Effect of melphalan and hyperthermia on p34cdc2 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 a melphalan-induced G2-phase cell block. In this study, we investigated the effect of melphalan and hyperthermia on proteins that regulate G2-M transition. Neither hyperthermia nor melphalan at a concentration of 2.5 micrograms ml-1, which had no antiproliferative effect at 37 degrees C, interfered with cyclin B1 expression or p34cdc2 kinase activity. At a concentration of 8.5 micrograms ml-1, which reduced cell growth by 50% at 37 degrees C, melphalan inhibited p34cdc2 kinase activity as a consequence of an increased tyrosine phosphorylation of the protein. A similar inhibitory effect on p34cdc2 kinase was obtained when the lowest melphalan concentration (2.5 micrograms 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 p34cdc2 kinase activity, which prevents cell progression into mitosis.