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Dual Role of Dextran Sulfate 5000 Da as Anti-Apoptotic and Pro-Autophagy Agent

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Abstract

Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.

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References

  1. Sunstrom, N. A., Gay, R. D., Wong, D. C., Kitchen, N. A., DeBoer, L., & Gray, P. P. (2000). Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells. Biotechnology Progress, 16, 698–702.

    Article  CAS  Google Scholar 

  2. Chang, K. H., Kim, K. S., & Kim, J. H. (1999). N-acetylcysteine increases the biosynthesis of recombinant EPO in apoptotic Chinese hamster ovary cells. Free Radical Research, 30, 85–91.

    Article  CAS  Google Scholar 

  3. Sung, Y. H., Song, Y. J., Lim, S. W., Chung, J. Y., & Lee, G. M. (2004). Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells. Journal of Biotechnology, 112, 323–335.

    Article  CAS  Google Scholar 

  4. Arden, N., & Betenbaugh, M. J. (2004). Life and death in mammalian cell culture: Strategies for apoptosis inhibition. Trends in Biotechnology, 22, 174–180.

    Article  CAS  Google Scholar 

  5. Oh, H. K., So, M. K., Yang, J., Yoon, H. C., Ahn, J. S., Lee, J. M., et al. (2005). Effect of N-acetylcystein on butyrate-treated Chinese hamster ovary cells to improve the production of recombinant human interferon-beta-1a. Biotechnology Progress, 21, 1154–1164.

    Article  CAS  Google Scholar 

  6. Martinet, W., De Meyer, G. R., Herman, A. G., & Kockx, M. M. (2005). Amino acid deprivation induces both apoptosis and autophagy in murine C2C12 muscle cells. Biotechnology Letters, 27, 1157–1163.

    Article  CAS  Google Scholar 

  7. Hwang, S. O., & Lee, G. M. (2008). Autophagy and apoptosis in Chinese hamster ovary cell culture. Autophagy, 4, 70–72.

    Google Scholar 

  8. Eng, C. H., Yu, K., Lucas, J., White, E., & Abraham, R. T. (2010). Ammonia derived from glutaminolysis is a diffusible regulator of autophagy. Science signaling, 3, ra31.

    Article  Google Scholar 

  9. Han, Y. K., Kim, Y. G., Kim, J. Y., & Lee, G. M. (2010). Hyperosmotic stress induces autophagy and apoptosis in recombinant Chinese hamster ovary cell culture. Biotechnology and Bioengineering, 105, 1187–1192.

    CAS  Google Scholar 

  10. Han, Y. K., Ha, T. K., Lee, S. J., Lee, J. S., & Lee, G. M. (2011). Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation. Biotechnology and Bioengineering, 108, 2182–2192.

    Article  CAS  Google Scholar 

  11. Levine, B., & Klionsky, D. J. (2004). Development by self-digestion: molecular mechanisms and biological functions of autophagy. Developmental Cell, 6, 463–477.

    Article  CAS  Google Scholar 

  12. Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., et al. (2004). The role of autophagy during the early neonatal starvation period. Nature, 432, 1032–1036.

    Article  CAS  Google Scholar 

  13. Lee, J. S., & Lee, G. M. (2012). Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells. Biotechnology Progress, 28, 349–357.

    Article  CAS  Google Scholar 

  14. Lee, J. S., & Lee, G. M. (2012). Rapamycin treatment inhibits CHO cell death in a serum-free suspension culture by autophagy induction. Biotechnology and Bioengineering, 109(12), 3093–3102.

    Article  CAS  Google Scholar 

  15. Stanley, M. J., Liebersbach, B. F., Liu, W., Anhalt, D. J., & Sanderson, R. D. (1995). Heparan sulfate-mediated cell aggregation. Syndecans-1 and -4 mediate intercellular adhesion following their transfection into human B lymphoid cells. The Journal of Biological Chemistry, 270, 5077–5083.

    Article  CAS  Google Scholar 

  16. Zanghi, J. A., Renner, W. A., Bailey, J. E., & Fussenegger, M. (2000). The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures. Biotechnology Progress, 16, 319–325.

    Article  CAS  Google Scholar 

  17. Li, L., Qin, J., Feng, Q., Tang, H., Liu, R., Xu, L., et al. (2011). Heparin promotes suspension adaptation process of CHO–TS28 cells by eliminating cell aggregation. Molecular Biotechnology, 47, 9–17.

    Article  CAS  Google Scholar 

  18. Jing, Y., Egan, S. E., Qian, Y., Borys, M. C., Abu-Absi, N. R., & Li, Z. J. (2011). Dextran sulfate inhibits staurosporine-induced apoptosis in Chinese hamster ovary (CHO) cells: Involvement of the mitochondrial pathway. Process Biochemistry, 46, 427–432.

    Article  CAS  Google Scholar 

  19. Takagi, T., Sakakura, C., Kin, S., Nakase, Y., Fukuda, K., Shimomura, K., et al. (2005). Dextran sulfate suppresses cell adhesion and cell cycle progression of melanoma cells. Anticancer Research, 25, 895–902.

    CAS  Google Scholar 

  20. Araki, Y., Sugihara, H., & Hattori, T. (2006). In vitro effects of dextran sulfate sodium on a Caco-2 cell line and plausible mechanisms for dextran sulfate sodium-induced colitis. Oncology Reports, 16, 1357–1362.

    CAS  Google Scholar 

  21. Rubio, E., Valenciano, A. I., Segundo, C., Sanchez, N., de Pablo, F., & de la Rosa, E. J. (2002). Programmed cell death in the neurulating embryo is prevented by the chaperone heat shock cognate 70. The European journal of Neuroscience, 15, 1646–1654.

    Article  Google Scholar 

  22. Eskelinen, E. L., Prescott, A. R., Cooper, J., Brachmann, S. M., Wang, L., Tang, X., et al. (2002). Inhibition of autophagy in mitotic animal cells. Traffic, 3, 878–893.

    Article  CAS  Google Scholar 

  23. Tasdemir, E., Maiuri, M. C., Tajeddine, N., Vitale, I., Criollo, A., Vicencio, J. M., et al. (2007). Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy. Cell Cycle, 6, 2263–2267.

    Article  CAS  Google Scholar 

  24. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., et al. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO journal, 19, 5720–5728.

    Article  CAS  Google Scholar 

  25. Mizushima, N. (2004). Methods for monitoring autophagy. The international Journal of Biochemistry and Cell Biology, 36, 2491–2502.

    Article  CAS  Google Scholar 

  26. Ravikumar, B., Berger, Z., Vacher, C., O’Kane, C. J., & Rubinsztein, D. C. (2006). Rapamycin pre-treatment protects against apoptosis. Human Molecular Genetics, 15, 1209–1216.

    Article  CAS  Google Scholar 

  27. Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew, D. S., et al. (2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 4, 151–175.

    CAS  Google Scholar 

  28. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., & Ohsumi, Y. (2004). In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Molecular Biology of the Cell, 15, 1101–1111.

    Article  CAS  Google Scholar 

  29. Maiuri, M. C., Zalckvar, E., Kimchi, A., & Kroemer, G. (2007). Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nature Reviews Molecular Cell Biology, 8, 741–752.

    Article  CAS  Google Scholar 

  30. Vousden, K. H., & Ryan, K. M. (2009). p53 and metabolism. Nature Reviews Cancer, 9, 691–700.

    Article  CAS  Google Scholar 

  31. Tasdemir, E., Maiuri, M. C., Orhon, I., Kepp, O., Morselli, E., Criollo, A., et al. (2008). p53 represses autophagy in a cell cycle-dependent fashion. Cell Cycle, 7, 3006–3011.

    Article  CAS  Google Scholar 

  32. Crighton, D., Wilkinson, S., O’Prey, J., Syed, N., Smith, P., Harrison, P. R., et al. (2006). DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell, 126, 121–134.

    Article  CAS  Google Scholar 

  33. Feng, Z., Zhang, H., Levine, A. J., & Jin, S. (2005). The coordinate regulation of the p53 and mTOR pathways in cells. Proceedings of the National Academy of Sciences of the United States of America, 102, 8204–8209.

    Article  CAS  Google Scholar 

  34. Amaravadi, R. K., Yu, D., Lum, J. J., Bui, T., Christophorou, M. A., Evan, G. I., et al. (2007). Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. The Journal of Clinical Investigation, 117, 326–336.

    Article  CAS  Google Scholar 

  35. Lee, I. H., Kawai, Y., Fergusson, M. M., Rovira, I. I., Bishop, A. J., Motoyama, N., et al. (2012). Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress. Science, 336, 225–228.

    Article  CAS  Google Scholar 

  36. Massey, A. C., Kaushik, S., Sovak, G., Kiffin, R., & Cuervo, A. M. (2006). Consequences of the selective blockage of chaperone-mediated autophagy. Proceedings of the National Academy of Sciences of the United States of America, 103, 5805–5810.

    Article  CAS  Google Scholar 

  37. Kaushik, S., Massey, A. C., Mizushima, N., & Cuervo, A. M. (2008). Constitutive activation of chaperone-mediated autophagy in cells with impaired macroautophagy. Molecular Biology of the Cell, 19, 2179–2192.

    Article  CAS  Google Scholar 

  38. Agarraberes, F. A., & Dice, J. F. (2001). A molecular chaperone complex at the lysosomal membrane is required for protein translocation. Journal of Cell Science, 114, 2491–2499.

    CAS  Google Scholar 

  39. Wei, Y. Y., Naderi, S., Meshram, M., Budman, H., Scharer, J. M., Ingalls, B. P., et al. (2011). Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation. Cytotechnology, 63, 663–677.

    Article  CAS  Google Scholar 

  40. Burg, D. L., Harrison, M. L., & Geahlen, R. L. (1993). Cell cycle-specific activation of the PTK72 protein–tyrosine kinase in B lymphocytes. The Journal of Biological Chemistry, 268, 2304–2306.

    CAS  Google Scholar 

  41. Tessner, T. G., Cohn, S. M., Schloemann, S., & Stenson, W. F. (1998). Prostaglandins prevent decreased epithelial cell proliferation associated with dextran sodium sulfate injury in mice. Gastroenterology, 115, 874–882.

    Article  CAS  Google Scholar 

  42. Liang, J., Shao, S. H., Xu, Z. X., Hennessy, B., Ding, Z., Larrea, M., et al. (2007). The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nature Cell Biology, 9, 218–224.

    Article  CAS  Google Scholar 

  43. Jenkins, N., & Hovey, A. (1993). Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotechnology and Bioengineering, 42, 1029–1036.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Dr. Francesca Demarchi for the anti-LC3 antibody, Dr. Isei Tanida for the construct HcRed–hLC3, Dr. Rodolfo Garcia for the anti Hsc70 antibody, Dr. Andrea Predonzani and Dr. Ramino Mendoza-Maldonado for assistance with flow cytometry, Dr. Yuna Ayala for assistance with fluorescent microscope. Special thanks to Prof. Francisco E. Baralle and Dr. Emanuele Buratti for helpful discussions and suggestions.

Conflict of Interest

The authors have no conflict of interest to declare.

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Authors and Affiliations

  1. International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34149, Trieste, Italy

    J. Pedro Menvielle, Najete Safini, Sergio G. Tisminetzky & Natasa Skoko

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  1. J. Pedro Menvielle
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  2. Najete Safini
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Correspondence to Natasa Skoko.

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Menvielle J. Pedro, Safini Najete: joint first authors.

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12033_2012_9620_MOESM1_ESM.tif

Supplement Figure S1. The effect of DS on p53, Hsc70 and LC3 levels in HeLa cells. a Western blot detection of p53, Hsc70 and LC3II/LC3I in extracts of HeLa cells treated or not with DS. The statistical significance was evaluated using t test (n = 3, independent experiments, *p < 0.05, **p < 0.01). b Relative level of LC3II/LC3I ratio, quantified in DS-treated versus control HeLa cells after blocking autophagosome maturation with NH4Cl. DS-induced autophagy is shown by an additional increase in LC3II/LC3I ratio with respect to control cells, after NH4Cl treatment (lane 4 vs lane 2). The statistical significance was evaluated using t test (n = 3, independent experiments, *p < 0.05). (TIFF 1963 kb)

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Menvielle, J.P., Safini, N., Tisminetzky, S.G. et al. Dual Role of Dextran Sulfate 5000 Da as Anti-Apoptotic and Pro-Autophagy Agent. Mol Biotechnol 54, 711–720 (2013). https://doi.org/10.1007/s12033-012-9620-x

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