The goals of this project are to characterize the protein/polypeptide growth factors required for estrogen- responsive and autonomous rat and human breast cancer growth and to apply this information to development of new therapeutic approaches to control of this disease. The growth factors involved may originate from the plasma (endocrine factors) or they may be secreted locally by tumor cells/stromal (autocrine/paracrine factors). We propose to use a combination of polyclonal and monoclonal antibodies (MAbs) to growth factors and their receptors to define the mitogens most closely related to estrogen-responsive and autonomous breast tumor growth. Autocrine/paracrine factors will be studied under serum-free defined culture conditions and the factors secreted characterized biochemically, immunologically and structurally (amino acid sequencing). Estrogen-inducible growth factors will be sought from clones of the MTW9/PL2 rat line and from the human MCF-7, T-47D and ZR-75-1 cells; factors secreted by autonomous cells will be characterized using the estrogen receptor negative human MDA- MB-231, BT-20, HBL-100 and Hs578t lines and autonomous MTW9B rat cell clones. Our experiments show insulin-like growth factor 1 (IGF-1) is the most potent (ED50 30-50 pg/ml) mitogen identified for breast cancer cells. IGF-1 is estrogen-inducible into the medium of MCF-7 cells. The second most potent factor identified is basic fibroblast growth factor (b-FGF). Both IGF-1 and FGF-like factors are found in the medium of autonomous cells. Our plan is to continue complete identification of the most important factors, and to use antibodies to inhibit estrogen- promoted or autonomous growth in culture. MAbs to growth factor receptors will be used to confirm an autocrine role, or where indicated, as endocrine role of blood borne activities. When the cell culture studies are complete, we will use receptor directed MAbs to attempt inhibition of estrogen-responsive and autonomous tumor formation in athymic nude mice. These studies will directly test of the role in growth factors as mediators of estrogen-responsive and autonomous tumor growth in vivo, and establish whether blocking their action has potential as a new therapeutic approach to control of breast cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Molecular Cytology Study Section (CTY)
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University of Texas Health Science Center Houston
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Eby, J E; Sato, H; Sirbasku, D A (1993) Apotransferrin stimulation of thyroid hormone dependent rat pituitary tumor cell growth in serum-free chemically defined medium: role of FE(III) chelation. J Cell Physiol 156:588-600
Eby, J E; Sato, H; Sirbasku, D A (1992) Preparation of iron-deficient tissue culture medium by deferoxamine-sepharose treatment and application to the differential actions of apotransferrin and diferric transferrin. Anal Biochem 203:317-25
Sato, H; Eby, J E; Pakala, R et al. (1992) Apotransferrins from several species promote thyroid hormone-dependent rat pituitary tumor cell growth in iron-restricted serum-free defined culture. Mol Cell Endocrinol 83:239-51
Sirbasku, D A; Pakala, R; Sato, H et al. (1992) Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium. In Vitro Cell Dev Biol 28A:67-71
Sato, H; Eby, J E; Sirbasku, D A (1991) Iron is deleterious to hormone-responsive pituitary cell growth in serum-free defined medium. In Vitro Cell Dev Biol 27A:599-602
Sirbasku, D A; Stewart, B H; Pakala, R et al. (1991) Purification of an equine apotransferrin variant (thyromedin) essential for thyroid hormone dependent growth of GH1 rat pituitary tumor cells in chemically defined culture. Biochemistry 30:295-304
Sirbasku, D A; Pakala, R; Sato, H et al. (1991) Thyroid hormone dependent pituitary tumor cell growth in serum-free chemically defined culture. A new regulatory role for apotransferrin. Biochemistry 30:7466-77
Sirbasku, D A; Pakala, R; Sato, H et al. (1991) Thyroid hormone regulation of rat pituitary tumor cell growth: a new role for apotransferrin as an autocrine thyromedin. Mol Cell Endocrinol 77:C47-55
Karey, K P; Sirbasku, D A (1989) Human platelet-derived mitogens. II. Subcellular localization of insulinlike growth factor I to the alpha-granule and release in response to thrombin. Blood 74:1093-100
Karey, K P; Sirbasku, D A (1989) Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis. Anal Biochem 178:255-9

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