Control of DNA Replication in Breast Cancer

Institution: Stanford University
Investigator(s): Dieter Wolf, M.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3FB-0021 Award: $47,543
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1997)
The vast majority of human breast cancers have an increased, abnormal DNA content (ploidy) per cell. Increased ploidy caused by gene amplification has been shown to play an important part in the pathogenesis and prognosis of various solid tumors. The exceptionally high frequency of gene amplification in human breast cancers suggests that events that increase the DNA content play an important role in the genesis and progression of this disease. Our working hypothesis is that alterations in factors controlling DNA synthesis are important for the development of human breast cancers.

In previous work we have identified a human gene called ORC1, which has the capacity to cause an increase in ploidy in experiments we conducted using yeast cells. ORCI and its relative Cdc6 are both part of a large protein complex that regulates the initiation of DNA synthesis. The ordered assembly and disassembly of the ORC/Cdc6 complex appears to be necessary to ensure that each segment of the genome (all the chromosomal DNA of a cell) is duplicated only once during each cell division cycle. Defects in this process may lead to increased amounts of cellular DNA, or ploidy.

The work here focuses on the specific roles of ORC1 and Cdc6 in the ploidy increases in breast cancer. We will perform this work on breast cancer and other mammalian cells in culture, and using protein extracts from amphibian eggs. We will determine both the potential and the structural requirements of these proteins to induce DNA ploidy increases in mammalian cells. Our strategies will include: (i) a detailed quantitative and qualitative analysis of ORC1 and Cdc6 in breast cancer cells to uncover potential increases in protein abundance or aberrant cell cycle timing of their interaction with nuclear structures, (ii) overproduction of ORC1 and Cdc6 in mammalian cells and examination for increased DNA content or cellular transformation, and (iii) a biochemical characterization of Cdc6 in the Xenopus (amphibian) in vitro DNA replication system, in order to study the role of purine nucleotide binding and hydrolysis in its replication function.

Despite numerous recent advances in the genetic identification of breast cancer susceptibility genes, there is a fundamental gap in our knowledge regarding global chromosomal changes. The novel features of our approach promises a potential breakthrough that could eventually allow for either the screening for or rational design of inhibitors that may be valuable in breast cancer therapy

Final Report (1998)
This research explored a potential connection between Cdc6, a conserved DNA replication protein, and breast cancer. We are trying to understand how Cdc6 is regulated in normal cells, and whether defects in regulation of Cdc6 contributes to abnormal DNA synthesis in breast cancer cells.

During the past year, we have generated and extensively characterized antisera against Cdc6. The sera is able to specifically detect Cdc6 in a variety of assays that will now be used to study its expression in breast cancer cells. Our initial findings in normal mouse cells indicate that the level of Cdc6 protein in cell nuclei varies during the cell cycle. We will now determine whether this regulation is disturbed in breast cancer cell lines, and how this may relate to abnormalities in DNA synthesis.

We have so far been unable to detect nucleotide binding and hydrolysis activity of Cdc6, but we have identified a pathway involved in regulating Cdc6 protein levels in the cells. Based on our findings in yeast, which show that degradation of the Cdc6 protein is critical for normal replication. Further, we have isolated a novel human gene, hPOP, that is likely to regulate Cdc6 protein stability. In future studies, we will address a potential connection between hPOP, the intracellular level of Cdc6 protein, and defects in replication control in breast cancer cells. We plan to look for mutations in hPOP in cancer cell lines, and address the biochemical consequences of such mutations for Cdc6 protein accumulation and replication control.

Finally, we have started to develop a system to screen for chemicals that influence the activity of the Cdc6 proteolysis pathway. Such compounds may later be used to change the level and activity of Cdc6 in breast and other cancer cells.

Concurrent paclitaxel and radiation therapy for breast cancer
Periodical:Seminars in Radiation Oncology
Index Medicus: Semin Radiat Oncol
Authors: Formenti SC, Symmans WF, Volm M, Skinner K, Cohen D, Spicer D, Danenberg PV
Yr: 1999 Vol: 9 Nbr: 2 Suppl 1 Abs: Pg:34-42

F-box/WD-repeat proteins Pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of Cdc18p
Periodical:Current Biology
Index Medicus: Curr Biol
Authors: Wolf DA, McKeon F, and Jackson PK
Yr: 1999 Vol: 9 Nbr: Abs: Pg:373-376

Budding yeast Cdc6p induces re-replication in fission yeast by inhibition of SCF(Pop)-mediated proteolysis
Periodical:Molecular and General Genetics
Index Medicus: Mol Gen Genet
Authors: Wolf DA, McKeon F, Jackson PK
Yr: 1999 Vol: 262 Nbr: 3 Abs: Pg:473-80