Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage.

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.

Dynamics of DNA repair suggested by the subcellular localization of Brca1 and Brca2 proteins.

The localization of proteins to specific subcellular compartments often reveals clues regarding their biological functions. Although significant progress has been made towards understanding how damaged DNA is repaired, experiments to date have primarily focused on signal transduction pathways that activate DNA repair protein complexes and on how these complexes are assembled. Current evidence suggests that certain DNA repair processes are spatially organized such that aberrant DNA structures can be brought into proximity with DNA repair proteins at fixed sites. Since biochemical evidence suggests that the tumor suppressor proteins, Brca1 and Brca2, may mediate the assembly of protein complexes involved in the repair of damaged DNA, we have performed subcellular fractionation experiments to determine the subnuclear localization of these proteins. The majority of Brca1 and Brca2 proteins were found to interact tightly with the nuclear matrix. Furthermore, within the limits of detection, localization of Brca1 and Brca2 to the nuclear matrix was not altered following treatment of cells with DNA damaging agents that activate homology-mediated double-stranded DNA break and transcription-coupled repair pathways. Our findings suggest that Brca1 and Brca2 may perform their DNA repair-related functions from positions that are anchored to the nuclear matrix. These data are consistent with proposed models that suggest that components of specific repair complexes residing on the nuclear matrix function to recruit damaged DNA.

Brca1-deficient murine mammary epithelial cells have increased sensitivity to CDDP and MMS.

In this report we describe the isolation of an isogenic pair of Brca1(++) and Brca1(-/-) murine mammary epithelial cells (MMECs). These cells were isolated from Brca1 conditional knock out mice which contained loxP sites flanking exon 11 of the Brca1 gene (Brca1(fl/f1)) and then immortalized by infection with HPV-16E6 retrovirus to degrade p53 protein. Brca1(-/-) MMECs were generated by deletion of exon 11 following transduction of Brca1(fl/f1) MMECs with a retroviral vector expressing Cre recombinase. Brca1-deficiency rendered MMECs sensitive to cis-platinum (II) diamine dichloride (CDDP) and methylmethane sulfonate (MMS). The Brca1(+/+) and Brca1(-/-) MMECS is the only known pair of isogenic mammary epithelial cell lines. The understanding of the mechanisms of the CDDP sensitivity of the BRCA1-deficient mammary epithelial cells would be very important in understanding how BRCA1-deficiency plays out in tissue specific breast cancer chemotherapy. These studies support the role of BRCA1 in the CDDP-induced and MMS-induced DNA damage and repair by p53-independent pathways.

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a targeted mutation of the Chk2 phosphorylation site in Brca1.

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.