Contributions of cytoplasmic factors to in vitro cellular senescence

Abstract

Mass populations of normal human lung fibroblasts were enucleated by centrifugation at ≥25,000 g in 4 μg/ml cytochalasin B. The 1% of cells that did not enucleate were rendered nonviable by treatment with mitomycin C. Whole cells were poisoned with a 99% lethal dose of the sulfhydryl reagent iodoacetate. The washed cells were then mixed with the anucleate cytoplasms, fused with inactivated Sendai virus, and planted in rotenone for 20 hours. Whereas normal cells are able to survive this rotenone treatment, the 1% surviving iodoacetate treated cells cannot withstand this additional stress. However, iodoacetate treated cells that fuse to untreated cytoplasms receive sufficient amounts of active enzymes to allow them to survive. Since this selective system does not rely on using enzymatic mutants, it should permit the selection of hybrids between anucleate cytoplasms and any type of whole cell. Cytoplasmic hybrids were cultured in order to determine their proliferative capacity. The life spans of cytoplasmic hybrids between young and old cells were compared to those of young/young and old/old controls. Cytoplasmic factors do not appear to control in vitro cellular senescence.