Differentiation of neuroblastoma cells induced by an inhibitor of mevalonate synthesis: Relation of neurite outgrowth and acetylcholinesterase activity to changes in cell proliferation and blocked isoprenoid synthesis

Abstract

Mevinolin, a competitive inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase, stimulates neurite outgrowth and acetylcholinesterase (ACE) activity in C1300 (Neuro‐2A) murine neuroblastoma cells. Sprouting of neurites began within 4–8 h, before changes in cell proliferation could be detected by [³H]thymidine incorporation or flow cytometry. In contrast, the increase in ACE activity was temporally correlated with suppression of DNA synthesis, which occurred after 8 h. The activity of the membrane marker enzyme phosphodiesterase I was not stimulated by mevinolin. Suppression of protein synthesis with cycloheximide blocked the induction of ACE activity but only partially inhibited neurite outgrowth in the mevinolin‐treated cultures. When mevinolin was removed from the culture medium, most of the cells retracted their neurites within 2 h, but ACE activity did not decline until DNA synthesis began to return to control levels after 10 h. Similarly, retraction of neurites in differentiated cells exposed to colchicine was not accompanied by a decrease in ACE activity. DNA histograms suggested that mevinolin arrests neuroblastoma cells in both the G₁ and G₂/M compartments of the cell cycle. Other cytostatic drugs that arrest cells at different stages of the cell cycle did not cause Neuro‐2A cells to form neurites such as those seen in the mevinolin‐treated cultures. When incorporation of [³H]acetate into isoprenoid compounds was studied in cultures containing mevinolin in concentrations ranging from 0.25 μM to 25 μM, the labeling of cholesterol, dolichol, and ubiquinone was suppressed by 90% or more at all concentrations. However, significant growth arrest and cell differentiation were observed only at the highest concentrations of mevinolin. Supplementing the medium with 100 μM mevalonate prevented the cellular response to mevinolin, but additions of cholesterol, dolichol, ubiquinone, or isopentenyl adenine were generally ineffective. The cholesterol content of neuroblastoma cells incubated with 25 μM mevinolin for 24 h was not diminished, and protein glycosylation, measured by [³H]mannose incorporation, was decreased only after 24 h at high mevinolin concentration. These studies suggest that the stimulation of neurite outgrowth and the increase in ACE activity induced by mevinolin are independent phenomena. Whereas neurite outgrowth is not related directly to the effects of mevinolin on cell cycling, the induction of ACE is correlated with the inhibition of cell proliferation. The cellular changes produced by blocking mevalonate synthesis do not appear to be causally linked to depletion of total cellular cholesterol or perturbation of glycoprotein synthesis. However, the possibility that depletion of ubiquinone or novel isoprenoid proteins triggers the arrest of cell proliferation and the expression of differentiated properties has not been ruled out.