The Development and Assay of an S-Phase Synchronized Cell System Using Skin Fibroblast Cultures of the Indian Muntjac

Abstract

An in vitro mammalian cell system which enables direct observation of DNA replication forks is essential to the study and understanding of the mammalian cell cycle S-phase. The prerequisite work of establishing S-phase synchronized cell cultures and an assessment of synchrony levels attained is the object of this study. Skin fibroblasts of the male Indian muntjac line which possesses large chromosomes and a low diploid number were selected for this work. Initial block-release experiments using mitosis as an assay point indicated that 1.5 mM hydroxyurea provided marked levels of synchrony in the absence of detectable cytotoxicity. Premature chromosome condensation patterns of interphase cells fused with mitotic cells indicated a substantially higher S-phase portion of cell populations subjected to hydroxyurea block-release. This was observed in contrast to lower S-phase portions in non-treated controls. These results were supported autoradiographic assays which showed a higher percentage of 3H-thymidine-labeled nuclei in hydroxyurea-treated populations than in controls. It is expected that large quantities of prematurely condensed replicating chromosomes as obtained by this synchronized fusion method would effectively expose sites of DNA replication forks under the electron microscope. This would allow DNA synthesis to be more completely quantified and characterized under normal conditions as well as under conditions which alter this critical phase.