The specific aim of this proposed research is to develop and extensively characterize human mammary epithelial cells (HMEC) transformed in vitro by chemical carcinogens, as a means of understanding the number and nature of the events occurring during immortal and/or malignant transformation. The relationship of these events to expression of functional differentiation, involvement of known oncogenes, and response to growth controlling factors will be explored in order to suggest possible mechanisms contributing to the development of the immortal and/or malignant phenotype. We have already demonstrated that benzo(a)pyrene (BaP) can induce an extended life in culture as well as the immortal transformation of HMEC from at least one specimen donor and that the infection of immortal cells with the Kirsten mouse sarcoma virus (K-MSV) will render them tumorigenic in nude mice. We will continue these studies by (1) characterizing the extended life, immortally transformed, and malignantly transformed HMEC, for a variety of properties associated with malignant progression and/or state of functional differentiation. We will look for the presence or absence of coordinate expression of these different markers with each other, and with the growth properties of the various cell types. (2) Obtaining a larger number and variety of transformed cell types by exposing HMEC from at least two other individual donors to BaP, and by exposing the existing extended life and immortal cells to further chemical carcinogens and/or promoters. (3) Fusing combinations of immortal cell lines, extended life, and normal cells to determine dominance or recessiveness, and possible complementation groups, for immortality and abnormal properties. (4) Screening the various HMEC substrates for alteration or activation of known oncogenes, cellular processing of the p21 protein, and susceptibility to K-MSV induced malignant transformation. (5) Providing HMEC substrates to other investigators interested in applying their specific expertise to this system. It is hoped that this research will provide a well characterized human epithelial cell system for the study of the etiology and mechanisms of human cellular carcinogenesis.

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National Cancer Institute (NCI)
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Chemical Pathology Study Section (CPA)
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Lawrence Berkeley National Laboratory
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Garbe, James C; Vrba, Lukas; Sputova, Klara et al. (2014) Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations. Cell Cycle 13:3423-35
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