Breast Cancer Research and Treatment 66: 1–7, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. Report Identification of Cripto-1 in human milk Caterina Bianco 1 , Christian Wechselberger 1 , Andreas Ebert 1,2 , Nadia I. Khan 1 , Youping Sun 1 , and David S. Salomon 1 1 Tumor Growth Factor Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, Na- tional Institute of Health, Bethesda, MD, USA; 2 Department of Obstetric and Gynecology, Free University of Berlin, Berlin, Germany Key words: Cripto-1, human milk, mammary epithelial cells Summary Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related peptide that plays an important role in normal mam- mary gland development. CR-1 is expressed in the growing terminal end buds in the virgin mouse mammary gland and its expression increases during pregnancy and lactation. Furthermore, CR-1 is involved in the early stages of mouse mammary tumorigenesis and in the pathogenesis of human breast cancer. Since CR-1 is expressed in the mouse mammary gland at high levels during pregnancy and lactation, we have evaluated whether this protein is present in human milk. In the present study we demonstrate that a 28 kDa immunoreactive CR-1 protein is present in 24 human milk samples as assessed by western blot analysis and that by enzyme-linked immunosorbent assay the concentration of CR-1 ranges between 62 and 118 ng/ml. In addition, CR-1 that had been purified from human milk is able to stimulate the phosphorylation of mitogen activated protein kinase in nontransformed NMuMG mouse mammary epithelial cells. These results suggest that CR-1 in human milk may be important in regulating mammary gland development during pregnancy and lactation. Introduction Human CR-1 is a member of a larger family of struc- turally related proteins, the EGF-CFC family that includes mouse Cr-1, mouse cryptic, Xenopus FRL- 1 and zebrafish one-eyed pinhead (oep) [1–5]. These proteins are characterized by the presence of a mod- ified EGF-like domain and by a second cysteine-rich region called CFC domain [6]. They also share a conventional signal sequence and a hydrophobic C- terminus that is essential for membrane-anchorage by a glycosylphosphatidylinositol (GPI) moiety [7]. The human CR-1 and mouse Cr-1 genes encode for gly- coproteins of 188 and 171 amino acids respectively, with molecular weights of 28 and 24 kDa [8]. The EGF-CFC proteins perform an essential role during early vertebrate embryogenesis by promoting meso- derm formation and cell migration during gastrulation [5, 9, 10]. CR-1 mRNA and immunoreactive protein are expressed in several human breast cancer cell lines, in approximately 80% of human primary breast car- cinomas and in mammary tumors that arise in mice that overexpress different transgenes in the mammary gland such as c-neu, transforming growth factor α (TGFα), int-3, polyoma middle T gene or SV-40 large T gene [11–13]. Cr-1 can also be detected in the devel- oping mouse mammary gland with different levels of expression in the virgin, pregnant, lactating and aged mammary gland [14]. In the virgin mammary gland Cr-1 expression is found primarily in the cap stem cells of the growing terminal end buds and Cr-1 ex- pression increases several fold in ductal epithelial cells during pregnancy and lactation and in the aged mam- mary gland [14, 15]. In addition, Cr-1 can modulate the expression of milk proteins in a mouse mammary epithelial cell line (HC-11) and in primary mouse mammary explant cultures [16]. Human milk contains a variety of different pro- teins and peptides that possess biological activity [17]. Among the many bioactive substances present in milk,