BRCA1 and GADD45 mediated G2/M cell cycle arrest in response to antimicrotubule agents Paul B Mullan 1,5 , Jennifer E Quinn 1,5 , Paula M Gilmore 1 , Stewart McWilliams 1 , Heather Andrews 1 , Celine Gervin 1 , Nuala McCabe 1 , Sarah McKenna 1 , Pat White 1 , Young-Han Song 2 , Shyamala Maheswaran 3 , Edison Liu 4 , Daniel A Haber 2 , Patrick G Johnston 1 and D Paul Harkin* ,1 1 Department of Oncology, Cancer Research Centre, The Queen’s University of Belfast, Belfast, N. Ireland; 2 Massachusetts General Hospital Cancer Centre and Harvard Medical School, Charlestown, Massachusetts, MA 02129 USA; 3 Department Pediatric Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA; 4 Division of Clinical Sciences, NIH, Bethesda, Maryland, MD 20892-2440, USA BRCA1 is a tumour suppressor gene implicated in the predisposition to early onset breast and ovarian cancer. We have generated cell lines with inducible expression of BRCA1 to evaluate its role in mediating the cellular response to various chemotherapeutic drugs commonly used in the treatment of breast and ovarian cancer. Induction of BRCA1 in the presence of Taxol and Vincristine resulted in a dramatic increase in cell death; an eect that was preceded by an acute arrest at the G2/ M phase of the cell cycle and which correlated with BRCA1 mediated induction of GADD45. A proportion of the arrested cells were blocked in mitosis suggesting activation of both a G2 and a mitotic spindle checkpoint. In contrast, no specific interaction was observed between BRCA1 induction and treatment of cells with a range of DNA damaging agents including Cisplatin and Adria- mycin. Inducible expression of GADD45 in the presence of Taxol induced both G2 and mitotic arrest in these cells consistent with a role for GADD45 in contributing to these eects. Our results support a role for both BRCA1 and GADD45 in selectively regulating a G2/M checkpoint in response to antimicrotubule agents and raise the possibility that their expression levels in cells may contribute to the toxicity observed with these compounds. Oncogene (2001) 20, 6123 – 6131. Keywords: BRCA1; GADD45; antimicrotubule agents; G2/M arrest; cell death Introduction BRCA1 was isolated in 1994 as one of the genes conferring genetic predisposition to breast and ovarian cancer (Miki et al., 1993). Germline mutations of BRCA1 are found in approximately 50% of breast- ovarian cancer pedigrees and in 10% of early onset breast cancer, irrespective of family history (reviewed in Gayther et al., 1998). Although the exact function of BRCA1 still remains obscure, roles in both the regulation of transcription and in mediating the cellular response to DNA damage have been implied (reviewed in Haber, 2000; Zheng et al., 2000). Substantial evidence exists to support a role for BRCA1 in transcriptional regulation. The C-terminal domain of BRCA1 is highly acidic, a feature of many transcription factors and has also been shown to mediate transcriptional activation when fused to a heterologous DNA binding domain, suggesting that BRCA1 may function as a transcriptional cofactor (Chapman and Verma, 1996; Monteiro et al., 1996). Furthermore, BRCA1 co-purifies with RNA poly- merase II holoenzyme complex through an association with RNA helicase A (Scully et al., 1997a; Anderson et al., 1998), suggesting that BRCA1 is a component of the core transcriptional machinery. A number of other reports have demonstrated that BRCA1 associates with a range of dierent transcription factors including p53 (Ouchi et al., 1998), c-Myc (Wang et al., 1998), ATF1 (Houvras et al., 2000) and STAT1 (Ouchi et al., 2000) and modulate their activity. What is clear from these studies is that BRCA1 alone does not physically interact with DNA but rather acts as a transcriptional co- activator or co-repressor. We previously identified GADD45 as being upregulated in response to inducible expression of exogenous BRCA1, an eect that was independent of p53 (Harkin et al., 1999) and subse- quently a BRCA1 responsive site was identified in the proximal GADD45 promoter (Jin et al., 2000). A potential model through which BRCA1 regulates expression of GADD45 has come from a number of related studies. It has previously been demonstrated that c-Myc mediated transactivation is repressed by its association with BRCA1 (Wang et al., 1998). In addition, it has been reported that c-Myc can repress GADD45 expression through the C/EBP element within the GADD45 promoter (Amundson et al., 1998). It is postulated therefore that BRCA1 mediated induction of Oncogene (2001) 20, 6123 – 6131 ª 2001 Nature Publishing Group All rights reserved 0950 – 9232/01 $15.00 www.nature.com/onc *Correspondence: P Harkin, Department of Oncology, Cancer Research Centre, Queen’s University Belfast, University Floor, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, N. Ireland; E-mail: d.harkin@qub.ac.uk 5 These authors contributed equally to this work Received 23 March 2001; revised 4 June 2001; accepted 8 June 2001