Transcriptional Regulation of the Base Excision Repair Pathway by BRCA1

Abstract

Inactivation of the breast cancer susceptibility gene BRCA1 plays a significant role in the development of a subset of breast cancers, although the major tumor suppressor function of this gene remains unclear. Previously, we showed that BRCA1 induces antioxidant-response gene expression and protects cells against oxidative stress. We now report that BRCA1 stimulates the base excision repair pathway, a major mechanism for the repair of oxidized DNA, by stimulating the activity of key base excision repair (BER) enzymes, including 8-oxoguanine DNA glycosylase (OGG1), the DNA glycosylase NTH1, and the apurinic endonuclease redox factor 1/apurinic endonuclease 1 (REF1/APE1), in human breast carcinoma cells. The increase in BER enzyme activity appears to be due, primarily, to an increase in enzyme expression. The ability of BRCA1 to stimulate the expression of the three BER enzymes and to enhance NTH1 promoter activity was dependent upon the octamerbinding transcription factor OCT1. Finally, we found that OGG1, NTH1, and REF1/APE1 each contribute to the BRCA1 protection against oxidative stress due to hydrogen peroxide and that hydrogen peroxide stimulates the expression of BRCA1 and the three BER enzymes. These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage. The base excision repair (BER)2 pathway is an evolutionarily conserved mechanism for repair of several types of DNA lesions, including oxidative lesions, alkylation, and incorporation of inappropriate bases (1). The primary source of these lesions is reactive oxygen species, whether generated endogenously or due to genotoxic agents. The BER pathway functions to maintain genomic integrity via a high fidelity repair process and is thus anti-mutagenic and anti-carcinogenic (2). This pathway usually has four or five enzymatic steps, involving a DNA glycosylase (e.g. OGG1 or NTH1), an AP endonuclease (REF1/APE1), a DNA polymerase (e.g. polymerase B and D), and a DNA ligase (ligase I or III) (3).DNA glycosylases recognize specific subsets of damaged bases, excise the damaged base, and may also incise at the site of the excised base due to an intrinsic lyase activity (e.g. OGG1 and NTH1). REF1/APE1 (the only mammalian AP endonuclease) then cleaves the abasic site to form a 3Ј-OH end and a 5Ј-deoxyribose phosphate end. The remaining steps may utilize a "long patch" or "short patch" pathway involving repair DNA synthesis and strand ligation by different sets of proteins. The preferred substrates for the DNA glycosylases OGG1 (8-oxoguanine glycosylase 1) and NTH1 (homolog of Escherichia coli endonuclease III) are 8-oxoguanine and thymine glycol (TG) lesions, respectively. REF1/APE1 (redox factor 1/apurinic endonuclease 1) is a multifunctional enzyme with AP endonuclease activity and 3Ј,5Ј-exonuclease, 3Ј-diesterase, and 3Ј-phosphatase activities. REF1/APE1 also has transcriptional regulatory activity independently of its function in BER (4, 5). Finally, the XRCC1 protein (x-ray repair, cross-complementing defective, ...

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

“…29,30 Cell survivability was measured as described under 'Methods.' Overexpression of LAMP2A, in both MCF-7 and T47D cells, induced cell survivability approximately by 15-20%, when compared with background vector and vehicle ( Fig.…”

“…MCF-7 and T47D human mammary adenocarcinoma cell lines were obtained from the American Type Culture Collection (ATCC, HTB-22, HTB-133) and cultured as described previously. 30 MCF-7 cells were isolated from a 69-y-old Caucasian woman suffering from invasive breast ductal carcinoma. This epithelial lineage cell line has wild-type TP53, posseses both estrogen and progesterone receptors and is only tumorigenic in mice if supplemented with estrogen.…”

“…Transient transfections were done as described previously. 30,[51][52][53] For overexpression, cells were transfected with LAMP2A plasmid (tagged with or without GFP) for 48 h before being harvested. In contrast, for underexpression of LAMP2A using LAMP2A siRNA, cells were harvested 10% FBS for 1 h at 5% CO 2 /37°C.…”

“…Whole cell lysates from transfected/nontransfected cells were prepared after the indicated treatments as described previously. 29,30 The primary antibodies were as follows: LAMP2A (Abcam, ab18528, 1 μg/ml); LAMP2B, (Abcam, ab18529 1 μg/ml); LAMP1 (Cell Signaling, C54H11, 1:1000); HSPA8 (Santa Cruz Biotechnology, sc1059, 1:200); CASP3 (Imgenex, IMG-144A, 5 μg/ml); BAX (Santa Cruz Biotechnology, sc526, 1:200); BCL2 (Santa Cruz Biotechnology, sc-7382, 1:200); GAPDH (Imgenex, IMG5143A, 1 μg/ml); PKM (Cell Signaling, 3186, 1:1000); AKT1 (Cell Signaling, 9272, 1:1000); phospho-AKT1 (ser473) (Cell Signaling, 4051S, 1:1000); TUBB (Imgenex, IMG-5810A, 1 μg/ml) and ACTB (Imgenex, IMG-5142A, 1 μg/ml).…”

BRCA1 affects global DNA methylation through regulation of DNMT1

“…We identified a common motif (AACGTAA) in both mouse and human fragments that are potentially responsive to OCT1, a wellstudied transcription factor ( Figure 6A). Previous investigations indicated that BRCA1 and OCT1 interact with each other and regulate downstream gene expression through binding to the OCT1 consensus sequence [42], and Oct1 deficiency impairs transcriptional activity of BRCA1 [43]. Next, we performed following experiments to investigate whether the regulation of DNMT1 by BR-CA1 is indeed mediated by the OCT1 consensus site.…”