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(2008) 27, 7083–7093 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE -dependent kinase 4/6 activity is a critical determinant of pre-replication complexassembly

WA Braden, AK McClendon and ES Knudsen

Department of 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 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 , CDK2/ and the replication apparatus has been extensively studied, the CDK2/ 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 needed for S-phase activity, we demonstrate that CDK4/6 kinase activity entry and subsequent DNA replication (Woo and Poon, promotes 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/ promotes entry into , whereas CDK2 inhibition had no effect on the pre-RC assembly. subsequent degradation of A and B mediates The inhibition of pre-RC assembly is dependent on (Sherr, 1996; Sullivan and Morgan, 2007). a functional retinoblastoma (RB) , 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 and expression during G1 is critical CDK4/6 inhibition had no effect on 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/ 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 factor , and subsequent transcrip- in regulating replication protein activity. Finally, CDK4/6 tion of E2F target (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 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 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- 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 ) 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. - 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 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 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. Interestingly, when CDK4/6 activity was and relatively unstable nature of cdc6 and cdt1proteins inhibited in mitosis, cells entered the subsequent G1, but coalesce to mediate the downregulation of the proteins. were unable to form new pre-RCs, suggesting CDK4/6 From these results, we conclude that CDK4/6 activity is is involved in replication apparatus assembly as cells required for MCM7 loading onto the pre-RC, and that progress through the cell cycle. Combined, these studies this effect is proximally dependent on the inhibition of provide new insight into the complex role of CDK/ cdt1and cdc6 expression. cyclin activity in regulating DNA replication.

CDK4/6 dependent inhibition of the pre-RC requires Results a functional RB pathway It is well established that the primary target of CDK4/6 Targeted CDK4/6 inhibition elicits a G1 cell cycle arrest phosphorylation is the RB tumor suppressor protein. and prevents pre-RC loading CDK4/6 functions to inactivate RB, releasing E2F To investigate the specific role of CDK4/6 activity transcription factors, resulting in the transcription in pre-RC assembly, a small molecule inhibitor of of genes necessary for progression into S phase.

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7085 U2OS 70 U2OS U2OS 100 80 60 80 50 60 40 60 40 30 40 % BrdU 20 20 20 % MCM7 positive % PCNA positive 10 0 0 0 wt PD rvt wt PD rvt wt PD rvt

U2OS U2OS U2OS wt PD rvt wt PD wt PD PD/MG pRB RB cdc6 cdc6 cdt1 RB-Ser780 cdt1 GAPDH β- cdc6 1 2 1 2 3 cdt1

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β-tubulin

1 2 3 Figure 1 Pharmacological inhibition of cyclin-dependent kinase 4/6 (CDK4/6) activity elicits a cell cycle arrest and prevents pre- replication complex (pre-RC) loading. (a) U2OS cells were treated with PD-332991, roscovitine (rvt) or dimethyl sulfoxide (DMSO) for 24 h and pulsed with BrdU for 4 h. Coverslips were fixed and immunostained for BrdU incorporation. Data represent three independent experiments with at least 200 cells scored per experiment. (b, c) U2OS cells were treated with PD-332991, roscovitine (rvt) or DMSO for 24 h, subjected to in situ extraction, fixed and immunostained for mini-chromosome maintenance 7 (MCM7) or proliferating cell nuclear antigen (PCNA). Shown is percent of nuclei positive for MCM7 (b) or PCNA (c). (d) U2OS cells were treated with PD-332991, rvt or DMSO for 24 h. After treatment, cells were lysed, proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and the indicated proteins were detected by immunoblotting. (e) U2OS cells were treated with PD-332991or DMSO for 24 h, RNA was isolated, cDNAs were generated and reverse transcriptase (RT)–PCR was performed using primers to amplify cdc6, cdt1and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). ( f) U2OS cells were treated with PD-332991, DMSO and/or MG132 for 24 h. After treatment, cells were lysed, proteins were resolved by SDS–PAGE and the indicated proteins were detected by immunoblotting.

To determine if signaling through RB was required for ciated kinase activity directly regulates PCNA chroma- CDK4/6-associated inhibition of the pre-RC, Saos-2 tin association (Sever-Chroneos et al., 2001). In cells (RB negative) were treated with PD-332991and addition, neither compound had a significant effect on roscovitine. As shown in Figure 2a, PD-332991had very cdc6 or cdt1expression in the absence of RB little effect on cell cycle progression, whereas roscovitine (Figure 2d). Correspondingly, there is no attenuation treatment significantly impeded progression into S of cdc6 or cdt1RNA levels with PD-0332991treatment phase. To determine if CDK4/6-associated kinase in RB-deficient cells, demonstrating that this regulation activity was necessary for pre-RC loading, Saos-2 cells is RB dependent (Figure 2e). Strikingly, treatment of were treated with PD-332991or roscovitine- and Saos-2 cells with MG132 results in a massive induction chromatin-associated levels of MCM7 and PCNA were of cdc6 and cdt1protein levels, indicating that, examined. Although neither compound had a significant regardless of RB status, cdc6 and cdt1are relatively effect on MCM7 loading (Figure 2b), roscovitine was unstable proteins. If PD-332991were inhibiting CDK4/ able to inhibit PCNA tethering (Figure 2c), in agreement 6, but not CDK2 activity as a consequence of the loss of with previous studies that demonstrate CDK2-asso- RB, one would expect no change in CDK2-associated

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7086 Saos-2 60 Saos-2 Saos-2 80 80 50 40 60 60 30 40 40

% BrdU 20 20 10 20 % PCNA positive % MCM7 positive 0 0 0 wt PD rvt wt PD rvt wt PD rvt

Saos-2 Saos-2 Saos-2 wt PD rvt wt PD wt PD PD/MG

cdc6 cdc6 cdc6

cdt1 cdt1 cdt1

GAPDH β tubulin -tubulin 1 2 1 2 3 1 2 3 Figure 2 A functional retinoblastoma (RB) pathway is required for cyclin-dependent kinase 4/6 (CDK4/6) action on the pre- replication complex (pre-RC). (a) Saos-2 cells were treated with PD-332991, roscovitine (rvt) or dimethyl sulfoxide (DMSO) for 24 h and pulsed with BrdU for 4 h. Coverslips were fixed and immunostained for BrdU incorporation. Data represent three independent experiments with at least 200 cells scored per experiment (b) and (c) Saos-2 cells were treated with PD-332991, rvt or DMSO for 24 h and subjected to in situ extraction, fixed and immunostained for mini-chromosome maintenance 7 (MCM7) or proliferating cell nuclear antigen (PCNA). Depicted is percent of nuclei staining positive for MCM7 (b) or PCNA (c). (d) Cells were treated with PD-332991, rvt or DMSO. After treatment, cells were lysed, proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE), and the indicated proteins were detected by immunoblotting. (e) Saos-2 cells were treated with PD-332991or DMSO for 24 h, RNA was isolated, cDNAs were generated and reverse transcriptase (RT)–PCR was performed using primers to amplify cdc6, cdt1and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (f) Saos-2 cells were treated with PD-332991, DMSO and/or MG132 for 24 h. After treatment, cells were lysed, proteins were resolved by SDS–PAGE and the indicated proteins were detected by immunoblotting.

kinase activity. Indeed, phosphorylation of Loss of D-type cyclins sensitize cells to CDK2-mediated was unchanged in the presence of PD-332991or cell cycle arrest and pre-RC disruption p16ink4a (data not shown). Combined, these results Although the preceding pharmacological studies sup- indicate that a functional RB pathway is required to port an important role for CDK4/6 in modulating pre- inhibit pre-RC loading, and suggest that this association RC assembly, a genetic model was utilized to further is likely dependent on both CDK4/6 and CDK2 kinase interrogate this action of CDK4/6. For these studies, activity and the capacity to downregulate cdc6 and cdt1 murine fibroblasts derived from the D1, D2, and D3 expression. triple knockout (TKO) were utilized (kindly To further interrogate the influence of RB in response provided by Dr Piotr Sicinski). These TKO cells have to CDK4/6-mediated pre-RC inhibition, U2OS cells been immortalized by the use of a dominant negative were transduced with retrovirus encoding a short- (Kozar et al., 2004) and were utilized to address the hairpin RNA against RB. Three days after selection, specific influence of D-type cyclins. As expected, TKO RB protein could not be detected in these cells cells express no D-type cyclins compared with matched (Figure 3a). When treated with PD-332991, cells wild-type (WT) murine fibroblast cultures (Figure 4a). expressing a scrambled hairpin (shDON) elicited a In addition, these cells have been shown to progress strong arrest in G1 of the cell cycle (Figure 3b), as well through the cell cycle independently of CDK4/6- as a decrease in RB target expression (Figure 3c), associated activity (Kozar et al., 2004). When treated whereas those cells in which RB had been knocked with PD-332991, WT cells elicited a significant G1 down elicited a modest decrease in BrdU incorporation arrest, whereas TKO cells did not respond to CDK4/6 (Figure 3b) and no change in protein expression of RB inhibition (Figure 4b). To determine if PD-332991was targets (Figure 3c). In concordance with these observed disrupting DNA replication in TKO cells, BrdU effects on cell cycle, MCM7 chromatin binding was incorporation assays were performed. Although WT significantly attenuated with PD-332991in RB-profi- cells responded to both PD-332991and roscovitine, cient cells, whereas only marginally downregulated in exhibiting a decrease in DNA replication (Figure 4c), RB-deficient cells (Figure 3d). From these results we TKO cells responded only to roscovitine treatment conclude that the effects of CDK4/6 on the pre-RC are (Figure 4c). This finding suggests that not only is RB dependent and not via direct interaction. CDK4/6 the sole target of PD-332991, but in this

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7087 104 (Singlets)104 (Singlets)

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shDonor 102 102 FL1-BrdU FL1-BrdU 101 101 39.91% 3.26% 100 100 shDON shRB 0 200 400 600 800 1000 0 200 400 600 800 1000 RB FL3-PI FL3-PI

104 (Singlets) 104 (Singlets) B 3 103 10

1 2 2 shRB 102 10 FL1-BrdU FL1-BrdU 101 101 47.07% 25.58% shDON shRB 100 100 0 200 400 600 800 1000 0 200 400 600 800 1000 - + - + :PD FL3-PI FL3-PI DMSO PD cdc6 U2OS - shDON U2OS - shRB

cdt1 MCM7 Extracted MCM7 Extracted 100 120 cyclin A 80 100 80 60 CDK2 60 40 40 % positive β-tubulin % positive 20 20 1 2 3 4 0 0 DMSO PD DMSO PD Figure 3 Retinoblastoma (RB) loss compromises cyclin-dependent kinase 4/6 (CDK4/6) signaling to the pre-replication complex (pre- RC). (a) shDON U2OS and shRB U2OS cells were lysed, proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and the indicated proteins were detected by immunoblotting. (b) shDON and shRB U2OS cells were treated with dimethyl sulfoxide (DMSO) or PD-332991for 24 h and pulsed with BrdU for 1h. Cells were then subjected to fluorescence-activated cell sorter (FACS) analysis and percent S-phase populations were quantified. (c) shDON and shRB U2OS cells were treated with DMSO or PD-332991 for 24 h, cells were lysed, proteins were resolved by SDS–PAGE and the indicated proteins were detected by immunoblotting. (d) shDON and shRB U2OS cells were treated with DMSO or PD-332991for 24 h, subjected to in situ extraction, fixed and immunostained for mini-chromosome maintenance 7 (MCM7). system, CDK2 is the predominant kinase modulating CDK4/6 activity is required for accumulation of licensing replication apparatus assembly. factors and S phase entry To interrogate the impact of D-type cyclin deletion on Pre-RCs assemble as cells exit mitosis, in a stage of the the replication apparatus, WT and TKO cells were cell cycle when no mitotic CDK activity is present, and treated with the CDK2 inhibitor roscovitine for 24 h. before CDK2 activity begins to rise. However, it is Although roscovitine had no effect on cdc6 and cdt1in apparent that nuclear cyclin D1is present in mitotic cells WT cells, roscovitine-treated TKO cells exhibited a and contributes to facets of G1 control (Yang et al., marked decrease in cdc6 and cdt1protein expression 2006). To assess the formation of new pre-RCs, U2OS (Figure 5a). To determine if diminished cdc6 and cdt1 cells were treated with to enrich for mitotic protein levels affected chromatin loading of replication populations. Cells were then subjected to mitotic shake- factors, MCM7 chromatin retention was analysed. In off and replated in the presence or absence of PD- the presence of roscovitine, chromatin-associated levels 332991. In the absence of PD-332991, mitotic cells could of MCM7 were not affected, whereas chromatin- progress into the subsequent G1 and S phases; however, associated PCNA was greatly diminished (Figure 5b). treatment with PD-332991resulted in continued arrest Interestingly, in TKO cells that lack CDK4/6 activity, with a 2N DNA content (Figure 6a). Furthermore, the roscovitine disrupted both MCM7 and PCNA chroma- expression of cdt1and cdc6 was specifically attenuated tin association (Figure 5b). MCM7 and PCNA chro- in PD-332991treated cells (Figure 6b). To investigate matin association was disrupted at 10 mM in TKO cells, the temporal coordination of cdt1and cdc6 expression whereas even higher doses did not affect MCM7 and cell cycle progression, cells were harvested at tethering in WT cells (Figure 5c). These results confirm various time points following mitotic shake-off. As that MCM loading is dependent on cdc6 and cdt1 shown in Figure 7a, cells reenter the subsequent G1 expression, and that CDK4/6 and CDK2 cooperate in phase within 2 h post-mitotic shake-off, and this occurs regulating pre-RC assembly. irrespective of PD-332991treatment. However,

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7088 DMSO PD

1000 800

800 600 wt 600 400 400 wt TKO Cell Number 200 200 2N 4N 2N 4N 360 β-tubulin 240 1 2 270 180 TKO 180 120 Cell Number 90 60

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wt TKO 60 60 50 50 40 40 30 30 % BrdU % BrdU 20 20 10 10 0 0 DMSOPD rvt DMSOPD rvt Figure 4 Loss of D-type cyclins sensitizes cells to cyclin-dependent kinase 2 (CDK2)-mediated cell cycle arrest. (a) Wild-type (WT) and cyclin D1, D2, D3-null (triple knockout, TKO) immortalized murine fibroblasts were lysed, proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and the indicated proteins were detected by immunoblotting. (b) WT and TKO cells were treated with dimethyl sulfoxide (DMSO) or PD-332991for 24 h and pulsed with BrdU for 1h. Cells were then subjected to fluorescence-activated cell sorter (FACS) analysis. (c) WT and TKO cells were treated with PD-332991, roscovitine (rvt) or DMSO for 24 h and pulsed with BrdU for 4 h. Coverslips were fixed and immunostained for BrdU incorporation. Data represent three independent experiments with at least 200 cells scored per experiment.

although there is a significant accumulation of phos- CDK activity is important in suppressing re-replication phorylated RB in the absence of PD-332991by 2 h post- of the genome by inhibiting reinitiation of DNA mitotic shake-off, RB remains unphosphorylated in the replication during S phase and latter phases of the cell PD-332991treated cells (Figure 7b). This finding is cycle (Hua et al., 1997; Coverley et al., 2002; Ekholm- consistent with previous studies demonstrating that RB Reed et al., 2004; Mimura et al., 2004). Much of the phosphorylation is only modestly reduced following emphasis regarding CDK activity and positive regula- mitotic progression in U2OS cells, and suggests that the tion of replication has focused on roles for CDK2 in maintenance of RB phosphorylation during this transi- modulating the firing of origins in S phase. However, we tion is CDK4/6 dependent (Broceno et al., 2002). and others have recently found that CDK/cyclin activity Protein levels of cdt1and cdc6 are detectable within is important in establishing the pre-RC in G1 (Mailand 2 h following plating (Figure 7c), and the accumulation and Diffley, 2005; Braden et al., 2006; Geng et al., 2007). of cdt1and cdc6 was dependent on CDK4/6 activity, Specifically, the accumulation of both cdt1and cdc6, as PD-332991resulted in a consistent decrease in which mediate the assembly of the MCM complex on both proteins. These findings indicate that CDK4/6 chromatin, are influenced by the activity of CDK/ is important in S phase entry and mediating the cyclins. Here we extend these prior findings by accumulation of these critical licensing factors following demonstrating that CDK4/6 activity is important in mitotic exit. modulating the levels of cdt1and cdc6 to facilitate the establishment of the pre-RC. A multitude of studies have analysed the importance Discussion of CDK2 activity in modulating DNA replication. These studies have suggested an important role for this The activity of CDK/cyclin complexes is critical for the kinase in stimulating the initiation of S phase and initiation DNA replication. It is well established that replication origin firing. Consistent with previous

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7089

wt TKO Chromatin

- + : rvt - + : rvt wt TKO - + : rvt - + : rvt cdc6 MCM7 cdt1 PCNA cyclin A

Lamin B β-tubulin 12 3 4 12 34

wt

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Extracted Extracted 100 70 90 60 80 70 50 60 40 50 40 30 % positive % positive 30 20 20 10 10 0 0 0 2.5 5 10 20 40 : uM 0 2.5 5 10 20 40 : uM

TKO

MCM7 PCNA

Extracted Extracted 100 70 90 60 80 70 50 60 40 50 40 30 % positive % positive 30 20 20 10 10 0 0 0 2.5 5 10 20 40 : uM 0 2.5 5 10 20 40 : uM Figure 5 Loss of cyclin-dependent kinase 4/6 (CDK4/6) activity sensitizes cells to CDK2-mediated pre-replication complex (pre-RC) assembly. (a) Wild-type (WT) and cyclin D1, D2, D3-null (triple knockout, TKO) immortalized murine fibroblasts were treated with dimethyl sulfoxide (DMSO) or roscovitine (rvt) for 24 h and lysed, proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and the indicated proteins were detected by immunoblotting. (b) WT and TKO cells were treated with DMSO or rvt for 24 h and subjected to in situ extraction. Cells were lysed, proteins resolved by SDS–PAGE and the indicated proteins were detected by immunoblotting. (c) WT and TKO cells were treated with DMSO or varying doses of rvt for 24 h, subjected to in situ extraction and immunostained for mini-chromosome maintenance 7 (MCM7) and proliferating cell nuclear antigen (PCNA). studies from our laboratory and others, we find that activity. Strikingly, inhibition of CDK4/6 activity results inhibition of CDK2 activity does not influence the in the strong suppression of cdt1and cdc6 protein levels loading of MCM proteins on chromatin, nor the and a corresponding loss of chromatin-associated MCM expression of cdt1and cdc6, which are required for proteins. In this context, previous studies have shown pre-RC assembly (Braden et al., 2006; Savio et al., that ectopic expression of cdt1and cdc6 allows for pre- 2006). However, inhibition of CDK2 activity does RC assembly even with CDK activity inhibited, inhibit ongoing replication in S phase as determined indicating a causal function for these factors (Mailand by retention of PCNA on chromatin (Sever-Chroneos and Diffley, 2005; Braden et al., 2006). However, et al., 2001; Angus et al., 2004; Braden et al., 2006; Savio restoration of pre-RC assembly is not sufficient to et al., 2006). Thus, it is becoming increasingly clear that stimulate DNA replication due to additional require- assembly of the pre-RC is independent of CDK2 ments for CDK activity in the initiation and progression

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7090 Mitotic shake-off

016:hr

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2N 4N 8N 2N 4N Figure 6 Cyclin-dependent kinase 4/6 (CDK4/6) activity is required for accumulation of licensing factors. U2OS cells were treated with nocodazole in the presence or absence of PD-332991. Cells were then subjected to mitotic shake-off and replated in the presence or absence of PD-332991. (a) Cells were harvested for fluorescence-activated cell sorter (FACS) analysis at the indicated time points post- mitotic shake-off. (b) Cells were harvested and lysed at the indicated time points post-mitotic shake-off. Proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and the indicated proteins were detected by immunoblotting. An asynchronous (Asynch.) population of U2OS cells is included for comparison.

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Mitotic shake-off Mitotic shake-off 2 4 8 : hr 1 2 :hr

Asynch. - + - + :PD - + - + - + :PD cdc6 pRb Rb cdt1 β -tubulin β-tubulin

123 45 12 3456 Figure 7 Cyclin-dependent kinase 4/6 (CDK4/6) activity is required for pre-replication complex (pre-RC) assembly in telophase. U2OS cells were treated with nocodazole in the presence or absence of PD-332991. Cells were then subjected to mitotic shake-off and replated in the presence or absence of PD-332991. (a) Cells were harvested for fluorescence-activated cell sorter (FACS) analysis at the indicated time points post-mitotic shake-off. (b and c) Cells were harvested and lysed at the indicated time points post-mitotic shake- off. Proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE), and the indicated proteins were detected by immunoblotting. An asynchronous (Asynch.) population of U2OS cells is included for comparison.

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7091 of S phase (Vaziri et al., 2003; Braden et al., 2006). Thus, potential target through which CDK4/6 and CDK2 combined these findings indicate CDK inhibition has could exert direct, yet redundant, control over the distinct implications on the replication apparatus. replication apparatus. The mechanism by which suppression of CDK4/6 Presumably, the activity of CDK4/6 in facilitating activity impacts the cell cycle remains controversial. pre-RC assembly could be critical in two distinct facets From genetic models, it is clear that deficiency of cyclin of cell cycle control. First, in asynchronous cells it is well D-associated kinase activity can be tolerated, as cells appreciated that cyclin D1is nuclear and present at high from genetically modified mice deficient in D-type concentrations as cells progress from S phase into cyclins or CDK4/6 activity remain competent for G2/M. Furthermore, the G2 pool of cyclin D1is proliferation (Kozar et al., 2004; Malumbres et al., required for -independent progression in a 2004). In this context, deletion of D-type cyclins or continuous cell cycle (Guo et al., 2002). This accumula- CDK4/6 allows for compensation by other CDK tion of cyclin D1could be particularly important for activities. This contrasts significantly with the pharma- facilitating the loading of the pre-RC as cells exit cological attenuation of CDK4/6 activity resulting in a mitosis. Such a function of cyclin D1is consistent with reduction of CDK2 function and cell cycle arrest. Here our data that suggest inhibition of mitotic CDK4/6 we demonstrate that suppression of CDK4/6 activity is activity prevents accumulation of cdc6 and cdt1, thus associated with a significant attenuation in CDK2 blocking pre-RC assembly. Second, as cells transition activity, which is dependent on the RB protein. There- from quiescence into the cell cycle, the phosphorylation fore, in an RB-proficient cell, CDK4/6 inhibition leads of RB, and subsequent activation of CDK2, is likely to the inhibition of CDK2 activity. The basis for this critical in enabling the synthesis of multiple factors distinction between deletion and inhibition of kinase involved in DNA replication. As such, targeting CDK4/ activity has not been clearly resolved. In the context of 6 activity acts through the RB pathway to influence our study, the cyclin D TKO cells lack the target of DNA replication. PD-0332991. Correspondingly, because CDK2 is now Accumulating evidence suggests that appropriate mediating functions normally achieved by cyclin coordination of the DNA replication machinery is D-associated kinase activity, the downregulation of important for the suppression of tumorigenesis (Tanaka cdc6 and cdt1is occurring as a consequence of inhibiting and Diffley, 2002; Vaziri et al., 2003). Aberrant pre-RC other kinase complexes. Combined these results suggest assembly has been shown to induce re-replication and that the composite activity of CDK4/6 and CDK2 , and correspondingly, enforced expression are overlapping in nature and support the assembly of of cyclin D1or cyclin E influences the replication pre-RC complexes. machinery to induce genome instability (Ekholm-Reed The mechanism through which CDK activity im- et al., 2004). In this study, we demonstrate that targeted pinges on cdt1and cdc6 levels remains an area of intense inhibition of CDK4/6 can prevent pre-RC assembly as a study. Both cdt1and cdc6 are E2F-regulated genes means to enforce cell cycle arrest. Although the specific (Dyson, 1998; Hateboer et al., 1998; Markey et al., 2002; mechanism for how G1-CDK activity in influencing Yan et al., 1998), and therefore their RNA levels can be DNA replication control remains the subject of ongoing modulated with the activation of the RB growth research, the data presented herein provide new insight inhibitory pathway. In addition, cdt1and cdc6 are also into specific roles for CDK4/6 in pre-RC assembly. responsive to post-translation modifications and regu- Interestingly, CDK4/6 activity is frequently deregulated lated turnover (Hateboer et al., 1998; Nguyen et al., in cancer due to loss of p16ink4a or aberrant cyclin D1 2001; Liu et al., 2004). Specifically, cdt1is ubiquitinated expression. Thus, targeted inhibition of CDK4/6 may be and degraded as cells progress through S phase in a particularly important for suppressing aberrant DNA Cul4a/PCNA-dependent fashion (Senga et al., 2006). replication and tumorigenesis. Interestingly, recent studies indicate that this degrada- tion process is modulated by nuclear localization of cyclin D1, such that CDK4/6/cyclin D1 activity Materials and methods stabilizes cdt1(Aggarwal et al., 2007). Here we , drug treatments, and synchronization demonstrate that inhibition of CDK4/6 in RB-proficient U2OS and Saos-2 osteosarcoma cell lines were utilized due to cells results in decreased cdc6 and cdt1RNA levels, the presence (U2OS) or absence (Saos-2) of functional RB. consistent with the idea that cdc6 and cdt1are E2F WT and cyclin D1, D2, D3-null immortalized murine regulated. In addition, treatment of cells with the fibroblasts were obtained from P Sicinski. Cells were cultured proteosome inhibitor MG132 stabilizes the two pro- in Dulbecco’s modified Eagle’s medium supplemented with teins, indicating that cdc6 and cdt1are relatively 10% heat-inactivated fetal bovine serum, 100 units/ml unstable in nature, a characteristic that enhances their penicillin–streptomycin and 2 mML-glutamine at 37 1Cin5% downregulation in RB-proficient cells. In addition, the CO2. Cells synchronized in mitosis were treated with 50 ng/ml phosphorylation of cdc6 by CDK2 on three serine sites nocodazole (CalBiochem, La Jolla, CA, USA). To release the nocodazole block, cells were washed 3 Â with phosphate- in the N-terminal domain is postulated to protect the buffered saline (PBS) and cultured in fresh media. Mitotic protein from -promoting complex mediated shake-off experiments were performed by incubating cells in degradation (Mailand and Diffley, 2005). Whether nocodazole overnight, followed by perturbation of the culture CDK4 directly catalyses phosphorylation of these sites plate and replating of mitotic cells into fresh media. Drug is not currently known, but provides a provocative treatments were performed with dimethyl sulfoxide (DMSO;

Oncogene CDK4/6 regulates pre-RC assembly WA Braden et al 7092 vehicle), 50 nM PD-332991(Pfizer Corp., New York, NY, protocols. Superscript reverse transcriptase (Invitrogen, USA), 1 mM MG132 (Sigma, St Louis, MO, USA) and 10 mM Carlsbad, CA, USA) and 5 mg of total RNA were used to roscovitine (CalBiochem) unless specified otherwise. generate cDNAs with random hexamer primers. PCR was carried out using the following primers: 50-GCGATGAC Viral transduction AACCTATGCAACACTC-30 and 50-GATGACATCCATCT U2OS and Saos-2 cells were transduced with adenovirus CCCTTTCCCT-30 (human cdc6), 50-AAGGATCCCGCCT expressing p16ink4a at an approximate multiplicity of infec- ACCAGCGCTTCC-30 and 50-CCAAGCTTGAAGGTGGG tion of 10 for 24 h. Retrovirus encoding an shRB plasmid GACACTG-30 (human cdt1), and 50-GGTCATCAATGGGA (murine virus (MSCV)-LMP Rb88; targeted AACCCATCAC-30 and 50-AGTACTGGTGTCAGGTACG sequence: 50-GAAAGGACATGTGAACTTA-30) or control GTAGT-30 (glyceraldehyde-3-phosphate dehydrogenase, plasmid (MSCV donor) were utilized to create RB knockdown GAPDH). PCR conditions were as follows: 35 cycles or control U2OS and IMR90 cell lines. Following selection (denaturation at 94 1C for 30 s, annealing at 57 1C for 30 s, with 2.5 mg/ml puromycin for 4 days, polyclonal cell lines were elongation at 72 1C for 30 s) for cdc6 and cdt1, and 30 cycles characterized. (denaturation at 94 1C for 30 s, annealing at 53 1C for 30 s, elongation at 72 1C for 30 s) for GAPDH. Biochemical fractionation and western blotting To analyse chromatin-bound proteins, the protocol from Immunofluorescence microscopy Fujita et al. (1996) was adapted. Cells were cultured in 15 cm For PCNA and MCM7 staining, approximately 105 cells were plates, washed 3 Â with PBS, trypsinized and centrifuged at seeded onto coverslips, washed with ice-cold PBS and fixed 5000 r.p.m. for 5 min at 4 1C. Soluble proteins were then with methanol for 5 min. To visualize chromatin-tethered extracted with ice-cold CSK buffer (10 mM (piperazine N, proteins coverslips were washed 3 Â with PBS and extracted N-bis(2-ethanesulfonic acid) (PIPES), pH 6.8), 100 mM NaCl, with a modified CSK buffer as described above and fixed in 300 mM sucrose, 1m M MgCl2,1mM EGTA, 1m M dithio- ice-cold methanol for 5 min. The following were threitol, 1m M phenylmethylsulfonyl fluoride), supplemented used for immunofluorescence: PCNA (PC-10 Santa Cruz) and with 0.1% Triton X-100 for 15 min at 4 1C. Extracted cells MCM7 (141.2 Santa Cruz). Immunofluorescence data are were centrifuged at 5000 r.p.m. for 5 min at 4 1C and the from at least three independent experiments with at least 200 supernatant collected. Total cell extracts were harvested cells scored per experiment. BrdU incorporation assays were as previously described (Angus et al., 2004). Lysates performed as previously described (Braden et al., 2006). were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA). Membranes were incubated Flow cytometry with the following antibodies: Santa Cruz—anti-cyclin A Cells were harvested by trypsinization, fixed with ethanol and (C19 mouse, H432 human), anti-cyclin D1, anti-cyclin D2, incubated with propidium iodide (PI) or PI and anti-BrdU anti-cyclin D3, anti-b tubulin (D10), anti-PCNA (PC10), FITC-conjugated secondary . Histograms represent anti-MCM7 (141.2), anti-Lamin B (M20), anti-Cdc6 (180.2), at least 10 000 events. anti-CDK2 (M2), anti-CDK4 (C22), anti-p27KIP1, anti- CDK1(17),anti-RB (Ser780) (Cell Signaling, Danvers, MA, USA) and cyclin D1(Neomarkers, Union City, CA, USA). Acknowledgements Antibodies against RB (J Wang, University of California, San Diego, La Jolla, CA, USA) and Cdt1(J Cook, University of We thank Dr Karen Knudsen and members of both Knudsen North Carolina at Chapel Hill, NC, USA) were generous gifts. laboratories for critical review of the paper. Critical reagents were provided by Dr Piotr Sicinski and Pfizer Corp. This study RT–PCR was funded by a grant to ESK from the NCI CA106471. WAB RNA was harvested from U2OS and Saos-2 cell cultures by and AKM are supported by NIEHS training grant T32 using TRIzol according to the manufacturer’s suggested ES07250-16.

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