The long-term goals of the applicant's research efforts are the eventual determination of the regulatory mechanisms controlling human cell DNA synthesis, and the development of a clear understanding of how these regulatory processes control both non-malignant and malignant cell proliferation in the hope that eventually new therapeutics can be identified which may selectively target cancer cells. Because of the broad scope of this type of investigation, she chose to focus her initial efforts on characterizing a multiprotein complex isolated from human cells which is fully capable of mediating DNA replication in vitro. She is the first to successfully isolate, extensively purify and characterize an intact human breast cell multiprotein complex that is both stable and fully functional. Her data also indicate that the transformation of a non-malignant human breast cell to a malignant state is accompanied by a significant alteration in the abundance and mobility of specific protein components of the multiprotein complex following two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Furthermore, it was observed that the multiprotein complex from malignant breast cells has a higher DNA synthetic activity and a low DNA replication fidelity than the complex purified from non-malignant breast epithelial cells. The higher DNA synthetic activity of the breast cancer cell derived multiprotein complex, coupled with the decrease in replication fidelity and the dramatically different 2D-PAGE gel protein profiles between non-malignant cell and cancer cell derived replication complexes clearly suggest critical differences in the make-up of the replication machinery of these cells. The applicant's observations are therefore unique in that this is the first time it has become possible to link specific changes in the activity and fidelity of cellular DNA replication with specific alterations in the components of the intact DNA synthetic apparatus of breast cancer cells. It therefore becomes crucially important to (1) rigorously identify those components of the breast cancer cell DNA synthetic apparatus which undergo structural modification; (2) validate that these changes occur in DNA synthesis complexes isolated from human breast tumors; and (3) determine whether specific post-translational modifications contribute to the alterations in the level and fidelity of the DNA synthesis reaction mediated by the breast cancer cell DNA replication complex.
