The growth of primary breast carcinoma and pre-cancerous cells in culture has proved difficult, and, as a result, there is a paucity of both short term and immortal primary breast carcinoma cell lines. Primary breast carcinoma cultures, though able to be initiated in selective media, undergo terminal differentiation, senescence, or apoptosis after only a few passages. The reasons for this are unclear. Because primary breast carcinomas and -pre-cancerous diseases states (DCIS) arise in via in the setting of surrounding myoepithelial cells, cells which exert important paracrine effects through both direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix, we hypothesized that the growth of primary breast carcinomas and precancerous cells in vitro might still be dependent on this type of myoepithelial interaction. Using a novel human myoepithelial cell line, HMS-1, and a urea/guanidine hydrochloride gel extract (Humatrix) of HMS-X, a transplantable human xenograft of HMS-1 which secretes a copious extracellular matrix, we have demonstrated in preliminary experiments dramatic effects on primary breast carcinoma cell morphogenesis. Using HMS-1 as a feeder layer, specific induction of spheroid formation occurred in 5 different cases of primary breast cancer. Using the extracted gel, Humatrix, specific induction of primary breast carcinoma cell glandular morphogenesis also occurred. This small grant proposal will exploit the effects of HMS-1 and Humatrix on primary DCIS and primary breast carcinomas to establish both short term and long term cultures which hopefully will lead to immortalization with the help of athymic/Scid mice. The initial observations of glandular and spheroid morphogenesis will be expanded with objective studies of the effects of HMS-l and Humatrix on primary breast carcinoma mitogenesis, morphogenesis, genetic stability, and apoptosis. Mitogenesis studies will exploit Ki-67 antigen expression, bromodeoxyuridine uptake, and flow cytometric studies. Morphogenesis studies will exploit known breast differentiation markers including gross cystic fluid protein-IS, milk fat globule-2, laminin, type IV collagen, BRST-2, CU18, casein, other milk proteins and cadherins. Measurements of genetic stability will include karyotype analyses. Apoptosis studies will include measurements of nucleosomal DNA laddering, nuclear condensation, and expression of the apoptosis-associated genes SGP-2 and ICE (interleukin-1 converting enzyme) by Northern blot. If the studies of this pilot proposal prove successful in demonstrating a myoepithelial paracrine effect on primary breast carcinoma growth, a subsequent R01 application will address the molecular mechanism of this effect by exploiting a subtractive expression library approach.
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