[Frontiers in Bioscience 3, d944-960, September 1, 1998]
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BIOLOGICAL AND MOLECULAR BASIS OF HUMAN BREAST CANCER

Jose Russo, Xiaoqi Yang, Yun-Fu Hu, Betsy A. Bove, Yajue Huang, Ismael D.C.G. Silva, Quivo Tahin, Yuli Wu, Nadia Higgy, Abdel Zekri, and Irma H. Russo

Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

Received 12/17/97 Accepted 7/21/98

3. HUMAN BREAST EPITHELIAL CELLS IN CULTURE

It has been shown that the life span of HBEC cultured in adequate medium is comparable to that of adult human fibroblasts (30-40 doublings) (15, 16) and is profoundly affected by the concentration of calcium (Ca++) in culture medium (13). Extended growth of HBEC for over 1000 days and more than 50 generations without expressing terminal differentiation has been maintained by lower Ca++ level in culture medium (13). HBEC cultured under low-Ca++ conditions maintain their normal diploid karyotype, form domes and duct-like structures in collagen, express specific keratin filaments and milk fat globule membrane antigen, and contain all the other structural features of breast epithelial cells (17-19). In addition, a higher number of doublings has been observed in HBEC derived from breast tissues with a lower differentiation grade and a higher proliferation rate (6), indicating that the growth characteristics of HBEC in primary culture reflect the in vivo degree of lobular development and the rate of cell proliferation in vitro (6).

Like all normal diploid and differentiated somatic cells, normal HBEC have a limited capacity to divide both in vivo and in vitro. Cellular mortality of normal HBEC is characterized by a progressive cessation of cell growth manifested in cell culture by senescence that typically occurs after 10-20 passages in vitro (approximately 100 to 200 population doublings) (13, 16, 20). In contrast, transformed or tumor cells are able to escape from senescence as a result of genetic and epigenetic changes that disrupt the regulatory mechanisms of limited growth potential and are thereby considered immortal (21). Induction of immortality or immortalization involves abrogation of cellular programs for limiting the rate and the number of cell replication (22) and is generally perceived as the key event of an oncogenic process (13, 23).

Spontaneous immortalization of HBEC is a rarely-occurring event. Numerous investigators have tried to induce immortalization of HBEC using various physical (e.g., radiation), chemical (e.g., benzo(a)pyrene) and biological (e.g., viruses, gene transfer) approaches, the last being the most consistent (20). Immortalization of HBEC has been successfully induced by introduction of the human papilloma virus 16 (HPV-16) oncogenes E6 and/or E7 (24-26), simian virus 40 (SV40) DNA (27). However, immortalization of HBEC experimentally induced with viral oncogenes is often accompanied by expression of phenotypes indicative of neoplastic transformation, such as an increase in anchorage-independent growth and tumorigenesis in nude mice (27, 28). Clearly, a normal HBEC without expressing any transformed phenotypes is essential to any studies on experimentally-induced cell transformation.

We have reported that a mortal HBEC line #130, when cultured in medium containing 0.04 mM Ca++ (low Ca++) for over 2 years, became immortalized spontaneously giving rise to MCF-10F (12, 17). Immortalization of these cells is characterized by their continuous growth in culture medium containing the conventional level of Ca++ (1.05 mM; also called high Ca++) without entering senescence and expressing transformed phenotypes such as colony formation in agar or in agar-methocel (12, 13). Both the mortal and immortal cells are bona fide normal HBEC in nature, expressing genetic, cytogenetic, ultrastructural and phenotypic characteristics of normal human breast epithelia (12, 13) and represent cell lines closest to normal HBEC available.

The availability of normal HBEC in primary cultures, the mortal HBEC line (e.g., #130) and the immortal HBEC line MCF-10F provides us with a unique system to address some of the most important questions concerning the molecular mechanisms of cell transformation, such as: 1. What are the factors influencing susceptibility to cell transformation? 2. What are the molecular mechanisms responsible for cell immortalization? 3. What are the mechanisms associated with cell transformation?