25-Hydroxycholesterol-induced elevation in 45Ca uptake: correlation with depressed DNA synthesis

The mechanism whereby 25-hydroxycholesterol, an inhibitor of the synthesis of cholesterol, depresses DNA synthesis in cycling P815 mastocytoma cells was investigated. The uptake of 45Ca into P815 cells treated with 1 microgram/ml 25-hydroxycholesterol began to rise above control levels by 6 hours after initiation of treatment and was increased tenfold by 15 hours. Kinetic data of calcium uptake indicated the presence of at least two components of calcium uptake, fast and slow. The fast phase of calcium exchange at the cell surface was changed little by treatment with 25-hydroxycholesterol. The slow phase of calcium exchange with the intracellular compartment was markedly affected by treatment with the inhibitor, there being a large increase in the flux and half-time of uptake, and a fall in the rate constant. This resulted in a large elevation of the intracellular compartment size. Incorporation of [3H]thymidine into DNA began to decline between 9 and 12 hours posttreatment in these cultures. Uptake of calcium and depression of DNA synthesis were shown to be directly related to the dose of 25-hydroxycholesterol used. The changes in 45Ca uptake and DNA synthesis due to 25-hydroxycholesterol treatment were abolished by addition of exogenous cholesterol to the incubation medium. The results are consistent with the hypothesis that 25-hydroxycholesterol, by inhibiting cholesterol production, depresses DNA synthesis via an elevation in the uptake of calcium into the cell to a level incompatible with continued DNA replication.