Nucleolar fragmentation in L cells exposed to quinacrine in vitro.

heat-inactivated fetal calf serum in an atmosphere of 5% CO2 and air at 37°C and subcultured every 4 days. Cells were grown it) monolayers on flying coverslips in Leighton tubes or in plastic T flasks for 2 days before the addition of 15 pg/nil of quinacrine to the culture medium. Cells were exposed to quinacrine for periods up to 2 hours ; some specimens were allowed to recover in medium without quinacrine for 24 hours. Phase Microscopic Observations Studies were performed on two types of specimens: unfixed vital preparations on coverslips mounted in medium and sealed with a paraffin-vaseine mixture; similar preparations fixed 10 minutes in 2.5% glutaraldehyde (Fischer Scientific Co., Fair lawn, New Jersey) in 0. 1 M phosphate buffer pH 7.4, mounted and sealed with the paraffin mixture. Electron Microscopy Fixation of cell monolayers was performed in the cold for 30 minutes with a freshly prepared mixture of 1 part 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 and 2 parts 1% osmium tetroxide in the same buffer by the method de scribed elsewhere (6). Five minutes after the addition of the fixative, the cells were then scraped off the plastic surface, centrifuged at 300 X g for 2 minutes in a dinical centrifuge (International Equipment Co.), and washed twice in cold isotonic saline before postfixation in cold 0.25% uranyl ace tate in acetate buffer 0.1 M pH 6.3 for 30 minutes. The cells were then washed in cold saline, the pellet suspended in @-ml molten 2% Nobel agar, and centrifuged in a warm water jacket at 750 X g for 2 minutes. The cell pellet was then chilled, trimmed into blocks, dehydrated in alcohol, and embedded in Epon (14). Thin sections were cut on a Porter Blum micro tome, stained with uranyl acetate and lead citrate (24), and examined in a Siemens Elmiskop I at 80 kv. The protein content of cells grown in T flasks was deter mined by a modified Lowry procedure (13). SUMMARY Quinacrine (15 pg/mI) has been demonstrated by phase con trast and electron microscopy to produce nuclear changes in L strain fibroblasts in vitro. Within 30 minutes after exposure to quinacrine, apparent fragmentation of nucleoli is visible under phase microscopy. Early ultrastructural changes in nuclei after quinacrine exposure show segregation of the fibrillar compo nents in the nucleolus and margination of chromatin around the nuclear membrane. Two hours after drug exposure …