AMIDINOPIPERIDINE-4-CARBOXYLIC ACID 4-TERT-BUTYLPHENYL ESTER, A TRYPSIN-INHIBITOR, SUPPRESSES THE ONSET OF DNA-SYNTHESIS IN HELA-CELLS SYNCHRONIZED BY A DOUBLE-THYMIDINE BLOCK

Release of HeLa cells arrested at the G1/S boundary by double-thymidin e block caused abrupt uptake of [methyl-H-3]thymidine into DNA after 5 min, and two sharp high activity peaks, peak I and peak II, were obse rved 8 and 23 min after removal of the thymidine block and this was fo llowed by a gradual uptake of [H-3]thymidine. The duration of the cell cycle was 23h, and definite changes in cell density were observed bet ween 12 and 13h and also between 35 and 36 h after removal of the thym idine. Addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphe nyl ester (APCA-OPh(t)Bu), a trypsin inhibitor, immediately after remo val of the arrest strongly suppressed DNA synthesis and mitosis. In co ntrast, addition of APCA-OPh(t)Bu 10 min after removal of the arrest, and hence also after the appearance of peak I, had no effect on peak I I nor on the uptake of thymidine occurring during the remainder of the first cell cycle, nor on mitosis. However, it strongly suppressed the second DNA synthesis and mitosis. These results suggest participation of a trypsin-like proteinase at the onset of DNA synthesis. Removal o f thymidine from the arrested cells at a cell density of 2% (4 x 10(3) cells/cm2) induced an immediate and rapid rise in trypsin-like protei nase activity. However, the activity decreased with increasing cell de nsity. No clear increase in the activity was seen at a cell density of 20% (4 x 10(4) cells/cm2). However, both trypsin-like proteinases obt ained at cell densities of 2% and 20% were strongly inhibited by APCA- OPh(t)Bu and these inhibitory effects were similar. The proteinase was named proteinase In.