Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-16
2004-12-07
Nickol, Gary (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210
Reexamination Certificate
active
06828111
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention in the field of biology and medicine relates to a cell culture model system that mimics human preneoplastic breast disease and uses thereof in screening agents that inhibit development of this disease and its progression to breast cancer.
2. Description of the Background Art
Reciprocal cellular interactions between epithelial and stromal cells have been demonstrated as a key determinant in the morphogenesis, proliferation and cyto-differentiation of both endocrine and non-endocrine target organs (I. Hom, Y. K. et al., Endocrinology, 139: 913-921, 1998; Donjacour, A. A. et al., Cancer Treatment Res., 53: 335-364, 1991; Cunha, G. R. et al., Cell Differ., 17:137-148, 1985). Carcinomas of the breast are composed of not only tumor epithelial cells but also of infiltrating endothelial cells (“EC's”), fibroblasts, macrophages and lymphocytes (Gregoire, M. et al., Cancer Metast. Rev., 14: 339-350, 1995). The stroma provides vascular supply and specific soluble and extracellular matrix (ECM) molecules that are required for tumor growth and progression (Hanahan, D. et al., Cell, 86: 353-364, 1996). Several lines of evidence indicate that stromal cells play a central role via ECM remodeling in tumor invasion and dissemination (Camps, J. L. et al., Proc. Natl. Acad., Sci. USA, 87: 75-79, 1990; Picard, O., et al. Cancer Res., 46: 3290-3294, 1986; Grey, A. M. et al., Proc. Natl. Acad. Sci. USA, 86: 2438-2442, 1989). However, a recent report has shown that stromal alteration(s) precede the malignant conversion of tumor cells (Moinfar, F. et al., Cancer Res., 60: 2562-2566, 2000).
Although there is experimental evidence supporting the involvement of angiogenesis in pathogenesis of breast cancer, the influence of functional interactions between human breast epithelial cells (also referred to as mammary epithelial cells) and endothelial cells have not been defined. Analysis of such cell interactions requires a culture/assay system that permits growth and differentiation of both epithelial cells and endothelial cells.
The present invention provides such a system and describes its utility as a model for studying the progress of preneoplastic lesions to cancer and for testing the activity of agents that can inhibit this process.
Growth and formation of capillary blood vessels or neovascularization is an essential component of solid tumor growth (Folkman, J. et al., Int. Rev. Exp. Pathol. 16: 207, 1976; Gimbrone, M. A. Jr et al., J. Natl. Cancer Inst., 52: 413, 1974). Any increase in the size of the tumor cell population must be preceded by an increase in new capillaries that converge upon the tumor; such angiogenesis has been directly correlated with tumor growth and metastasis (Folkman, J, J. Nat'l Canc Inst 82:4-6, 1990). Tumor cell products and products of various non-neoplastic mediator systems have been implicated in this vasoproliferative response (Gimbrone et al., supra; Auerbach, R., In: E. Pick (ed),
Lymphokines
. Vol 4, pp. 69-84, New York: Academic Press, 1981). Several growth factors, cytokines, molecules of the extracellular matrix (ECM) or physical conditions induce or regulate endothelial cell growth and/or migration in vitro (Klagsbrun, M. et al., Ann. Rev. Physiol. 53:217-239, 1991). These include several well characterized polypeptide growth factors, proteolytic enzymes, interferon, cyclic nucleotides, prostaglandins, heparin, lowered oxygen tension, histamine and other vasoactive amines, and several low molecular weight endothelial mitogens and chemotactic factors (Klagsbrun et al., supra).
Vascular endothelial growth factor (Ferrara, N. et al., Biochem. Biophys. Res. Commun., 161:851-858, 1989) or vascular permeability factor (Connolly, D. T. et al., J. Biol. Chem., 264:20017-20024, 1989) (abbreviated VEGF/PF or VEGF) is an endothelial cell-specific mitogen that mediates physiological and pathological neovascularization (Leung, D. W. et al., Science 246:1306-1309, 1989). VEGF acts as a survival factor, preventing the apoptotic death of microvascular endothelial cells (Alon, T. et al., Nat. Med., 1:1024-1028, 1995, 1995; Watanabe, Y. et al., Exp. Cell. Res., 233:340-349, 1997). The human VEGF gene encodes a dimeric glycoprotein comprising four possible monomers as a result of differential splicing of eight exons that make up the gene product. The four VEGF subtypes are 121-, 165-, 189-, and 206-amino acids in length (Neufeld, G. et al., FASEB J., 13:9-22, 1999). The smaller forms are secreted whereas VEGF
189
and VEGF
206
are bound to heparan proteoglycans and thus retained close to the membrane of producing cells. Three receptors for VEGF have been described:
VEGFR-1 (=Flt-1) binds VEGF;
VEGFR-2 (=Flk-1/KDR) binds VEGF; and
VEGFR-3 (=Flt-4) appears to be specific for VEGF-C (Neufeld et al., supra).
Expression of Flk-1/KDR is confined to endothelial cells, accounting for the selective nature of VEGF-induced mitogenesis (Neufeld, supra). VEGF is expressed at high levels in a wide range of tumors and tumor cell lines (Berse, B. et al., Mol. Biol. Cell, 3:211-220, 1992) and is believed to be a key mediator of (1) tumor angiogenesis (Connolly, D. T. et al., J. Clin. Invest., 84:1470-1477, 1989; Kim, K. J. et al., Nature (London), 362:841-844, 1993; Plate, K. H. et al., Nature (London), 359:845-848, 1992) and (2) the high blood vessel permeability characteristic of tumors (Senge, D. R. et al., Science 219:983-985, 1983; Yeo, K. T. et al., Cancer Res., 53:2912-2918, 1983). Expression of VEGF in the uterus was rapidly and strongly stimulated by estrogen (Cullinan-Bove, K. et al., Endocrinology, 133:829-837, 1993), suggesting that VEGF mediates the normal, estrogen-induced increase in vascular permeability and blood vessel growth in the uterus. Similarly, expression of VEGF is rapidly induced by 17 &bgr;-estradiol (E
2
) in dimethylbenzanthracene (DMBA)-induced estrogen-dependent mammary tumors (Nakamura, J. et al., Endocrinology, 137:5589-5596, 1996).
Using the MCF10AT1 xenograft model for human proliferative breast disease, the present inventors and their colleagues previously demonstrated that E
2
exerts a growth promoting effect on benign or premalignant ductal epithelium by enhancing the speed of transformation from simple/mild hyperplasia (grades 0/1) to atypical hyperplasia (grade 3) and ductal carcinoma in situ (grade 4) (Shekhar, P. V. M. et al., Amer. J. Pathol., 152:1129-1132, 1998). Table 1, below, summarizes the criteria for grading proliferative breast lesions (from Dawson, P. J. et al., Am. J. Pathol., 148:313-319, 1996).
Much of this growth promoting effect appeared to arise from effects of E
2
on angiogenesis since lesions from unsupplemented animals were either simple or hyperplastic without atypia and lack angiogenesis. The dramatic increase in growth and advanced histological grades of progression concomitant with its remarkable effect on angiogenesis suggested to the present inventors that one of the mechanisms by which E
2
acts as a breast cancer promoter could be through its effect on expression of angiogenesis-regulating factors.
The extracellular matrix (ECM) acts locally to modulate the responsiveness of endothelial cells and mammary epithelial cells to external factors. Besides providing a scaffolding during capillary morphogenesis, the ECM, by virtue of its ability to mediate both biochemical and biomechanical signaling events, exerts complex local controls on the functions of endothelial cells (Polverini, P. J., Eur. J. Cancer, 32A:2430-2437, 1996). For example, the ECM controls growth, differentiation and apoptosis of normal murine and human breast epithelial cells (Barcellos-Hoff, M. H. et al., Development, 105:223-235, 1989; Boudreau, N. et al., Science 267:891-893, 1995).
Collagenolytic degradation of endothelial and parenchymal basement membranes is an essential step in the process of tumor invasion and angiogenesis (Liotta, L. A. et al., Cell, 64:327-336, 1991). Proteolysis and interruption of the basement membrane and ECM require the activ
Shekhar Malathy P. V.
Tait Larry
Livnat Sandy
Nickol Gary
Venable LLP
Wayne State University
Yaen Christopher
LandOfFree
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