Density-dependent stimulation and inhibition of cell growth by agents that disrupt microtubules

We have previously reported that agents that disrupt microtubules, such as colchicine, inhibit the growth stimulation of lymphocytes and arrested fibroblasts; other workers have recently reported enhanced stimulation of fibroblasts in the presence of the same drugs. In the present studies, we resolve this conflict by demonstrating that the stimulatory and inhibitory effects of microtubule disruption depend upon the density and the cell type of the treated cultures. Our analysis included an examination of three variables: (i) cell density (sparse or confluent), (ii) cell type (resting fibroblasts from mouse or chicken embryos or from the permanent 3T3 mouse fibroblast line), and (iii) treatment with colchicine and related drugs in the presence or absence of various mitogens such as serum, insulin, and epidermal growth factor. We found that colchicine augmented mitogenesis in confluent cultures of all cell types. Colchicine by itself appeared to be mitogenic only for confluent chicken embryo fibroblasts. In sparse cultures with minimal cell-cell contacts, however, there were differences between embryonic cells and the 3T3 cell line. In confirmation of our previous reports, disruption of microtubules by colchicine inhibited the mitogenic stimulation of sparse cultures of embryonic chicken or mouse fibroblasts. In contrast, fibroblasts of the permanent 3T3 line in sparse cultures were stimulated by some mitogens despite the presence of colchicine. The augmentative effects of colchicine on the stimulation of confluent cultures were synergistic with the mitogens, and colchicine allowed response to otherwise submitogenic doses of growth factors. Kinetic studies indicated that the stimulatory and inhibitory effects of colchicine are separable and that both can operate simultaneously. The experiments suggest that the regulation of growth by nutrient deprivation and the regulation by density dependence proceed at least in part by different mechanisms. All of the results suggest that microtubular integrity can be associated with the expression of either negative or positive controls on cell growth, depending upon the confluence or lineage of the cells in culture.