The influence of growth factors on the proliferative potential of normal and primary breast cancer-derived human breast epithelial cells.

In previous studies, we developed serum-free, bovine pituitary extract (BPE)-free culture conditions for the growth of normal and neoplastic rat mammary epithelial cells. The present studies were aimed at determining if these culture methods could be used to study the influence of specific growth factors on the proliferative potential of normal human mammary epithelial (HME) cells and cells derived from human breast cancer (HBC) specimens. Our results indicate that normal HME cells in primary culture express stringent requirements for insulin (IN), epidermal growth factor (EGF), and cholera toxin (CT). Of these factors, EGF is most important, with essentially no proliferation taking place in the absence of this factor. By contrast, when cells are grown in serum-free primary culture in the presence of a full complement of growth factors and then subcultured, growth in secondary culture is not influenced by the removal of individual growth factors. Growth in secondary culture in the absence of EGF is mediated by autocrine factors secreted by the cells. However, there is no evidence for autocrine activity that mediates growth in the absence of IN in secondary cultures. Primary culture of HBC cells in serum-free, BPE-free medium revealed two patterns of growth factor requirements. One set of HBC cells expressed identical requirements for IN and EGF in primary culture as normal cells. Likewise, these cells grew in secondary culture in the absence of either factor. The second set of tumors expressed independence of IN for growth in primary culture. These cells grew to confluence in primary culture in the absence of IN and could be subcultured in this medium. All tumor cells examined expressed a requirement for EGF for primary culture growth, whereas none of the HBC cells examined expressed a significant CT requirement. In many cases, growth in the absence of CT exceeded that observed in its presence. Thus, our culture system allows analysis of the growth factor requirements of HME and HBC cells in primary culture. Our results indicate significant differences between HME and HBC cells in this regard. However, the results of secondary culture experiments indicate that the growth factor milieu from which cells are taken can have a profound effect on the requirements for growth factors in culture.

cAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo.

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10(6) cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10(6) cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.

Importance of extended growth potential and growth factor independence on in vivo neoplastic potential of primary rat mammary carcinoma cells.

Recently developed culture systems that allow extended growth of normal rat mammary epithelial (RME) cells were used to directly compare the proliferative potentials and growth factor requirements of primary normal and primary neoplastic rat mammary cells. RME cells were obtained from 45- to 55-day-old inbred female Lew rats and rat mammary tumor (RMT) cells from 7,12-dimethylbenz(a)anthracene- or N-nitroso-methylurea-induced mammary carcinomas. To compare the proliferative lifespan of RME and RMT cells, colony forming efficiencies were determined after consecutive passages over a 70-day culture period. Whereas the proliferative potential of RME cells declined with time in culture, RMT cells from five separate mammary carcinomas had colony forming efficiencies that increased with serial passage. By the end of the 70-day culture period, colony forming efficiencies for RMT cells were 100- to 1000-fold higher than those for RME cells. To compare the growth factor requirements for RME and RMT cells, a serum-free culture medium that supports RME cell growth was used and the influence of specific growth factors was examined by deletion experiments. Cells from 5 of 18 primary mammary carcinomas exhibited requirements for insulin, epidermal growth factor, and cholera toxin identical to those of RME cells. In contrast, cells from 9 of 18 tumors expressed independence of one, two, or all three of these factors for growth in serum-free culture. To examine the in vivo growth potential of primary RMT cells, samples of the cell suspensions used to initiate primary cultures were transplanted into the interscapular white fat pads of syngeneic female recipients. Transplantation of cells from growth factor dependent tumors yielded nonneoplastic mammary outgrowths. In contrast, transplantation of growth factor independent tumor cells yielded grossly visible tumors in 100% of the recipients within 4 weeks of transplantation. Thus, our results indicate that cells from all primary mammary carcinomas have dramatically enhanced growth potential in long-term culture relative to RME cells. Furthermore, a subset of these tumors are also independent of growth factors required by RME cells, and expression of growth factor independence is associated with high neoplastic potential in vivo.

Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cells in vitro.

These experiments were aimed at using a recently developed serum-free culture system for growth of normal rat mammary epithelial (RME) cells in vitro to examine the interactions of specific hormones and growth factors on the proliferative potential of these cells. RME cells were obtained by enzymatic dissociation of mammary tissues of Lewis rats. Primary cultures were started by plating 2 X 10(5) RME cells per 60-mm type I collagen-coated tissue culture dish. Cultures were maintained in a basal medium that consisted of Ham's F-12 medium supplemented with bovine serum albumin (BSA), ethanolamine (EA), and transferrin (Tf), which, by itself, did not support RME cell proliferation. Insulin (I), hydrocortisone (HC), and epidermal growth factor (EGF), when added to the basal medium interacted synergistically to stimulate RME cell proliferation, but this effect was dependent on the additional presence of cholera toxin (CT). Under these conditions a greater-than-tenfold increase in cell number over a 10-day culture period was obtained. Insulin could be replaced by physiological levels of insulin-like growth factor-I (IGF-I). CT could be replaced by other agents that elevate intracellular levels of cyclic adenosine 3':5' monophosphate (cAMP) such as dibutyryl-cAMP (db-cAMP), prostaglandin E1 (PGE-1), and/or isobutylmethylxanthine (IBMX). Prolactin (M) or progesterone (P) potentiated the effect of I, HC, EGF, and CT, resulting in an additional twofold increase in cell number over that found in their absence. However, addition of both hormones was no more effective than either one alone. Furthermore, addition of M or P in the absence of EGF had no effect on RME cell proliferation. Addition of 17-B-estradiol (E2) to the I-, HC-, EGF-, and CT-containing medium also resulted in enhanced RME cell proliferation. These results point to a number of hormone and growth factor interactions that influence the proliferation of normal RME cells in vitro.