Effect of melphalan and hyperthermia on p34(cdc2) kinase activity in human melanoma cells

We previously reported that combined treatment with melphalan and mild hyperthermia (1 h at 42 degrees C) caused a synergistic cytotoxic effect in JR8 melanoma cells, paralleled by a stabilisation of melphalan-induced G(2)-phase cell block. In this study, we investigated the effect of melphalan and hyperthermia on proteins that regulate G(2)-M transition. Neither hyperthermia nor melphalan at a concentration of 2.5 mu g ml(-1), which had no antiproliferative effect at 37 degrees C, interfered with cyclin B-1 expression or p34(cdc2) kinase activity. At a concentration of 8.5 mu g ml(-1), which reduced cell growth by 50% at 37 degrees C, melphalan inhibited p34(cdc2) kinase activity as a consequence of an increased tyrosine phosphorylation of the protein. A similar inhibitory effect on p34(cdc2) kinase was obtained when the lowest melphalan concentration (2.5 mu g ml(-1)) was used under hyperthermic conditions. Our results indicate that thermal enhancement of melphalan cytotoxicity could be mediated at least in part by an inhibition of p34(cdc2) kinase activity, which prevents cell progression into mitosis.