Cyclin D- and E-Dependent Kinases and the p57KIP2 Inhibitor: Cooperative Interactions In Vivo

This study examines in vivo the role and functional interrelationships of components regulating exit from the G₁ resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57^KIP2 inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57^KIP2 inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G₁, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57^KIP2-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57^KIP2 function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57^KIP2 to regulate the G₁/S transition in a cell type highly dependent upon pRb.     

Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0

The cell cycle is at the center of growth, productivity, and death of mammalian cell cultures. There exists a need to identify and quantify major landmarks in the cell cycle of industrially relevant mammalian cell lines and its association with productivity; central for designing productivity optimization strategies. Herein, we studied the expression of three cyclins, under both perturbed and unperturbed growth, by flow cytometry in batch cultures of GS-NS0. The perturbed systems involved two different DNA synthesis inhibitors, thymidine and dimethyl sulfoxide (DMSO). This approach enables the establishment of characteristic cyclin profiles, timings, and thresholds. In particular, two G₁ class cyclins (D1 and E1), and one G₂ cyclin (B1) were investigated. Cyclin B1 showed a clear cell cycle phase-specific expression increasing during G₂ phase where it was approximately 40% higher when compared to G₁ phase. Similarly, cyclin E1 showed a clear pattern being expressed approximately 10% higher in G₁ compared to G₂ phase and decreased through S phase. Cyclin D1 expression was fairly invariable throughout the cell cycle phases. The observed patterns provide a blueprint of the cell line's cell cycle, which can be used for the development of biologically accurate and experimentally validated distributed cell cycle models.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1

Background

The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G₁ phase.

Methodology/Principal Findings

In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G₀-G₁ phase arrest, but also reduced the number of cells at S and G₂-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G₀-G₁ phase despite the reduction of G₀-G₁ regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G₀-G₁ regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC.

Conclusions/Significance

This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G₀-G₁ phase.     

Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: Differential induction mechanism of cyclin D1 and D3

D-type cyclins are involved in the regulation of the G₁/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17β-estradiol (E₂) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G₀ to G₁ and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E₂-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E₂-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E₂ administration. Interestingly, the ³H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E₂ at G₁ phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression. Mol. Reprod. Dev. 46:450–458, 1997. © 1997 Wiley-Liss, Inc.     

PP2ACdc55 regulates G1 cyclin stability

Maintaining accurate progression through the cell cycle requires the proper temporal expression and regulation of cyclins. The mammalian D-type cyclins promote G₁-S transition. D1 cyclin protein stability is regulated through its ubiquitylation and resulting proteolysis catalyzed by the SCF E3 ubiquitin ligase complex containing the F-box protein, Fbx4. SCF E3-ligase-dependent ubiquitylation of D1 is trigged by an increase in the phosphorylation status of the cyclin. As inhibition of ubiquitin-dependent D1 degradation is seen in many human cancers, we set out to uncover how D-type cyclin phosphorylation is regulated. Here we show that in S. cerevisiae, a heterotrimeric protein phosphatase 2A (PP2A^Cdc55) containing the mammalian PPP2R2/PR55 B subunit ortholog Cdc55 regulates the stability of the G₁ cyclin Cln2 by directly regulating its phosphorylation state. Cells lacking Cdc55 contain drastically reduced Cln2 levels caused by degradation due to cdk-dependent hyperphosphorylation, as a Cln2 mutant unable to be phosphorylated by the yeast cdk Cdc28 is highly stable in cdc55-null cells. Moreover, cdc55-null cells become inviable when the SCF^Grr1 activity known to regulate Cln2 levels is eliminated or when Cln2 is overexpressed, indicating a critical relationship between SCF and PP2A functions in regulating cell cycle progression through modulation of G₁-S cyclin degradation/stability. In sum, our results indicate that PP2A is absolutely required to maintain G₁-S cyclin levels through modulating their phosphorylation status, an event necessary to properly transit through the cell cycle.     

Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system.

Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.     

Selective inhibition of proteins regulating CDK/cyclin complexes: strategy against cancer—a review

Cancer prevention is a global priority, but history indicates that the journey towards achieving the goal is difficult. Various cyclin dependent kinase complexes (CDKs/cyclins) operate as major cell signaling components in all stages of cell cycle. CDK/cyclin protein complexes, regulating the cell cycle, are conserved during evolution. In cancer cells, cell division is uncontrolled and CDKs/cyclins become ‘check-points’ or targets. Keeping this in view the proteins cyclin C, cyclin D2, CDKN1C, and Growth Arrest and DNA Damage (GADD45α) which play a major role in regulating CDK/cyclin complexes and operate in the initial stages of cell cycle (G₀ phase–S phase), have been identified as promising targets. Targeting critical regulators of cell-cycle signaling components by applying modern computational techniques is projected to be a potential tool for future cancer research.     

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34^cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

Increased stringency of the 1,25-dihydroxyvitamin D3-induced G1 to S phase block in polyploid HL60 cells

Treatment of mammalian cells with 1,25-dihydroxyvitamin D₃ (1,25D₃) produces a G1 to S (G1/S) phase cell cycle block. In addition, it has been noted that a smaller proportion of cells accumulates in the G2/M compartment in 1,25D₃-treated cultures. Since cyclins have a major influence on the regulation of cell cycle progression, we determined the expression of cyclins A and B as markers of the G2 phase and of cyclin E as the marker of G1/S transition. No increase in the steady-state levels of cyclin A or cyclin B mRNA was detected in the total cell population or in the cyclin B1 protein in the G2/M cell cycle compartment. In contrast, immunodetectable cyclin E protein was increased in cell cultures as a whole and specifically in the G2/M compartment cells. Determination of BrdU incorporation into DNA by flow cytometry showed marked inhibition of DNA replication in cells with DNA content higher than 4C, and autoradiography of ³H-TdR-pulsed cells showed that polynucleated cells did not replicate DNA after 96 h of treatment with 1,25D₃ or analogs. Taken together, these experiments show that at least a portion of the G2/M compartment in 1,25D₃-arrested cultures of HL60 cells represents G1 cells at a higher ploidy level, which are blocked from entering the high ploidy S phase. © 1996 Wiley-Liss, Inc.     

Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G₁ phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ~120 and ~200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.     

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for beter understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G₁ phase, cyclins C, D and E, or in S, G₂ and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G₁ phase, cyclin A in late G₁/early S phase and cyclin B1 in G₂. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones—oestrogens and progestins—and growth factors—insulin-like investigated for their effects on G₁ cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G₁ arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.     

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G₁ to S phase transition of cells by enhancing CDK inhibitor p21^Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB^Ser-780) followed by the up-regulation of E2F1 target genes required to promote G₁ to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB^Ser-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G₁ phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21^Cip-1/Waf1 pathway.     

Cyclin D1 Promotes Cell Cycle Progression through Enhancing NDR1/2 Kinase Activity Independent of Cyclin-dependent Kinase 4

Cyclin/cyclin-dependent kinases (Cdks) are critical protein kinases in regulating cell cycle progression. Among them, cyclin D1/Cdk4 exerts its function mainly in the G₁ phase. By using the tandem affinity purification tag approach, we identified a set of proteins interacting with Cdk4, including NDR1/2. Interestingly, confirming the interactions between NDR1/2 and cyclin D1/Cdk4, we observed that NDR1/2 interacted with cyclin D1 independent of Cdk4, but NDR1/2 and cyclin D1/Cdk4 did not phosphorylate each other. In addition, we found that NDR1/2 did not affect the kinase activity of cyclin D1/Cdk4 upon phosphorylation of GST-Rb. However, cyclin D1 but not Cdk4 promoted the kinase activity of NDR1/2. We also demonstrated that cyclin D1 K112E, which could not bind Cdk4, enhanced the kinase activity of NDR1/2. To test whether cyclin D1 promotes G₁/S transition though enhancing NDR1/2 kinase activity, we performed flow cytometry analysis using cyclin D1 and cyclin D1 K112E Tet-On inducible cell lines. The data show that both cyclin D1 and cyclin D1 K112E promoted G₁/S transition. Importantly, knockdown of NDR1/2 almost completely abolished the function of cyclin D1 K112E in promoting G₁/S transition. Consistently, we found that the protein level of p21 was reduced in cells overexpressing cyclin D1 K112E but not when NDR1/2 was knocked down. Taken together, these results reveal a novel function of cyclin D1 in promoting cell cycle progression by enhancing NDR kinase activity independent of Cdk4.     

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G₂ nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G₁ phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM–chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G₁, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G₁ quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM–chromatin association.