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J. Biol. Chem., Vol. 276, Issue 41, 37747-37753, October 12, 2001

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Gleevec (STI571) Influences Metabolic Enzyme Activities and Glucose Carbon Flow toward Nucleic Acid and Fatty Acid Synthesis in Myeloid Tumor Cells^*

Joan Boren^§, Marta Cascante, Silvia Marin, Begoña Comín-Anduix, Josep J. Centelles, Shu Lim^¶, Sara Bassilian^¶, Syed Ahmed^¶, Wai-Nang Paul Lee^¶, and László G. Boros^¶

From the  Department of Biochemistry and Molecular Biology, Institut d'Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Marti i Franques 1, Barcelona 08028, Spain and ^¶ UCLA School of Medicine, Harbor-UCLA Research and Education Institute, Torrance, California 90502

Received for publication, June 22, 2001, and in revised form, July 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chronic myeloid leukemia cells contain a constitutively active Bcr-Abl tyrosine kinase, the target protein of Gleevec (STI571) phenylaminopyrimidine class protein kinase inhibitor. Here we provide evidence for metabolic phenotypic changes in cultured K562 human myeloid blast cells after treatment with increasing doses of STI571 using [1,2-¹³C₂]glucose as the single tracer and biological mass spectrometry. In response to 0.68 and 6.8 µM STI571, proliferation of Bcr-Abl-positive K562 cells showed a 57% and 74% decrease, respectively, whereas glucose label incorporation into RNA decreased by 13.4% and 30.1%, respectively, through direct glucose oxidation, as indicated by the decrease in the m₁/m_n ratio in RNA. Based on the in vitro proliferation data, the IC₅₀ of STI571 in K562 cultures is 0.56 µM. The decrease in ¹³C label incorporation into RNA ribose was accompanied by a significant fall in hexokinase and glucose-6-phosphate 1-dehydrogenase activities. The activity of transketolase, the enzyme responsible for nonoxidative ribose synthesis in the pentose cycle, was less affected, and there was a relative increase in glucose carbon incorporation into RNA through nonoxidative synthesis as indicated by the increase in the m₂/m_n ratio in RNA. The restricted use of glucose carbons for de novo nucleic acid and fatty acid synthesis by altering metabolic enzyme activities and pathway carbon flux of the pentose cycle constitutes the underlying mechanism by which STI571 inhibits leukemia cell glucose substrate utilization and growth. The administration of specific hexokinase/glucose-6-phosphate 1-dehydrogenase inhibitor anti-metabolite substrates or competitive enzyme inhibitor compounds, alone or in combination, should be explored for the treatment of STI571-resistant advanced leukemias as well as that of Bcr-Abl-negative human malignancies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Bcr-Abl fusion oncoprotein with protein tyrosine kinase activity is primarily implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias (1). Significant defects in cytoskeletal architecture are observed in Bcr-Abl-transfected human fibroblasts that result in the loss of stress fibers and focal adhesions (2). In a recent clinical study, the new low molecular weight phenylaminopyrimidine class protein kinase inhibitor STI571 (CGP57148) was administered to 61 chronic myeloid leukemia (CML)¹ patients who demonstrated desirable hematologic and cytogenetic responses with no dose-limiting toxicities (3). The human myeloid leukemia cell line K562, which was isolated from a patient with CML-related blast crisis, is currently undergoing extensive studies to determine the molecular mechanisms of the role of the Bcr-Abl fusion oncoprotein in the progression of CML. It is evident that K562 cells strongly depend on the N-glycosylation of their universal high affinity glucose transporter, Glut-1, for disease progression because they show decreased proliferation and [³H]thymidine incorporation into DNA after inhibition of the N-glycosylation of Glut-1 in culture (4). K562 cells also exhibit a concentrative mechanism consistent with Na⁺-dependent transport of glucose that shows similarities with Glut-2 low affinity, high capacity glucose transport (5, 6).

Based on the fact that glucose provides the primary source of carbons for de novo nucleic acid, lipid, and amino acid synthesis in tumor cells, recent studies analyzing glucose metabolic pathways using biological mass spectrometry have revealed the mechanism by which transforming growth factor-2 and the tyrosine kinase inhibitor genistein oppositely influence glucose intermediate metabolism and cell proliferation (7, 8). In this study, we demonstrate that STI571 treatment decreases the proliferation and glucose-derived synthesis of RNA and palmitate in K562 leukemia cells. Metabolic enzymes, which control glucose carbon flow through the oxidative reactions of the pentose cycle, such as hexokinase and glucose-6-phosphate 1-dehydrogenase (G6PDH), are primary targets of STI571.

Our study reveals the importance of glucose-derived nucleic acid and fatty acid production in K562 cells as potential target pathways for STI571 via inhibition of the Bcr-Abl fusion protein.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Line and Culture-- K562 human leukemia cells and MIA pancreatic adenocarcinoma cells (American Type Culture Collection) were grown in minimum essential medium in the presence of 10% fetal bovine serum at 37 °C in 95% air/5% CO₂. K562 leukemia cells were selected for the study because they represent a bone marrow-originated pure CML cell line isolated from a 53-year-old female patient with blast crisis, and they also express the Bcr-Abl tyrosine kinase protein, the primary target kinase of STI571 (1). Metabolic changes observed in K562 cells in response to STI571 treatment were compared with the metabolic response of MIA pancreatic adenocarcinoma cells (9), which lack the Bcr-Abl fusion construct and also tested negative for functioning platelet-derived growth factor receptors (B type) and basic fibroblast growth factor (10). Platelet-derived growth factor receptors have also been reported as potential target sites of STI571 (11). MIA cells were selected as negative controls because their metabolism has been studied extensively using the single [1,2-¹³C₂]glucose label and biological mass spectrometry in the presence of various antiproliferative compounds in previous experiments (8, 9). MIA negative control cells exhibit a comparable glucose consumption rate and bear the same G6PDH izoenzyme and doubling time in culture as K562 cells.

To compare glucose utilization rates, ribose synthesis, lactate production, and fatty acid synthesis, K562 and MIA cells were incubated in [1,2-¹³C₂]glucose-containing media (180 mg%, 50% isotope enrichment) in the presence of increasing doses of STI571. Cultures for the study were performed with the same cell number (1 × 10⁷), which was achieved using standard cell counting techniques as described below. STI571 was kindly provided by Novartis Pharma AG (Basel, Switzerland) according to a material/chemical transfer agreement.

Cell number was determined using a Beckman Coulter (Miami, FL) Z₁ Dual 2.2 counter-top particle counter before and after incubations with STI571. The gates were set at 38.02 µm (upper size) and 10 µm (lower size) during counts. Apoptotic and disintegrated cell figures were gated below the 10-µm size limit. One hundred µls of the trypsinized single cell suspension of each culture suspended in 9.9 ml of Isoton II balanced electrolyte solution (Beckman) was counted, and the number of surviving cells was displayed by the onboard computer interface. Trypsin (0.05%) was added to minimize adhesions between single cells during counting in the Isoton II solution.

Media glucose and lactate levels were measured using a Cobas Mira chemistry analyzer (Roche Diagnostics). For glucose utilization/lactate production studies, we used short incubation period (12 h) cultures with reduced media volume (5 ml) to eliminate differences originating from the antiproliferative response of K562 cells to STI571 treatment. Glucose oxidation was determined by media ¹³C/¹²C ratios in released CO₂ by a Finnegan Delta-S ion ratio mass spectroscope (GC/C/IRMS). Release of ¹³CO₂ was measured to estimate glucose carbon utilization through oxidation by the cell lines and expressed as atom percent excess, which is the portion of ¹³C produced by the cultured cells above background in calibration standard samples (12).

RNA ribose was isolated by acid hydrolysis of cellular RNA after Trizol purification of cell extracts. The mRNA and rRNA of tumor cells were separated using the Qiagen RNA purification kit (Qiagen, Valencia, CA) to determine differential glucose tracer uptake by the structural species (rRNA) and the continuously synthesized species (mRNA) of cellular RNA. Ribose isolated from rRNA and mRNA was derivatized to its aldonitrile acetate form using hydroxyl amine in pyridine and acetic anhydride. We monitored the ion cluster around the m/z 256 (carbons 1-5 of ribose; chemical ionization), m/z 217 (carbons 3-5 of ribose), and m/z 242 (carbons 1-4 of ribose; electron impact ionization) to find molar enrichment and positional distribution of ¹³C labels in ribose (13).

Stable [1,2-¹³C₂]D-glucose isotope was purchased with >99% purity and 99% isotope enrichment for each position (Isotec, Inc., Miamisburg, OH). For isotope incubation and drug treatment studies, K562 and MIA pancreatic adenocarcinoma cells were seeded in T-75 tissue culture flasks after adjusting the number of cells to the values reported above. During the study, the cultures were supplied with 50% [1,2-¹³C₂]glucose dissolved in otherwise glucose- and sodium pyruvate-free Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The final glucose concentration was adjusted to 180 mg/100 ml. Glucose mass isotope analysis of the medium before cell incubations showed that the actual labeled glucose enrichment was 56.8% in the culture media, and this number was used for further calculations to accurately determine the maximum labeled glucose incorporation into biomolecules.

Fig. 1 demonstrates ¹³C label incorporation into various metabolites of the glycolytic pathway using [1,2-¹³C₂]glucose as the tracer. In Fig. 1A, the [1,2-¹³C₂]glucose label is incorporated as the second and third ¹³C-labeled carbons of dihydroxyacetone-phosphate; in Fig. 1B, [2,3-¹³C₂]dihydroxyacetone-phosphate is then converted to [2,3-¹³C₂]glyceraldehyde-3-phosphate through triosephosphate isomerase (EC 5.3.1.1), which will produce [2,3-¹³C₂]pyruvate and [2,3-¹³C₂]lactate labeled on the second and third positions.

View larger version (48K): [in this window] [in a new window]   Fig. 1.   A, possible rearrangements of 13C in various metabolites of glycolysis using [1,2-13C2]glucose as the single tracer. B, formation of [2,3-13C2]lactate. C, the rearrangement of 13C in pentose cycle metabolites due to direct glucose oxidation. D, [1-13C]ribose 5-phosphate formation in the nonoxidative pentose cycle. E, the formation of nucleic acid ribose through the nonoxidative reactions of the pentose cycle. F, formation of [1,2-13C2]acetic acid, precursor of fatty acid synthesis. 13C-labeled carbon positions derived from [1,2-13C2]glucose are shown in bold, boxed, uppercase letters, whereas 12C native-labeled carbon positions are shown in lowercase letters.

The alternate routes for glucose degradation include direct glucose oxidation and ¹³C carbon labeling of pentose cycle metabolites (Fig. 1C). Direct oxidation of glucose in the pentose cycle separates ¹³C label on the first and second carbon positions and produces ribulose 5-phosphate that is labeled only on the first position with ¹³C. Ribulose 5-phosphate can be incorporated into nucleotides by ribose isomerase (EC 5.3.1.20) (top bent arrow, Fig. 1D), or it can be recycled back into the glycolytic pathway by pentose-5-phosphate 3-epimerase (EC 5.1.3.1), carrying back only a single ¹³C label on the first carbon of [1-¹³C]xylulose 5-phosphate (bottom bent arrow, Fig. 1D). The loss of ¹³C from the first carbon of glucose in cellular RNA allows the measurement of direct glucose oxidation and of the contribution of the oxidative steps of the pentose cycle to nucleic acid ribose synthesis (m₁/m_n). If ribose supplies exceed the need for nucleic acid synthesis, consecutive enzymatic steps in the nonoxidative pentose cycle produce [1-¹³C]fructose, which is cleaved by aldolase (EC 4.1.2.13) and further metabolized into [3-¹³C]pyruvate and [3-¹³C]lactate, as described above. The relative ratio of [3-¹³C]lactate to [2,3-¹³C₂]lactate (m₁/m₂) is a reliable index of glucose oxidation through the pentose cycle in mammalian cells (13).

Nucleic acid ribose is also formed in mammalian cells through the nonoxidative reactions of the pentose cycle (Fig. 1E). When the cell culture medium contains only [1,2-¹³C₂]glucose as the tracer and no isotope dilution is observed, nonoxidative ribose synthesis involves [1,2-¹³C₂]fructose-6-phosphate and natural glyceraldehyde-3-phosphate or [2,3-¹³C₂]glyceraldehyde-3-phosphate as primary substrates for ribose synthesis. Ribose formation through the nonoxidative steps of the pentose cycle yields [1,2-¹³C₂]ribose 5-phosphate or [1,2,4,5-¹³C₄]ribose 5-phosphate, depending on the amount of label accumulation from labeled glyceraldehyde through triosephosphate isomerase (m₂ + m₄/m_n). Because ribose is an obligatory precursor of nucleotide formation in all mammalian cells, the ratios of [1-¹³C]ribose to [1,2-¹³C₂]ribose and [1,2,4,5-¹³C₄]ribose in nucleic acid hydrolysates can be used to estimate the contribution of the oxidative and nonoxidative steps of the pentose cycle to de novo nucleotide ribose synthesis.

Lactate from the cell culture media (0.2 ml) was extracted by ethyl acetate after acidification with HCl. Lactate was derivatized to its propylamine-heptafluorobutyric anhydride form using n-propylamine heptafluorobutyrate, and the m/z 328 (carbons 1-3 of lactate; chemical ionization) was monitored for the detection of m₁ (recycled lactate through the pentose cycle) and m₂ (lactate produced by the Embden-Meyerhof-Parnas pathway) for the estimation of pentose cycle activity (13-15).

Fatty acids were extracted with saponification of the Trizol cell extract after removal of the RNA-containing supernatant. Cell debris was treated with 30% KOH and 100% ethanol overnight, and the extraction was performed using petroleum ether. Fatty acids were converted to their methylated derivative using 0.5 N methanolic-HCl (Supelco, Bellefonte, PA). Palmitate was monitored at m/z 270, and stearate was monitored at m/z 298. The enrichment of acetyl units and the synthesis of the new lipid fraction in MIA and K562 cells in response to STI571 treatment were determined using the mass isotopomer distribution analysis approach of different isotopomers of palmitate, an abundant cell membrane lipid readily recovered by biological mass spectrometry from cell pellets (16). Lipid synthesis is also dependent on glucose carbons because they are the primary source of acetyl-CoA (Fig. 1F), which is then incorporated into fatty acids through de novo synthesis (C16, palmitate) or chain elongation (C18, stearate). The process involves pyruvate dehydrogenase (EC 1.2.4.1) as the catalyzing enzyme and results in the formation of [1,2-¹³C₂]acetyl-CoA, which is readily incorporated into palmitate (8).

Gas Chromatography/Mass Spectrometry-- Mass spectral data were obtained on the HP5973 mass selective detector connected to an HP6890 gas chromatograph. The settings are as follows: gas chromatograph inlet, 230 °C; transfer line, 280 °C; mass spectrometer source, 230 °C; mass spectrometer Quad, 150 °C. An HP-5 capillary column (30 m, length; 250 µm, diameter; 0.25 µm, film thickness; Supelco) was used for glucose, ribose, glutamate, and lactate analysis. A Bpx70 column (25 m, length; 220 µm, diameter; 0.25 µm, film thickness; SGE Inc., Austin, TX) was used for fatty acid analysis with specific temperature programming for each compound studied.

Determination of Hexokinase, G6PDH, and Transketolase Activities-- These enzymes have been shown to bear high (0.6-0.8) flux control coefficients in glycolysis and the pentose cycle in various mammalian cells (17-19). Therefore, changes in their activities in response to increasing doses of STI571 treatment likely influence glucose uptake and the synthesis of nucleic acid ribose, lactate, palmitate, and glutamate in MIA and K562 cells. For enzyme activity studies, ribose 5-phosphate, xylulose 5-phosphate, Triton X-100, sodium deoxycholate, phenylmethylsulfonyl fluoride, dithiothreitol, NADH, triosephosphate isomerase/-glycerophosphate dehydrogenase, glucose, ATP, thiamine pyrophosphate, glucose-6-phosphate, NADP⁺, and G6PDH were purchased from Sigma Chemical Co. (St. Louis, MO); MgCl₂ and EDTA were obtained from Panreac Quimica S.A. (Barcelona, Spain); Tris was obtained from ICN Pharmaceuticals Inc. (Costa Mesa, CA); and the Bio-Rad Protein Assay was obtained from Bio-Rad (Hercules, CA). Enzyme activities were measured using colorimetric assays on a spectrophotometer (Shimadzu Cell Positioner CPS-260). Protein concentration in cell extracts was determined using the Bio-Rad Protein Assay to calculate the specific activity of these enzymes after STI571 treatment in cultured MIA and K562 cells.

Preparation of Cell Extracts for Enzyme Activity Studies-- MIA and K562 cells were plated, cultured, and treated as described for the isotope incubation studies above. After the 72-h treatment period using 0.68 and 6.8 µM doses of STI571, cells were washed with ice-cold phosphate-buffered saline, detached from the flask using 0.025% trypsin EDTA, and then resuspended in lysis buffer. Cells were homogenized using a laboratory sonicator and ultracentrifuged at 35,000 rpm for 1 h at 4 °C. The supernatant was separated and used for enzyme activity determinations.

Transketolase (EC 2.2.1.1) activity was determined using the enzyme-linked method (20). One-ml aliquots of transketolase-free buffer were measured in spectrophotometry cuvettes containing 50 mM Tris-HCl, pH 7.6, 2 mM ribose 5-phosphate, 1 mM xylulose-5-phosphate, 5 mM MgCl₂, 0.2 unit/ml triosephosphate isomerase/-glycerophosphate dehydrogenase, 0.2 mM NADH, and 0.1 mM thiamine pyrophosphate. Transketolase reaction was initiated by the addition of 10 µl of cell extract at 37 °C. The oxidation of NADH, which is directly proportional to transketolase activity, was measured by the decrease of 340 nm absorbance. Transketolase activity is expressed as nmol × min¹ × (mg protein)¹.

G6PDH (EC 1.1.1.49) activity was measured as described by Tian et al. (21). Briefly, cuvettes were prepared with a buffer of 50 mM Tris-HCl, pH 7.6, 2 mM glucose-6-phosphate, and 0.5 mM NADP⁺. Reactions were initiated by the addition of 10 µl of cell extract at 37 °C. The reduction of NADP, which is directly proportional to G6PDH activity, was quantitated by the increase of 340 nm absorbance, and G6PDH activity is expressed as nmol × min¹ × (mg protein)¹.

Hexokinase (EC 2.7.1.1) activity was determined by a modification of the method described by Grossbard et al. (22) and Grossbard and Shimke (23). Hexokinase reaction is coupled with the release of NADPH by glucose-6-phosphate dehydrogenase after glucose activation by hexokinase. The reaction cuvette contained 1 mM NADP, 5 mM glucose, and 2 mM MgATP in 50 mM Tris-HCl buffer, pH 7.6, at 37 °C. In this system, NADPH release is only possible upon the activation (phosphorylation) of glucose by hexokinase, in the presence of 1 unit/ml glucose-6-phosphate dehydrogenase as the secondary enzyme. The reaction was initiated by adding 10-µl aliquots of cell supernatant. We recorded 340 nm absorbance changes due to NADPH formation, and hexokinase activity is expressed as nmol × min¹ × (mg protein)¹.

Data Analysis and Statistical Methods-- In vitro experiments were carried out using three cultures each time for each treatment regimen and then repeated twice. Mass spectral analyses were carried out by three independent automatic injections of 1-µl samples by the automatic sampler and were accepted only if the standard sample deviation was <1% of the normalized peak intensity. Enzyme activity measurements were determined after correction for total protein content in cell extracts. Statistical analysis was performed using the parametric unpaired, two-tailed independent sample t test with 99% confidence intervals (µ ± 2.58), and p < 0.01 was considered to indicate significant differences in glucose carbon metabolism and enzyme activities in K562 and MIA cell cultures treated with increasing doses of STI571. Because of the two human cell lines involved, a clearance was obtained from the Institutional Review Board for the use of these commercially available cells for the experiments reported.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

For the present report, K562 myeloid blast and MIA pancreatic adenocarcinoma cells were treated with increasing amounts of STI571 (0.68 and 6.8 µM) for 72 h in the presence of [1,2-¹³C₂]glucose. STI571 doses were selected for the study because the plasma levels of STI571 inducing hematologic and cytogenetic response in patients with CML are in the range of 0.1-3.4 µg/ml (0.17-5.68 µM) after treatment with 25-600 mg STI571/day (11). Changes in metabolic enzyme activities, which bear high glucose carbon flow control characteristics through glycolysis and the pentose cycle, are also reported.

The two cell lines showed similar rates of proliferation because their untreated control cultures contained the same cell number after 72 h of culture (2.75 × 10⁷ cells; Fig. 2A). Increasing doses of STI571 did not alter the final cell number in MIA cell cultures but decreased the K562 cell number in a dose-dependent fashion. Based on the in vitro proliferation data, the IC₅₀ concentration of STI571 in K562 cultures is 0.56 µM. Both MIA and K562 cultures maintained an adequate lactate production rate, indicating viable and metabolically active cells throughout the experiment (Fig. 2B). Glucose consumption (Fig. 3A) and ¹³CO₂ release (Fig. 3B) in K562 cells indicated that glucose uptake and CO₂ production from glucose are significantly decreased after STI571 treatment but are unaffected in MIA cells. The negative  ¹³CO₂/¹²CO₂ values in K562 cultures treated with 6.8 µM STI571 indicate the oxidation of carbon substrates that have ¹³C enrichment below the calibration standard sample.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Comparison of cell proliferation and lactate production in K562 leukemia and MIA pancreatic adenocarcinoma cell cultures in response to increasing doses of STI571 treatment. A, MIA pancreatic adenocarcinoma cell proliferation was not affected by STI571 treatment, whereas K562 cells showed a significant decrease in final cell number in a dose-dependent fashion after 72 h of culture. B, both cell lines demonstrated well maintained lactate production throughout the study period with no significant differences between the treatment groups ( + S.D.; n = 6; *, p < 0.05; **, p < 0.01).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Comparison of glucose consumption and 13CO2 production from glucose in K562 leukemia and MIA pancreatic adenocarcinoma cultures in response to increasing doses of STI571 treatment. A, glucose consumption decreased in a dose-dependent manner in K562 cells in response to STI571 treatment (12-h culture). B, MIA pancreatic adenocarcinoma cell 13CO2 release did not respond to STI571 treatment; on the other hand, 13CO2 release from [1,2-13C2]glucose in K562 leukemia cells exhibited a dose-dependent significant decrease after treatment with 0.68 and 6.8 µM STI571 (72-h culture) ( + S.D.; n = 9; *, p < 0.05; **, p < 0.01).

¹³C content (known as m_n) of rRNA and mRNA, respectively, demonstrated a dose-dependent decrease in response to escalating doses of STI571 in K562 cells in comparison with MIA cells. The decrease in RNA ¹³C content was strictly confined to limited direct glucose oxidation (Fig. 4A) because m₁/m_n showed a characteristic decrease, whereas nonoxidative ribose synthesis (Fig. 4B) demonstrated a relative increase (m₂/m_n). As is evident, STI571 treatment uniformly affected the synthesis of both mRNA and rRNA from glucose with a rapid, 13% and 30% decrease through direct glucose oxidation. MIA cell total RNA ¹³C content did not indicate a dose-dependent change after STI571 treatment.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Comparison of RNA ribose synthesis through direct glucose oxidation and the nonoxidative steps of the pentose cycle in K562 leukemia and MIA pancreatic adenocarcinoma cells in response to increasing doses of STI571. A, incorporation of 13C glucose into mRNA and rRNA ribose through direct oxidation is expressed as m1/molar enrichment (m1/mn). Glucose utilization for RNA synthesis in MIA pancreatic adenocarcinoma cells remained unchanged. On the other hand, K562 cell ribose synthesis from glucose decreased in a dose-dependent fashion, affecting both the ribosomal and messenger fractions of RNA (72-h culture). B, nonoxidative ribose synthesis increased through transketolase and transaldolase relative to direct glucose oxidation ( + S.D.; n = 9; *, p < 0.05; **, p < 0.01).

Likewise, the activities of G6PDH (Fig. 5A) and hexokinase (Fig. 5B), the enzymes primarily in control of direct glucose oxidation, were decreased by about 30% and 50% in 0.68 and 6.8 µM STI571-treated K562 myeloid cells, but not in MIA cells. The activity of transketolase, the primary enzyme in nonoxidative ribose synthesis, showed a less pronounced (only 4.8%) decrease after 0.68 µM STI571 treatment in K562 cells (Fig. 4C). Based on the calculated 0.56 µM concentration of STI571 corresponding to the IC₅₀ value in cell proliferation, it is evident that hexokinase and G6PDH, but not transketolase, are the enzymes responding to STI571 treatment and alter pentose cycle carbon flow as well as the proliferation of K562 cells.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Comparison of metabolic enzyme activities in K562 leukemia and MIA pancreatic adenocarcinoma cells in response to increasing doses of STI571. G6PDH (A) and hexokinase (B) activities decreased by about 30% and 50% after treatment with 0.68 and 6.8 µM STI571, respectively, whereas transketolase (C) activity decreased less ( + S.D.; n = 9; *, p < 0.05; **, p < 0.01).

¹³C enrichment of acetyl units from glucose was significantly higher (4-fold) in untreated MIA cultures than in K562 cells. On the other hand, K562 cells demonstrated about 50% higher de novo palmitate synthesis from glucose carbons. K562 cells exhibited a significant decrease in acetyl enrichment after all doses of STI571 (Fig. 6A). The fraction of newly synthesized palmitate was also significantly decreased after all doses of STI571 in K562 cells (Fig. 6B).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Comparison of acetyl-CoA 13C enrichment and newly synthesized palmitate in K562 leukemia and MIA pancreatic adenocarcinoma cells in response to increasing doses of STI571. A, 13C enrichment of acetyl units from glucose decreased in a dose-dependent fashion in K562 cells after treatment with increasing doses of STI571. B, the fraction of newly synthesized palmitate from glucose was less affected in MIA cells by STI571 treatment, whereas K562 leukemia cells showed a significant decrease in the synthesis of new palmitate after all doses of STI571 (72-h culture) ( + S.D.; n = 9; *, p < 0.05; **, p < 0.01).

STI571 treatment did not affect ¹³C incorporation into glutamate in either cell line, indicating a limited role of the Bcr-Abl tyrosine kinase signal transducer pathway and STI571 in the regulation of tricarboxylic acid cycle enzyme activities and carbon flow. Lactate m₁/m₂ ratios also responded weakly to STI571 treatment in both cells lines (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study investigates the metabolic responses to STI571, a potent low molecular weight inhibitor of the Bcr-Abl tyrosine kinase construct, in cultured K562 chronic myeloid leukemia cells in comparison with MIA pancreatic adenocarcinoma cells. Using the [1,2-¹³C₂]glucose tracer in in vitro metabolic studies has enabled us to study a broad range of intracellular glucose intermediates simultaneously and to investigate their label distribution to determine carbon flow through various metabolic pathways in response to this leukemia growth-modifying agent. Activity changes of three important metabolic enzymes involved in hexose/glucose phosphorylation (hexokinase), direct glucose oxidation (G6PDH), and nonoxidative glucose utilization (transketolase) were also reported. Our studies revealed profound metabolic differences directly affecting substrate utilization toward the synthesis of nucleic acid ribose and new palmitate in K562 myeloid leukemia cells.

Previous studies revealed that transforming growth factor-2, a prominent tyrosine kinase stimulator ligand, induces profound metabolic changes in invasive lung epithelial carcinoma cells characterized by increased glucose utilization and increased nucleic acid ribose synthesis through the nonoxidative steps of the pentose cycle but decreases direct glucose oxidation and pentose cycle activity (7). On the other hand, genistein, the isoflavonoid of the soy plant, is known to inhibit epidermal growth factor and transforming growth factor-2 signaling through tyrosine kinase in various tumors, and it is a strong inhibitor of nucleic acid synthesis from glucose, which is the mechanism underlying its antiproliferative action in MIA cells (8). Other natural tumor-fighting compounds, such as the fermented wheat germ extract Avemar, also inhibit glucose-derived nucleic acid synthesis, glucose uptake, and cell proliferation in a dose-dependent manner (24).

In the present study, we demonstrated that Bcr-Abl-positive K562 cells respond to STI571 treatment with decreased glucose uptake and decreased proliferation. Both these features are present in metastatic tumors of stromal gastric cancer in response to STI571 treatment (25). In a case report, multiple liver metastases and increased accumulation of [¹⁸F]fluorodeoxyglucose were demonstrated on a positron emission tomography scan, but 1 month after treatment with STI571 no abnormal uptake of the tracer was seen in the liver metastases of stromal tumor cells. In a finding consistent with the hypodense appearance of metastases on magnetic resonance imaging, "cold" areas with less uptake of [¹⁸F]fluorodeoxyglucose than the surrounding liver parenchyma were seen at the sites of liver metastases on the positron emission tomography scan obtained 2 months after STI571 treatment was started. Data acquired in our study indicate that STI571 regulates leukemia cell proliferation by altering the rate of glucose utilization for various intermediary metabolic steps including the synthesis of nucleic acid ribose through the oxidative reactions of the pentose cycle. This can be deduced from the dose-dependent significant decrease of hexokinase and G6PDH activities in K562 cells after STI571 treatment. Furthermore, stable glucose isotope incorporation into both mRNA and rRNA fractions decreased in a dose-dependent manner. Because ribose is a close metabolite of glucose, and ribose nucleotides are essential for de novo DNA and RNA synthesis, it is evident that inhibiting the formation of ribose from glucose through enzymes with high carbon flow control coefficients in glycolysis and the pentose cycle (18,19) is a central metabolic event by which STI571 regulates leukemia cell growth.

It is likely that the oncogenic process mediated by the Bcr-Abl tyrosine kinase signaling pathway is closely associated with increased enzyme protein synthesis and enzyme activities through either transcriptional regulation or direct phosphorylation that control carbon flow toward nucleic acid and fatty acid synthesis in myeloid tumor cells. STI571 has a remarkable effect on the oxidation of the first carbon of glucose through the oxidative steps of the pentose cycle during oxidative ribose synthesis; therefore, it acts as an important agent in controlling NADPH production and fatty acid synthesis in Bcr-Abl-positive tumor cells. Although a relative increase in transketolase-derived ribose production is documented in this study, it is likely that increased nonoxidative ribose synthesis is not able to supplement tumor cell metabolic needs for reducing equivalents. These reducing equivalents would be used intensively for de novo fatty acid synthesis as well as the reduction of ribonucleotides to deoxyribonucleotides during DNA replication.

We acknowledge a potential limitation of this study. Whereas there are clearly major changes in glucose metabolism in cells being treated with STI571, it remains unknown whether this is the major cause of cell death. A short review of reported changes in glucose metabolism in response to hydroxyurea treatment (a potent DNA/RNA synthesis inhibitor also used to treat CML) is given here to address whether decreased glucose uptake, activation, and oxidation are unique to direct targeting of the kinase activity of Bcr-Abl by STI571. Glucokinase activity of regenerating liver showed a dose-dependent increase (24 h) or no change (48 and 96 h) after 250 and 1000 mg/kg hydroxyurea treatment in rats (26). In the same study, hexokinase activity was decreased after 250 mg/kg hydroxyurea treatment but increased after 1000 mg/kg hydroxyurea treatment in hepatoma 7777 and 3924-A cells. In a later study, it was demonstrated that although the transport systems for thymidine and uridine are rapidly lost upon inhibition of RNA synthesis by hydroxyurea, the transport systems for choline and 2-deoxy-D-glucose are active and stable (27) in Novikoff rat hepatoma cells. Additional experiments indicate that deoxy-D-glucose and 3-O-methylglucose uptakes, which are associated with S-phase fraction, actually increase in a time-dependent manner in human SW620 colon tumor cells after hydroxyurea treatment (28). Whereas hydroxyurea treatment seems to increase G6PDH and hexokinase activities, resulting in increased glucose activation and glucose oxidation rates in various tumor cells, the Bcr-Abl inhibitor STI571 inhibits these key enzymes, which are critically important for nucleic acid ribose synthesis from glucose. On the other hand, the activation of Bcr-Abl protein tyrosine kinase is associated with the stimulation of glucose transport in a multipotent hemopoietic cell line of chronic myeloid leukemia (29). The observation that Bcr-Abl regulates glucose transport in CML raises the possibility that glucose metabolism plays a pivotal role in the aberrant survival/proliferation of stem cells in CML and that the effective inhibition of Bcr-Abl by STI571 ultimately affects glucose transport, activation, and nucleic acid ribose synthesis.

In conclusion, the Bcr-Abl tyrosine kinase construct is an important signaling mechanism for the regulation of myeloid tumor cell macromolecule synthesis, metabolic pathway activities, proliferation, and growth. As demonstrated herein, regulation of metabolic enzymes involved in glucose carbon redistribution between proliferation-related structural and functional macromolecules (RNA, DNA, and fatty acids) is an effective mechanism to control tumor cell growth. Inhibiting the Bcr-Abl tyrosine kinase signal transducer pathway with STI571 results in profound intracellular metabolic changes that bring devastating consequences for the survival/proliferation of leukemia cells of the Bcr-Abl-positive myeloid lineage. Here we stipulate that similar growth control could be achieved in a wide variety of human tumors by the combination of glucose metabolic enzyme-inhibitory compounds. These compounds would exert their effect directly, without the need of the myeloid cell-specific Bcr-Abl signal transducer pathway (30), on glucose metabolic enzymes that control nucleic acid synthesis, carbon flow through the pentose cycle, and, ultimately, cell proliferation.

	FOOTNOTES

^* This work was supported in part by Grant PHS M01-RR0045 of the General Clinical Research Unit, Grant P01-CA42710 of the UCLA Clinical Nutrition Research Unit Stable Isotope Core through its preliminary/feasibility grant program, grants from the Spanish government Science and Technology Ministry (PPQ2000-0688-C05-04) and Health Ministry (FISS00/1120), and a grant from the NATO Science Program (SA.LST.CLG.976283).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by Grant AP39369265 from the Spanish Government.

To whom correspondence should be addressed: Stable Isotope Research Laboratory, UCLA School of Medicine, Harbor-UCLA Research and Education Institute, 1124 W. Carson St. RB1, Torrance, CA 90502. Tel.: 310-222-1886; Fax: 310-222-3887; E-mail: boros@gcrc.humc.edu.

Published, JBC Papers in Press, August 6, 2001, DOI 10.1074/jbc.M105796200

	ABBREVIATIONS

The abbreviations used are: CML, chronic myeloid leukemia; G6PDH, glucose-6-phosphate 1-dehydrogenase.

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