Confluence-induced cell cycle exit involves pre-mitotic CDK inhibition by p27Kip1 and cyclin D1 downregulation

Tissue homeostasis requires precise control of cell proliferation and arrest in response to environmental cues. In situation such as wound healing, injured cells are stimulated to divide, but as soon as confluence is reached proliferation must be blocked. Such reversible cell cycle exit occurs in G1, requires pRb family members, and is driven by p27Kip1-dependent Cdk inactivation. This implies that, while dividing, cells should simultaneously prepare the exit once mitosis is accomplished. For a long time, the decision to cycle or not was presumed to occur in G1, prior to the restriction point, beyond which the cells were bound to divide even in the absence of mitogens, before finally arresting after mitosis. However, more recent reports suggested that the commitment to cycle in response to serum occurs already in G2 phase and requires the Ras-dependent induction of cyclin D1, which promotes following G1/S transition. To test whether this hypothesis applies to arrest induced by contact inhibition, we used an in vitro wounding model where quiescent human dermal fibroblasts, stimulated to proliferate by mechanical injury, synchronously exit cell cycle after mitosis due to renewed confluence. We show that this exit is preceded by p27-dependent inhibition of cyclin A-Cdk1/2, cyclin D1 downregulation and reduced pre-mitotic pRb pocket protein phosphorylation. Over-expression of cyclin D1 but not p27 depletion reversed this phenotype and compromised confluence-driven cell cycle exit. Thus, a balance between cyclin D1 and p27 may provide sensitive responses to variations in proliferative cues operating throughout the cell cycle.     

Multiple Functions of D-Type Cyclins Can Antagonize pRb-Mediated Suppression of Proliferation

The most well understood function of the D-type cyclins is to activate the G1kinases, cdk4 and cdk6, and target the retinoblastoma gene product (pRb) forphosphorylation and inactivation. pRb can suppress S phase entry, cause a transientG1 arrest following DNA damage, and is critical in establishing terminal cell cyclewithdrawal in cells exposed to differentiation or senescence-inducing signals. Each ofthese functions of pRb can be demonstrated in cultured cells derived from humantumors that have suffered RB1 gene inactivation. In such in vitro assays, coexpressionof D type cyclins has been shown to inhibit the function of pRb, likelyreflecting an oncogenic role of cyclin D1 in vivo. Two regions of cyclin D, the LxCxEpRb-binding motif, and the cyclin box, are thought to be critical for the proper functionof cyclin D. Here we show that the LxCxE motif is dispensable in cyclin D1 for allfunctions tested, but is required by cyclin D2. This observation suggests that there isa functional difference between cyclins D1 and D2 in pRb regulation, and arguesagainst complete functional redundancy of these D cyclins. In addition, the ability ofcyclins D1 and D2 to activate cdk partners is required for induction of pRbphosphorylation and S phase entry. However, mutant forms of cyclins D1 and D2that are incapable of activating kinase partners were still able to prevent pRb-inducedsenescence. Thus, D cyclins have both kinase-dependent and kinase-independentmechanisms of interfering with proliferation arrest and senescence.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1.

We have created fibroblast cell lines that express cyclin A under the control of a tetracycline-repressible promoter. When stimulated to reenter the cell cycle after serum withdrawal, these cells were advanced prematurely into S phase by induction of cyclin A. In an asynchronous population, induction of cyclin A caused a decrease in the percentage of cells in G1. These results demonstrate that expression of cyclin A is rate limiting for the G1-to-S transition and suggest that cyclin A can function as a G1 cyclin. Although the level of exogenous cyclin A was constant throughout the cell cycle, its associated kinase activity increased as cells approached S phase. Low kinase activity in early G1 was found to correlate with the presence of p27Kip1 in cyclin A-associated complexes, while high kinase activity in late G1 was correlated with its absence. These results suggest that a function of p27Kip1 in G1 is to prevent premature activation of cyclin A-associated kinase. Cyclin A expression in early G1 led to phosphorylation of the product of the retinoblastoma susceptibility gene (pRb). Thus, cyclin A expression can be rate limiting for pRb phosphorylation, implicating pRb as a physiological substrate of the cyclin A-dependent kinase. Taken together, these results demonstrate that deregulated expression of cyclin A can perturb the normal regulation of the G1-to-S transition.     

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.     

D-type cyclin-dependent kinase activity in mammalian cells.

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.     

Coordinated regulation of M phase exit and S phase entry by the Cdc2 activity level in the early embryonic cell cycle

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.     

Direct inhibition of G(1) cdk kinase activity by MyoD promotes myoblast cell cycle withdrawal and terminal differentiation

MyoD has been proposed to facilitate terminal myoblast differentiation by binding to and inhibiting phosphorylation of the retinoblastoma protein (pRb). Here we show that MyoD can interact with cyclin-dependent kinase 4 (cdk4) through a conserved 15 amino acid (aa) domain in the C-terminus of MyoD. MyoD, its C-terminus lacking the basic helix-loop-helix (bHLH) domain, or the 15 aa cdk4-binding domain all inhibit the cdk4-dependent phosphorylation of pRb in vitro. Cellular expression of full-length MyoD or fusion proteins containing either the C-terminus or just the 15 aa cdk4-binding domain of MyoD inhibit cell growth and pRb phosphorylation in vivo. The minimal cdk4-binding domain of MyoD fused to GFP can also induce differentiation of C2C12 muscle cells in growth medium. The defective myogenic phenotype in MyoD-negative BC3H1 cells can be rescued completely only when MyoD contains the cdk4-binding domain. We propose that a regulatory checkpoint in the terminal cell cycle arrest of the myoblast during differentiation involves the modulation of the cyclin D cdk-dependent phosphorylation of pRb through the opposing effects of cyclin D1 and MyoD.     

Role for non-proteolytic control of M-phase-promoting factor activity at M-phase exit

M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.     

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1)

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.     

Residual Cdc2 activity remaining at meiosis I exit is essential for meiotic M-M transition in Xenopus oocyte extracts

To investigate the regulatory mechanisms of the cell cycle transition from M phase to M phase in meiotic cycles, a XENOPUS: oocyte extract that performs the M-M transition has been developed. Using the meiotic extract, we found that a low level of Cdc2 activity remained at the exit of meiosis I (MI), due to incomplete degradation of cyclin B. The inactivation of the residual Cdc2 activity induced both entry into S phase and tyrosine phosphorylation on Cdc2 after MI. Quantitative analysis demonstrated that a considerable amount of Wee1 was present at the MI exit and Cdc2 inhibitory phosphorylation during this period was suppressed by the dominance of Cdc2 over Wee1. Consistently, the addition of more than a critical amount of Wee1 to the extract induced Cdc2 inhibitory phosphorylation, changing the M-M transition into an M-S-M transition. Thus, the Cdc2 activity remaining at MI exit is required for suppressing entry into S phase during the meiotic M-M transition period.     

Increased G1 cyclin/cdk activity in cells overexpressing the candidate oncogene, MCT-1.

We have recently identified a novel candidate oncogene, MCT-1, in the HUT 78 T-cell line. When overexpressed in NIH3T3 fibroblasts, the MCT-1 gene shortens the G1 phase of the cell cycle and promotes anchorage-independent growth. Progression of cells through a late G1 phase restriction point is regulated by G1 cyclins whose phosphorylation of the retinoblastoma gene product facilitates entry into S phase. Deregulated expression of G1 cyclins and their cognate cdk partners is often found in human tumor cells. In order to address the potential relationship of MCT-1 to cell cycle regulatory molecules, we analyzed the ability of MCT-1 overexpression to modulate cdk4 and cdk6 kinase activity in NIH3T3 fibroblasts constitutively overexpressing MCT-1. We observed an increase in the kinase activity of both cdk4 and cdk6 in asynchronously growing transformed cells compared with the parent cells. This increased kinase activity was accompanied by an elevated level of cyclin D1 protein and increased G1 cyclin/cdk complex formation. We also observed a correlation between increased protein levels of MCT-1 with cyclin D1 expression in a panel of lymphoid cell lines derived from T-cell malignancies. These results demonstrate that constitutive expression of MCT-1 is associated with deregulation of protein kinase-mediated G1 phase checkpoints.     

Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression

The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.     

Functional ablation of pRb activates Cdk2 and causes antiestrogen resistance in human breast cancer cells

Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER) positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb) family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT), and cdk activity was inhibited using the cdk inhibitors p16(INK4A) and p21(Waf1/Cip1). Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.