Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress

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Abstract

The adenomatous polyposis coli (APC) tumor suppressor traffics between nucleus and cytoplasm to perform distinct functions. Here we identify a specific role for APC in the DNA replication stress response. The silencing of APC caused an accumulation of asynchronous cells in early S phase and delayed S phase progression in cells released from hydroxyurea-mediated replication arrest. Immunoprecipitation assays revealed a selective binding of APC to replication protein A 32 kDa subunit (RPA32), and the APC–RPA32 complex increased at chromatin after hydroxyurea treatment. Interestingly, APC knock-down prevented accumulation at chromatin of the stress-induced S33- and S29-phosphorylated forms of RPA32, and reduced the expression of ATR-phosphorylated forms of S317-phospho-Chk1 and γ-H2AX. Using RPA32-inducible cells we showed that reconstitution of RPA32 diminished the S-phase delay caused by loss of APC. In contrast to full-length APC, the truncated APC mutant protein expressed in SW480 colon cancer cells was impaired in its binding and regulation of RPA32, and failed to regulate cell cycle after replication stress. We propose that APC associates with RPA at stalled DNA replication forks and promotes the ATR-dependent phosphorylation of RPA32, Chk1 and γ-H2AX in response to DNA replication stress, thereby influencing the rate of re-entry into the cell cycle.

Introduction

The adenomatous polyposis coli (APC) gene encodes a large 310 kDa tumor suppressor protein expressed in most tissues and whose expression in the colorectal epithelium contributes to normal growth and differentiation (Nathke, 2006, Senda et al., 2007). Germ-line mutations in one allele of APC give rise to the intestinal polyp disorder, familial adenomatous polyposis (FAP). The mutation of both APC alleles occurs in tumors of FAP patients and the majority of sporadic colorectal cancers, and is an early event in tumorigenesis (Senda et al., 2007). Most APC cancer mutations cause deletion of the C-terminus and target a centrally located mutation hotspot called the mutation cluster region (MCR) (Polakis, 2007, Senda et al., 2007). APC comprises multiple protein interaction domains and shuttles to different subcellular locations where it acts as a scaffold to regulate cytoskeleton assembly and other processes (Brocardo and Henderson, 2008), some of which are disrupted by cancer mutations. The role of APC in regulation of cell cycle has been extensively described in mitotic cells (Fodde et al., 2001, Kaplan et al., 2001) and interphase cells (Olmeda et al., 2003, Heinen et al., 2002, Ishidate et al., 2000, Baeg et al., 1995). It has been known for some time that APC can translocate to the nucleus and bind to A and T rich sequences in vitro through C-terminal sequences (Deka et al., 1999, Henderson and Fagotto, 2002) but only recently have specific nuclear functions been described. One recent finding correlates APC with DNA replication. The transient overexpression of full-length APC or C-terminal APC sequence (aa2140–2421) inhibited DNA replication in an in vitro assay using Xenopus laevis oocyte extracts. The repressive effect was thought to involve the non-specific binding of APC to DNA through S(T)PXX motifs and was impaired by CDK phosphorylation of APC (Qian et al., 2008). By contrast, Schneikert and Behrens (2006) analysed the proliferation of cells in which endogenous APC was silenced by RNAi and observed an impairment of progression through S phase after release from aphidicolin-induced S-phase arrest. These studies could be taken to suggest that disruption of APC by RNAi or peptide competition have a negative effect on DNA replication and S phase progression, however the mechanism remains poorly defined.

APC is most often detected in the cytoplasm, however it is known to shuttle into the nucleus (Henderson, 2000, Zhang et al., 2000) to regulate nuclear functions, which include not only DNA replication but also DNA repair. Indeed, APC was reported to associate in the nucleus with DNA polymerase β and PCNA to mediate DNA base excision repair (Narayan et al., 2005), with DNA-dependent protein kinase (DNA-PK) as part of the DNA damage response (Kouzmenko et al., 2008), and with topoisomerase II to potentially regulate the G2/M transition (Wang et al., 2008).

In this study we explored the role of APC in regulating S phase progression in the context of the cellular response to stress at replication sites. We report a novel association between APC and replication protein A (RPA), which itself has been described as a prognostic indicator of colon cancer and has commenced usage as a target of cytotoxins designed to inhibit cellular proliferation (Givalos et al., 2007, Peters et al., 2001). RPA is a nuclear heterotrimer composed of three subunits of ∼70, 32, 14 kDa (referred as to RPA70, RPA32 and RPA14, or RPA1, RPA2 and RPA3, respectively) and is a component of the replisome required for stabilizing single-stranded DNA (ssDNA) at replication forks to ensure efficient DNA synthesis (Fairman and Stillman, 1988, Wold and Kelly, 1988). RPA is also a regulator of the DNA damage checkpoint (Wold, 1997, Zou et al., 2006), and undergoes phosphorylation during replication stress or DNA damage stress (Binz et al., 2004) as part of its transition to sites of DNA damage (Vassin et al., 2004). These intriguing characteristics of RPA overlap the recently described roles of APC in DNA replication stress and repair (Schneikert and Behrens, 2006, Kouzmenko et al., 2008). Here we describe experiments which demonstrate that APC stimulates chromatin association of phosphorylated RPA32 and expression levels of Chk1 and γ-H2AX in response to hydroxyurea-induced replication stress, and use functional reconstitution experiments to show that RPA32 contributes to the regulation of S phase progression by APC following replication arrest.

Section snippets

Cell culture, transfections and treatment

Human SW480 colon cancer cells (APC protein truncated at amino acid 1338), and human U2OS osteosarcoma cells and HEK293T epithelial kidney cells and (both express full-length APC) were cultured in Dulbecco's modified Eagles medium (DMEM) with 10% fetal bovine serum (FBS). The cell lines were verified negative for mycoplasma contamination. To carry out transient transfections, the cells were seeded at 75% confluence and transfected with 2 μg/ml of DNA in 100 μl of optimal medium using FuGENE HD

Full-length APC contributes to S phase progression after DNA replication arrest

To study the effect of endogenous APC on S phase progression, we silenced APC for 48 h in cells synchronized at G1/S phase by hydroxyurea (HU) and the cell cycle profile was analysed by flow cytometry. The effectiveness of the APC silencing was validated by Western blot using two different siRNA (Fig. 1A). U2OS cells transfected with control siRNA responded to HU treatment by accumulating in G1 phase (64%), and after drug release cells progressed through S phase and into mitosis over a 9 h period

Discussion

The DNA replication checkpoint operates to maintain integrity and stability of the replication forks during periods of genetic stress. In this study we demonstrate for the first time a specific role for APC in the DNA replication checkpoint. Under drug-induced DNA replication stress conditions, we found that APC contributed to the post-stress cell cycle recovery and present evidence that the mechanism involves RPA32: (a) the knock-down of APC reduced chromatin association of ATR-phosphorylated

Acknowledgements

We thank other lab members for useful discussions and Dr. Bert Vogelstein for supplying pCMV-APC plasmids. This work was supported by the National Health and Medical Research Council of Australia (NHMRC) and the Australian Research Council. BRH is a NHMRC Senior Research Fellow.

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