Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor Elena Yu. Blishchenko a, *, Olga A. Kalinina a , Olga V. Sazonova a , Sergei V. Khaidukov a , Natalya S. Egorova a , Andrei Yu. Surovoy a , Marina M. Philippova a , Arpad A. Vass b , Andrei A. Karelin a , Vadim T. Ivanov a a Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117871 Moscow, GSP-7, Russia b Oak Ridge National Laboratory, P.O.Box 2008,4500S, MS6101 Oak Ridge, TN, USA Received in revised form December 20, 2000; accepted May 11, 2001 Abstract It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Neokyotorphin; Tissue-specific peptide pool; Tumor cells; Proliferation 1. Introduction Cell growth and differentiation are regulated in tissues/ organs by a number of growth factors. Until recently the niche of “maintenance” of tissue homeostasis, i.e. the ap- propriate ratio of functional cells in a given tissue has been occupied exclusively by these small proteins performing, as a rule auto- and para-endocrine regulation of cell number. Such regulation involves induction of cell growth, differen- tiation, cell cycle arrest and cytolysis. Growth/differentia- tion factors are present in very low amounts in tissues and bind to their receptors with high affinity (K d of 10 -10 -10 -12 M). The majority of growth/differentiation factors are formed by specific proteolysis and released, in contrast to the hormones by non-specialized cells. The typical levels of growth factors at normal conditions are in the g per kg of tissue range. Their levels increase several fold in tumors and in regenerating tissues [1,2,19]. At the same time, there is a growing evidence that all above mentioned effects (i.e. regulation of proliferation, differentiation and cytolysis) are also mediated by classic regulatory peptides and endogenous fragments of functional proteins [11]. In contrast to growth factors and peptide hormones, the latter are present in high amounts (up to mg per kg of tissues), their content and composition in tissues being highly stable at normal conditions on one hand and tissue specific on the other [11,13]. An important member of that group is neokyotorphin, a peptide arising from endog- enous proteolysis of hemoglobin [13]. Neokyotorphin, a pentapeptide with the amino acid se- quence Thr-Ser-Lys-Tyr-Arg corresponding to the 137–141 C-terminal segment of mammalian -globins has been ini- tially isolated from bovine brain and defined as a novel neuropeptide [17]. This peptide demonstrates some neuro- specific effects, such as induction of naloxone-dependent analgesia [6] and inhibition of enkephalin-degrading en- zymes [8]. On the other hand, neither receptor binding nor Abbreviations: FBS - fetal bovine serum; FALS - forward angle light scattering; MTT - 3-(4;5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide; SRB - sulforhodamine B. * Corresponding author. Fax: +7-095-3361155. E-mail address: karelin@ibch.siobc.ras.ru (E.Y. Blishchenko). Peptides 22 (2001) 1999 –2008 0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0196-9781(01)00565-4