The DNA synthesome: A model for studying breast cancer cell DNA replication and the mechanisms of action of anti-breast cancer agents

Abstract

We have isolated a multiprotein complex for DNA synthesis, designated the DNA synthesome, from human breast cancer (MDA MB-468) cells, biopsied human breast tumor tissue and xenografts from nude mice injected with the human breast cancer cell line MCF-7. The breast cell DNA synthesome was shown to fully support the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Moreover, our results obtained from a forward mutagenesis assay indicate that the DNA synthesome isolated from malignant breast cells possesses a lower fidelity for DNA replication in vitro than the complex from a nonmalignant breast cell line. The proteins and enzymes found to copurify with the breast cell DNA synthesome include: DNA polymerases alpha, delta, and epsilon, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), DNA ligase, DNA topoisomerases I and II and poly(ADP-ribose) polymerase. To begin to determine the organization of these DNA synthetic proteins within the breast cell DNA synthesome, we performed co-immunoprecipitation experiments with antibodies directed against DNA polymerases alpha, delta and PCNA. We found that DNA polymerases alpha, delta, DNA primase, RF-C and PCNA tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP and several other components interact with the synthesome via an interaction with only PCNA or DNA polymerase alpha. Furthermore, we employed the breast cell DNA synthesome as a model to study the mechanisms of action of two anti-breast cancer agents that target the DNA synthetic process, irinotecan (CPT-11/SN-38) and etoposide (VP-16). We obtained novel data suggesting that both SN-38 and VP-16 stabilized cleavable complexes represent blocks to replication fork progression, as each agent caused an accumulation of short DNA products during synthesome mediated in vitro replication. Overall, our results indicate that breast cancer cells utilize an asymmetric multiprotein complex to mediate DNA synthesis and that utilization of the DNA synthesome as a drug model may provide important new insights into the mechanisms of action of SN-38 and VP-16.