Promotion-sensitive epidermal and mammary epithelial cells maintained in suspension over agarose

The molecular basis of tumour promotion is still largely unknown. In in vitro model of tumour promotion, the promotion-sensitive cells are induced to grow under anchorage-independent conditions in the presence of promoting agent. The customary way of providing such conditions is to immobilize these cells in soft agar, but such cells cannot be readily recovered to study the induced biochemical and molecular events. In the present report, we analysed these events using JB6 mouse epidermal cells maintained in suspension in liquid medium over agarose. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent synthesis of DNA in promotion-sensitive P+ (but not in promotion-resistant P-) JB6 cells and this TPA-induced synthesis of DNA positively correlated with TPA-induced formation of colonies in soft agar. The TPA-induced synthesis of DNA began on or shortly before 24 h after the introduction of TPA, peaked at about 48 h and then declined to the control levels over the next several days. All trans-retinoic acid and dexamethasone inhibited and calcitriol (1 alpha,25-dihydroxy-vitamin D3) synergistically stimulated this TPA-induced DNA synthesis. Western immunoblot analysis of cyclins (A, B1, D1 and E) and p27Kip1, a cyclin-dependent kinase inhibitor, indicated that TPA induced cyclin A and cyclin B1 expression in P+ (but not in P-) JB6 cells and this induction coincided in time with TPA-induced synthesis of DNA. TPA also strongly induced cyclin D1 expression in P+ (but not in P-) JB6 cells, but this induction started prior to the expression of cyclin A and cyclin B1. TPA did not affect the expression of either cyclin E or p27Kip1 to any significant extent. We also found that NMU38 rat mammary epithelial cells were operationally equivalent to the promotion-sensitive P+ JB6 cells, but in these cells 17 beta-oestradiol exerted a strong synergistic effect on TPA-induced synthesis of DNA. Based on these observations, we tentatively propose a sequence of molecular events which possibly lead to the anchorage-independent synthesis of DNA in these cells.