- Email: [email protected]
Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-α
Life Sciences,
Vol. 65, No. 15, pp. PL 215-220, Copyright
0
1999 Elswier
Printed in the USA.
1999
Science Inc.
All rights reserwd
0024_3205/99/E_see
front matter
PII SOO24-3205(99)00408-7
ELSEVIER
PHARMACOLOGY LETTERS Accelerated Communication
INHIBITION OF GLUCOSE UPTAKE AND SUPPRESSION OF GLUCOSE TRANSPORTER 1 mRNA EXPRESSION IN L929 CELLS BY TUMOUR NECROSIS FACTOR-u E. LIONG, SK. KONG, K.K. AU, J.Y.LI, G.Y. XU, Y.L.LEE, Y.M. CHOY, C.Y. LEE andK.P. FUNG Department
of Biochemistry, The Chinese University Shatin, N.T., Hong Kong, China
T.T. KWOK,
of Hong Kong,
(Submitted March 19, 1999; accepted April 8, 1999; received in final form June 8, 1999) Recombinant human tumour necrosis factor-a (rhTNF-a) arrested the growth and Abstract: suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-a for 24 hours, the mRNA level of glucose transporter 1 (GLUT l), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dosedependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNFa inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells. 0 1999Elsevier Science inc. Key Words:
TNF-a,
glucose transporter, L929 cell
Introduction Recombinant human TNFa (rhTNF-a), either alone or combined with other cytokines or drugs, can kill many tumour cells in vitro (reviewed in ref. 1). The antitumour mechanism of rhTNF-a is not completely understood. The rhTNF-a bioactivity is conventionally measured by its inhibition of growth on mouse fibrosarcoma L929 cells (1). rhTNFa induces both necrotic cell death (2) and apoptosis (3, 4) in these cells. It is now known that a crucial step in the cytotoxic action of rhTNF-a on L929 cells involves perturbation of mitochondrial function leading to the formation of reactive oxygen intermediates (2, 5). Goossens et al. (2) reported that this free radicals-mediated perturbation is a bioenergetic reaction. Whether this action of rhTNFCYon L929 or on other mmour cells is mediated by glucose is not clear. In this connection, we have observed that rhTNF-a suppresses glucose uptake in Ehrlich ascites tumour (EAT) cells and causes inhibition of proliferation of these tumour cells in vitro (6). In present study, we examined the effect of rhTNF-a on the expression of mRNA of glucose transporter of L929 cells in order to obtain more insights on the tumoricidal mechanism of this qtokine. Facilitative transport of glucose across the mammalian cell membrane k mediated by six tissue-specific types of glucose transporters (GLUT) (7). Increased levels of mRNA and/or Corresponding author: Dr. K.P. Fung, Department Hong Kong, Shatin, N.T., Hong Kong, [email protected]
of Biochemistry, China. FAX:
The Chinese University of (852)-26035123, e-mail:
PL-216
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GLUT 1 mRNA Expression
Vol. 65, No. 15, 1999
protein of GLUT 1 or of both GLUT 1 and GLUT 3 isoforms have been observed in many malignant human tumours including tumours developed in gastrointestine (8) brain (9, lo), headand-neck (1 l), bladder (12) breast (12) kidney (12) lung (12) and ovary (12). In animal studies, we (13) also found that the mRNA levels of GLUT 1 and GLUT 3 increase progressively in Ehrlich ascites tumour during development. It is of interest to examine whether anti-tumour agents such as rhTNP-a can exert any effect on the expression of GLUT mRNA in tumour cells. In present study, we tested this possibility by using L929 cell line as an in vitro model. We first characterized the isoform of GLUT mRNA in L929 cells and then tested the effect of rhTNP-a on the change of GLUT mRNA expression in these cells. Methods Materials: Recombinant human tumour necrosis factor-a (rhTNP-a, 10 @ml) was purchased from R&D Systems (Abingdon, U.K.). 2-deoxy-D-glucose and protein assay kit were obtained from Sigma Co. (St. Louis, USA). RPMI-1640 medium, fetal calf serum ,;penicillin and streptomycin were obtained from Gibco (NY, USA). 2-deoxy-D-[3 HI-glucose, [ P]dCTP and Megaprime labeling kit were purchased from Amersham (IL., USA). Nylon membrane (Zeta Probe) for electrophoresis was purchased from BioRad (USA). Human-GLUT 3 plasmid DNA GLUT 5 and g-actin were obtained from American Type Culture Collection (Maryland, USA). Hep2-GLUT 1 probe was kindly provided by Dr. B. Thorens (University of. Lausanne, Switzerland). Rat-GLUT 2 probe and rat-GLUT 4 probe were kindly provided by Dr. G.I. Bell (University of Chicago, USA). Tumour Cell preparation: L929 cells were grown in RPMI-1640 medium supplemented with 5% (v/v) fetal calf serum, 2 mM glutamine, 30 mM D-glucose, 50 units/ml penicillin and 100 pg/ml streptomycin and buffered with 25 mM HEPES and 25 mM NaI-ICOs. Cells were grown in culture plates or in culture flasks in a 5% COz-incubator at 37’C. Treatment of cultured L929 cells with rhKAF-a: To examine the effect of rhTNP-a on the viability of L929 cells, cells at 1.5 x lo4 cells/ml were seeded in culture plates in culture media containing 0.025 - 0.4 ng/ml rh TNP-a and incubated at 37°C for 24h. Viability of cells was determined by neutral red uptake assay. In experiments studying the effects of rhTNF-a on glucose uptake and GLUT mRNA expression, L929 cells were seeded at 4 x lo5 cells/ml in culture media in culture plates and culture flasks respectively. Cells were allowed to grow at 37’C for 24h after which the media were replaced by media containing rhTNF-a. Cells were further incubated at 37°C for 24h in the presence of rhTNP-a. Measurement of 2-akox.vD-glucose uptake: The glucose uptake of L929 cells was measured as described before (6) with minor modifications After treatment with rhTNP-a, the medium was removed and cells were washed twice with pre-warmed phosphate buffered saline, pH 7.4 (PBS). Half ml of pre-warmed glucose reaction mixture (0.1 mM 2-deoxy-D-glucose and 1 @i/ml [311J-2-deoxy-D-glucose in PBS) was then added to the cells. The reaction mixture was further incubated at 37°C for different time intervals. The reaction was terminated by aspirating the reaction mixture and gently washing the cells twice with ice cold 10 mM 2-deoxy-D-glucose in PBS. Cells were then solubilized with 200 pl of 0.1% Triton-X 100 and 190 pl of which was transferred to scintillation vial containing 2 ml Triton-X-toluene scintillant. The radioactivity was counted in a Beckman LS6500 liquid scintillation counter. The remaining 10 ul of cell homogenate was used for protein determination study. Protein determination: Protein determination of the cell homogenate was performed by using a protein assay kit (Sigma, USA) according to the manufacturer’s recommendation.
Vol. 65, No. 15, 1999
TNF-a Suppresses GLUT 1 mRNA Expression
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RNA preparation:
L929 cells were washed with ice-cold PBS and the cell pellets were resuspended in 10 volume of solution containing 4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 4, 0.5% sarkosyl, 0.01% 2-mercaptoethanol, pH 4.0. Total RNA was isolated by method described by Chomczynski and Sacchi (14).
Northern hybridization:
The GLUT mRNA expression in L929 cells was quantified by Northern blot hybridization as described (13). RNA samples were denatured by heating in 6% formaldehyde and 50% formamide at 60°C for 15 min, separated on 1% agarose gel containing 2.2 M formaldehyde by electrophoresis (20 ug/lane) and subsequently transferred to a nylon membrane. The membranes were prehybridized at 65°C for 3 h in the prehybridization solution (1 .O M NaCl, 1% SDS) and then hybridized in the hybridization solution (10% dextran sulfate, 1.O M NaCl, 1% SDS, 100 Kg/ml denatured salmon sperm DNA) with 32P labeled cDNA probes for 16 h according to the method modified by Hardy et al. (15). The human-GLUT 3 was prepared from corresponding plasmid DNA. The cDNA of l3-actin was used to monitor the e?rression of the internal control gene. The cDNAs of GLUTS’ and g-actin were labeled with [ P]dCTP by megaprime labeling system. Following hybridization, membranes were washed for 1 h at 65°C in low stringency wash (2 X SSPE, 0.1% SDS; where 1 X SSPE fi 150 mM NaCl, 10 mM NaH2PG4, 1 mM EDTA, pH 7.4) or medium stringency wash (0.5 X SSPE, 0.1% SDS). Membranes were exposed to X-ray film at -70°C with intensifying screen. The abundance of mRNA was quantitated by densitometry of autoradiographic bands using a laser densitometer. The relative levels of GLUT-l mRNA expressed in the cells after rhTNF-a treatments were expressed as percentages of the GLUT-l mRNA level of the control. Results Fig. 1 shows the inhibitory effect of different concentrations of rhTNF-a on the growth of cultured L929 cells. Cells were seeded at 1.5 x lo4 cells/ml in culture medium and treated with rhTNF-a at 37°C for 24 h. The viability of the cells was suppressed by rhTNF-a in a dosedependent manner. The If& was found to be 0.15 @ml. Fig. 2 shows the result of the effect of rhTNF-a on the 2-deoxyglucose uptake of L929 cells. It was found that rhTNF-a at concentration of 0.15 @ml (i.e. concentration at 1Cs0) could significantly inhibit the glucose uptake in the cells during the time course of study as compared with control. Northern hybridization was used to characterize the isoform of GLUT in L929 cells. We found that only GLUT 1 mRNA of 2.7 kb exists in L929 cells. No mRNA of GLUT 2, GLUT 3 and GLUT 4 could be found (results not shown). We also tested the effect of rhTNF-a on the change of GLUT 1 mRNA in L929 cells. Fig. 3 shows the relative levels of GLUT-l mRNA expressed in the cells after treatments with different concentrations of rhTNF-a compared with control. The expression of GLUT 1 mRNA in L929 cells was suppressed by rhTNF-a in a dosedependent manner. When cells were treated with 0.15 ng/ml rhTNF-a (i.e. concentration at IC50) for 24 h at 37°C 40% of suppression of GLUT 1 mRNA was observed. When the cells were treated with 0.075 @ml rhTNF-a (i.e. concentration at half of IC~O), approximately 20% suppression of GLUT1 mRNA was found, and when cells were treated with 0.3 @ml rhTNF-a (i.e. concentration at 2 x ICSO), complete inhibition in expression of GLUT 1 mRNA was observed. In control experiments, the expression of mRNA of B-actin was not affected by rhTNF-a treatments (results not shown). Discussion
Tumour necrosis factor, as its name implies, is an antitumour agent (1). L929 cells have been used as an in vitro model to study the effect of rhTNF-a on tumour cells. The antitumour mechanism of rhTNF-a is not completely known. Goossens et al. (2) found that rhTNF-a elicits formation of reactive oxygen species via glutamine oxidation in mitochondria of L929 cells. Bouchelouche et al. (16) reported that rhTNFa increases the cytosolic free Ca2’ and may then
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05 0
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0.1
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0.2
0.25
Concentration
0.3
0.35
0.4
(nglml)
Fig. 1 Effect of rhTNF-a on the viability of L929 cells. Cells at 1.5 x lo4 cells/ml were seeded in culture media containing various amounts of rhTNF-a and incubated at 37°C for 24 h. Viability of cells after treatment was determined by neutral red uptake assay. Results are expressed as mean + SD. for triplication determinations.
0
20
40
60
80
100
, 120
Time (min.)
Fig. 2 Effect of rhTNF-a on 2-deoxy-glucose uptake of L929 cells. Cells at 4 x lo5 cells/ml were seeded in culture media for 24 h at 37°C and were further incubated with (0) or without (0) rhTNF-a at 0.15 r&ml for another 24 h at 37°C. The uptake of [3H]-2-deoxy-D-glucose was performed as described in text. Results are expressed as mean + SD. for triplicate determinations.
‘DIP-a Suppresses GLUT 1 mRNA Expression
Vol. 65, No. 15, 1999
2
s0
‘;5 g
PL-219
100 80 60
2 40 2 ‘, 20 CL 0 1
2
3
4
Treatment
Fig. 3 Effect of rhTNF-a on the expression of GLUT 1 mRNA in L929 cells. L929 cells were treated with rhTNF-a at 0.075, 0.15 and 0.3 ng/ml for 24 h at 37°C. The GLUT-l mRNA levels were estimated by Northern hybridization as described in Methods. The expression of GLUT-l mRNA after each TNF-a treatment was expressed as percentage of that in control. Column 1, control (no rhTNF-a treatment), column 2, 0.075 ng/ml rhTNF-a; column 3, 0.15 @ml rhTNF-a; column 4,0.3 ng/ml rhTNF-a. cause Ca”-mediated necrosis in L929 cells. In other in vitro tumour model, namely Ehrlich ascites tumour (EAT) cell which is also very susceptible to rhTNF-a treatment (17,6), we found that rhTNF-a suppresses cellular glucose uptake and decreased the membrane density of glucose transporters as measured by cytochalasin B binding method (6). In present study, we further confirmed the inhibitory effect of rhTNF-a on glucose uptake in L929 cells. We found that rhTNFa arrested L929 in vitro with an ICso of 0.15 @ml (Fig. 1). At this concentration, it significantly suppresses cellular glucose uptake (Fig. 2). The suppression of expression of GLUT 1 niRNA (Fig. 3) in L929 cells by rhTNF-a treatment is in a dose-dependent manner. However, it should be noted that although 0.3 rig/ml rhTNF-a exhibited complete suppression of GLUT 1 mRNA in L929 cells after 24 h treatment (column 4, Fig. 3) it could only cause 60% decrease in viability compared with control (Fig. 1). The reason is not known at present but one of the possibilities is that since the intracellular half-life of GLUT 1 mRNA is about 8 hours (7), within the initial phase of this 24 h treatment of rhTNF-a the originally existing GLUT 1 mRNA were still active and could translate some functional glucose transporter 1 proteins to secure glucose for energy. It is also of interest to investigate whether at the later phase of rhTNF-a treatment, further suppression of glucose uptake rate in L929 cells may cause enhancement of the uptake rates of lipids or amino acids as a compensation of supply of nutrients. In human cancer extracts, overexpressions of mRNA and/or protein of GLUT 1 alone or of both GLUT 1 and GLUT 3 compared with normal biopsies are commonly observed (8-12). We found that only GLUT 1 mRNA predominates in L929 cells whereas the expression of GLUT 3 mRNA was not observed in these cells. Gverexpression of GLUT 1 mRNA without the concommitant increase in GLUT 3 mRNA was also observed in human breast tumour cell lines The reason why GLUT 3 is not detectable in some tumour cells is not known. (18). Nevertheless, our findings that rhTNF-a suppresses the expression of GLUT-l mKNA in L929 cells is of significant importance in terms of understanding the tumoricidal property of rhTNF-a. The mechanism by which rhTNF-a inhibits GLUT-l mRNA expression in tumour cells is also not clear. Recently, Kafert e? al. (19) reported that a 33 nucleotide region of the 55 kDa TNF+-a receptor mI0JA exhibits homology to a 38 nucleotide regulatory region of GLUT 1 rrKNA. This finding implies that rhTNF-a may be capable of binding on the regulatory region of GLUT 1
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mRNA resulting in enhancement of intracellular degradation of GLUT 1 mRNA. Since most tumour cells depend mainly upon metabolizing glucose for growth (20) the cytotoxicity of rhTNF-a on L929 cells should, at least in part, attributable to its ability to suppress the expression of GLUT-l mRNA resulting in inhibition of glucose uptake in these cells. Acknowledgements This work was supported by earmarked grants from the Research Grants Council, Hong Kong (CUHK 366/95M & CUHK 4148/98M), a direct grant and a grant from Strategic Research Programme of The Chinese University of Hong Kong (CUHK) and a postdoctoral fellowship to Dr. E. Liong from CUHK, Hong Kong, China. We are deeply grateful to Dr. B. Thorens and Dr. G.1 Bell for providing us the probes of GLUTS. References 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
R. BEYAERT and W. FLERS, FEBS Lett. 340: 9-16 (1994). V. GOOSSENS, J. GROOTEN and W. FIERS, J. Biol. Chem. 271: 192-196 (1996). M. ZINETTI, G. GALLI, M.T. DEMITRI, G. FANTUZZI, M. MINTO, P. GHEZZI, R. ALZANI, E. COZZI AND M. FRATELLI, Immunology 86: 4 16-42 1 (1995). C. FADY, A. GARDNER F. JACOBY, K. BRISKIN, Y. TU, I. SCHMID and A. LICHTENSTEIN, J. Interferon Cytokine Res. 15: 7 l-80 ( 1995). V.B. O’DONNELL, S. SPYCHER and A. AZZI, Biochem. J. 310: 133-141(1995). K.P. FUNG, W.P. LAM, Y.M. CHOY and C.Y. LEE, Life Sci. 57: PL l-6 (1995). M. MUECKLER, Eur. J. Biochem. 219: 713-725 (1994). T. YAMAMOTO, Y. SEINO, H. FUKUMOTO, G. KOH, H. YANO, N. INAGAKI, Y. YAMADA, K. INOUE, T. MANABE and H. IMURA, Biochem. Biophys. Res. Commun. 172: 223-230 (1990). S. NAGAMATSU, H. SAWA, A. WAKIZAKA and T. HOSHINO, J. Neurochem. 61: 2048-2053 (1993). R. J. BOADO, K.K. BLACK and W.M. PARDRIDGE, Brain Res. Mol. Brain Res. 27: 5 l57 (1994). P. MELLANEN, H. MINN, R. GRENMAN and P. HARKONEN, Int. J. Cancer 56: 622629 (1994). M. YOUNES, L.V. LECHAGO, J.R. SOMOANO, M. MOSHARAF and J. LECHAGO, Cancer Res. 56: 1164-l 167 (1996). K.K. AU, E. LIONG, J.Y. LI, P.S. LI, C.C. LIEW, T.T. KWOK, Y.M. CHOY and K.P. FUNG, J. Cell. Biochem. 67: 131-135 (1997). P.K. Jr. CHOMCZYNSKI and N. SACCHI, Anal. Biochem. 162: 156-159 (1987). K.J. HARDY, B.M. PETERLIN, R.E. ATCHISON and J.D. STOBO, Proc. Natl. Acad. Sci. 82: 8173-8177 (1985). P.N. BOUCHELOUCHE, K. BENDTZEN, S. BAK and O.H. NIELSEN, Cell. Signalling 2: 479-487 (1990). K.P. FUNG. S.W. LEUNG. D.K.K. HA. Y.M. CHOY and C.Y. LEE, Cancer Lett. 27: 269276 (1985). ’ R.S. BROWN and R.L. WAHL, Cancer 72: 2979-2985 (1993). S. KAFERT, R. WINZEN, A. LOOS, F. BOLIG, K. RESCH and H. HOLTMAN, FEBS Lett. 421: 2-6 (1998). N.W. MERRALL, R. PLEVIN and G.W. GOULD, Cell. Signalling 5: 667-675 (1993).
Vol. 65, No. 15, pp. PL 215-220, Copyright
0
1999 Elswier
Printed in the USA.
1999
Science Inc.
All rights reserwd
0024_3205/99/E_see
front matter
PII SOO24-3205(99)00408-7
ELSEVIER
PHARMACOLOGY LETTERS Accelerated Communication
INHIBITION OF GLUCOSE UPTAKE AND SUPPRESSION OF GLUCOSE TRANSPORTER 1 mRNA EXPRESSION IN L929 CELLS BY TUMOUR NECROSIS FACTOR-u E. LIONG, SK. KONG, K.K. AU, J.Y.LI, G.Y. XU, Y.L.LEE, Y.M. CHOY, C.Y. LEE andK.P. FUNG Department
of Biochemistry, The Chinese University Shatin, N.T., Hong Kong, China
T.T. KWOK,
of Hong Kong,
(Submitted March 19, 1999; accepted April 8, 1999; received in final form June 8, 1999) Recombinant human tumour necrosis factor-a (rhTNF-a) arrested the growth and Abstract: suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-a for 24 hours, the mRNA level of glucose transporter 1 (GLUT l), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dosedependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNFa inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells. 0 1999Elsevier Science inc. Key Words:
TNF-a,
glucose transporter, L929 cell
Introduction Recombinant human TNFa (rhTNF-a), either alone or combined with other cytokines or drugs, can kill many tumour cells in vitro (reviewed in ref. 1). The antitumour mechanism of rhTNF-a is not completely understood. The rhTNF-a bioactivity is conventionally measured by its inhibition of growth on mouse fibrosarcoma L929 cells (1). rhTNFa induces both necrotic cell death (2) and apoptosis (3, 4) in these cells. It is now known that a crucial step in the cytotoxic action of rhTNF-a on L929 cells involves perturbation of mitochondrial function leading to the formation of reactive oxygen intermediates (2, 5). Goossens et al. (2) reported that this free radicals-mediated perturbation is a bioenergetic reaction. Whether this action of rhTNFCYon L929 or on other mmour cells is mediated by glucose is not clear. In this connection, we have observed that rhTNF-a suppresses glucose uptake in Ehrlich ascites tumour (EAT) cells and causes inhibition of proliferation of these tumour cells in vitro (6). In present study, we examined the effect of rhTNF-a on the expression of mRNA of glucose transporter of L929 cells in order to obtain more insights on the tumoricidal mechanism of this qtokine. Facilitative transport of glucose across the mammalian cell membrane k mediated by six tissue-specific types of glucose transporters (GLUT) (7). Increased levels of mRNA and/or Corresponding author: Dr. K.P. Fung, Department Hong Kong, Shatin, N.T., Hong Kong, [email protected]
of Biochemistry, China. FAX:
The Chinese University of (852)-26035123, e-mail:
PL-216
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Suppresses
GLUT 1 mRNA Expression
Vol. 65, No. 15, 1999
protein of GLUT 1 or of both GLUT 1 and GLUT 3 isoforms have been observed in many malignant human tumours including tumours developed in gastrointestine (8) brain (9, lo), headand-neck (1 l), bladder (12) breast (12) kidney (12) lung (12) and ovary (12). In animal studies, we (13) also found that the mRNA levels of GLUT 1 and GLUT 3 increase progressively in Ehrlich ascites tumour during development. It is of interest to examine whether anti-tumour agents such as rhTNP-a can exert any effect on the expression of GLUT mRNA in tumour cells. In present study, we tested this possibility by using L929 cell line as an in vitro model. We first characterized the isoform of GLUT mRNA in L929 cells and then tested the effect of rhTNP-a on the change of GLUT mRNA expression in these cells. Methods Materials: Recombinant human tumour necrosis factor-a (rhTNP-a, 10 @ml) was purchased from R&D Systems (Abingdon, U.K.). 2-deoxy-D-glucose and protein assay kit were obtained from Sigma Co. (St. Louis, USA). RPMI-1640 medium, fetal calf serum ,;penicillin and streptomycin were obtained from Gibco (NY, USA). 2-deoxy-D-[3 HI-glucose, [ P]dCTP and Megaprime labeling kit were purchased from Amersham (IL., USA). Nylon membrane (Zeta Probe) for electrophoresis was purchased from BioRad (USA). Human-GLUT 3 plasmid DNA GLUT 5 and g-actin were obtained from American Type Culture Collection (Maryland, USA). Hep2-GLUT 1 probe was kindly provided by Dr. B. Thorens (University of. Lausanne, Switzerland). Rat-GLUT 2 probe and rat-GLUT 4 probe were kindly provided by Dr. G.I. Bell (University of Chicago, USA). Tumour Cell preparation: L929 cells were grown in RPMI-1640 medium supplemented with 5% (v/v) fetal calf serum, 2 mM glutamine, 30 mM D-glucose, 50 units/ml penicillin and 100 pg/ml streptomycin and buffered with 25 mM HEPES and 25 mM NaI-ICOs. Cells were grown in culture plates or in culture flasks in a 5% COz-incubator at 37’C. Treatment of cultured L929 cells with rhKAF-a: To examine the effect of rhTNP-a on the viability of L929 cells, cells at 1.5 x lo4 cells/ml were seeded in culture plates in culture media containing 0.025 - 0.4 ng/ml rh TNP-a and incubated at 37°C for 24h. Viability of cells was determined by neutral red uptake assay. In experiments studying the effects of rhTNF-a on glucose uptake and GLUT mRNA expression, L929 cells were seeded at 4 x lo5 cells/ml in culture media in culture plates and culture flasks respectively. Cells were allowed to grow at 37’C for 24h after which the media were replaced by media containing rhTNF-a. Cells were further incubated at 37°C for 24h in the presence of rhTNP-a. Measurement of 2-akox.vD-glucose uptake: The glucose uptake of L929 cells was measured as described before (6) with minor modifications After treatment with rhTNP-a, the medium was removed and cells were washed twice with pre-warmed phosphate buffered saline, pH 7.4 (PBS). Half ml of pre-warmed glucose reaction mixture (0.1 mM 2-deoxy-D-glucose and 1 @i/ml [311J-2-deoxy-D-glucose in PBS) was then added to the cells. The reaction mixture was further incubated at 37°C for different time intervals. The reaction was terminated by aspirating the reaction mixture and gently washing the cells twice with ice cold 10 mM 2-deoxy-D-glucose in PBS. Cells were then solubilized with 200 pl of 0.1% Triton-X 100 and 190 pl of which was transferred to scintillation vial containing 2 ml Triton-X-toluene scintillant. The radioactivity was counted in a Beckman LS6500 liquid scintillation counter. The remaining 10 ul of cell homogenate was used for protein determination study. Protein determination: Protein determination of the cell homogenate was performed by using a protein assay kit (Sigma, USA) according to the manufacturer’s recommendation.
Vol. 65, No. 15, 1999
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RNA preparation:
L929 cells were washed with ice-cold PBS and the cell pellets were resuspended in 10 volume of solution containing 4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 4, 0.5% sarkosyl, 0.01% 2-mercaptoethanol, pH 4.0. Total RNA was isolated by method described by Chomczynski and Sacchi (14).
Northern hybridization:
The GLUT mRNA expression in L929 cells was quantified by Northern blot hybridization as described (13). RNA samples were denatured by heating in 6% formaldehyde and 50% formamide at 60°C for 15 min, separated on 1% agarose gel containing 2.2 M formaldehyde by electrophoresis (20 ug/lane) and subsequently transferred to a nylon membrane. The membranes were prehybridized at 65°C for 3 h in the prehybridization solution (1 .O M NaCl, 1% SDS) and then hybridized in the hybridization solution (10% dextran sulfate, 1.O M NaCl, 1% SDS, 100 Kg/ml denatured salmon sperm DNA) with 32P labeled cDNA probes for 16 h according to the method modified by Hardy et al. (15). The human-GLUT 3 was prepared from corresponding plasmid DNA. The cDNA of l3-actin was used to monitor the e?rression of the internal control gene. The cDNAs of GLUTS’ and g-actin were labeled with [ P]dCTP by megaprime labeling system. Following hybridization, membranes were washed for 1 h at 65°C in low stringency wash (2 X SSPE, 0.1% SDS; where 1 X SSPE fi 150 mM NaCl, 10 mM NaH2PG4, 1 mM EDTA, pH 7.4) or medium stringency wash (0.5 X SSPE, 0.1% SDS). Membranes were exposed to X-ray film at -70°C with intensifying screen. The abundance of mRNA was quantitated by densitometry of autoradiographic bands using a laser densitometer. The relative levels of GLUT-l mRNA expressed in the cells after rhTNF-a treatments were expressed as percentages of the GLUT-l mRNA level of the control. Results Fig. 1 shows the inhibitory effect of different concentrations of rhTNF-a on the growth of cultured L929 cells. Cells were seeded at 1.5 x lo4 cells/ml in culture medium and treated with rhTNF-a at 37°C for 24 h. The viability of the cells was suppressed by rhTNF-a in a dosedependent manner. The If& was found to be 0.15 @ml. Fig. 2 shows the result of the effect of rhTNF-a on the 2-deoxyglucose uptake of L929 cells. It was found that rhTNF-a at concentration of 0.15 @ml (i.e. concentration at 1Cs0) could significantly inhibit the glucose uptake in the cells during the time course of study as compared with control. Northern hybridization was used to characterize the isoform of GLUT in L929 cells. We found that only GLUT 1 mRNA of 2.7 kb exists in L929 cells. No mRNA of GLUT 2, GLUT 3 and GLUT 4 could be found (results not shown). We also tested the effect of rhTNF-a on the change of GLUT 1 mRNA in L929 cells. Fig. 3 shows the relative levels of GLUT-l mRNA expressed in the cells after treatments with different concentrations of rhTNF-a compared with control. The expression of GLUT 1 mRNA in L929 cells was suppressed by rhTNF-a in a dosedependent manner. When cells were treated with 0.15 ng/ml rhTNF-a (i.e. concentration at IC50) for 24 h at 37°C 40% of suppression of GLUT 1 mRNA was observed. When the cells were treated with 0.075 @ml rhTNF-a (i.e. concentration at half of IC~O), approximately 20% suppression of GLUT1 mRNA was found, and when cells were treated with 0.3 @ml rhTNF-a (i.e. concentration at 2 x ICSO), complete inhibition in expression of GLUT 1 mRNA was observed. In control experiments, the expression of mRNA of B-actin was not affected by rhTNF-a treatments (results not shown). Discussion
Tumour necrosis factor, as its name implies, is an antitumour agent (1). L929 cells have been used as an in vitro model to study the effect of rhTNF-a on tumour cells. The antitumour mechanism of rhTNF-a is not completely known. Goossens et al. (2) found that rhTNF-a elicits formation of reactive oxygen species via glutamine oxidation in mitochondria of L929 cells. Bouchelouche et al. (16) reported that rhTNFa increases the cytosolic free Ca2’ and may then
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rhTNF - a
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Concentration
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Fig. 1 Effect of rhTNF-a on the viability of L929 cells. Cells at 1.5 x lo4 cells/ml were seeded in culture media containing various amounts of rhTNF-a and incubated at 37°C for 24 h. Viability of cells after treatment was determined by neutral red uptake assay. Results are expressed as mean + SD. for triplication determinations.
0
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, 120
Time (min.)
Fig. 2 Effect of rhTNF-a on 2-deoxy-glucose uptake of L929 cells. Cells at 4 x lo5 cells/ml were seeded in culture media for 24 h at 37°C and were further incubated with (0) or without (0) rhTNF-a at 0.15 r&ml for another 24 h at 37°C. The uptake of [3H]-2-deoxy-D-glucose was performed as described in text. Results are expressed as mean + SD. for triplicate determinations.
‘DIP-a Suppresses GLUT 1 mRNA Expression
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s0
‘;5 g
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2 40 2 ‘, 20 CL 0 1
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Treatment
Fig. 3 Effect of rhTNF-a on the expression of GLUT 1 mRNA in L929 cells. L929 cells were treated with rhTNF-a at 0.075, 0.15 and 0.3 ng/ml for 24 h at 37°C. The GLUT-l mRNA levels were estimated by Northern hybridization as described in Methods. The expression of GLUT-l mRNA after each TNF-a treatment was expressed as percentage of that in control. Column 1, control (no rhTNF-a treatment), column 2, 0.075 ng/ml rhTNF-a; column 3, 0.15 @ml rhTNF-a; column 4,0.3 ng/ml rhTNF-a. cause Ca”-mediated necrosis in L929 cells. In other in vitro tumour model, namely Ehrlich ascites tumour (EAT) cell which is also very susceptible to rhTNF-a treatment (17,6), we found that rhTNF-a suppresses cellular glucose uptake and decreased the membrane density of glucose transporters as measured by cytochalasin B binding method (6). In present study, we further confirmed the inhibitory effect of rhTNF-a on glucose uptake in L929 cells. We found that rhTNFa arrested L929 in vitro with an ICso of 0.15 @ml (Fig. 1). At this concentration, it significantly suppresses cellular glucose uptake (Fig. 2). The suppression of expression of GLUT 1 niRNA (Fig. 3) in L929 cells by rhTNF-a treatment is in a dose-dependent manner. However, it should be noted that although 0.3 rig/ml rhTNF-a exhibited complete suppression of GLUT 1 mRNA in L929 cells after 24 h treatment (column 4, Fig. 3) it could only cause 60% decrease in viability compared with control (Fig. 1). The reason is not known at present but one of the possibilities is that since the intracellular half-life of GLUT 1 mRNA is about 8 hours (7), within the initial phase of this 24 h treatment of rhTNF-a the originally existing GLUT 1 mRNA were still active and could translate some functional glucose transporter 1 proteins to secure glucose for energy. It is also of interest to investigate whether at the later phase of rhTNF-a treatment, further suppression of glucose uptake rate in L929 cells may cause enhancement of the uptake rates of lipids or amino acids as a compensation of supply of nutrients. In human cancer extracts, overexpressions of mRNA and/or protein of GLUT 1 alone or of both GLUT 1 and GLUT 3 compared with normal biopsies are commonly observed (8-12). We found that only GLUT 1 mRNA predominates in L929 cells whereas the expression of GLUT 3 mRNA was not observed in these cells. Gverexpression of GLUT 1 mRNA without the concommitant increase in GLUT 3 mRNA was also observed in human breast tumour cell lines The reason why GLUT 3 is not detectable in some tumour cells is not known. (18). Nevertheless, our findings that rhTNF-a suppresses the expression of GLUT-l mKNA in L929 cells is of significant importance in terms of understanding the tumoricidal property of rhTNF-a. The mechanism by which rhTNF-a inhibits GLUT-l mRNA expression in tumour cells is also not clear. Recently, Kafert e? al. (19) reported that a 33 nucleotide region of the 55 kDa TNF+-a receptor mI0JA exhibits homology to a 38 nucleotide regulatory region of GLUT 1 rrKNA. This finding implies that rhTNF-a may be capable of binding on the regulatory region of GLUT 1
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mRNA resulting in enhancement of intracellular degradation of GLUT 1 mRNA. Since most tumour cells depend mainly upon metabolizing glucose for growth (20) the cytotoxicity of rhTNF-a on L929 cells should, at least in part, attributable to its ability to suppress the expression of GLUT-l mRNA resulting in inhibition of glucose uptake in these cells. Acknowledgements This work was supported by earmarked grants from the Research Grants Council, Hong Kong (CUHK 366/95M & CUHK 4148/98M), a direct grant and a grant from Strategic Research Programme of The Chinese University of Hong Kong (CUHK) and a postdoctoral fellowship to Dr. E. Liong from CUHK, Hong Kong, China. We are deeply grateful to Dr. B. Thorens and Dr. G.1 Bell for providing us the probes of GLUTS. References 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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