCr/P(i) after 24 h in the presence of 100 µM 6AN(31) . The cells used in their experiments, however, were analyzed by NMR following trypsinization rather than by using a perfusion apparatus. It has been shown in human muscle that cellular PCr concentrations act as a buffer for NTP in order to maintain the cellular NTP concentration(43) . PCr, as a buffer for NTP levels, would be expected to be severely depleted before any significant reduction in NTP was observed, during inhibition of glycolysis. A number of different mechanisms for the cytotoxicity of 6AN have been demonstrated(5, 6, 7, 8) , and all of these mechanisms could contribute to the effects observed here. The inhibition of glycolysis, however, is sufficient to explain the reduction of PCr/P(i) observed here.

6AN as a Radiosensitizer

The surviving fraction experiments indicated that 6AN enhanced the effect of x-irradiation when cells were exposed to 6AN for 15 h prior to irradiation. There was no such effect when 6AN treatment followed x-irradiation, which may be attributed to the fact that DNA repair was complete at 3 h before addition of the drug, or when 6AN was given alone. These data indicate, therefore, that 6AN alone has no significant cytotoxic effect but does act as a radiosensitizer. 6AN inhibits 6PG dehydrogenase, which is involved in the production of both ribose units and NADPH, both of which are required for DNA repair and synthesis. 6AN, therefore, could act as a radiosensitizer through the indirect inhibition of DNA repair, which is required following x-irradiation. Alternatively, the reduction in cellular PCr in the presence of 6AN, as measured by NMR, and the increased efficacy of radiation treatment, suggest that 6AN may potentiate other treatments, including radiation (4) and 1,3-bis(2-chloroethyl)-1-nitrosourea(5) , by reducing the amount of cellular energy available for DNA repair.

Conclusions

This study demonstrates the value of NMR for study of the effects of antimetabolites on cellular metabolism. The use of C NMR has enabled us to simultaneously monitor the accumulation of 6PG due to the inhibition of PPP by 6ANADP and the secondary inhibition of glycolysis. We were also able to identify 6PGL, a metabolite that had not previously been observed in studies of the drug 6AN using other techniques. 6AN has also been shown to be a radiosensitizer, acting either by inhibition of ribose and NADPH synthesis or by compromising cellular energy levels.


FOOTNOTES

*
This work was supported by NCI, National Institutes of Health, Grant USHPS PO 1 CA 25842. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medical Physics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.: 212 639 8835; Fax: 212 717 3010.

(^1)
The abbreviations used are: 6AN, 6-aminonicotinamide; 6ANAD, 6-amino-NAD; 6ANADP, 6-amino-NADP; 6PG, 6-phosphogluconate; 6PGL, 6-phosphoglucono--lactone; PCA, perchloric acid; PPP, pentose phosphate pathway; PCr, phosphocreatine; RIF-1, radiation induced fibrosarcoma 1.


REFERENCES

  1. Stolfi, R. L., Colofiore, J. R., Nord, L. D., Koutcher, J. A., and Martin, D. S. (1992) Cancer Res. 52, 4074-4081 [Abstract]
  2. Martin, D. S. (1987) in Metabolism and Action of Anti-cancer Drugs (Powis, G., and Prough, R. A., eds) pp. 91-140, Taylor and Francis, London
  3. Herter, F. P., Weissman, S. G., Thompson, H. G., Jr., Hyman, G., and Martin, D. S. (1960) Cancer Res. 21, 31-37
  4. Varnes, M. E. (1988) Natl. Cancer Inst. Monogr. 6, 199-202
  5. Berger, N. A., Catino, D. M., and Vietti, T. J. (1982) Cancer Res. 42, 4382-4386 [Abstract]
  6. Coper, H., and Neubert, D. (1964) Biochim. Biophys. Acta 82, 167-170
  7. Hunting, D., Gowans, B., and Henderson, J. F. (1985) Biochem. Pharmacol. 34, 3999-4003 [Medline] [Order article via Infotrieve]
  8. Ofori-Nkansah, N., and Von Bruchhausen, F. (1972) Naunyn-Schmiedeberg's Arch. Pharmacol. 272, 156-168
  9. Warburg, O. (1930) The Metabolism of Tumours , Constable, London
  10. Reitzer, L. J., Wice, B. M., and Kennell, D. (1980) J. Biol. Chem. 255, 5616-5626 [Free Full Text]
  11. Singer, S., Okunieff, P., Gostin, C., Thilly, W. G., Chen, L. B., and Neuringer, L. J. (1993) Surg. Oncol. 2, 7-18 [Medline] [Order article via Infotrieve]
  12. Navon, G., Lyon, R. C., Kaplan, O., and Cohen, J. S. (1989) FEBS Lett. 247, 86-90 [CrossRef][Medline] [Order article via Infotrieve]
  13. Bhujwalla, Z. M., Shungu, D. C., Chatham, J. C., Wehrle, J. P., and Glickson, J. D. (1994) Magn. Reson. Med. 32, 303-309 [Medline] [Order article via Infotrieve]
  14. Shinkarenko, L., Kaye, A. M., and Degani, H. (1994) NMR Biomed. 7, 209-217 [Medline] [Order article via Infotrieve]
  15. Rothman, D. L., Magnusson, I., Katz, L. D., Shulman, R. G., and Shulman, G. I. (1991) Science 254, 573-576 [Medline] [Order article via Infotrieve]
  16. Kunnecke, B., and Seelig, J. (1991) Biochim. Biophys. Acta 1095, 103-113 [Medline] [Order article via Infotrieve]
  17. Cohen, S. M. (1987) Biochemistry 26, 581-589 [Medline] [Order article via Infotrieve]
  18. Malloy, C. R., Sherry, A. D., and Jeffrey, F. M. H. (1990) Am. J. Physiol. 259, H987- 995
  19. Kunnecke, B., Cerdan, S., and Seelig, J. (1993) NMR Biomed. 6, 264-277 [Medline] [Order article via Infotrieve]
  20. Galons, J. P., Fantini, J., Vion-Dury, J., Cozzone, P. J., and Canioni, P. (1990) Int. J. Cancer 45, 168-173 [Medline] [Order article via Infotrieve]
  21. Singer, S., Neuringer, L. J., Thilly, W. G., and Chen, L. B. (1993) Cancer Res. 53, 5808-5814 [Abstract]
  22. Ben-Horin, H., Tassini, M., Vivi, A., Navon, G., and Kaplan, O. (1995) Cancer Res. 55, 2814-2821 [Abstract]
  23. Mahmood, U., Alfieri, A. A., Ballon, D., Traganos, F., and Koutcher, J. A. (1995) Cancer Res. 55, 1248-1254 [Abstract]
  24. Ballon, D., Mahmood, U., Jakubowski, A., and Koutcher, J. A. (1993) Magn. Reson. Med. 30, 754-758 [Medline] [Order article via Infotrieve]
  25. Navon, G., Ogawa, S., Shulman, R. G., and Yamane, T. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 87-91 [Abstract]
  26. Bell, J. D., Cox, I. J., Sargentoni, J., Peden, C. J., Menon, D. K., Foster, C. S., Watanapa, P., Iles, R. A., and Urenjak, J. (1993) Biochim. Biophys. Acta 1225, 71-77 [Medline] [Order article via Infotrieve]
  27. Street, J. C., Delort, A.-M., Braddock, P. S. H., and Brindle, K. M. (1993) Biochem. J. 291, 485-492 [Medline] [Order article via Infotrieve]
  28. Brodie, A. F., and Lipmann, F. (1955) J. Biol. Chem. 212, 677-685 [Free Full Text]
  29. Blazer, R. M., and Whaley, T. W. (1980) J. Am. Chem. Soc. 102, 5082-5085
  30. Dietrich, L. S., Friedland, I. M., and Kaplan, L. A. (1958) J. Biol. Chem. 233, 964-968 [Free Full Text]
  31. Keniry, M. A., Hollander, C., and Benz, C. C. (1989) Biochem. Biophys. Res. Commun. 164, 947-953 [Medline] [Order article via Infotrieve]
  32. Köhler, E., Barrach, H.-J., and Neubert, D. (1970) FEBS Letts. 6, 225-228 [CrossRef][Medline] [Order article via Infotrieve]
  33. Lange, K., and Proft, E. R. (1970) Naunyn-Schmiedeberg's Arch. Pharmacol. 267, 177-180
  34. Bauer, H. P., Srihari, T., Jochims, J. C., and Hofer, H. W. (1983) Eur. J. Biochem. 133, 163-168 [Abstract]
  35. Kauffman, F. C., and Johnson, E. C. (1974) J. Neurobiol. 5, 379-392 [Medline] [Order article via Infotrieve]
  36. Kahana, S. E., Lowry, O. H., Schulz, D. W., Passonneau, J. V., and Crawford, E. J. (1960) J. Biol. Chem. 235, 2178-2184 [Medline] [Order article via Infotrieve]
  37. Lowry, O. H., Passonneau, J. V., Hasselberger, F. X., and Schulz, D. W. (1964) J. Biol. Chem. 239, 18-30 [Free Full Text]
  38. Levintow, L., and Eagle, H. (1961) Ann. Rev. Biochem. 30, 605-640
  39. Rheinwald, J. G., and Green, H. (1964) Cell 2, 287-293
  40. Miller, M. W., Wilke, C. R., and Blanch, H. W. (1989) Biotech. Bioeng. 33, 477-486
  41. McComb, R. B., and Yushok, W. D. (1964) Cancer Res. 24, 198-203 [Medline] [Order article via Infotrieve]
  42. Dietrich, L. S., Leatrice, A. K., Friedland, I. M., and Martin, D. S. (1958) Cancer Res. 18, 1272-1280 [Medline] [Order article via Infotrieve]
  43. Radda, G. K. (1986) Science 233, 640-645 [Medline] [Order article via Infotrieve]

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