The supernatant contained all activity for NADP+-dependent. CO2 release from glucose and 2-dGlc. a scintillation counter with toluene-based fluor.

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp , November 1978 Biochemistry Catabolism of 2-deoxyglucose by phagocytic leukocytes in the presence of 12-0-tetradecanoyl phorbol- 13-acetate (oxidative decarboxylation/tumor promotion/inflammation/sugar transport) PETER ZABOS*, DAVID KYNER*, NAOMI MENDELSOHNt, CAROL SCHREIBERt, SAMUEL WAXMANI, JUDITH CHRISTMAN*t, AND GEORGE Acs*t Departments of t Biochemistry, * Pediatrics, and * Medicine, The Mount Sinai Medical Center, 1 Gustave L. Levy Place, New York, New York Communicated by Philip Siekevitz, August 24, 1978 ABSTRACT We have found that phagocytic leukocytes exposed to the tumor-promoting agent, 12-O-tetradecanoyl phorbol-13-acetate, efficiently release carbon-i of 2-deoxyglucose in the form of CO2 with concurrent intracellular accumulation of a phosphorylated 5-carbon intermediate. In the absence of 12-atetradecanoyl phorbol-13-acetate, these cells release barely detectable amounts of CO2 from 2-deoxyglucose Tetradecanoyl phorbol-13-acetate, at a concentration of 1 ng/ml, has an immedate effect on CO2 release, which is temperature-dependent and linear with time and cell number. The ability of a number of phorbol ester-like compounds to enhance this catabolic pathway for 2-deoxyglucose correlates with their ability to act as tumor promotors and inflammatory agents. Although this effect of phorbol esters appears to be restricted to granulocytes, monocytes, and macrop a es, the possibility arises that other mammalian cells are capable of catabolizing or can be induced to catabolize 2-deoxyglucose. Thus, 2-deoxyglucose decarboxylation should be considered whenever this analog of mannose and glucose is used as an indicator for sugar transport, especially when pharmacodynamic agents are present. 12-O-Tetradecanoyl phorbol-1s-acetate (TPA), the most active tumor-promoting agent isolated from croton oil (1, 2), acts as an inflammatory agent (3), increases the level of proteases in mouse skin (4), and enhances the carcinogenicity of a number of agents applied to the skin (5). TPA is capable of evoking a multiplicity of changes in cultured cells which are generally considered to be characteristic of the transformed phenotype. Responses include an increase in plasminogen activator production (6-8), an increase in the rate of sugar transport (9), a decrease in LETS protein (10), altered morphology (11), and inhibition of terminal differentiation (12-17). TPA also increases the rate of decarboxylation of glucose in human granulocytes via the hexose monophosphate shunt (18, 19). We present data that indicate that TPA induces a dramatic increase in the rate of decarboxylation of 2-deoxyglucose (2- dglc) by granulocytes, monocytes, and macrophages. While it is generally accepted that 2-dGlc is not metabolized further than 2-deoxyglucose 6-phosphate, 2-deoxygluconic acid 6- phosphate, and 2-deoxyglucose nucleoside diphosphates, our findings indicate that mammalian cells are capable of an additional catabolic conversion of this compound. MATERIALS AND METHODS Cells. Granulocytes, erythrocytes, and lymphocytes were isolated from human blood by the Ficoll-Hypaque and dextran-sedimentation methods of B0yum (20). Human leukemic lymphocytic lines MOLT-3, RPMI-8402, and Daudi-Burkitt (21) were maintained in RPMI-1640 supplemented with 10% The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. fetal bovine serum. The human myeloid line HL-60 (22) was maintained in the same medium with a supplement of 20% heat-inactivated fetal bovine serum. For preparation of cell-free extracts, washed cells were suspended in 3 mm EDTA/30,uM NADP+/20 mm Hepes, ph 7.5, at a concentration of 107/ml. After three cycles of rapid freezing and thawing, particulate matter was removed by centrifugation at 15,000 rpm for 5 min. The supernatant contained all activity for NADP+-dependent CO2 release from glucose and 2-dGlc. Assay for Release of CO2. CO2 released by cells incubated under conditions described in figure and table captions was trapped in a Hyamine-soaked filter paper in a glass well suspended above the incubation mixture. The incubation was terminated by injection of concentrated sulfuric acid (100,ul/ml) and incubation was continued for 30 min at 37 C to ensure that all dissolved CO2 was released from the acidified medium. Radioactivity in the filter paper was determined in a scintillation counter with toluene-based fluor. Chromatographic Methods. For characterization of the intracellular metabolites, cells incubated with radiolabeled 2-dGlc were collected by centrifugation, washed, and extracted three times with cold 50% ethanol adjusted to ph 8.2 with triethylamine. The combined extracts (600,l total, per 107 cells) were lyophilized and redissolved in distilled H20. The material was chromatographed on a Dowex AG1X4 column (1 X 25 cm) and eluted with a concave gradient between 0.03 and 0.2 M NH4Cl (23). Radioactivity was determined directly in 0.2-ml aliquots from 8-ml fractions in an aqueous fluor. Peak fractions were pooled, diluted, and concentrated by binding to a small amount of Dowex AG1X4. After elution with 0.1 M HC1, the samples were brought to 0.05 M with acetate buffer (ph 5), 6 units of acid phosphatase (Sigma, 60 units/mg) was added per ml, and the mixture was incubated at 25 C for 30 min. Samples corresponding to an extract from 5 X 105 cells were chromatographed on cellulose thin-layer plates with water/ethyl acetate/pyridine (25:100:35 vol/vol) as solvent (24). Materials. Human leukemic lines were the generous gift of J. Holland, and HL-60 cells of S. Collins and R. Gallo. 2-dGlc, arabinose, erythrose, and other sugar standards, TPA, acid phosphatase, glucose-6-phosphate dehydrogenase, and 6- phosphogluconic acid dehydrogenase were obtained from Sigma; PEI-cellulose and cellulose thin-layer plates were from Brinkman; [G-3H]2-dGlc, [1,2-3H]2-dGlc, [1-14C]2-dGlc, [1-14C]glucose, and [U-14C]glucose were from New England Nuclear; [U-'4C]2-dGlc was from ICN; lymphocyte separation medium was from Bionetics; dextran sulfate-500 was from Pharmacia; Dowex AG1X4 was from Bio-Rad; sera were from Flow Laboratories; and culture media were from GIBCO. Abbreviations: TPA, 12-0-tetradecanoyl phorbol-1s-acetate; 2-dGlc, 2-deoxyglucose. 5422

2 Biochemistry: Zabos et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5423 Table 1. Effect of TPA on release of 14CO2 from 2-dGlc and glucose by cells of human origin [1-14C]2-dGlc [1-14C]Glucose Cell type -TPA +TPA -TPA +TPA Granulocytes , ,400 T lymphocytes* ,930 B lymphocytes (unfractionated)* 343 8, ,400 Nonadherent (B lymphocytes) , ,040 Adherent (monocytes) 489 6, ,300 Erythrocytes , ,842 HL , ,169 RPMI ,497 MOLT , ,980 Daudi-Burkitt ,294 Cells, suspended at a concentration of 106/ml in glucose-free minimal Eagle's medium buffered to ph 7.5 with Hepes and 2.5 mm NaHCO3 and supplemented with 40MjAM 2-mercaptoethanol, 1 mm sodium pyruvate, and 10% dialyzed fetal bovine serum, were incubated in the presence of 0.5 tci of [1-14C]2-dGlc (6.25 Ci/mol) or [1-14C]glucose (0.8 Ci/mol) per ml for 60 min at 370C in sealed tubes (12 X 75 mm). TPA (10 ng/ml) was added as indicated. 14CO2 release was determined. Values are cpm per 106 cells per 60 min; each value represents the mean of closely agreeing triplicate determinations. * T lymphocytes were separated from the total lymphocyte fraction by rosetting with sheep erythrocytes (25). The nonrosetting cells were further fractionated into adherent (monocytes) and nonadherent (B lymphocytes) populations after incubation on plastic plates for 2 hr at 37 C. All other cells were prepared or maintained as described in Materials and Methods. RESULTS When granulocytes are incubated in the presence of TPA, sugar transport, measured as the rate of entry of [1-'4C]2-dGlc into the intracellular pool, is markedly inhibited. However, if the rate of entry of [U-'4C]2-dGlc is measured, the inhibition is much less evident (19% compared to 79%). The data in Table 1 account for this difference. In the presence of TPA, granulocytes release a substantial amount of the radioactivity in [1-'4C]2-dGlc as 14CO2. The rate of "4CO2 liberation is linear with time and dependent on temperature and TPA concentration. Human monocytes, mouse macrophages, and HL-60 cells [a line established from a patient with acute myeloid leukemia (22)] also respond to TPA with increased release of 14CO2 from [1-"4C]2-dGlc. In contrast, T lymphocytes, B lymphocytes, erythrocytes, and platelets release low levels of "4CO2 both in the presence and absence of TPA. Cells from three human lymphoid lines are also unresponsive to TPA. In every case in which TPA causes an increased rate of release of CO2 from 2-dGlc, a parallel increase in the rate of release of CO2 from carbon-i of glucose is observed. The effect of other phorbol ester-like compounds on the release of "4CO2 from [1-14C]2-dGlc was tested. As can be seen in Table 2, only those compounds that possess inflammatory and tumor-promoting activity increase "4CO2 release. Analysis by Dowex AG1X4 chromatography of the intracellular pools of granulocytes incubated with [1-14C]2-dGlc in the presence or absenie of TPA reveals accumulation of four radiolabeled compounds (Fig. 1A). The first compound, eluted in the void volume, cochromatographs with 2-dGlc on cellulose thin-layers (24). The radiolabeled material in peak 3 cochromatographs with 2-deoxyglucose 6-phosphate on PEI-cellulose and, after enzymatic dephosphorylation, with 2-dGlc on cellulose thin layers (Fig. 1B). The material in peak 4 has been similarly identified as 2-deoxygluconic acid 6-phosphate. Peak 2, a minor metabolite, has not been characterized. When granulocytes are incubated with [U-"4C]2-dGlc in the absence of TPA the same chromatographic pattern is obtained. In the presence of TPA, however, a new metabolite derived from 2-dGlc is found, as indicated by the arrow in Fig. 1A. Our unpublished method. Sodium formate (1 M) saturated with boric acid and adjusted to ph 3.4 is used as solvent. After enzymatic dephosphorylation, the radiolabeled material in this peak can be clearly separated from 2-dGlc by cellulose thin-layer chromatography (Fig. 1B). Since this new compound (i) migrates faster than 2-dGlc; (ii) appears only under conditions where extensive release of carbon-i from 2-dGlc occurs; and (iii) is completely undetectable in cells incubated with [1-14C]2-dGlc even in the presence of TPA (Fig. 1A), we conclude that it represents the decarboxylation product of 2-dGlc. This compound cannot be found in cells incubated with [U-14C]glucose either in the presence or absence of TPA. Moreover, as increasing amounts of unlabeled glucose are added to the medium, release of CO2 from carbon-i of 2-dGlc is inhibited with concurrent accumulation of 2- deoxygluconic acid 6-phosphate. The unidentified intermediate reacts with phenylhydrazine, indicating that it may contain either a ketone or an aldehyde group. Although the characterization of this derivative of 2- dglc is not completed, the evidence presented in Fig. 2 is a strong indication that it contains five carbon atoms. The alcohol-soluble pool derived from granulocytes incubated simul- Table 2. Activity of phorbol derivatives and mezerein in stimulating 2-dGlc catabolism by human granulocytes Activity in 14CO2 release,* mouse skint cpm per 106 Tumor Inflam- Addition cells per 60 min promotion mation None 1,625 Phorbol 1, a-Phorbol didecanoate 1,665 Phorbol didecanoate 13, TPA 12, Mezereint 12,450 * Average of triplicate determinations. All conditions as in Table 1 except [1-14C]2-dGlc concentration (0.2 MCi/ml, 62.5 Ci/mol). Phorbol, phorbol derivatives, and mezerein were added at 100 ng/ml. t For description of the activities of these compounds in mouse skin, see ref. 26. Mezerein is an antileukemic agent which resembles TPA in its ability to inhibit cell differentiation (13) and to enhance plasminogen activator production (27).

3 5424 Biochemistry: Zabos et al. Proc. Natl. Acad. Sci. USA 75 (1978) v) x E 0 0 x C- E Fraction cm FIG. 1. Analysis of intracellular metabolites of 2-dGlc in human granulocytes. (A) Radiolabeled derivatives of [1-14C]2-dGlc (---) and [U- '4C]2-dGlc (-) isolated from granulocytes incubated in the presence of TPA. Extracts from 5 X 107 cells were prepared and chromatographed on Dowex AG1X4. Incubation was for 1 hr with 10 ng of TPA per ml and 0.5 uci of [1-14C]2-dGlc (62.5 Ci/mol) or 0.5 MCi of [U-14C]2-dGlc (8.2 Ci/mol) per ml. (B) Characterization of dephosphorylated intermediates by cellulose thin-layer chromatography. Material isolated from the fractions indicated (-) in A was digested with acid phosphatase and chromatographed on cellulose thin-layer plates with water/ethyl acetate/ pyridine (25:100:35 vol/vol) as solvent. Mobility of radiolabeled material from peak 3 (---) and unknown (-) relative to sugar standards is indicated. (Ara, arabinose; Ery, erythrose). Equal amounts of radioactivity were applied. taneously with [1-"4C]dGlc and [1,2-3H]2-dGIc in the presence of TPA was analyzed by Dowex AG1X4 chromatography. As can be seen, the ratio of 14C/6ih in 2-dGlc and 2-dGlc 6-phosphate is close to one. In 2-deoxygluconic 6-phosphate the ratio is significantly higher due to the loss of the hydrogen atom from carbon-i when 2-deoxyglucose 6-phosphate is oxidized to 2-deoxygluconic acid 6-phosphate. This change is also found in peak 2. Carbon-i of 2-dGlc is absent from the unknown compound, as indicated by the complete lack of 14C radioactivity. However, it still contains 3H, indicating the retention of carbon-2 of 2-dGlc. On dephosphorylation, the 3H-radiolabeled material from this peak has the same mobility on cellulose thin E - ~ ~ Fraction FIG. 2. Radiolabeled derivatives of [1-14C]2-dGlc (-)and [1,2-3H]2-dG1c (-) isolated from granulocytes incubated for 1 hr in the presence of 10 ng of TPA per ml. The incubation mixture contained 100 nmol of 2-dGlc per ml with a specific activity of 0.7 Ci/mol for both 14C and 3H. Chromatography on Dowex AG1X.

4 Table 3. Biochemistry: Zabos et al. Catabolism of glucose and 2-dGlc in cell-free extracts (nmol CO2 liberated) [l 14C]- [1-14C]2- Extract or enzymes Glucose dglc Untreated human granulocytes 1 X 106 cells X 106 cells X 106 cells plus TPA X 106 cells minus NADP X 106 cells minus ATP 0 0 Human granulocytes incubated 1 hr with TPA 1 X 106 cells Human erythrocytes 1 X 107 cells X 107 cells minus NADP X 107 cells minus ATP+ 0 0 Purified hexose monophosphate shunt enzymes* Minus NADP+ 0 0 Minus ATP 0 0 Extracts from the indicated number of cells were incubated for 60 min at 370C with 0.5 yci of [1-14C]glucose (8 Ci/mol) or [1-14C]2-dGlc (67.4 Ci/mol). Release of 14C02 was determined. The reaction mixture contained 40 mm Hepes (ph 8.0), 6 mm MgCl2, 2mM CaCl2, 2 mm dithiothreitol, 1 mm ATP, and 1 mm NADP+ in a final volume of 0.5 ml. * Hexokinase (0.5 unit); glucose-6-phosphate dehydrogenase (5 units); gluconic acid-6-phosphate (2 units). layers as the compound that accumulates when granulocytes are incubated with [U-'4C]2-dGlc and TPA (Fig. IB). The presence in the intracellular pool of 2-deoxyglucose 6- phosphate, 2-deoxygluconic acid 6-phosphate, and a 5-carbon phosphorylated compound that reacts with phenylhydrazine suggests that the mechanism of 2-dGlc catabolism in the presence of TPA is analogous to the catabolism of glucose via the hexose monophosphate shunt. In fact, several lines of evidence (Table 3) indicate that the enzymes of the hexose monophosphate shunt are capable of catabolizing both glucose and 2- dglc: (i) Erythrocyte lysates, which contain the enzymes of the hexose monophosphate shunt, can release CO2 from 2-dGlc; (ii) purified hexose monophosphate shunt enzymes from commercial sources can release CO2 from 2-dGlc; and (ifi) in both cases, decarboxylation of 2-dGlc requires ATP and NADP+, known cofactors for the enzymes of the hexose monophosphate shunt. However, the rate of decarboxylation of 2-dGlc in these cell-free reaction mixtures is approximately 1/40th of the rate of decarboxylation of glucose at the same substrate concentration. The same relative inefficiency of 2-dGlc decarboxylation is observed with cell-free extracts prepared from granulocytes (Table 3). In contrast to the marked effect of TPA on 2-dGlc catabolism in intact cells, pretreatment of granulocytes with TPA or addition of TPA to reaction mixtures has no effect on the catabolic activity of cell-free granulocyte extracts. This suggests that TPA acts to increase 2-dGlc catabolism without increasing the level or activity of the enzymes in the hexose monophosphate shunt. DISCUSSION The data presented above indicate that the enzymes of the hexose monophosphate shunt are able to mediate oxidative decarboxylation of 2-dGlc. This finding could account for the discrepancy which exists ii the literature regarding the effect of TPA on 2-dGlc transport. In a number of cell types, TPA has Proc. Natl. Acad. Sci. USA 75 (1978) 5425 been found to increase the rate of transport of 2-dGlc. On the other hand, using generally labeled [3H]2-dGlc, De Chatelet and his coworkers (19) found an inhibition of 2-dGlc transport in granulocytes. Our results, demonstrating efficient conversion of carbon-i of 2-dGlc to C02, together with the fact-that approximately 90% of the 3H label in [GI-3H]2-dGlc is attached to carbon-11 which is lost by oxidation of 2-deoxyglucose 6- phosphate, explain the basis for an apparent inhibition of 2-dGlc transport in granulocytes. Thus, before intracellular accumulation of 2-dGlc is used as an indicator for glucose transport, the rate of release of carbon-i from 2-dGlc should be determined. This is of special importance when agents that might affect the activity of the hexose monophosphate shunt are used. Even though we have been able to detect oxidative-decarboxylation of 2-dGlc by purified enzymes of the hexose monophosphate shunt, several lines of evidence indicate that some factor other than an increase in the intracellular level of these enzymes is necessary to explain the rapid effect of TPA on the oxidative-decarboxylation of 2-dGlc in granulocytes. The rate of decarboxylation of 2-dGlc in cell-free systems is extremely low relative to the rate of decarboxylation of glucose. The important mechanistic role played by the hydroxyl group on carbon-2 in -the oxidative-decarboxylation of gluconic acid 6-phosphate may be sufficient to explain this difference. In any case, if the concentration of 2-dGlc is increased above 50 mm, the efficiency of cell-free extracts of granulocytes approaches or surpasses that of intact cells. Our observation that cell-free extracts of erythrocytes decarboxylate 2-dGlc while intact erythrocytes, which cannot accumulate sugars against a concentration gradient, do not, might suggest that TPA acts through an effect on active transport. The possibility has also been suggested, however, that TPA increases oxidative metabolism of glucose by activating NADPH oxidase and thus generating NADP+, the rate-limiting substance for the hexose monophosphate shunt (19, 28). Such a mechanism would also explain why TPA has an effect in intact cells but not in cell-free extracts. In addition to demonstrating that 2-dGlc can be catabolized in mammalian cells, we have shown that TPA markedly enhances this catabolism, specifically in phagocytic leukocytes. The enhancement of 2-dGlc catabolism is always paralleled by a comparable increase in the oxidative-decarboxylation of glucose. The ability of other phorbol ester-like compounds to increase this catabolic activity correlates with their ability to cause inflammation and to act as tumor promotors. The relevance of this effect of tumor promotors on phagocytic leukocytes to carcinogenesis remains to be determined. This work was supported in part by Grants CA-16890, and AM (Chemotherapy Foundation) of the National Institutes of Health, U.S. Public Health Service and NP-36 of the American Cancer Society. J.C. is a Senior Investigator of the New York Heart Association. 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