Oncogene (2008) 27, 7083–7093 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Cyclin-dependent kinase 4/6 activity is a critical determinant of pre-replication complexassembly WA Braden, AK McClendon and ES Knudsen Department of Cancer Biology and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Cyclin-dependent kinases (CDKs) are important in regu- functions to phosphorylate the retinoblastoma (RB) lating cell cycle transitions, particularly in coordinating tumor suppressor (Sherr, 1996; Sherr and Roberts, DNA replication. Although the role of CDK2 activity on 1999). As cells enter into S phase, CDK2/cyclin E and the replication apparatus has been extensively studied, the CDK2/cyclin A activity accumulates (Koff et al., 1991, role of CDK4/6 in DNA replication control is less 1992; Hwang and Clurman, 2005), ultimately leading to understood. Through targeted inhibition of CDK4/6 phosphorylation of several proteins needed for S-phase activity, we demonstrate that CDK4/6 kinase activity entry and subsequent DNA replication (Woo and Poon, promotes cdc6 and cdt1 expression, and pre-replication 2003). Finally, as cells pass through G2, high levels complex(pre-RC) assembly in cycling cells. Conversely, of CDK1/cyclin B promotes entry into mitosis, whereas CDK2 inhibition had no effect on the pre-RC assembly. subsequent degradation of cyclins A and B mediates The inhibition of pre-RC assembly is dependent on mitotic exit (Sherr, 1996; Sullivan and Morgan, 2007). a functional retinoblastoma (RB) protein, which mediates CDK4/6 activity is positively regulated through downstream effects. As such, CDK4/6 inhibition has mitogenic signaling (Diehl, 2002; Knudsen et al., 2006) minimal effect on the replication apparatus in the absence and negatively regulated by stresses that inhibit G1 of RB. The requirement of CDK4/6 was further inter- progression (Sherr and Roberts, 1999; Sherr, 2001). The rogated using cells lacking D-type cyclins, in which induction of cyclin D1expression is a delayed early replication complexes form normally, and correspondingly response to mitogens and expression during G1 is critical CDK4/6 inhibition had no effect on cell cycle or replication for initiating cell cycle progression from quiescence. control. However, in the absence of D-type cyclins, CDK2 Under favorable growth conditions, CDK4/6/cyclin D inhibition resulted in the attenuation of cdc6 and cdt1 complexes phosphorylate RB, allowing for the release of levels, suggesting overlapping roles for CDK4/6 and CDK2 the transcription factor E2F, and subsequent transcrip- in regulating replication protein activity. Finally, CDK4/6 tion of E2F target genes (for example, cyclins E and A) inhibition prevented the accumulation of cdc6 and cdt1 as (Palmero and Peters, 1996; Sherr, 1996, 2000; Bartek cells progressed from mitosis through the subsequent G1. et al., 1997; Bartkova et al., 1997; Sherr and McCor- Combined, these studies indicate that CDK4/6 activity is mick, 2002). The accumulation of cyclin E contributes important in regulating the expression of these critical to the activation of CDK2, which further phosphor- mediators of DNA replication. ylates RB and promotes S phase entry (Hwang and Oncogene (2008) 27, 7083–7093; doi:10.1038/onc.2008.319; Clurman, 2005). As cells progress into S phase, high published online 8 September 2008 levels of cyclin A are involved in DNA replication control (Angus et al., 2004; Braden et al., 2006). Keywords: CDK; cyclin; pre-RC; tumor suppressor; DNA replication is a highly ordered process that cancer ensures cells replicate their genome only once per cell cycle (Diffley, 1994; Bell and Dutta, 2002; Blow and Hodgson, 2002; Diffley and Labib, 2002; Dimitrova et al., 2002). In the accepted model, two proteins, cdc6 Introduction and cdt1, are individually recruited to the origin recognition complex in late telophase before cells enter Cyclin-dependent kinases (CDKs) are important in cell G1 of the subsequent cell cycle (Dutta and Bell, 1997; proliferation. As eukaryotic cells progress through the Stillman, 2005). Together, these proteins serve as a cell cycle, CDK/cyclins phosphorylate a myriad of scaffold and are required to recruit mini-chromosome proteins crucial to cells entering each cell cycle stage. maintenance (MCM) proteins, MCM2–7 (Dimitrova In G1, CDK4/6 partners with D-type cyclins (D1, D2 et al., 1999; Mendez and Stillman, 2000; Okuno et al., and D3) to form a catalytically active complex, which 2001; Diffley and Labib, 2002). A large body of evidence suggests that cdc6 and cdt1are essential for MCM Correspondence: Dr ES Knudsen, Department of Cancer Biology, loading, and that loss of either protein will result in the Kimmel Cancer Center, 233 South 10th Street, BLSB-Room 1002, cessation of replication. Once MCMs are bound, this Philadelphia, PA 19107, USA. E-mail: [email protected] complex is termed the pre-replication complex (pre-RC), Received 4 November 2007; revised 22 July 2008; accepted 22 July 2008; and origins are licensed for replication due to the published online 8 September 2008 helicase activity associated with MCMs (Dimitrova et al., CDK4/6 regulates pre-RC assembly WA Braden et al 7084 1999, 2002; Mendez and Stillman, 2000; Diffley and Labib, CDK4/6, PD-332991(Pfizer), was utilized. PD-332991 2002; Diffley, 2004; Stillman, 2005). MCMs are necessary has been shown to selectively inhibit CDK4/6 without for DNA replication and disruption of any one of the affecting CDK1, CDK2 or CDK5 activity (Fry et al., subunits in the hexameric ring will prevent further 2004). In U2OS (human osteosarcoma) cells, PD-332991 replication factor recruitment (Labib et al., 2000, 2001). treatment elicited significant cell cycle inhibition The pre-RC will in turn recruit cdc45 and MCM10 to (Figure 1a). The CDK2 inhibitor, roscovitine, also become the pre-initiation complex as cells transition into S inhibited cell cycle progression, albeit to a lesser extent phase (Diffley and Labib, 2002). A multitude of additional (Figure 1a). To determine if this arrest involved the factors are then recruited (that is, replication factor C disruption of DNA replication apparatus, members of (RFC), replication protein A, proliferating cell nuclear the replication machinery were examined. Chromatin- antigen (PCNA) and polymerase) to form the fully associated MCM7 was disrupted by PD-332991 competent replisome, primed for genome duplication (Figure 1b), as was PCNA chromatin retention (Diffley and Labib, 2002). (Figure 1c). Consistent with previously published results, For replication origins to become active, the pre-RC roscovitine treatment disrupted PCNA chromatin asso- is modified at the G1/S transition by CDK2 and ciation (Figure 1c), although having no effect on MCM7 cdc7–dbf4 (Lei et al., 1997; Blow and Hodgson, 2002; chromatin levels (Figure 1b) (Savio et al., 2006). It is Coverley et al., 2002). Interestingly, these same kinases known that MCM chromatin loading is dependent on phosphorylate the pre-RC to prevent further origin cdc6 and cdt1origin binding. To determine if this loss of firing (Dahmann et al., 1995; Hua et al., 1997; Noton MCM loading was dependent on cdc6 and cdt1 and Diffley, 2000); however, the exact mechanism(s) expression, total protein of these two pre-RC compo- underlying this regulation remains unclear. The para- nents was examined. When U2OS cells were treated with doxical regulation of the pre-RC by CDKs has been PD-332991, cdc6 and cdt1 expression was downregu- termed the ‘replication switch’, in which basal CDK lated (averages of sixfold and fourfold, respectively), activity in early G1 is sufficient to license the pre-RC, whereas roscovitine treatment had minimal effects on whereas high levels of CDKs prevent formation of new cdc6 and cdt1levels (Figure 1d).PD-332991treatment pre-RCs and origin refiring (Blow and Hodgson, 2002). also resulted in hypophosphorylation of RB and down- Early studies demonstrated that high levels of CDK2 regulation of the E2F target genes, cyclin A and CDK2 function to prevent pre-RC assembly and further MCM (Figure 1d). However, CDK4/6 inhibition did not affect recruitment once cells have entered into S phase to expression of the CDK2 inhibitor p27KIP1. Since prevent re-replication. More recently, it has been shown CDK4/6 inhibition via PD-332991led to the down- that not only does CDK2 negatively regulate cdc6 regulation of CDK2 expression, PD-332991would be expression in S phase, but CDK2 activity stabilizes cdc6 expected to indirectly inhibit CDK2 kinase activity. To in G1. Despite these findings relating CDK2 or cyclin E test this hypothesis, cells were treated with PD-332991 to pre-RC assembly, the role of CDK4/6 function on the or transduced with adenovirus to p16ink4a and exam- pre-RC has not been explored in detail. ined for CDK2-dependent histone H1phosphorylation. Here we sought to determine the action of CDK4/6 Indeed, PD-332991treatment resulted in a marked activity on replication dynamics. Our findings indicate decrease in CDK2 kinase activity (data not shown). To that CDK4/6 function is required for pre-RC assembly. further elucidate the mechanism by which inhibition of Specifically, CDK4/6 inhibition was found to inhibit the CDK4/6 influences pre-RC formation, the transcription expression of cdc6 and cdt1, preventing MCM chromatin and protein stability of cdc6 and cdt1were examined. loading. A functional RB pathway is required for RNA levels of both cdc6 and cdt1were decreased in CDK4/6 to influence the pre-RC, thus facilitating U2OS cells treated with PD-332991(Figure 1e).In downregulation of cdc6 and cdt1, as well as the addition, stabilization of these proteins with the attenuation of CDK2 activity. These findings were proteosome inhibitor MG132 resulted in a restoration further supported by a cell system expressing no D-type of cdc6 and cdt1protein levels in the face of CDK4/6 cyclins in which CDK2 inhibition disrupted pre-RC inhibition (Figure 1f). Thus, the reduction in RNA levels formation.