Lithium, which is used to treat bipolar psychiatric disorders, can stimulate proliferation of a number of cells in tissue culture. Proliferation of MCF-7 human breast cancer cells, which also respond to EGF and estrogens, was stimulated by LiCl (1-5 mM) within the concentration range that is encountered during human therapy with lithium. Stimulation of growth was specific for lithium; rubidium, potassium, and sodium showed no such effect. In the presence of antiestrogen, lithium stimulated the growth of hormone-dependent breast cancer cells MCF-7, ZR-75-1, and T47D but not hormone-independent MDA-MB-231 cells or an estrogen-independent clone of MCF-7 cells. Lithium-stimulated proliferation was limited by cytotoxicity which could be moderated by added potassium chloride (5-20 mM) in the medium. Each of the mitogens lithium, 17 beta-estradiol, and EGF increased the rate of uptake of myo-inositol into MCF-7 cells. Whether normalized to inositol lipids, to protein, or to DNA, steady-state levels of inositol phosphates were elevated by each of the mitogens including lithium, which inhibits the breakdown of inositol phosphates in the phosphoinositide signaling pathway. These data indicate that therapeutic concentrations of lithium can stimulate the proliferation of human breast cancer cells by a mechanism that may involve the phosphoinositide pathway.

A commercial preparation of charcoal-stripped adult bovine serum was used to culture MCF-7 cells in estrogen-free media. Use of this stripped adult bovine serum represents an alternative to calf serum which is in more limited supply, and saves charcoal-stripping of serum in the laboratory, which can be a rate-limiting step in the preparation of materials for estrogen-free tissue culture. MCF-7 cell proliferation was controlled by estrogens, epidermal growth factor (EGF) and lithium chloride in adult bovine serum as well as in standard media prepared with charcoal-stripped calf serum, and approximately the same fold-increase in response to the tested agents was observed in the two sera. Although the growth rates were lower in media prepared with adult bovine serum, MCF-7 cells in both media exhibited the same sensitivities in dose-responses to these three mitogens. Levels of estrogen and progesterone receptors, and the magnitude of estrogen-dependent stimulation of the progesterone receptors, were similar in cells maintained in both sera. Therefore, a commercially stripped adult bovine serum can be used to replace calf serum in the study of estrogenic responses and the control of proliferation in MCF-7 breast cancer cells.

Lithium-stimulated MCF-7 cell proliferation was compared to proliferation stimulated by other mitogens for this cell line-estradiol (E2) and epidermal growth factor (EGF)-and lithium was found to be effective within a narrow concentration range. Mitogenic effects of lithium on proliferation stimulated by E2 and EGF were additive below maximum, but were not synergistic. The phosphoinositide pathway is a cell signaling system involved in cell proliferation, within which phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] leads to the production of the second messengers inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG), as well as to calcium mobilization. At mitogen concentrations which maximally stimulated cell growth, estradiol stimulated both growth and PLC activity, while EGF and lithium stimulated cell growth but had little effect on the activity of the enzyme. Dose-responses with EGF revealed that a low concentration (0.1 ng/ml, 0.017 nM) of EGF appeared to stimulate both PLC activity and cell growth, but that higher concentrations of EGF which stimulated greater proliferation inhibited PLC activity. Steady-state levels of inositol phosphates including inositol trisphosphate were increased by all three mitogens. In growth assays, the phorbol ester phorbol 12-myristate-13-acetate (PMA), which mimics the actions of DAG, stimulated some cell growth, but dioctanoylglycerol, an additional DAG analog, and the calcium ionophore A23187, alone or with the DAG analogs, had no effect. These results suggest that PLC-mediated PtdIns(4,5)P2 hydrolysis is not primarily associated with signaling proliferation by lithium or EGF in MCF-7 breast cancer cells.