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The Role of RASSF1A in Cell Cycle Control After DNA Damage
The Role of RASSF1A in Cell Cycle Control after DNA Damage
Background: RASSF1A is a candidate tumor suppressor gene (TSG) located in chromosome 3p21.3 region where loss of heterozygosity is extremely common in tumors of lung, breast and other tissues. RASSF1A is expressed in normal epithelial cells but the expression is lost in many tumor types due to promoter methylation. Re-expression of RASSF1A reversed the tumor phenotype both in vitro and in vivo. The mechanism by which RASSF1A inhibits the tumor growth is under investigation. RASSF1A inhibits the accumulation of cyclin D1 and induces cell cycle arrest in ectopically transfected lung cancer cell lines. There is a consensus ataxia-telangiectasia-mutated (ATM) protein phosphorylation site in RASSF1A (Serine131). The polymorphism near the ATM phosphorylation site (A133S) has been detected in breast and lung tumors and has been shown to have an impaired function in cell cycle control compared with wild type RASSF1A. ATM is a member of the phosphoinositide kinase-related kinase family, which plays important roles in signaling the presence of DNA damage and activating DNA repair response by phosphorylating a series of proteins including BRCA1 and p53. Phosphorylation of RASSF1A by ATM in response to DNA damage has not been shown.
Objective/Hypothesis: The objective of this proposal is to study the role of RASSF1A in cell cycle control after DNA damage. The hypothesis is that RASSF1A is phosphorylated by ATM in response to DNA damage, which enhances the activity of RASSF1A in cell cycle control, and that a polymorphism near the ATM phosphorylation site (A133S) has impaired its function in cell cycle control.
Specific Aims: 1) Determine if RASSF1A is phosphorylated by ATM in response to DNA damage. 2) Determine the cell cycle control function of RASSF1A that is activated by ATM in response to DNA damage and determine if loss of RASSF1A expression is correlated to defect in cell cycle check point control. 3) Determine if A133S RASSF1A protein has reduced activity in cell cycle control. 4) Screen for A133S polymorphism in DNA from breast cancer patients, and determine if the polymorphism is a risk factor for breast tumor development and if it is an adverse factor for poor prognosis.
Study Design: The phosphorylation of endogenous RASSF1A by ATM will be determined by Western blot after gamma irradiation (IR) using antibodies specific to phosphorylated RASSF1A. Phosphorylation at amino acid 131 serine will be further confirmed by ectopic expression of wild type RASSF1A as well as RASSF1A mutated in the phosphorylation site (S131F) after IR. Propidium Iodide staining and FACS analysis will be used to generate cell cycle profiles before or after IR. The data will be analyzed to determine the cell cycle control point where RASSF1A is active. SiRNA will be used for down-regulation of endogenous RASSF1A to study if the loss of RASSF1A expression is correlated with defection in cell cycle control. Standard PCR/restriction enzyme digestion method will be used to identify the polymorphism in ATM phosphorylation region in RASSF1A.
Potential Outcomes and Benefits of the Research: Since the initial cloning and characterization of RASSF1A as a candidate TSG by ourselves and others, there are over 80 papers studying the methylation and inactivation of RASSF1A in variety of cancers. The methylation of RASSF1A promoter has been best studied in breast tumors. Laser-assisted microdissected breast tissues were analyzed and the results are striking- RASSF1A promoter is extensively methylated in epithelial hyperplasia, papillloma and ducal carcinoma in situ but the methylation is not found in normal epithelium, stromal, apocrine metaplasia, and the highly proliferating lactating epithelium. Thus, these data strongly suggest the importance of the inactivation of RASSF1A in the pathogenesis of breast cancer. In contrast to the wealth of information regarding the inactivation of RASSF1A in tumors, there have been very few studies on the function of RASSF1A. The finding that an ATM substrate consensus sequence is present in RASSF1A provides probably the best clue for studying the physiological function of RASSF1A. The outlined proposal will help us to understand the DNA damage repair pathways and to assess whether the polymorphism A133S is a new risk factor for breast tumor development.
The Role of RASSF1A in Cell Cycle Control after DNA Damage
DNA damage is one of the major causes of cancer. Cells have developed a sophisticated system of repairing DNA damage caused by gamma-ray and UV radiation, by a variety of chemical carcinogens, and by other environmental insults. The consequence of defection in DNA repair pathway is serious—it allows cells with mutated DNA to divide in an uncontrolled fashion eventually leading to tumor development. A major player in the DNA damage response pathway is ataxia- telangiectasia-mutated (ATM) protein. ATM senses DNA damage, activates a series of proteins to stop cell division (cell cycle control), and allows cells to have enough time to repair damaged DNA before the next cell division starts. Mutations in either the ATM gene or in proteins that are activated by ATM predispose to breast cancer. The best example of the mutation is Breast Cancer Gene1(BRCA1), which is carried by fifty percent of breast cancer patients from high-risk families. BRCA1 is a protein activated by ATM and plays a critical role in cell cycle control. Because both BRCA1/2 and ATM proteins are cell cycle control proteins and are involved in breast tumor development, it is important to know if other proteins in cell cycle control play an equally important role in breast cancer development. Since more than 90 % of breast cancer patients do not carry mutations in ATM or BRCA1/2, answers to this question will help us to identify additional risk factors for breast cancer, particularly for those breast cancers with no BRCA1/2 mutations.
RASSF1A is a recently identified protein involved in cell cycle control. RASSF1A expression is lost in many tumors including breast cancer. Although RASSF1A expression is always present in normal epithelium, stromal and lactating breasts, recent studies including our own have shown that RASSSF1A expression has been lost specifically in breast tumors at a very early stage due to promoter methylation. We, as well as others, have reported the cloning and characterization of RASSF1A, and have found that re-introduction of RASSF1A into tumor cells reversed the tumor phenotype both in vitro and in vivo. The structure of RASSF1A suggests that it is activated by ATM and participates in DNA repair pathways. We further found an alteration near ATM activation site (A133S) in breast and lung tumors, which was defective in cell cycle control. Thus, RASSF1A is a prime candidate to be involved in the ATM mediated DNA repair pathway. The focus of this proposal is to study the function of RASSF1A as a tumor suppressor gene and to determine if the polymorphism A133S in RASSF1A is a risk factor for breast cancer development. Four studies will be conducted in this proposal. 1) Determine whether RASSF1A is activated by ATM upon DNA damage. 2) Determine the role that RASSF1A plays in ATM mediated DNA repair pathways and determine if the loss of RASSF1A expression is correlated with defection in cell cycle control after DNA damage. 3) Determine if A133S RASSF1A protein has reduced activity in cell cycle control. 4) Screen for A133S polymorphism in DNA from breast cancer patients and determine if the polymorphism is a risk factor for breast tumor development and if it is an adverse factor for poor prognosis.
In summary, the most effective treatment of breast cancer is early detection and intervention. A great effort had been made to search for markers to predict the individual risk for developing breast cancer. Since mutations in ATM/BRCA1/2 can only account for half of the breast cancer patients from the high risk families, or less than 10% from all breast cancer patients, mutations in genes other than ATM/BRCA1/2 must exist. We would like to know whether the newly discovered RASSF1A gene is one of the markers to predispose to breast cancer. Study on the function of RASSF1A will not only help to understand DNA repair pathways, a critical defense system for preventing tumor development, but may also help to develop a potential marker for early detection of breast cancer.