Disruption of Cyclin E/Cell Cycle In Breast Cancer Cells
| Institution: | Scripps Research Institute | ||
| Investigator(s): |
Kevin Sato , Ph.D. -
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| Award Cycle: | 1997 (Cycle III) | Grant #: 3FB-0084 | Award: $75,600 |
| Award Type: | Postdoctoral Fellowship | ||
| Research Priorities | |||
| Pathogenesis>Too much cell growth: defective messages and internal signaling | |||
Initial Award Abstract (1997)
The ability of a cell to divide is highly regulated, and this regulation becomes defective in cancer. The progression (temporal and physical) of a cell through cell division is termed the ‘cell cycle’. Numerous genes have been identified that control the cell cycle. A critical phase is the transition from either a stationary (G0) or a growing cell (G1) into the phase of active DNA synthesis (S) immediately prior to cell division. The cellular protein factors that regulate the forward progression of the cell cycle are called cyclins. Mutations in the genes encoding cyclins have been detected in clinical breast tumors. In normal cell growth and division, the temporal expression and destruction of specific cyclins coordinate cellular activities required for ordered cell cycle passage. Significantly, it is during the first phase of the cycle, G1, that the cell makes the commitment to either (i) exit the cycle to differentiate, or (ii) begin DNA replication (S). Our research focus is the G1-S transition, which appears to be regulated by a specific cyclin, cyclinE (cycE). Mutations in the cycE gene have been observed both in cultured human breast cancer cells and in clinical breast tumors. Also, comparing normal human breast cells and breast tumors showed that cycE protein either was either present at a high level or was abnormally stable in the tumors. Our research will further define the role of cycE activity and the proteins it regulates in breast cancer. The most direct means of studying cycE is to deplete the cells of the protein and compare them with normal cells for cell cycle abnormalities. The key approach we will use is to block cycE protein production at the RNA level using two types of inhibitory molecules called either ‘antisense’ or ‘ribozymes’. These inhibitory molecules block the production of cycE protein, and we anticipate a possible reversion of the breast cancer phenotype to more normal cells. The cell systems used in this project include both normal and tumorigenic human breast epithelial cells. An improved understanding of cell cycle regulatory genes is essential to explain how carcinogens and genetic mutations cause uncontrolled tumor cell growth. Ultimately, this will greatly aid in the design and development of measures to prevent the initiation of breast cancer and therapies to combat it once it has developed.
Final Report (1999)
A process called the cell cycle regulates normal cell division. The final commitment to replicate DNA, a major regulatory point in the cell cycle, is promoted by the protein kinase complex composed of two different proteins, cyclin E and Cdk2. Elevated and sustained production of cyclin E is observed in human breast cancer and is associated with poor prognosis. The objective of this project was to characterize the function of cyclin E in promoting cell commitment to DNA replication. To meet this objective, three specific aims were defined. The first was to develop a system to inhibit cyclin E protein production in non-cancerous human breast epithelial cells. The next two goals involved using this system to conduct comparative studies between normal and cyclin E-inhibited cells to determine cell cycle changes, and to identify phosphorylation targets o the cyclin E/Cdk2 kinase. During this fellowship, two inhibitory systems were developed. The first used an antisense oligonucleotide specific for the cyclin E mRNA, which was found to reduce the amount of this protein within cells. The second system involved constructing a cyclin E knockout vector that would remove the genes of cyclin E in the cell. All cell cycle studies were conducted using the antisense oligonucleotide system. This study demonstrated that a significant reduction of cyclin E protein levels by 95% only delayed entry of the cell into the DNA replication phase by two hours compared to control cells. The production and activation of the kinase activity of another important cyclin, cyclin A, in the DNA replication phase, was also delayed by two hours in the antisense treated cells. This demonstrated that it was not responsible for entry into the DNA replication phase. Although the amount of cyclin E protein was significantly lower than normal, the specific activity of the cyclin E/Cdk2 kinase complex increased dramatically. The increased specific activity appeared to be the result of a destruction of a novel Cdk inhibitor. This suggested that cyclin E is required for cell cycle progression and that the cell is capable of ensuring cyclin E/Cdk2 kinase activity by reducing the level of an inhibitor of cyclin E/Cdk2 kinase activity. Currently, studies are directed towards discovering the mechanism controlling the interplay between cyclin E production levels and inhibitor stability. Overall, these studies suggest that cells may have mechanisms to ensure that they retain activities that are critical for their viability, especially promotion of cell division. This puts into question the method of trying to target an individual protein to combat breast cancer prior to understanding all of the underlying regulatory systems present that cells have to ensure integrity of the cell cycle.
