No abstract

The binding and accumulation of the chitin synthesis inhibitor diflubenzuron (DFB) by a cell line derived from embryonic tissue of the tobacco hornworm, Manduca sexta (L.), was analyzed. A rapid and reversible binding to viable and nonviable cells suspended in the culture medium was observed at soluble concentrations of DFB for short exposure periods. Scatchard analysis gave no indication of a saturable uptake mechanism. The DFB-binding capacity of intact cells was found to be similar to that of a crude membrane preparation (70,OOOg pellet); however, plasma membrane-enriched fractions bound almost three times as much DFB as the homogenate. Repetitive shorttime incubations (up to 3 h) of suspended cells with DFB resulted in a stepwise intracellular accumulation of DFB. Treatment of growing cells with DFB at high concentrations (50 pM) of DFB for longer periods (up to 7 days) resulted in elevated intracellular accumulation of DFB, which exceeded the binding capacity of the cell membranes and the aqueous solubility of DFB. These results indicate that the intracellular crystals detected by transmission electron microscopy are precipitated DFB. N o metabolites or other chemically modified products of intracellular DFB were detected by high pressure liquid chromatography (HPLC) after a 7-day incubation.

In in vitro short-term (3 h) assays, the beta-chloroethyl-methyl-hydrazones B 1 and B 2 inhibit the uptake of 3H-thymidine by EAC and L 1210 leukemia cells, B 2 being 5 to 10 times more effective than B 1. The growth inhibitory effect of both compounds was also confirmed in long-term (7 days) clonal assays using agar-containing glass capillaries, B 2 again being more effective than B 1. In contrast to these differences in vitro, in vivo both substances showed remission to the same degree in EAC- and complete resistance in L 1210-bearing mice. The diverging in vitro/in vivo sensitivities were thought to result from differences in the affinity of the methylhydrazones to the tumor cells: using short exposure periods (3 h) B 1 was more inhibitory than B 2 on both EAC and L 1210 colony growth; i.e., the more hydrophilic B 2 could more easily be washed off. To further test the idea of different cell membrane affinities, the methylhydrazones ZB 1 and P 1 with increasing lipophilic properties were synthesized. In vitro, after both pulse and continuous exposure ZB 1 and P 1 showed enforced inhibitory effects on colony growth. In vivo, ZB 1 and P 1 reduced the tumor weight of EAC mice, while only P 1 increased the survival time of L 1210 mice. The results suggest that from the combination of in vitro/in vivo assays mechanistic conclusions can be derived that are valuable for further development of these cystostatics.