EFFECT OF MELPHALAN AND HYPERTHERMIA ON CELL-CYCLE PROGRESSION AND CYCLIN B-1 EXPRESSION IN HUMAN-MELANOMA CELLS

We have investigated the effect of mild hyperthermia (42 degrees C) on the cytotoxic activity of a 1h melphalan exposure in human melanoma c ell lines. Hyperthermia did not affect cell growth of any culture, but it increased, to a different extent, melphalan cytotoxicity in all ce ll lines, with a reduction in the IC50 of 1.7 to 2.6-fold. Flow cytome tric analysis showed that in normal temperature conditions melphalan c aused S phase cell accumulation, which was evident only at 24h in JR8, M14 and 2/21 cell lines and was still persistent at 72h in 2/60 cells . Moreover, in all cell lines, the delay in S phase was paralleled, or followed, by an accumulation of cells in G(2)+M, which was transient in JR8 and M14 cells and persisted until 72h in 2/21 and 2/60 melanoma clones. Hyperthermia caused a stabilization and prolongation of melph alan induced G(2)+M accumulation in JR8 and M14 cells. Conversely, in 2/21 and 2/60 clones, cell cycle perturbations induced by the drug wer e similar under normothermic or hyperthermic conditions. Specifically, in JR8, for which the maximum enhancement by hyperthermia on melphala n cytotoxicity was observed, cell accumulation in G(2)+M was still pre sent 120h after treatment. The accumulation was accompanied by an inhi bition in the G(2)-M transition, as demonstrated by the significant re duction in the mitotic index of cells exposed to combined treatment co mpared to controls. Moreover, a bivariate distribution of cells staine d for DNA and cyclin B-1 showed that, following melphalan and hyperthe rmia treatment, the fraction of cyclin B-1-expressing cells paralleled the fraction of G(2)+M phase cells, thus indicating that the inabilit y of cells to enter mitosis was not ascribable to a reduction of cycli n B-1 expression. On the whole, our results indicate that hyperthermia can stabilize the G(2) accumulation induced by melphalan in human mel anoma cells. Such a stabilization could contribute to the enhancement of melphalan cytotoxicity by heat, even though a strict correlation wa s not observed between the magnitude and persistence of the cell cycle perturbations and the extent of melphalan activity.