Abstract
Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50 %. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.
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References
Canellakis ES, Viceps-Madore D, Kariakidis DA, Heller JS (1979) The regulation and function of ornithine decarboxylase and of the polyamines. Curr Top Cell Regul 15:155–202
Chen KY, Canellakis ES (1977) Enzyme regulation in neuroblastoma cells in a salts-glucose medium: induction of ornithine decarboxylase by asparagine and glutamine. Proc Natl Acad Sci USA 74:3791–3795
Coffino P (2001) Regulation of cellular polyamines by antizyme. Nat Rev Mol Cell Biol 2:188–194
Dulloo I, Gopalan G, Melino G, Sabapathy K (2010) The antiapoptotic deltaNp73 is degraded in a c-jun-dependent manner upon genotoxic stress through the antizyme-mediated pathway. Proc Natl Acad Sci USA 107:4902–4907
Fausto N (1971) The control of ornithine decarboxylase activity during liver regeneration. Biochim Biophys Acta 238:116–128
Fong WF, Heller JS, Canellakis ES (1976) The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures. Biochem Biophys Acta 428:456–465
Fong LY, Feith DJ, Pegg AE (2003) Antizyme over expression in transgenic mice reduces cell proliferation, increases apoptosis, and reduces N-nitrosomethylbenzylamide-induced forestomach carcinogenesis. Cancer Res 63:3945–3954
Fujita K, Murakami Y, Hayashi S (1982) A macromolecular inhibitor of the antizyme to ornithine decarboxylase. Biochem J 204:647–652
Hovis JG, Stumpo DJ, Halsey DL, Blackshear PJ (1986) Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH3T3 fibroblasts. J Biol Chem 261:10380–10386
Kahana C (2009) Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor. Essays Biochem 46:47–61
Kay JE, Lindsay VJ, Cooke A (1972) Ornithine decarboxylase in phytohaemagglutinin stimulated lymphocytes: control of degradation by amino acids. FEBS Lett 21:123–126
Lim SK, Gopalan G (2007) Antizyme1 mediates AURKAIP1-dependent degradation of aurora-A. Oncogene 26:6593–6603
Lin Y, Martin J, Gruendler C, Farley J, Meng X, Li BY, Lechleider R, Huff C, Kim RH, Grasser WA, Paralkar V, Wang T (2002) A novel link between the proteasome pathway and the signal transduction pathway of the bone morphogenetic proteins (BMPs). BMC Cell Biol 3:15
Lopatin AN, Makhina EN, Nichols CG (1994) Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 372:366–369
Matsufuji S, Matsufuji T, Miyazaki Y, Murakami Y, Atkins JF, Gesteland RF, Hayashi S (1995) Auto-regulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80:51–60
McCormack SA, Viar MJ, Johnson LR (1993) Polyamines are necessary for cell migration by a small intestinal crypt cell line. Am J Physiol Gastrointest Liver Physiol 264:G367–G374
Murakami T, Matsufuji S, Miyazaki Y, Hayashi S (1994) Forced expression of antizyme abolishes ornithine decarboxylase activity, suppresses cellular levels of polyamines and inhibits cell growth. Biochem J 46:47–61
Newman RM, Mosbascher A, Mangold U, Koike C, Diah S, Schmidt M, Finley D, Zetter BR (2004) Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression. J Biol Chem 279:41504–41511
Olsen RR, Zetter BR (2011) Evidence of a role for antizyme and antizyme inhibitor as regulators of human cancer. Mol Cancer Res 9:1285–1293
Pegg AE (1988) Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 48:759–774
Pegg AE (2006) Regulation of ornithine decarboxylase. J Biol Chem 281:14529-14532 Quaroni A, Wands J, Trelstad RL, Isselbacher KJ (1988) Epithelial cell culture from rat small intestine. J Cell Biol 80:248–265
Pegg AE, McGovern KA, Weist L (1987) Decarboxylation of alpha-difluoromethylornithine by ornithine decarboxylase. Biochem J 241:305–307
Quaroni A, Wands J, Trelstad RL, Isselbacher KJ (1979) Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80:248–265
Ray RM, Viar MJ, Yuan Q, Johnson LR (2000) Polyamine depletion delays apoptosis of rat intestinal epithelial cells. Am J Physiol 278:C400–C489
Ray RM, Viar MJ, Johnson LR (2012) Amino acids regulate the expression of antizyme-1 to modulate ornithine decarboxylase activity. J Biol Chem 287:3674–3690
Ray RM, Bhattacharya S, Bavaria MN, Viar MJ, Johnson LR (2014) Spermidine, a sensor for antizyme 1 expression regulates intracellular polyamine homeostasis. Amino Acids. doi:10.1007/s00726-014-1757
Rinehart CA Jr, Viceps-Madore D, Fong WF, Ortiz JG, Canellakis ES (1985) The effect of transport system A and N amino acids and nerve and epidermal growth factors on the induction of ornithine decarboxylase activity. J Cell Physiol 123:435–441
Russell DH (1985) Ornithine decarboxylase. A key regulatory enzyme in normal and neoplastic growth. Drug Met Rev 16:1–88
Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53:749–790
Acknowledgments
This publication was made possible by grants (Number DK-16505) from the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) and by support from the Thomas A. Gerwin endowment. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Health.
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Ray, R.M., Bhattacharya, S., Bavaria, M.N. et al. Antizyme (AZ) regulates intestinal cell growth independent of polyamines. Amino Acids 46, 2231–2239 (2014). https://doi.org/10.1007/s00726-014-1777-0
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DOI: https://doi.org/10.1007/s00726-014-1777-0