Method for detecting the presence of malignant cells using a mul

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

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435 915, 435 9152, 435 911, 435 912, C12Q 168, C12P 1934

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06093543&

ABSTRACT:
We describe herein the isolation and purification of a multi-protein complex for DNA replication from MDA MB-468 human breast cancer cells as well as human breast tumor tissue and xenografts from nude mice injected with human breast cancer cell line MCF-7. This complex, designated the "DNA synthesome", fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome plays a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA proteins are incorporated into the DNA synthesome: DNA polymerase .alpha., DNA primase, DNA polymerase .delta., proliferating cell nuclear antigen (PCNA), replication protein A (RP-A), replication protein C (RF-C), DNA topoisomerase I and II, and DNA polymerase .epsilon.. Furthermore, our results obtained from a forward mutagenesis assay suggest that DNA isolated from a non-malignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism whereas the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo, with continued characterization of the DNA synthesome providing important insight into the molecular mechanisms regulating breast cancer call DNA replication.

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