Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma cells synchronized by centrifugal elutriation

Abstract

The S-phase-specific cytotoxicity of hydroxyurea (HU) was tested on synchronized murine fibrosarcoma (FSa) cells lodged in the lungs of C3Hf/Bu mice. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations. After elutriation, 8000 viable FSa cells from each fraction, along with 10(6) heavily irradiated tumour cells (unseparated) were injected i.v. into whole-body-irradiated mice (20 per group). Under these conditions, 95% of the injected cells, regardless of size or position in the cell cycle, are arrested in the lungs. Twenty minutes later, hydroxyurea (HU, 1 mg/g) was administered i.p. into 10 animals of each group. Fourteen days later the animals were killed, their lungs removed and fixed, and the number of macroscopic tumour nodules counted. Killing of the initially injected cells by HU, as evidenced by a reduction in lung colonies in treated animals, correlated with the precentage of S-phase cells in each fraction. The greatest effect, an 80% reduction in colony number, was seen in Fraction 8, containing the largest percentage of S-phase cells (65%). These results demonstrate the usefulness of this procedure as a rapid method for characterizing the phase specificity of chemotherapeutic drugs in vivo.