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.