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.

Abstract

Release of HeLa cells arrested at the G1/S boundary by double-thymidine block caused abrupt uptake of [methyl-3H]thymidine into DNA after 5 min, and two sharp high activity peaks, peak I and peak II, were observed 8 and 23 min after removal of the thymidine block and this was followed by a gradual uptake of [3H]thymidine. The duration of the cell cycle was 23 h, and definite changes in cell density were observed between 12 and 13 h and also between 35 and 36 h after removal of the thymidine. Addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester (APCA-OPh'Bu), a trypsin inhibitor, immediately after removal of the arrest strongly suppressed DNA synthesis and mitosis. In contrast, addition of APCA-OPh'Bu 10 min after removal of the arrest, and hence also after the appearance of peak I, had no effect on peak II 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 of 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 proteinase activity. However, the activity decreased with increasing cell density. 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 obtained at cell densities of 2% and 20% were strongly inhibited by APCA-OPh'Bu and these inhibitory effects were similar.(ABSTRACT TRUNCATED AT 250 WORDS)