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Anti-TNF Treatment Reverts Increased Muscle Ubiquitin Gene Expression in Tumour-Bearing Rats
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
221, 653–655 (1996)
0651
Anti-TNF Treatment Reverts Increased Muscle Ubiquitin Gene Expression in Tumour-Bearing Rats Marta Llovera,* Neus Carbó,* Celia García-Martínez,* Paola Costelli,† Luciana Tessitore,† Francesco M. Baccino,†,‡ Neus Agell,§ Gregory J. Bagby,¶ Francisco J. López-Soriano,* and Josep M. Argilés* *Departament de Bioquímica i Biologia Molecular, and §Departament de Biologia Cellular, Universitat de Universitat de Barcelona, Barcelona, Spain; ¶Department of Physiology, Louisiana State University Medical Center, New Orleans, Louisiana; †Dipartimento di Medicina ed Oncologia Sperimentale, Universita di Torino, Torino, Italy; and ‡Centro CNR di Immunogenetica ed Oncologia Sperimentale, Torino, Italy Received March 5, 1996 Implantation of the ascitic tumour Yoshida AH-130 hepatoma (a cachectic tumour) resulted in important increases in muscle ubiquitin gene expression. Administration of daily injections of 25 mg/kg b.w. polyclonal goat anti-murine TNF IgG preparation to tumour-bearing rats abolished the increase in muscle ubiquitin gene expression observed in the control (non-anti-TNF-treated) tumour-bearing rats. It is concluded that TNF can have an important role in the activation of the ubiquitin-dependent proteolytic system during tumour growth. © 1996 Academic Press, Inc.
The loss of body weight and development of cachexia are common signs associated with several diseases. Muscle wasting associated with infection, trauma or tumour growth, results in large part from accelerated protein breakdown (1), this process leading to weight loss (2,3). Tumour necrosis factor-a (TNF) and interleukin-1 (IL-1) are cytokines synthesized and released by blood monocytes and tissue macrophages in response to invasive stimuli, and exert diverse metabolic effects (see (4) for a review). Although a large body of evidence suggests that cytokines participate in the protein wasting and loss of nitrogen associated with cachectic situations (5,6), the mechanisms underlying such actions still remain obscure. However, we have demonstrated that cytokine treatment enhances protein degradation measured in vivo in rat skeletal muscle (7). In addition, we have described that, at least during tumour growth, muscle wasting is associated with the activation of non-lysosomal ubiquitin-dependent proteases (8) and that this activation seems to be mediated via cytokines (9,10). Bearing this in mind, the aim of the present investigation was to examine if TNF was the cytokine responsible for the activation of muscle proteolysis in a tumor cachexia model. In order to accomplish this objective, we administered anti-TNF antibodies to cachectic tumour bearing rats and examined muscle ubiquitin and C8 proteasome subunit gene expression. MATERIALS AND METHODS Animals. Male Wistar rats from our own colony at the Faculty of Biology, University of Barcelona, weighing 100–150 g, were used. They were housed in animal quarters with a daily photoperiod of 12 hrs light between 08:00 and 20:00 hrs, and were individually caged in polypropylene cages, maintained at 22 ± 2°C and fed standard laboratory chow (Panlab, S.A. Barcelona). Biochemicals. They were all reagent grade and obtained either from Boehringer Mannheim (Barcelona, Spain) or from Sigma Chemical Co. (St. Louis, MO, U.S.A). Tumour implantation. A suspension of Yoshida AH-130 ascites hepatoma cell (approx. 108 cells in 2 ml) was injected intraperitoneally, while the control rats received 2 ml of 0.9% (w/v) NaCl solution. Food intake and body weight were measured daily after tumour inoculation. Anti-TNF treatment. The tumour-bearing animals received either no treatment, or were given daily (09:00) subcutaneous injections of 25 mg/kg b.w. polyclonal goat anti-murine TNF IgG preparation (anti-TNF) prepared as previously described (11). The anti-TNF was 100% effective in neutralizing rat TNF using the L929 cytotoxicity assay. Treatments started the 653 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
day after tumour transplantation and lasted 6 days. No significant differences were observed in daily food intake among the tumour-bearing animals receiving the anti-TNF treatment or the control ones. RNA isolation and Northern blot analysis. The gastrocnemius muscles were removed under penthobarbital anaesthesia. Excised muscles were quickly weighed and rapidly frozen in liquid N2. Total RNA was extracted using the acid guanidinium isothiocyanate/phenol/chloroform method as previously described (12) and quantified by absorbance at 260 nm. RNA samples (20 mg) were denatured, subject to electrophoresis in 1.2% agarose gels containing 6.3% formaldehyde and transferred to Hybond N membranes (Amersham). RNA was fixed to membranes by illuminating with UV light for 4 min. The RNA in gels and in filters was visualized with ethidium bromide and photographed by UV transillumination to ensure the integrity of RNA, to check the loading of equivalent amounts of RNA and to confirm proper transfer. RNA was transferred in 20× standard saline citrate (SSC; 0.15 M NaCl and 15 mM sodium citrate, pH 7.0). Hybridization was done at 65°C overnight in the hybridization buffer (0.25 M Na2HPO4/7% SDS/1 mM EDTA/1% BSA/10% dextran sulfate), denatured labelled probes (106–107 cpm/ml) being added. Radiolabelled probes were prepared by the random primer method (Boehringer). The ubiquitin probe used was a cDNA clone containing 12 base pairs of the second ubiquitin coding sequence plus a complete third and fourth ubiquitin coding sequence and 120 base pairs of the 39-untranslated region of the chicken polyubiquitin gene UBI (13). The C8 proteasome subunit probe used was a cDNA clone containing 850 base pairs of the rat C8 proteasome gene (14). Ethidium bromide was used as a control of sample loading. Filters were exposed to Hyperfilm-MP (Amersham) at −70°C for 2–4 days and the films quantified by laser densitometry. Statistical analysis. Statistical analysis of the data was performed by means of the Student’s t test.
RESULTS AND DISCUSSION Previous studies have shown that in many experimental tumour models, protein waste in skeletal muscle is basically associated with an increase in protein degradation (15). Further studies have shown that the proteolytic response seems to be mediated, at least in part, by cytokines, TNF in particular (16). We have also recently identified the proteolytic system which is activated during cancer cachexia in skeletal muscle (8). It is a non-lysosomal, ubiquitin-dependent proteolytic pathway. The present investigation was carried out to determine if the cytokine was also responsible for the activation of the proteolytic system during cancer cachexia. In spite of several studies linking TNF with cachexia, a significant correlation between the severity of weight loss to serum TNF levels in cachectic patients, has not been found (17,18). The animals bearing the Yoshida AH-130 ascites hepatoma show high circulating TNF concentrations during all the tumour cycle (16). Anti-TNF treatment resulted in non-detectable circulating levels of the cytokine; it is thus a
FIG. 1. Northern blots of muscle extracts from tumour-bearing rats. Expression of both ubiquitin and C8 proteasome subunit mRNAs in skeletal muscle (gastrocnemius) extracts from (a) non-tumour-bearing rats, (b) tumour-bearing rats, and (c) tumour-bearing rats receiving an anti-TNF preparation. It was detected after hybridization with cDNA probes containing either a region of the chicken polyubiquitin gene UBI or the C8 proteasome subunit gene (see Materials and Methods). Autoradiographs were subject to scanning densitometry. The results of three separate experiments are shown and expressed as a percentage of control animals. 654
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
particularly interesting model for examining the possible involvement of the cytokine on cancer cachexia. The results presented in Figure 1 show that treatment of the animals with the polyclonal anti-TNF antibodies clearly abolished the increase in both the ubiquitin and C8 proteasome subunit gene expression that can be observed in the tumour-bearing animals. It can be thus concluded that in the present cachexia-inducing tumour model, TNF is involved in activating ubiquitin-dependent proteolysis. The mode of action of the cytokine upon skeletal muscle (either directly or though other cytokines or hormones) is at present under investigation. ACKNOWLEDGMENTS This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (F.I.S) (94/663) of the Spanish Ministry and from the DGICYT (PB95-0497) of the Spanish Ministry of Education and Science. We are very grateful to Dr. M. J. Schlesinger and to Dr. K. Tanaka for providing the ubiquitin and C8 subunit specific probes.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Lawson, D. H., Richmond, A., Nixon, D. W., and Rudman, D. (1982) Annu. Rev. Nutr. 2, 227–301. Strain, A. J. (1979) Invest. Cell Pathol. 2, 181–193. Theologides, A. (1979) Cancer 43, 2004–2012. Evans, R. D., Argilés, J. M., and Williamson, D. H. (1989) Clin. Sci. 77, 357–364. Argilés, J. M., López-Soriano, F. J., Wiggins, D., and Williamson, D. H. (1989) Biochem. J. 261, 355–362. Argilés, J. M., García-Martínez, C., Llovera, M., and López-Soriano, F. J. (1992) Med. Res. Rev. 12, 637–652. Llovera, M., López-Soriano, F. J., and Argilés, J. M. (1993) J. Natl. Cancer Inst. 85, 1134–1139. Llovera, M., García-Martínez, C., Agell, N., López-Soriano, F. J., and Argilés, J. M. (1994) FEBS Lett. 338, 311–318. García-Martínez, C., Agell, N., Llovera, M., López-Soriano, F. J., and Argilés, J. M. (1993) FEBS Lett. 323, 211–214. García-Martínez, C., Llovera, M., Agell, N., López-Soriano, F. J., and Argilés, J. M. (1994) Biochem. Biophys. Res. Commun. 201, 682–686. Lang, C. H., Obih, J. C. A., Bagby, G. J., Bagwell, J. N., and Spitzer, J. J. (1991) Amer. J. Physiol. 260, G548–G555. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Bond, U., and Schlesinger, M. J. (1985) Mol. Cell Biol. 5, 949–956. Tanaka, K., Kanamaya, H., Tamura, T., Lee, D. H., Kumatori, A., Fujiwara, T., Ichihara, A., Tokunaga, F., Aruga, R., and Iwanaga, S. (1990) Biochem. Biophys. Res. Commun. 171, 676–683. Tessitore, L., Bonelli, G., and Baccino, F. M. (1987) Biochem. J. 241, 153–159. Costelli, P., Carbó, N., Tessitore, L., Bagby, G. J., López-Soriano, F. J., Argilés, J. M., and Baccino, F. M. (1993) J. Clin. Invest. 92, 2783–2789. Selby, P., Hobbs, S., Viner, C., Jackson, E., Jones, A., Newell, D., Calvert, A. H., McElwain, T., Fearon, K., Humphreys, J., and Shiga, T. (1987) Br. J. Cancer 56, 803–808. Selby, P. J., Hobbs, S., Viner, C., Jackson, E., Smith, I. F., and McElwain, T. J. (1988) Lancet ii, 483.
655
221, 653–655 (1996)
0651
Anti-TNF Treatment Reverts Increased Muscle Ubiquitin Gene Expression in Tumour-Bearing Rats Marta Llovera,* Neus Carbó,* Celia García-Martínez,* Paola Costelli,† Luciana Tessitore,† Francesco M. Baccino,†,‡ Neus Agell,§ Gregory J. Bagby,¶ Francisco J. López-Soriano,* and Josep M. Argilés* *Departament de Bioquímica i Biologia Molecular, and §Departament de Biologia Cellular, Universitat de Universitat de Barcelona, Barcelona, Spain; ¶Department of Physiology, Louisiana State University Medical Center, New Orleans, Louisiana; †Dipartimento di Medicina ed Oncologia Sperimentale, Universita di Torino, Torino, Italy; and ‡Centro CNR di Immunogenetica ed Oncologia Sperimentale, Torino, Italy Received March 5, 1996 Implantation of the ascitic tumour Yoshida AH-130 hepatoma (a cachectic tumour) resulted in important increases in muscle ubiquitin gene expression. Administration of daily injections of 25 mg/kg b.w. polyclonal goat anti-murine TNF IgG preparation to tumour-bearing rats abolished the increase in muscle ubiquitin gene expression observed in the control (non-anti-TNF-treated) tumour-bearing rats. It is concluded that TNF can have an important role in the activation of the ubiquitin-dependent proteolytic system during tumour growth. © 1996 Academic Press, Inc.
The loss of body weight and development of cachexia are common signs associated with several diseases. Muscle wasting associated with infection, trauma or tumour growth, results in large part from accelerated protein breakdown (1), this process leading to weight loss (2,3). Tumour necrosis factor-a (TNF) and interleukin-1 (IL-1) are cytokines synthesized and released by blood monocytes and tissue macrophages in response to invasive stimuli, and exert diverse metabolic effects (see (4) for a review). Although a large body of evidence suggests that cytokines participate in the protein wasting and loss of nitrogen associated with cachectic situations (5,6), the mechanisms underlying such actions still remain obscure. However, we have demonstrated that cytokine treatment enhances protein degradation measured in vivo in rat skeletal muscle (7). In addition, we have described that, at least during tumour growth, muscle wasting is associated with the activation of non-lysosomal ubiquitin-dependent proteases (8) and that this activation seems to be mediated via cytokines (9,10). Bearing this in mind, the aim of the present investigation was to examine if TNF was the cytokine responsible for the activation of muscle proteolysis in a tumor cachexia model. In order to accomplish this objective, we administered anti-TNF antibodies to cachectic tumour bearing rats and examined muscle ubiquitin and C8 proteasome subunit gene expression. MATERIALS AND METHODS Animals. Male Wistar rats from our own colony at the Faculty of Biology, University of Barcelona, weighing 100–150 g, were used. They were housed in animal quarters with a daily photoperiod of 12 hrs light between 08:00 and 20:00 hrs, and were individually caged in polypropylene cages, maintained at 22 ± 2°C and fed standard laboratory chow (Panlab, S.A. Barcelona). Biochemicals. They were all reagent grade and obtained either from Boehringer Mannheim (Barcelona, Spain) or from Sigma Chemical Co. (St. Louis, MO, U.S.A). Tumour implantation. A suspension of Yoshida AH-130 ascites hepatoma cell (approx. 108 cells in 2 ml) was injected intraperitoneally, while the control rats received 2 ml of 0.9% (w/v) NaCl solution. Food intake and body weight were measured daily after tumour inoculation. Anti-TNF treatment. The tumour-bearing animals received either no treatment, or were given daily (09:00) subcutaneous injections of 25 mg/kg b.w. polyclonal goat anti-murine TNF IgG preparation (anti-TNF) prepared as previously described (11). The anti-TNF was 100% effective in neutralizing rat TNF using the L929 cytotoxicity assay. Treatments started the 653 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
day after tumour transplantation and lasted 6 days. No significant differences were observed in daily food intake among the tumour-bearing animals receiving the anti-TNF treatment or the control ones. RNA isolation and Northern blot analysis. The gastrocnemius muscles were removed under penthobarbital anaesthesia. Excised muscles were quickly weighed and rapidly frozen in liquid N2. Total RNA was extracted using the acid guanidinium isothiocyanate/phenol/chloroform method as previously described (12) and quantified by absorbance at 260 nm. RNA samples (20 mg) were denatured, subject to electrophoresis in 1.2% agarose gels containing 6.3% formaldehyde and transferred to Hybond N membranes (Amersham). RNA was fixed to membranes by illuminating with UV light for 4 min. The RNA in gels and in filters was visualized with ethidium bromide and photographed by UV transillumination to ensure the integrity of RNA, to check the loading of equivalent amounts of RNA and to confirm proper transfer. RNA was transferred in 20× standard saline citrate (SSC; 0.15 M NaCl and 15 mM sodium citrate, pH 7.0). Hybridization was done at 65°C overnight in the hybridization buffer (0.25 M Na2HPO4/7% SDS/1 mM EDTA/1% BSA/10% dextran sulfate), denatured labelled probes (106–107 cpm/ml) being added. Radiolabelled probes were prepared by the random primer method (Boehringer). The ubiquitin probe used was a cDNA clone containing 12 base pairs of the second ubiquitin coding sequence plus a complete third and fourth ubiquitin coding sequence and 120 base pairs of the 39-untranslated region of the chicken polyubiquitin gene UBI (13). The C8 proteasome subunit probe used was a cDNA clone containing 850 base pairs of the rat C8 proteasome gene (14). Ethidium bromide was used as a control of sample loading. Filters were exposed to Hyperfilm-MP (Amersham) at −70°C for 2–4 days and the films quantified by laser densitometry. Statistical analysis. Statistical analysis of the data was performed by means of the Student’s t test.
RESULTS AND DISCUSSION Previous studies have shown that in many experimental tumour models, protein waste in skeletal muscle is basically associated with an increase in protein degradation (15). Further studies have shown that the proteolytic response seems to be mediated, at least in part, by cytokines, TNF in particular (16). We have also recently identified the proteolytic system which is activated during cancer cachexia in skeletal muscle (8). It is a non-lysosomal, ubiquitin-dependent proteolytic pathway. The present investigation was carried out to determine if the cytokine was also responsible for the activation of the proteolytic system during cancer cachexia. In spite of several studies linking TNF with cachexia, a significant correlation between the severity of weight loss to serum TNF levels in cachectic patients, has not been found (17,18). The animals bearing the Yoshida AH-130 ascites hepatoma show high circulating TNF concentrations during all the tumour cycle (16). Anti-TNF treatment resulted in non-detectable circulating levels of the cytokine; it is thus a
FIG. 1. Northern blots of muscle extracts from tumour-bearing rats. Expression of both ubiquitin and C8 proteasome subunit mRNAs in skeletal muscle (gastrocnemius) extracts from (a) non-tumour-bearing rats, (b) tumour-bearing rats, and (c) tumour-bearing rats receiving an anti-TNF preparation. It was detected after hybridization with cDNA probes containing either a region of the chicken polyubiquitin gene UBI or the C8 proteasome subunit gene (see Materials and Methods). Autoradiographs were subject to scanning densitometry. The results of three separate experiments are shown and expressed as a percentage of control animals. 654
Vol. 221, No. 3, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
particularly interesting model for examining the possible involvement of the cytokine on cancer cachexia. The results presented in Figure 1 show that treatment of the animals with the polyclonal anti-TNF antibodies clearly abolished the increase in both the ubiquitin and C8 proteasome subunit gene expression that can be observed in the tumour-bearing animals. It can be thus concluded that in the present cachexia-inducing tumour model, TNF is involved in activating ubiquitin-dependent proteolysis. The mode of action of the cytokine upon skeletal muscle (either directly or though other cytokines or hormones) is at present under investigation. ACKNOWLEDGMENTS This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (F.I.S) (94/663) of the Spanish Ministry and from the DGICYT (PB95-0497) of the Spanish Ministry of Education and Science. We are very grateful to Dr. M. J. Schlesinger and to Dr. K. Tanaka for providing the ubiquitin and C8 subunit specific probes.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Lawson, D. H., Richmond, A., Nixon, D. W., and Rudman, D. (1982) Annu. Rev. Nutr. 2, 227–301. Strain, A. J. (1979) Invest. Cell Pathol. 2, 181–193. Theologides, A. (1979) Cancer 43, 2004–2012. Evans, R. D., Argilés, J. M., and Williamson, D. H. (1989) Clin. Sci. 77, 357–364. Argilés, J. M., López-Soriano, F. J., Wiggins, D., and Williamson, D. H. (1989) Biochem. J. 261, 355–362. Argilés, J. M., García-Martínez, C., Llovera, M., and López-Soriano, F. J. (1992) Med. Res. Rev. 12, 637–652. Llovera, M., López-Soriano, F. J., and Argilés, J. M. (1993) J. Natl. Cancer Inst. 85, 1134–1139. Llovera, M., García-Martínez, C., Agell, N., López-Soriano, F. J., and Argilés, J. M. (1994) FEBS Lett. 338, 311–318. García-Martínez, C., Agell, N., Llovera, M., López-Soriano, F. J., and Argilés, J. M. (1993) FEBS Lett. 323, 211–214. García-Martínez, C., Llovera, M., Agell, N., López-Soriano, F. J., and Argilés, J. M. (1994) Biochem. Biophys. Res. Commun. 201, 682–686. Lang, C. H., Obih, J. C. A., Bagby, G. J., Bagwell, J. N., and Spitzer, J. J. (1991) Amer. J. Physiol. 260, G548–G555. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Bond, U., and Schlesinger, M. J. (1985) Mol. Cell Biol. 5, 949–956. Tanaka, K., Kanamaya, H., Tamura, T., Lee, D. H., Kumatori, A., Fujiwara, T., Ichihara, A., Tokunaga, F., Aruga, R., and Iwanaga, S. (1990) Biochem. Biophys. Res. Commun. 171, 676–683. Tessitore, L., Bonelli, G., and Baccino, F. M. (1987) Biochem. J. 241, 153–159. Costelli, P., Carbó, N., Tessitore, L., Bagby, G. J., López-Soriano, F. J., Argilés, J. M., and Baccino, F. M. (1993) J. Clin. Invest. 92, 2783–2789. Selby, P., Hobbs, S., Viner, C., Jackson, E., Jones, A., Newell, D., Calvert, A. H., McElwain, T., Fearon, K., Humphreys, J., and Shiga, T. (1987) Br. J. Cancer 56, 803–808. Selby, P. J., Hobbs, S., Viner, C., Jackson, E., Smith, I. F., and McElwain, T. J. (1988) Lancet ii, 483.
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