1790

Several reports have shown that structural alterations of proteins involved in DNA replication and repair processes result in low replication fidelity, which is one of the hallmarks of malignancy. Decreased replication fidelity renders the DNA replication machinery itself error-prone with more mutations being introduced with every round of DNA replication. We have previously shown that DNA synthesis in mammalian cells is orchestrated by a multiprotein replication complex (designated the DNA synthesome). The DNA synthesome was successfully isolated, characterized, and extensively purified from normal and breast cancer cells and tissues. All of the components necessary for DNA replication including poly(ADP-ribose) polymerase (PARP) has been detected in the DNA synthesome. We have also shown that the malignant breast cell DNA synthesome exhibits lower replication fidelity relative to the non-malignant breast cell DNA synthesome. The main objective of our present study is to establish whether the malignant transformation process in breast cancer is accompanied by a structural and/or functional alteration of PARP. PARP from both malignant and non-malignant breast cells was analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blot analysis. PARP was also purified from the two cell lines using a combination of phosphocellulose and hydroxylapatite chromatography. Differences in the kinetic properties (Km and Vmax) of the purified PARP from both malignant and non-malignant breast cells were determined by measuring the initial rate of 32P-NAD incorporation into acid-insoluble material at distinct time intervals after the initiation of the reaction. Using 2D-PAGE we were able to identify an altered isoform(s) of PARP in breast cancer cells. PARP present in non-malignant breast cells had a basic pI and was resolved as a single spot; however, malignant cell PARP appeared as a basic spot in addition to less abundant species having less basic pI values. PARP isolated from malignant breast cells has different Km and Vmax values compared to the enzyme isolated from non-malignant cells. Mass spectrometry analysis is underway to identify the basis of PARP modification and its effect on the decreased replication fidelity observed in breast cancer cells. In addition, we are studying the 2D resolution profiles of altered PARP isoform(s) in response to doxorubicin treatment. These unique PARP isoform(s) may provide not only an early signature of malignancy but novel therapeutic targets as well.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]