Regulatory mechanisms that are clearly different between non-malignant and malignant cells can potentially be exploited as new targets for the early detection and treatment of malignancy. For the last few years the PI has been extensively characterizing a multiprotein complex isolated from human cells which is capable of mediating DNA replication in vitro. She was the first to successfully isolate and characterize the intact mammalian multiprotein DNA replication complex (termed the DNA synthesome) from a variety of mammalian cell lines and tissues and demonstrated that it is stable and fully functional. Recent data indicate that the transformation of a non-malignant human breast epithelial cell to a malignant state is accompanied by a significant alteration of a specific protein component of the synthesome, namely, proliferating cell nuclear antigen (PCNA). She has determined that it arises not because of a genetic mutation, but through an epigenetic mechanism. In normal breast cells PCNA is poly(ADP)-rybosilated, while in breast cancer cells and tissues the majority of PCNA molecules are free of this post-translational modification. Furthermore, she has been able to detect the non-rybosilated form of PCNA in the sera of breast cancer patients, prompting her to hypothesize that the non-rybosilated form of PCNA may be useful as a diagnostic marker for breast cancer. Another critical observation is that the synthesome isolated from breast cancer cells has a significantly decreased DNA replication fidelity, exhibiting a more error-prone replication process than the complex purified from non-malignant breast epithelial cells. This was the first direct experimental evidence indicating that the DNA replication machinery of breast cancer cells is, in itself, mutagenic. To date there has been a 100% correlation between the observed decrease of DNA replication fidelity and the non-rybosilation of PCNA in malignant breast cells and tissue. Dr Malkas data strongly suggest that the specific alteration of PCNA and the decreased replication fidelity in breast cancer cells are linked and may be hallmarks of breast cell malignancy. She proposes now to fully characterize the specific alteration of PCNA (both structurally and mechanically), which distinguishes the non-malignant and the breast cancer cell DNA synthetic apparatus from one another. This project has the unique potential to provide significant insight into the process(es) whereby tumor cells can override some of the control signals which regulate DNA replication fidelity in non-malignant cells and contribute to the emergence of a mutagenic DNA replication.
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