Study of HL-60 cells DNA synthetic machinery: Isolation, characterization and its biochemical status at differential cellular growth stages

Abstract

The mammalian cell DNA synthetic machinery has been isolated from human cervical carcinoma HeLa and murine FM3A cells, and characterized as a multiprotein replication complex (MRC). This complex is fully capable of supporting SV40 in vitro DNA replication in the presence of viral large T antigen. To further study the regulation of the activity of the MRC during cell differentiation, a human leukemia HL-60 cell model was used. HL-60 cells are able to undergo macrophage-like differentiation after exposure to a phorbol ester. The differentiation is accompanied by the cessation of DNA synthesis. In the attempt to isolate and characterize the HL-60 cell DNA synthetic machinery, we found that several essential DNA replication proteins such as DNA polymerases alpha and delta, DNA primase, replication factor C (RF-C), replication protein A (RP-A), proliferating cell nuclear antigen (PCNA), topoisomerases I and II form a multiprotein complex in HL-60 cells, which is consistent with the finding in HeLa and FM3A cells. The DNA synthetic machinery is therefore believed to be commonly present in mammalian cells as an organized protein complex, and is designated the DNA synthesome. The DNA synthesome is absent from HL-60 cells induced to differentiate by TPA. Western blot analysis showed that TPA-induced differentiation is accompanied by the disassembly of the DNA synthesome, which is consistent with the observed cessation of DNA synthesis. RNA analysis suggested that the disassembly of the DNA synthesome is regulated at the gene expression level. Studies of the integrity and activity of the DNA synthesome in aphidicolin-arrested HL-60 cells showed that the synthesome is present in the temporarily arrested cells but in an inactive form. Taken together, these evidence suggested that the disassembly of the DNA synthesome is a differentiation-specific cellular event, and that the biochemical status of the DNA synthesome can distinguish the difference between temporary and permanent cell growth arrest.