trans-4-amidinocyclohexanecarboxylic acid 4-tert-butylphenyl ester, a trypsin inhibitor, blocks entry of HeLa cells from G2 phase into mitosis.

Release of HeLa cells arrested at the G1/S boundary by double-thymidine block immediately caused uptake of [3H]thymidine into DNA. The duration of the cell cycle time was 23 h and definite changes in cell density were observed between 12 h and 13 h and also between 35 h and 36 h after removal of thymidine. Addition of trans-4-amidino-cyclohexanecarboxylic acid 4-tert-butylphenyl ester (ACHCA-OPhBut), immediately after removal of the arrest had no effect on DNA synthesis, although it dose-dependently suppressed the first mitosis and the next round of DNA synthesis. While the addition of ACHCA-OPhBut at any time from 0 to 10 h after removal of thymidine suppressed mitosis, its addition after 11 h did not. A trypsin-like proteinase sharply appeared around 10 h 30 min and vanished within a few minutes. The proteinase activity seemed to be density dependent and was strongly inhibited by ACHCA-OPhBut. The effects of trans-4-amidinocyclohexanepropionic acid 4-tert-butylphenyl ester (ACHPA-OPhBut), another trypsin inhibitor, on the proteinase activity and mitosis were more potent than those of ACHCA-OPhBut. These results suggest the involvement of the proteinase in the entry of HeLa cells from the G2 late phase into mitosis. The proteinase was named late G2 proteinase.