The Role of Chk1 in Breast Cancer DNA Damage Repair

Institution: Scripps Research Institute
Investigator(s): Jennifer  Scorah , Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12FB-0068 Award: $80,740
Award Type: Postdoctoral Fellowship
Research Priorities
Pathogenesis>Too much cell growth: defective messages and internal signaling



Initial Award Abstract (2006)

Damage to chromosomal DNA constantly occurs in normal cells due to everyday metabolic processes and exposure to environmental insults. These changes are normally detected and corrected by special genes working in processes known as DNA “damage checkpoints.” Significantly, cancer cells have defects in these checkpoint processes that detect and correct DNA damage, and these defects allow DNA mutations to occur and become passed on the daughter cells. An example of failure of this system is the breast cancer gene BRCA1 that is mutated in over 50% of familial breast and ovarian cancers. BRCA1 is involved in the DNA auditing process but the mechanism of its action and association with other proteins is not clear. Our interest is a gene named Chk1 that produces a protein that is regulated in part by BRCA1. Chk1 is an important messenger that relays the signal generated when DNA damage occurs. This signal tells the cell to pause and make time for repair or removal of the damaged DNA. Chk1 is essential for cells to survive normally, even in the absence of DNA damage. We believe that the ways in which Chk1 is regulated in the presence or absence of DNA damage differ in cancer cells and these functions represents targets to control the progression of the disease.

We have developed an experimental system where we can remove normal Chk1 protein from cells and replace it with a mutant of Chk1 that cannot receive the signal it normally responds to after DNA damage. We will test how this affects cell survival before and after the induction of DNA damage. In addition, we will replace Chk1 with mutants that function as if the DNA damage signal were continually present. We will test how this altered regulation affects cell survival and normal processes in the cell. This type of experimental approach has not previously been used to investigate the regulation of Chk1 although it has been successfully used to answer similar questions for other checkpoint genes.

We predict that these experiments will provide information that could lead to the design of strategies to target specific aspects of Chk1 regulation for better breast cancer therapies. By targeting specific functions of Chk1 it might be possible to make breast cancer cells more sensitive radiation and chemotherapy whilst reducing the damage suffered by non-cancerous cells. Patients could be treated with lower doses of therapeutic agents making the overall treatment less harsh, decreasing the chance that normal cells will be affected and therefore diminishing the side-effects suffered.




Final Report (2008)

Changes in DNA that occur through natural processes are recognized and corrected by genes functioning in DNA damage checkpoints. Cells that have defects in checkpoint processes often attain the ability to multiply 'unchecked' and become cancerous. In addition, cells must duplicate their DNA every cell cycle and this replication process must be tightly regulated to maintain the proper DNA content in cells and to prevent errors becoming propagated. Our interests are genes named Chk1 and Claspin that function in the DNA damage checkpoints and have roles in replication. These genes are essential for cells to survive normally, and it is thought that it is their replication functions that are crucial for cell viability. Claspin is required for efficient activation of Chk1 in a cell cycle checkpoint response but it is unknown whether the roles of Claspin in replication are dependent on Chk1.

This project aimed to determine the specific roles of Chk1 and Claspin in replication and establish whether replication roles of Claspin are independent of Chk1. To address our aims we used DNA fiber technology, a method that can analyze many parameters of DNA replication at the level of individual molecules, rather than the whole genome. It is quantitative and results can be subject to statistical analysis providing unambiguous data. One of the major accomplishments of project was to successfully establish conditions for the DNA fiber technology. We performed studies in which Chk1 or Claspin expression was inhibited by specific knockdown of protein expression, then examined DNA fibers from these cells to determine the effect of inhibition on replication. We found that progression of replication was decreased when Chk1 or Claspin expressed was reduced. In addition, we found that Claspin, like Chk1, regulates replication fork stability and density in a normal cell cycle. We determined that Chk1 regulates origin firing predominantly by controlling it's downstream effectors Cdk2-Cdc25. By contrast, Claspin functions independently of the Cdc25-Cdk2 pathway in mammalian cells. Our findings support a model in which Claspin plays a role regulating replication fork stability that is independent of its function in mediating Chk1 phosphorylation.

Such detailed analysis of Claspin and Chk1 replication functions at the level of individual molecules had not been previously performed. Characterising the replication functions of these proteins in such detail contributes significant knowledge to the replication field and might allow us to design novel ways of targeting Chk1 or Claspin to make breast cancer cells more sensitive to therapeutic intervention whilst increasing the viability of non-cancerous cells. Such treatments have the potential to reduce mortality and significantly decrease morbidity.



A conserved proliferating cell nuclear antigen-interacting protein sequence in Chk1 is required for checkpoint function.
Periodical:Journal of Biological Chemistry
Index Medicus: J Biol Chem
Authors: Scorah J, Dong MQ, Yates JR 3rd, Scott M, Gillespie D, McGowan CH
Yr: 2008 Vol: 283 Nbr: 25 Abs: Pg:17250-9