Regulation of the Rad1 Checkpoint Complex in Breast Cancer

Institution: Stanford University
Investigator(s): Patrick Lupardus, B.S. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8GB-0091 Award: $59,987
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2002)
Cells are constantly exposed to DNA damaging agents, and if a cell does not properly respond to this threat it runs the risk of having cancer-causing mutations incorporated into its DNA. The responsibility of sensing and responding to DNA damage falls upon a group of proteins, called checkpoint proteins, that activate repair proteins and slow cell growth until the damage is repaired. Failure of these DNA surveillance mechanisms is found to correlate with a higher incidence of cancer. For example, mutations in the checkpoint proteins ATM (mutated in the human genetic disorder ataxia-telangiectasia) and BRCA1 have both been found to be inherited genetic factors that contribute to familial breast cancer. The reason for this is that loss of "policing" of DNA in these patients causes chromosome instability at the cellular level, which contributes to the complex genetic abnormalities that characterize cancers seen in the clinical stages.

The aim of this project is to study how two DNA damage response checkpoint proteins, ATR and Rad1, interact to initiate the cellular processes necessary to repair DNA damage. We have evidence that ATR regulates Rad1, by attaching small molecules, called phosphates, to specific sites on the protein. My specific goal is to determine what the exact sites of modification are on Rad1, and to test this by altering the protein sequence to confirm their existence. Finally, we will test the mutant forms of Rad1 for their ability to support the cell's responses to DNA damage. The initial phases of this project will be performed in Xenopus (frog) egg extracts, and the final phase of testing the Rad1 mutants will be performed in breast cancer cells. The use of both a Xenopus in vitro cell cycle model system and breast tissue cell lines allows me to exploit the advantages of each system to advance this research more rapidly.

In conclusion, a better understanding of DNA damage checkpoint processes and how they are defective in cancer will provide new insights into tumor initiation and progression. Understanding these proteins and their interactions may provide new opportunities to arrest and reverse the disease, particularly in ways that might not impact healthy tissue.


Final Report (2004)
The focus of my research is to understand how a cell protects itself against DNA damage. Failure of the mechanisms that protect a cell against DNA damage leads to an accumulation of mutations that can cause cancer. In this CBCRP-funded project, I studied a group of three proteins, called the RHR (Rad1/Hus1/Rad9) complex, that are involved in activating repair mechanisms and slowing cell growth until DNA damage is repaired. We have evidence that another protein, called ATR, modifies the RHR complex after DNA damage by attaching small molecules, called phosphates, to specific sites on the proteins. To understand how modification of the RHR complex by ATR helps activate the mechanisms that protect a cell from DNA damage, I have developed an experimental approach to “probe” the function of the RHR complex and its modifications. Basically, our approach is to deplete Xenopus (frog) egg extracts of the RHR complex. Then, we can add back recombinant, purified, and mutated forms of the key checkpoint proteins to test the effect on DNA polymerase. We are currently using this experimental system to “dissect” at the molecular level how this complex contributes to a cell’s response to DNA damage.

A better understanding of how these processes work will provide new insight into how an organism protects itself from cancer-causing mutations, and may provide new targets for cancer chemotherapeutics. We recently reviewed some of the emerging molecular mechanisms in cell cycle checkpoints (Lupardus PJ, Cimprich KA. “Checkpoint adaptation; molecular mechanisms uncovered.” 2004 Cell 28;117:555-6). Yeast cells (unicellular organisms) have the ability to show “adaptation” to allow cell division in the presence of DNA damage. Evidence is accumulating that cells from higher animals might have corresponding adaptor functions. This means that understanding these processes in Xenopus would be a first-step and might prove very informative in explaining why cancer cells can continue to divide in the presence of DNA damage, which causes normal cells to self-destruct.


Symposium Abstract (2003)
Cells are constantly exposed to DNA damaging agents. If a cell does not properly respond to this threat, then it runs the risk of having cancer-causing mutations incorporated into its DNA. The responsibility of sensing and responding to DNA damage falls upon a group of proteins, called checkpoint proteins, that activate repair proteins and slow cell growth until the damage is repaired. Failure of these DNA surveillance mechanisms has been found to correlate with a higher incidence of cancer. For example, mutations in the checkpoint proteins ATM (mutated in the human genetic disorder ataxia-telangiectasia) and BRCA1 have both been found to be inherited genetic factors that contribute to familial breast cancer. The reason for this is that loss of “policing” DNA in these individuals with ATM and BRCA1 mutations causes chromosome instability at the cellular level, which lead to the complex genetic abnormalities that are characteristic of breast cancers seen in the clinical stages.

The aim of this project is to study how several DNA damage response checkpoint proteins, ATR, Rad1, and Hus1, interact to initiate the cellular processes necessary to repair DNA damage. Using the Xenopus (a frog species used for research) model system, our lab has recently shown that active DNA replication is required for ATR and Rad1 to sense ultraviolet (UV) and methyl-methanesulfonate (MMS) induced damage. We suggest that the disruption of DNA replication by these agents may create the signal that activates ATR and Rad1, which then can cooperate to initiate an arrest to the cell cycle until this damage can be repaired. We also have evidence that ATR regulates Rad1 and Hus1, and I am currently developing techniques to study these interactions. Previous results suggest that ATR attaches small molecules, called phosphates, to specific sites on Rad1 and Hus1. I hope to use these assays to study how these regulatory events influence the cell’s responses to DNA damage.

A requirement for replication in activation of the ATR-dependent DNA-damage checkpoint. Genes Dev. 16, 2327-2332. No breast cancer
Periodical:Genes and Development
Index Medicus: Genes Dev
Authors: Lupardus PJ, Byun T, Yee MC, Hekmat-Nejad M, Cimprich KA (2002)
Yr: 2002 Vol: 16 Nbr: Abs: Pg:2327-2332