EFFECTS OF SYNTHETIC TRYPSIN-INHIBITORS ON THE CELL-CYCLE OF SYNCHRONIZED HELA-CELLS

HeLa cells were synchronized by a double-thymidine block. After remova l of thymidine, the cells immediately caused the uptake of [H-3]thymid ine into DNA and reached a half-maximum. The duration of the cell cycl e was 23 h, and definite changes in cell density were observed between 12 and 13 h and between 35 and 36 h after removal of thymidine. Thus, the initiation time of S phase could be fixed. A trypsin-like protein ase appearing at around 17 h 17 min after removal of thymidine and cor related with the onset of the second S phase, tryptase 17:17 [cf., M. Muramatu et al., Biochim. Biophys. Acta, 1087, 87 (1990)], was obtaine d. 4-tert-Butylphenyl and 4-biphenyl esters of trans-4-guanidinomethyl cyclohexanecarboxylic acid (GMCHA) and amidinopiperidine-4-alkanoic ac ids, trypsin inhibitors, strongly inhibited the tryptase activity, and these esters exert different effects on the cell cycle of HeLa cells at concentrations showing complete inhibition or maximal inhibitory ef fect on the tryptase. Both esters of GMCHA elongated the onset of the second S phase for 3h. Esters of amidinopiperidine-4-acetic and 4-prop ionic acids showed a similar effect at lower concentrations than GMCHA esters. 4-tert-Butylphenyl esters of amidinopiperidine-4-propionic ac id and butyric acids strongly suppressed the second S and M phases by probably affecting the G1 late phase, since they have no effect on the first S and M phases. The addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester 0 min after removal of thymidine into th e cells completely suppressed the first S and M phases. Addition 10 mi n after removal of the arrest had no effect on the first S and M phase s, but completely suppressed the second S and M phases, suggesting tha t this ester inhibits the onest of DNA synthesis.