Cyclin D


Table of contents

Inhibitors of Cyclin D-CDK

Mouse protein p27 is related to the p21 cyclin-CDK inhibitor (See Drosophila Dacapo). In mouse fibroblasts, p27 is associated predominantly with cyclinD1-CDk4. Recombinant p27 is a potent inhibitor of CyclinD1-cdk4 and Cyclin A-cdk2 protein kinase, and a weaker inhibitor of Cyclin B1-cdc2. p27 is identical to p27Kip1, a cyclin-cdk inhibitor present in TGFß-treated cells. p27 has the hallmarks of a negative regulator of G1 progression and may mediate TGFß-induced G1 arrest (Toyoshima, 1994).

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. The CDK inhibitors p21CIP, p27KIP, and p57KIP2 all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase association constant, respectively, mostly because of a decrease in disassociation constant. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, the assembly of endogenous complexes was examined in mammary epithelial cells after release from a G0 arrest. Cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. All three CIP/KIP inhibitors are found to target cdk4 and cyclin D1 to the nucleus. It has been suggested that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions. Although all three of the inhibitors behave similarly with respect to promoting complex assembly, only p21 promotes the assembly of complexes with active RB kinase activity. Under the same conditions as those used for p21, both p27 and p57 cause only a gradual loss of cdk4-associated RB kinase activity. Possibly p27 and p57 may act only as inhibitors, revealing some fundamental difference between the way they interact with cdk/cyclin D complexes and the way p21 interacts (LaBaer, 1997).

Although p27(Kip1) has been considered a general inhibitor of G1 and S phase cyclin-dependent kinases, the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27 level completely inhibits Cdk2 and cell cycle progression. In contrast, the same or a larger increase in p27 levels does not inhibit Cdk4 or its homolog Cdk6, despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed in vitro is essentially inactive, whereas a p27-cyclin D2-Cdk4 complex is active as a retinoblastoma kinase and serves as a substrate for the Cdk-activating kinase Cak. High concentrations of p27 inhibit cyclin D2-Cdk4, apparently through the conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 are similarly inhibited by bound p21(Cip1/Waf1). The role of cyclin A-Cdk2 as a p27 target, and the role of cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes (Blain, 1997).

The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), it has been shown that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells it has been found that activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells (Ladha, 1998).

Loss-of-function mutations of p16(INK4a) have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro; this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4(N158), designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, is unable to arrest the cell cycle of the same cells. Functional differences have now been determined between p16 and Cdk4(N158). p16 and Cdk4(N158) inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociates cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) forms complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 forms complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities are correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E become resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression (Jiang, 1998).

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules (Parry, 1999).

The retinoblastoma protein (pRb) acts to constrain the G1-S transition in mammalian cells. Phosphorylation of pRb in G1 inactivates its growth-inhibitory function, allowing for cell cycle progression. Phosphorylation of S780 results in a lose of Rb's ability to bind to E2F. Phosphorylation of S807 and/or S811 is required to abolish Rb binding to c-Abl, while modification of threonine 821 and or T826 is required to abolish Rb binding to LXCXE-containing proteins such as simian virus 40 large T antigen. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By selectively inhibiting either cdk4/6 or cdk2, it has been shown that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after the sequential action of at least two distinct G1 cyclin kinase complexes (Lundberg, 1998).

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activates extracellular signal-regulated protein kinases (ERKs) and induces expression of cyclin D1. Newly synthesized cyclin D1 assembles with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that inefficiently phosphorylate the retinoblastoma protein. Activation of the MEK1/ERK pathway triggers neither degradation of the CDK inhibitor kinase inhibitory protein-1 (p27Kip1) nor leads to activation of cyclin E- and A-dependent CDK2, and such cells do not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits, generates levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27Kip1 is mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes are activated, and serum-starved cells enter S phase. Thus, although the activity of p27Kip1 normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequester p27Kip1 and reduce the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells complete S phase and divide, but they are unable to continuously proliferate, indicating that other serum-responsive factors ultimately become rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27Kip1-mediated inhibition (Cheng, 1998).

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts were used, capable of expressing cyclin E, cyclin D1, or both in an inducible manner. Ectopic expression of cyclin D1 is sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrest in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells does not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 is down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts (Resnitzky, 1997).

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that are prevented from spreading in medium containing soluble growth factors exhibit normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells fail to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlates with the failure of three activities: (1) an increase in cyclin D1 protein levels; (2) the down-regulation of the cell cycle inhibitor p27(Kip1), and (3) the phosphorylation of the retinoblastoma protein in late G1. A similar block in cell cycle progression is induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension have any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, these events alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation is controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition (Huang, 1998).

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. The 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 from which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment. This location permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibit clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins is more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation requires their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins is most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induces a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E does 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 on pRb (Gomez Lahoz, 1999).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. This sequestration is shown to be mediated by the protein synthesis rate of induction of cyclin D1 and/or cyclin D2. Consistent with this, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant that forms kinase-defective, Cki-binding cyclin-cdk complexes. Thus, the sequestration function of D cyclins appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).

The rate of the induction of cyclin D1 and/or cyclin D2 protein synthesis leads to the preferential association of p27Kip1 and p21Cip1 with cyclin D-Cdk complexes. At the same time Myc also induces cyclin E protein synthesis; the rates of induction help to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These complexes become active concomitant with phosphorylation of the kinase subunit by CAK. Consistent with this model of dynamic equilibrium, cyclin E-Cdk2 kinase activity can be controlled by changes in the rates of cyclin D synthesis. Moreover, as shown with a cyclin D mutant that forms kinase-defective Cki-binding cyclin D-Cdk complexes, this link between cyclins D-Cdk and cyclin E-Cdk2 is independent of cyclin D-Cdk activity, but correlates with the ability of cyclin D-Cdk complexes to bind or sequester Ckis. This is strongly supported by the fact that the deficiency of cyclin D1-/- mouse embryo cells to respond to Myc with increased proliferation is restored by expression of the same cyclin D mutant. Consistent with these findings, transient over-expression of either catalytically inactive cyclin D-Cdk, or cyclin E-Cdk2 complexes can rescue the cell cycle inhibitory effect of a dominant-negative Mad-Myc chimera. It is concluded that due to the nature of physical interactions between cyclin D-Cdks and the cell cycle inhibitors p27Kip1 and p21Cip1, cyclin D-Cdk complexes can fulfil a dual function as cell cycle kinases and as buffers for sequestration or release of cell cycle inhibitors (Perez-Roger, 1999 and references therein).

The cyclin-dependent kinases 4 and 6 (Cdk4/6) that drive progression through the G1 phase of the cell cycle play a central role in the control of cell proliferation, and CDK deregulation is a frequent event in cancer. Cdk4/6 are regulated by the D-type cyclins, which bind to CDKs and activate the kinase, and by the INK4 family of inhibitors. INK4 proteins can bind both monomeric CDK, preventing its association with a cyclin, and also the CDK-cyclin complex, forming an inactive ternary complex. In vivo, binary INK4-Cdk4/6 complexes are more abundant than ternary INK4-Cdk4/6-cyclinD complexes, and it has been suggested that INK4 binding may lead to the eventual dissociation of the cyclin. The 2.9-Å crystal structure of the inactive ternary complex between Cdk6, the INK4 inhibitor p18INK4c, and a D-type viral cyclin is presented here. The structure reveals that p18INK4c inhibits the CDK-cyclin complex by distorting the ATP binding site and misaligning catalytic residues. p18INK4c also distorts the cyclin-binding site, with the cyclin remaining bound at an interface that is substantially reduced in size. These observations support the model that INK4 binding weakens the cyclin's affinity for the CDK. This structure also provides insights into the specificity of the D-type cyclins for Cdk4/6 (Jeffrey, 2000).

Cyclin D mutation

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. The requirement for D-cyclins in mouse development and in proliferation was tested by generating mice lacking all D-cyclins. These cyclin D1-/-D2-/-D3-/- mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. This analyses reveal that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. The proliferation of cyclin D1-/-D2-/-D3-/- cells is resistant to the inhibition by p16INK4a, but proliferation critically depends on CDK2. Lastly, it was found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. These results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion (Kozar, 2004).

In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, a disproportionate loss of parvalbumin (PV) interneurons was found in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. It is proposed that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons (Glickstein, 2007).

Expression of cyclins D1 (cD1) and D2 (cD2) in ventricular zone and subventricular zone (SVZ), respectively, suggests that a switch to cD2 could be a requisite step in the generation of cortical intermediate progenitor cells (IPCs). However, direct evidence is lacking. In this study, cD1 or cD2 was seen to colabel subsets of Pax6-expressing radial glial cells (RGCs), whereas only cD2 colabeled with Tbr2. Loss of IPCs in cD2(-/-) embryonic cortex and analysis of expression patterns in mutant embryos lacking cD2 or Tbr2 indicate that cD2 is used as progenitors transition from RGCs to IPCs and is important for the expansion of the IPC pool. This was further supported by the laminar thinning, microcephaly, and selective reduction in the cortical SVZ population in the cD2(-/-)cortex. Cell cycle dynamics between embryonic day 14-16 in knock-out lines showed preserved parameters in cD1 mutants that induced cD2 expression, but absence of cD2 was not compensated by cD1. Loss of cD2 was associated with reduced proliferation and enhanced cell cycle exit in embryonic cortical progenitors, indicating a crucial role of cD2 for the support of cortical IPC divisions. In addition, knock-out of cD2, but not cD1, affected both G(1)-phase and also S-phase duration, implicating the importance of these phases for division cycles that expand the progenitor pool. That cD2 was the predominant D-cyclin expressed in the human SVZ at 19-20 weeks gestation indicated the evolutionary importance of cD2 in larger mammals for whom expansive intermediate progenitor divisions are thought to enable generation of larger, convoluted, cerebral cortices (Glickstein, 2009).

Effects of Cyclin D overexpression

Stable overexpression of cyclin D1 in R6 rat embryo fibroblasts shortens the G1 phase and impairs growth control. The effects of cyclin D1 overexpression were examined on other events involved in the G1 to S progression, utilizing the overexpressor cell line R6-ccnD1. When compared to R6 control cells, serum-starved quiescent R6-ccnD1 cells have not only increased levels of the cyclin D1 protein but also increased levels of the cyclin E protein. The latter protein is complexed to phosphorylated cyclin-dependent kinase 2 (CDK2). However, in quiescent serum-starved R6-ccnD1 cells, this cyclin E-CKD2 complex lacks in vitro kinase activity due to the presence of a heat-stable inhibitor. This appears to be a reflection of the inhibitory effects of the CDK inhibitors (CDKIs) p21WAF1 and p27KIP1. Serum stimulation of the quiescent R6-ccnD1 cells is associated with a loss of this inhibitory activity and a decrease in the levels of the latter two proteins, as the cells progress through the G1 phase. In contrast, serum stimulation of the control R6 cells is associated with both induction of cyclin E and increased levels of phosphorylated CDK2 proteins and decreased levels of p21WAF1 and p27KIP1, as the cells progress through the G1 phase. Thus, even though overexpression of cyclin D1 can induce the expression of cyclin E and phosphorylated CDK2, premature activation of cyclin E-CDK2 kinase activity in quiescent cells or during progression through G1 appears to be blocked by CDKIs. Nevertheless, the R6ccnD1 cells have a shorter G1 phase than the control cells, presumably due to the high levels of both cyclin D1 and cyclin E. Taken together, these results indicate that overexpression of cyclin D, which is frequently seen in human tumors, can have complex effects on the expression of other genes that control cell cycle progression (Imoto, 1997).

Cyclin D2 is member of the family of D-type cyclins whose members are implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5- to 20-fold) are seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies show that cyclin D2 protein localizes to the nucleus in G0, suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 is also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increase in amounts but are inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrate that overexpression of cyclin D2 protein efficiently inhibits cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit (Meyyappan, 1998).

Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, clonal MCF-7 breast cancer cell lines were derived in which c-Myc or cyclin D1 is expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 is sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression is not accompanied by increased cyclin D1 expression or Cdk4 activation, nor is cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 is sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21 following estrogen treatment. Interestingly, this is accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130 (a pRB-related pocket protein), identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis, which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21 (Prall, 1998).

Patterns of gene expression in human tumors have been deconvoluted to reveal a mechanism of action for the cyclin D1 oncogene. Computational analysis of the expression patterns of thousands of genes across hundreds of tumor specimens suggest that a transcription factor, C/EBPß/Nf-Il6 (Drosophila homolog: Slbo), participates in the consequences of cyclin D1 overexpression. Functional analyses confirmed the involvement of C/EBPß in the regulation of genes affected by cyclin D1 and established this protein as an indispensable effector of a potentially important facet of cyclin D1 biology. This work demonstrates that tumor gene expression databases can be used to study the function of a human oncogene in situ (Lamb, 2003).

It has been assumed that the established ability of cyclin D1 to activate cdk4/6, leading to phosphorylation of pRb with consequent derepression of E2F-mediated transcription, and the resulting promotion of cell cycle progression, underlies the tumorigenic consequences of cyclin D1 overexpression. However, conventional analyses of human tumor material have frequently failed to find correlative evidence in support of this model. For example, breast cancers overexpressing cyclin D1 do not show correspondingly high levels of the canonical E2F target gene cyclin E. Similarly, hyperphosphorylation of pRb is not observed in B cell lymphomas overexpressing cyclin D1. Such data have led some to suggest that the oncogenic activity of cyclin D1 must be exerted through pathways other than cdk-dependent and E2F-mediated acceleration of the cell cycle (Lamb, 2003).

The similarity of the transcriptional consequences of ectopic overexpression of cyclin D1 and the cyclin D1 KE mutant (which is unable to activate cdk4), the paucity of E2F target genes in this cyclin D1 expression signature, and the absence of a correlation between the expression patterns of these E2F target genes and cyclin D1 argue directly against activation of cdk4 or sequestration of cdk inhibitor proteins by catalytically inactive cyclin D1/cdk4 complexes as the mechanism of cyclin D1 action in human tumors. Therefore, a data-mining strategy (KSS) was developed to exploit the experimentally determined expression signature for the identification of potential participants in the mechanism of cyclin D1 oncogenicity ab initio. A transcription factor previously unconnected with cyclin D1 was ranked very highly across a number of independent tumor gene expression databases. Subsequent functional analyses confirmed that C/EBPβ is indeed a direct effector of the activity of cyclin D1 encoded in its expression signature. These findings indicate that the pathways connecting cyclin D1 with E2F-mediated transcription are perhaps not so germane as previously thought and instead implicate modulation of C/EBPβ function as a major mechanism of cyclin D1 action in human cancer (Lamb, 2003).

Table of contents

Cyclin D: Biological Overview | Regulation | Developmental Biology | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.