Cyclin D

EVOLUTIONARY HOMOLOGS


Table of contents

Regulation of Cyclin D transcription

Phosphatidylinositol (PI) 3-kinase is required for G1 to S phase cell cycle progression stimulated by a variety of growth factors and is implicated in the activation of several downstream effectors, including p70(S6K; see Drosophila RPS6-p70-protein kinase). However, the molecular mechanisms by which PI 3-kinase is engaged in activation of the cell cycle machinery are not well understood. The expression of a dominant negative form of either the p110alpha catalytic or the p85 regulatory subunit of heterodimeric PI 3-kinase strongly inhibits epidermal growth factor (EGF)-induced upregulation of cyclin D1 protein in NIH 3T3(M17) fibroblasts. The PI 3-kinase inhibitors LY294002 and wortmannin completely abrogate increases in both mRNA and protein levels of cyclin D1 and phosphorylation of pRb, inducing G1 arrest in EGF-stimulated cells. By contrast, rapamycin, which potently suppresses p70(S6K) activity throughout the G1 phase, has little inhibitory effect, if any, on either of these events. PI 3-kinase, but not rapamycin-sensitive pathways, is also indispensable for upregulation of cyclin D1 mRNA and protein by other mitogens in NIH 3T3 (M17) cells and in wild-type NIH 3T3 cells as well. An enforced expression of wild-type p110 is sufficient to induce cyclin D1 protein expression in growth factor-deprived NIH 3T3(M17) cells. The p110 induction of cyclin D1 in quiescent cells is strongly inhibited by coexpression of either of the PI 3-kinase DN forms, and by LY294002, but is independent of the Ras-MEK-ERK pathway. Unlike mitogen stimulation, the p110 induction of cyclin D1 is sensitive to rapamycin. These results indicate that the catalytic activity of PI 3-kinase is necessary, and could also be sufficient, for upregulation of cyclin D1, with mTOR signaling being differentially required depending upon cellular conditions (Takuwa, 1999).

Orderly cell cycle progression is regulated by coordinated interactions among cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylates the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending on the cell type. In these studies, overexpression of E2F-1 inhibits cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 are required for full repression of cyclin D1. Overexpression of pRB activates the cyclin D1 promoter, and a dominant interfering pRB mutant is defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter are required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at -127 binds Sp1, Sp3, and Sp4, and the distal region at -143 binds E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induces cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell (Watanabe, 1998).

Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1 (See Drosophila Ras). Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until approximately 2 h before the G1/S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade (Aktas, 1997).

Expression of the fos family of transcription factors is stimulated by growth factors that induce quiescent cells to reenter the cell cycle, but the cellular targets of the Fos family that regulate cell cycle reentry have not been identified. To address this issue, mice that lack two members of the fos family, c-fos and fosB, were derived. The fosB-/- c-fos-/- mice are similar in phenotype to c-fos-/- mice but are 30% smaller. This decrease in size is consistent with an abnormality in cell proliferation. Fibroblasts derived from fosB-/- c-fos-/- mice have a defect in proliferation that results at least in part from a failure to induce cyclin D1 following serum-stimulated cell cycle reentry. Although definitive evidence that c-Fos and FosB directly induce cyclin D1 transcription will require further analysis, these findings raise the possibility that c-Fos and FosB are either direct or indirect transcriptional regulators of the cyclin D1 gene and may function as a critical link between serum stimulation and cell cycle progression (Brown, 1998).

The persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts and it has been postulated that MAPKs control the activation of G1 cyclin-dependent complexes. This study examined the mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression have been compared in CCL39 cells, using a co-transfection procedure. Inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibits expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increases cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) reveals that in the absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade shows an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3), together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression is observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhances cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities (Lavoie, 1996).

Stable IIC9 cell lines, Goa1 and Goa2, were generated that overexpress full-length antisense Goalpha RNA. Expression of antisense Goalpha RNA ablates the alpha subunit of the heterotrimeric G protein, Go, resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G1/S phase progression were characterized. Ablation of Goalpha results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. These characteristics are abolished by the transient overexpression of the transducin heterotrimeric G protein alpha subunit, strongly suggesting the transformation of Goalpha-ablated cells involves Gobetagamma subunits. This is the first study to implicate a heterotrimeric G protein in tumor suppression (Weber, 1997a).

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulates mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induces G1 phase progression. ERK1 activation is biphasic and is sustained throughout the G1 phase of the cell cycle. PDGF induces cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) is correlated with growth arrest. In contrast, growth is unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, results in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1-cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreases ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression is observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also results in G1 growth arrest. These data provide evidence of a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression (Weber, 1997b).

Cyclin D1 controls the timing of S phase onset in mammalian cells, and acts as a positive regulator for the transcription factor E2F. Cyclin D1 overexpression leads to the activation of the dihyrofolate reductase gene promoter, acting through the E2F binding site in the promoter. P16INK4 represses this interaction, a repression that can be released by overexpression of cdk4. Thus Cyclin D1 and its associated kinase has a direct role in cell cycle regulation of E2F activity and consequently of S phase-specific gene expression. E2F binding sites bind complexes containing the retinoblastoma protein. In Rb-deficient cell lines, overexpression of Cyclin D1 fails to activate E2F-dependent transcription, suggesting that Rb may be involved in promoter activation (Schulze, 1994).

The cell cycle-dependent expression of Cyclin D1 is dependent on the presence of a functional Rb protein. Rb-deficient tumor cell lines as well as cells expressing viral oncoproteins (large tumor antigen of simian virus 40, early region 1A of adenovirus, early region 7 of papillomavirus) have low or barely detectable levels of cyclin D1. Expression of Cyclin D1, but not of Cyclins A and E, is induced by transfection of the Rb gene into Rb-deficient tumor cells. Cotransfection of a reporter gene under the control of the D1 promoter, together with the Rb gene, into Rb-deficient cell lines demonstrates stimulation of the D1 promoter by Rb. This parallels the stimulation of endogenous Cyclin D1 gene expression (Muller, 1994).

In the human, accumulation of G1 cyclins is regulated by E2F1. E2F binding sites are found in both the Cyclin E and Cyclin D1 promoters: both promoters are activated by E2F gene products, and in the case of Cyclin E, the E2F sites contribute to cell cycle control (Ohtani, 1995).

Conditional expression of an activated ras mutant in Balb/c-3T3 fibroblasts fails to stimulate S phase entry in the absence of plasma-derived progression factors, but does shorten the G1 interval from 12 to 6 h and abrogates the normal proliferative requirement for platelet-derived growth factor. Ras-dependent alteration of the 3T3 cell cycle is accompanied by a dramatic increase in the expression of, Cyclin D1. Cyclin/cdk complexes assembled in response to ectopic ras expression in the absence of growth factor stimulation bind Kip1, the cdk inhibitory factor, and are inactive. However, plasma-stimulated regulatory pathways function co-operatively with the oncogenic ras molecule to decrease Kip1 levels, induce kinase activities associated with Cyclins D, E and A, and trigger the initiation of DNA replication. Therefore, a ras-activated signal transduction pathway may link environmental mitogenic stimuli to the cell cycle machinery via modulation of G1 cyclin expression (Winston, 1996).

Mutations in the adenomatous polyposis coli (APC) tumor-suppressor gene occur in most human colon cancers. Loss of functional APC protein results in the accumulation of beta-catenin. Mutant forms of beta-catenin have been discovered in colon cancers that retain wild-type APC genes, and also in melanomas, medulloblastomas, prostate cancer and gastric and hepatocellular carcinomas. The accumulation of beta-catenin activates genes that are responsive to transcription factors of the TCF/LEF family, with which beta-catenin interacts. Beta-catenin activates transcription from the cyclin D1 promoter, and sequences within the promoter that are related to consensus TCF/LEF-binding sites are necessary for activation. The oncoprotein p21ras further activates transcription of the cyclin D1 gene, through sites within the promoter that bind the transcriptional regulators Ets or CREB. Cells expressing mutant beta-catenin produce high levels of cyclin D1 messenger RNA and protein constitutively. Furthermore, expression of a dominant-negative form of TCF in colon-cancer cells strongly inhibits expression of cyclin D1 without affecting expression of cyclin D2, cyclin E, or cyclin-dependent kinases 2, 4 or 6. This dominant-negative TCF causes cells to arrest in the G1 phase of the cell cycle; this phenotype can be rescued by expression of cyclin D1 under the cytomegalovirus promoter. Abnormal levels of beta-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1 (Tetsu, 1999).

beta-Catenin plays a dual role in the cell: it links the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and it acts in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation, result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. The cyclin D1 gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Three inhibitors of beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin, suppress cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels are augmented by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle (Shtutman, 1999).

STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin-3 (IL-3)-dependent cell line F-36P-mpl, the induced expression of dominant-negative (dn)-STAT5 and of dn-ras leads to inhibition of IL-3-dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6-STAT5A) and ras (H-rasG12V) enable F-36P-mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6-STAT5A and H-rasG12V individually and cooperatively transactivate the cyclin D1 promoter in luciferase assays. Both dn-STAT5 and dn-ras suppress IL-3-induced cyclin D1 promoter activities in F-36P-mpl cells. Using a series of mutant cyclin D1 promoters, 1*6-STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT-binding sequence at -481 bp. STAT5 binds to the element in response to IL-3. Furthermore, the inhibitory effect of dn-STAT5 on IL-3-dependent growth is restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine-dependent growth of hematopoietic cells (Matsumura, 1999).

During the past several years, reports have accumulated indicating that human tumor samples contain constitutively activated Stats (1, 3, and 5 most frequently). Likewise, a consistent activation of Stat proteins (see Drosophila Marelle), particularly Stat3, has been described in cells transformed in culture by known oncogenes or in cell lines started from human tumors. The requirement of Stat3 activation for the maximal transformation of cells by v-src was demonstrated using dominant-negative Stat3 to suppress transformation frequency and soft agar colony formation. In addition, maximal growth rates of squamous carcinoma cell lines were suppressed by expression of Stat3 dominant-negative constructs. All of these observations suggest at least a supplementary role for constitutively active wild-type Stat3 in tumorgenesis. However, until the present studies, it remained unsettled whether constitutively active Stat3 could by itself act as a transforming agent (Bromberg, 1999).

The events downstream from constitutively active Stat3 (Stat3-C) that promote tumorgenesis are unclear but could include enhancing conditions for cell cycle progression and/or providing protection against apoptosis. A large number of cell cycle regulators, both inhibitors or promoters of cycling, are now known and form one important group of genes that might be affected by an activated Stat3 molecule. In examining this class of molecules it was found that both cyclin D1 mRNA and c-myc mRNA were elevated 3- to 5-fold in Stat3-C transformed cells, as compared to untransformed 3Y1 cells (cyclin A mRNA was not elevated). In addition, transfection of the cyclin D1 promoter appended to a luciferase reporter gene was transcriptionally activated by Stat3-C in transfection experiments. In addition to positive effectors (or inhibitors of negative effectors) of cell cycle regulation, inducible genes that affect apoptosis are also crucial in the survival of transformed cells. Stat3 has been clearly demonstrated to have an antiapoptotic role in T cells and in monocytes during their differentiation. Induction of Stat3-/- precursor cells in these lineages leads to cell death of the Stat3-/- cells. Likewise, cultured human multiple myeloma cells that have incorporated a Stat3 dominant-negative gene undergo apoptosis. And it was found that Stat3-C transformed cells have elevated levels of Bcl-XL mRNA. Thus, transformation by constitutively active Stat3 may generally contribute to stimulating cell cycle progression and provide protection against apoptosis (Bromberg, 1999 and references).

The transcription factor AP-1, composed of Jun and Fos proteins, is a major target of mitogen-activated signal transduction pathways. However, little is known about AP-1 function in normal cycling cells. The quantity and the phosphorylation state of the c-Jun and JunB proteins vary at the M-G1 transition. Phosphorylation of JunB by the p34cdc2-cyclin B kinase is associated with lower JunB protein levels in mitotic and early G1 cells. In contrast, c-Jun levels remain constant while the protein undergoes N-terminal phosphorylation, increasing its transactivation potential. Since JunB represses and c-Jun activates the cyclin D1 promoter, these modifications of AP-1 activity during the M-G1 transition could provide an impetus for G1 progression by a temporal increase in cyclin D1 transcription. These findings constitute a novel example of a reciprocal connection between transcription factors and the cell cycle machinery (Bakiri, 2000).

The decrease in the concentration of JunB relative to c-Jun and the N-terminal phosphorylation of c-Jun at the beginning of the G1 phase could result in increased AP-1 activity and the induction of genes that respond better to c-Jun. A number of putative AP-1 target genes are known. Among them, cyclin D1 is interesting since it is synthesized early in the G1 phase and is required for progression to S phase. c-Jun has also been reported to induce cyclin D1 transcription in transient transfection assays. The human cyclin D1 transcriptional control sequences have been cloned and shown to contain different regulatory elements, including a TRE site and a CRE site, located 935 and 52 bp upstream of the initiation site, respectively. In exponentially cycling cells, transcriptional activation by c-Jun has been shown to be mediated essentially by these two sites, and Jun, Fos and ATF proteins might be part of a complex bound to this site. Using cyclin D1 promoter constructs containing both sites for transient transfection in HeLa cells, different responses to JunB and c-Jun were observed. While c-Jun strongly activates the cyclin D1 reporter, JunB repressed it in a dose-dependent manner. In contrast, JunB reproducibly activates a collagenase promoter construct containing the canonical TRE element, although this activation is weak compared with c-Jun. Furthermore, the strong activation by c-Jun is inhibited by co-expression of JunB. However, the inhibition of the cyclin D1 promoter is more pronounced than that of the collagenase promoter. When c-Jun N-terminal phosphorylation was mimicked by using a c-Jun protein in which serines 63 and 73 are replaced by aspartic acid, the ability of c-Jun to activate cyclin D1 transcription increases. In contrast, replacing these residues with leucines, which blocks c-Jun phosphorylation, strongly decreases its transcriptional activation of the cyclin D1 promoter. This c-JunLL protein exhibites activity similar to JunD, which is a weak activator of cyclin D1. Finally, c-Jun and JunB seem to affect the same sites on the cyclin D1 promoter since only the mutation of both the proximal CRE and the distal TRE could abolish the effect of the two proteins (Bakiri, 2000).

Another important point with respect to the effect of AP-1 on transcription is that, in the same cellular system and depending on the promoter context, two opposite effects of JunB have been observed: JunB-ER activation reproducibly induces a collagenase reporter construct while it represses the cyclin D1 reporter and RNA. This is an interesting example of differential regulation of two target genes by the same AP-1 component. JunB, like c-Jun, can form both active and inactive AP-1 dimers, depending not only on the nature of its partner (Fos, Jun or ATF) but also on the targeted promoter sequence. Since the AP-1-responsive elements in cyclin D1 and in the collagenase promoters are different, the opposing effects of JunB on these two genes could be explained by differences in cooperation with other transcription factors. The fact that JunB can activate or inhibit transcription depending on the promoter and partner context may extend to other genes and could contribute to the complexity of the genetic response to AP-1 (Bakiri, 2000).

ß-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of ß-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in ß-catenin itself. Another tumor suppressor, PTEN, is also involved in the regulation of nuclear ß-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. Nuclear ß-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a ß-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of ß-catenin and enhances its rate of degradation. A pathway is defined that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of ß-catenin stability, nuclear ß-catenin expression, and transcriptional activity. These data indicate that ß-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression (Persad, 2001).

Cyclin D1 is known to be one of the oncogenic targets of ß-catenin. In PC3 cells, the expression level of cyclin D1 is upregulated in a constitutive, serum-independent manner. More importantly, replacement of PTEN or inhibition of ILK results in dramatic suppression of the expression levels of cyclin D1. Furthermore, transient overexpression of GSK-3-WT also suppresses cyclin D1 expression. These results are supported by the observation that overexpression of ILK stimulates cyclin D1 expression. Also, ILK has been shown recently to regulate cyclin D1 transcription via a pathway involving GSK-3 and the cyclic AMP response element binding protein transcription factor. Cyclin D1 expression in PC3 cells changes in a parallel manner to ß-catenin expression in response to reexpression of PTEN or expression of ILK-KD and GSK-3. Therefore, it is proposed that the alterations in cyclin D1 expression very likely represent the physiological end result of the regulation of ß-catenin by PTEN and ILK via GSK-3. It should be pointed out that although nuclear ß-catenin and cyclin D1 expressions undergo parallel alterations due to reexpression of PTEN or inhibition of ILK, the expression of the CDK inhibitors p27Kip and p21Cip remain unchanged. This illustrates the specificity of the alterations induced upon ß-catenin and cyclin D1. PTEN, ILK-KD, and GSK-3-WT all dramatically reduce cyclin D1 promoter activity. Furthermore, Northern blot analysis demonstrates that ILK-KD, PTEN-WT, and GSK-3 induce dramatic inhibitory effects upon cyclin D1 transcriptional expression. This supports the working hypothesis that PTEN and ILK can regulate nuclear ß-catenin through GSK-3. This is in agreement with the fact that ß-catenin is known to be regulated by GSK-3 and recent studies have identified GSK-3 as a critical regulatory component for the transcriptional activity and binding of the TCF-LEF-1-ß-catenin complex transcription factors (Persad, 2001).

In conclusion, a novel pathway involving PI-3 kinase/PTEN, ILK, and GSK-3 has been demonstrated that maintains tight control over the levels and localization of ß-catenin. In prostate cancer cells, as well in other malignancies where PTEN is either lost or inactive, this control may be eliminated, resulting in elevated ß-catenin levels, its accumulation in the nucleus, and increased transcription of its oncogenic targets. Cyclin D1 is a known target of ß-catenin and it is also the first participant of the chain of cyclins and CDKs that control progression through the G1 and S phase of the cell cycle. Therefore, it is conceivable that PTEN and ILK, by virtue of their capacity to regulate ß-catenin and subsequently cyclin D1, may ultimately regulate the progression of cells through the cell cycle. By virtue of their ability to regulate the expression of E-cadherin, PTEN/PI-3 kinase and ILK may also control the metastatic potential of cancer cells. Therefore, the inhibition of a potent regulator such as ILK may present a feasible alternative means of treating the numerous forms of tumors where the PI-3 kinase-dependent signal transduction pathway is dysregulated due to mutations of the tumor suppressor PTEN (Persad, 2001).

Beta-catenin can function as an oncogene when it is translocated to the nucleus, binds to T cell factor or lymphoid enhancer factor family members, and transactivates its target genes. Cyclin D1 is one of the targets of beta-catenin in breast cancer cells. Transactivation of beta-catenin correlates significantly with cyclin D1 expression both in eight breast cell lines in vitro and in 123 patient samples. Beta-catenin activity significantly correlates with poor prognosis of the patients and is a strong and independent prognostic factor in breast cancer. These studies, therefore, indicated that beta-catenin can be involved in breast cancer formation and/or progression and may serve as a target for breast cancer therapy (Lin, 2000).

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. A mechanism is described by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. Misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy; this is accompanied by elevated levels of cyclin D1 expression. It is proposed that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. This model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis (Hu, 2001).

Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. TRRAP is a component of TIP60 and PCAF/GCN5 histone acetyl transferase (HAT) complexes. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc (Bouchard, 2001).

The aim of this study was to resolve the role of MBII (an effector domain of Myc that binds TRRAP) and TRRAP in gene activation by Myc, using an endogenous target gene of Myc, cyclin D2, as model. Upon binding to the cyclin D2 promoter, Myc recruits TRRAP and induces the preferential acetylation of histone H4 at a single nucleosome. Conversely, loss of endogenous Myc binding correlates with histone deacetylation and loss of TRRAP binding during the TPA-induced differentiation of a human promyelocytic cell line, HL60. The integrity of MBII is required for TRRAP recruitment, histone acetylation, and transcriptional activation at the cyclin D2 locus. Therefore, previous suggestions that MBII has no role in transcriptional activation based on transient reporter assays need to be reevaluated. Deletion of the entire N terminus of Myc up to MBII (s-Myc) renders Myc unable to induce cell cycle progression and expression of either cyclin A or cyclin D2 in 3T3 fibroblasts, consistent with recent results that the N terminus of Myc is required for regulation of proliferation and induction of gene expression in a cell-type-dependent manner. Most likely, this is because stable association with TRRAP requires sequences in the N terminus of Myc in addition to MBII (Bouchard, 2001 and references therein).

Mad proteins are thought to antagonize the function of Myc by recruiting a repressor complex that contains histone deacetylase activity. Observations suggest that this model applies to the cyclin D2 promoter: (1) repression of the cyclin D2 promoter by Mad1 requires the integrity of an N-terminal domain, which mediates recruitment of histone deacetylase complexes through interaction with Sin3A (see Drosophila Sin3A); (2) during HL60 differentiation, Mad1 and HDAC1 are corecruited to the cyclin D2 promoter, correlating with histone deacetylation of both histones H3 and H4 at the cyclin D2 promoter. Taken together, these data strongly support a model in which endogenous Myc/Max and Mad/Max complexes contribute to the regulation of transcription of the cyclin D2 gene through their antagonistic effects on histone acetylation. In addition, these findings show the functional relevance of the switch between Myc/Max and Mad/Max complexes during differentiation of hematopoietic cells. Recent work on the gene encoding the catalytic subunit of telomerase, htert, suggests that this model also may apply to this promoter (Bouchard, 2001 and references therein).

Up-regulation of the CAD (carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase) gene by Myc does not involve changes in histone acetylation. Instead, high levels of histone acetylation at the promoter were found in both quiescent and proliferating cells, showing that Myc can control at least one step in addition to histone acetylation to promote active transcription. Additional proteins have been identified that bind to different domains of Myc and that are candidates for such an activity: for example, the C terminus of Myc binds to Ini1, a component of the Swi/Snf family of chromatin-remodeling complexes. Clearly, a detailed analysis of the role of Myc in activation of individual promoters will be required before the role of each interaction in Myc biology can be resolved fully (Bouchard, 2001 and references therein).

Notch genes encode a family of transmembrane proteins that are involved in many cellular processes such as differentiation, proliferation, and apoptosis. Although it is well established that all four Notch genes can act as oncogenes, the mechanism by which Notch proteins transform cells remains unknown. Transformation of RKE cells can be conditionally induced by hormone activation of Notchic-estrogen receptor (ER) chimeras. Using this inducible system, it has been shown that Notchic activates transcription of the cyclin D1 gene with rapid kinetics. Transcriptional activation of cyclin D1 is independent from serum-derived growth factors and de novo synthesis of secondary transcriptional activators. Moreover, hormone activation of Notchic-ER proteins induces CDK2 activity in the absence of serum. Upregulation of cyclin D1 and activation of CDK2 by Notchic result in the promotion of S-phase entry. These data demonstrate the first evidence that Notchic proteins can directly regulate factors involved in cell cycle control and affect cellular proliferation. Furthermore, nontransforming Notchic proteins do not induce cyclin D1 expression, indicating that the mechanism of transformation involves cell cycle deregulation through constitutive expression of cyclin D1. A CSL binding site has been identified within the human and rat cyclin D1 promoters, suggesting that Notchic proteins activate cyclin D1 transcription through a CSL-dependent pathway (Ronchini, 2001).

One receptor-ligand pair that could mediate NF-kappaB activation during mammary gland development consists of RANK (receptor activator of NFkappa-B), a member of the TNF receptor (TNFR) family and RANK ligand (RANKL, also known as OPGL, ODF, and TRANCE), a member of the TNF family. As its namesake indicates, RANK is an efficient NF-kappaB activator. Although RANK and RANKL were originally characterized as playing important roles in lymphocyte and osteoclast differentiation and activation, they were recently found to be essential for mammary gland development. The biochemical pathway by which RANK controls mammary gland development has not been defined. IKKalpha activity is required for NF-kappaB activation. To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, an IkkalphaAA knockin allele was created containing alanines instead of serines in the activation loop. IkkalphaAA/AA mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the IkkalphaAA mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1 (Cao, 2001).

Cell cycle progression and exit must be precisely patterned during development to generate tissues of the correct size, shape and symmetry. Evidence that dorsal-ventral growth of the developing spinal cord is regulated by a Wnt mitogen gradient. Wnt signaling through the ß-catenin/TCF pathway positively regulates cell cycle progression and negatively regulates cell cycle exit of spinal neural precursors in part through transcriptional regulation of cyclin D1 and cyclin D2. Wnts expressed at the dorsal midline of the spinal cord, Wnt1 and Wnt3a, have mitogenic activity, while more broadly expressed Wnts do not. Several lines of evidence are presented suggesting that dorsal midline Wnts form a dorsal to ventral concentration gradient. A growth gradient that correlates with the predicted gradient of mitogenic Wnts emerges as the neural tube grows, with the proliferation rate highest dorsally, and the differentiation rate highest ventrally. These data are rationalized in a 'mitogen gradient model' that explains how proliferation and differentiation can be patterned across a growing field of cells. Computer modeling demonstrates that this model is a robust and self-regulating mechanism for patterning cell cycle regulation in a growing tissue (Megason, 2002).

Potential transcriptional targets of mitogenic Wnt signaling in neural precursors were investigated to address the mechanism through which they regulate the cell cycle and to determine the pattern of mitogenic Wnt response. Since the activities of Wnt1 and Wnt3a suggest that they directly impinge on the cell cycle rather than regulate cell fate specification, key components of the cell cycle were screened including cyclins, cyclin dependent kinases (CDKs), and CDK inhibitors (CKIs) as candidate targets. cyclin D1 is expressed throughout the early period of neural tube development in a dorsal to ventral gradient (highest dorsally) in mitotically active medial neural precursors in both mouse and chick. D-type cyclins are key regulators of G1 exit. When the spinal cord is small, the gradient of cyclin D1 expression extends all the way across the ventricular zone but as the spinal cord grows the expression gradient becomes more dorsally restricted relative to the size of the DV axis (Megason, 2002).

To investigate the mechanism by which dorsal midline Wnts regulate the cell cycle of neural precursors, the expression of cell cycle regulators was examined in the neural tube of embryos transfected with Wnt signaling components. Transfection of dominant active ß-catenin upregulates transcription of the G1 cyclins cyclin D1 and cyclin D2 but not the G2/M cyclins cyclin A1 or cyclin B3 in neural precursors. The locations of transfected cells were first determined by visualizing GFP and then the same sections were processed by in situ hybridization. Regions of the neural tube that ectopically express ß-catenin have increased levels of cyclin D1 and cyclin D2. Previous reports have found that ß-catenin signaling upregulates cyclin D1 but not cyclin D2 in cultured cell lines and that the cyclin D1 promoter contains consensus Lef/TCF binding sites required for this activity. The transcriptional regulation of cyclin D1 by Wnt signaling in neural precurors could thus be direct (Megason, 2002).

Ectopic expression of Wnt1 or Wnt3a also upregulated cyclin D1 expression. However, in contrast to dominant active ß-catenin, expression of Wnt1 or Wnt3a only upregulated cyclinD1 at intermediate to ventral levels. These data again suggest that endogenous mitogenic Wnts are saturating at dorsal levels. Ventral precursors have a stronger mitogenic response to ectopic Wnt expression than do dorsal precursors. This dorsal-ventral pattern of mitogenic responsiveness of neural precursors to ectopic expression of Wnt1 correlates with the pattern of cyclin D1 upregulation. If activating Wnt signaling upregulates cyclin D1 transcription, then attenuating Wnt signaling should downregulate cyclin D1 transcription. Accordingly, high levels of expression of dominant negative TCF4 downregulates expression of cyclin D1 (Megason, 2002).

To address how major a role transcriptional control of D-cyclins plays in the mitogenic response of Wnts, dominant negative and wild-type versions of cyclin D1 were ectopically expressed. Transfection of a dominant negative cyclin D1 construct that forms abortive complexes with the G1 cyclin dependent kinases CDK4 and CDK6 reduce neural precursor expansion but do not block neural cell cycle progression as severely as dominant negative TCF4. Ectopic expression of wild-type cyclin D1 is not sufficient to cause overgrowth of the neural tube as does Wnt1, Wnt3a, and dominant active ß-catenin. Additionally, mice mutant for cyclin D1 have small eyes and reduced body size but are viable. These data show that two key components of the cell cycle, cyclin D1 and cyclin D2, are transcriptional targets of Wnt signaling in neural precursors but suggest other targets are also involved in the mitogenic response of neural precursors to Wnts. Conversely, it is likely that other regulatory elements in addition to the TCF binding sites contribute to the regulation of cyclin D1 in the neural tube, especially its transient expression in apparently exiting cells. Taken together, these results support a model in which Wnt1 and Wnt3a expressed in the dorsal midline form a dorsal to ventral mitogen gradient that controls the graded expression of cell cycle regulators including D-type cyclins through the ß-catenin pathway (Megason, 2002).

tob (Drosophila homolog: Tob) is a member of an emerging family of genes with antiproliferative function. Tob is rapidly phosphorylated at Ser 152, Ser 154, and Ser 164 by Erk1 and Erk2 upon growth-factor stimulation. Oncogenic Ras-induced transformation and growth-factor-induced cell proliferation are efficiently suppressed by mutant Tob which carries alanines but not glutamates, thereby mimicking phosphoserines at these sites. Wild-type Tob inhibits cell growth when the three serine residues are not phosphorylated but is less inhibitory when the serines are phosphorylated. Because growth of Rb-deficient cells is not affected by Tob, Tob appears to function upstream of Rb. Intriguingly, cyclin D1 expression is elevated in serum-starved tob-/- cells. Reintroduction of wild-type Tob and mutant Tob with serine-to-alanine but not to glutamate mutations on the Erk phosphorylation sites in these cells restores the suppression of cyclin D1 expression. Finally, the S-phase population is significantly increased in serum-starved tob-/- cells as compared with that in wild-type cells. Thus, Tob inhibits cell growth by suppressing cyclin D1 expression, which is canceled by Erk1- and Erk2-mediated Tob phosphorylation. It is proposed that Tob is critically involved in the control of early G1 progression (Suzuki, 2002).

Although mutations that activate the Hedgehog (Hh) signaling pathway have been linked to several types of cancer, the molecular and cellular basis of Hh's ability to induce tumor formation is not well understood. A mutation in patched (ptc), an inhibitor of Hh signaling, was identified in a genetic screen for regulators of the Retinoblastoma (Rb) pathway in Drosophila. Hh signaling promotes transcription of Cyclin E and Cyclin D, two inhibitors of Rb, and principal regulators of the cell cycle during development in Drosophila. Upregulation of Cyclin E expression, accomplished through binding of Cubitus interruptus (Ci) to the Cyclin E promoter, mediates the ability of Hh to induce DNA replication. Upregulation of Cyclin D expression by Hh mediates the distinct ability of Hh to promote cellular growth. The discovery of a direct connection between Hh signaling and principal cell-cycle regulators provides insight into the mechanism by which deregulated Hh signaling promotes tumor formation (Duman-Scheel, 2002).

During eye development in Drosophila, initiation of neural differentiation, marked by an indentation referred to as the morphogenetic furrow, begins at the posterior end of the disc and passes anteriorly. Cells within the furrow arrest in G1 phase before differentiating. Cells located just posterior to the furrow exit G1 arrest and enter a synchronous S phase referred to as the second mitotic wave. Overexpression of the Drosophila Retinoblastoma-family gene (Rbf), an inhibitor of the S phase promoting transcription factor E2F, produces a 'rough' adult eye phenotype, characterized by loss of bristles and fusion of ommatidia. This phenotype results from delay of S phase progression in cells of the second mitotic wave as a consequence of inhibited E2F target gene expression. Loss of one copy of the ptc gene suppresses this rough eye phenotype and restores E2F target gene expression. The observed genetic interaction between Rbf and ptc suggests that the Hh signaling pathway might regulate the cell cycle during eye development (Duman-Scheel, 2002).

Hh is secreted from differentiating neurons located just posterior to cells entering S phase in the second mitotic wave. This expression pattern is consistent with the idea that reception of the Hh signal might be required for S phase entry in the second mitotic wave. To test this hypothesis, the effect of blocking Hh signaling during eye development was assessed. Cells with a mutated smoothened (smo) gene cannot respond to the Hh signal and fail to enter S phase in the second mitotic wave. Conversely, when Ci, the transcription factor that mediates Hh signaling, is overexpressed in the furrow, cells normally arrested in G1 enter S phase. Ectopic expression of Ci can also promote S phase in G1-arrested cells located in the wing margin and in the brain. Thus, Ci can induce S phase in a variety of tissues (Duman-Scheel, 2002).

Cyclin D, a G1/S cyclin, promotes S phase by inhibiting Rb. During eye development, Cyclin D is expressed in the furrow, and its highest level of expression overlaps with Ci expression. This expression pattern is consistent with the idea that Ci protein, which is stabilized in response to reception of the Hh signal from posterior neurons, promotes expression of Cyclin D in the eye. In support of this idea, Cyclin D levels are drastically reduced in smo mutant clones that extend through the furrow. Conversely, overexpression of Ci induces high levels of Cyclin D transcript and protein expression in the furrow and in cells immediately surrounding the furrow. In the eye, the ability of Ci to induce high levels of Cyclin D expression is limited to the vicinity of the furrow, the only region where it is capable of inducing S phase. Overexpression of Ci also induces high levels of Cyclin D transcript and protein expression when expressed ectopically in the wing disc. Thus, in addition to promoting S phase, Ci induces expression of Cyclin D in both the eye and wing (Duman-Scheel, 2002).

The ability of Hh signaling to induce expression of Cyclin D may explain why increased Hh signaling suppresses phenotypes associated with RBF overexpression. In support of this idea, overexpression of Ci, which induces Cyclin D expression, also induces ectopic expression of PCNA, an E2F target gene, in both the furrow and in G1-arrested cells located in the wing margin. Coexpression with Ci of RBF-280, a constitutively active form of RBF that cannot be regulated by Cyclin D or Cyclin E, blocks the ability of Ci to induce ectopic PCNA expression in the furrow and wing margin. However, although RBF-280 can block the ability of Ci to induce E2F target gene expression, it does not block the ability of Ci to promote S phase in the eye. These results indicate that although Hh signaling induces E2F target gene expression, it must also be capable of inducing S phase independently of E2F (Duman-Scheel, 2002).

Cell growth is thought to be regulated independently of cell proliferation. For example, overexpression of several well-characterized cell-cycle regulators induces cell proliferation but fails to stimulate growth (defined as the accumulation of mass). In contrast, Cyclin D induces both cell proliferation and promotes the accumulation of mass. Thus, it seems likely that Hh, which induces expression of Cyclin D, may also have a distinct function in regulating cell growth. To test how the modulation of Hh signaling influences growth the effects of overexpression of Ptc or Ci in clones of undifferentiated wing disc cells werre examined. Estimation of clone size was used as a measure of growth. Ptc overexpression clones are significantly smaller than control clones and cover only 65% of the area covered by control clones. In contrast, Ci overexpression clones are significantly larger than control clones and cover 143% of the area covered by control clones. These data indicate that, in addition to promoting S phase, Hh signaling has a distinct function in regulating cell growth. The observed ability of Hh signaling to promote cellular growth correlates with previous experiments indicating that Ptc overexpression results in reduced tissue growth (Duman-Scheel, 2002).

It is likely that Cyclin D, which is upregulated upon overexpression of Ci in the wing , might mediate the ability of Hh to promote growth. Consistent with this hypothesis, overexpression of Ci, like overexpression of Cyclin D-Cyclin-dependent kinase 4 (Cdk4), induces growth and accelerates cell proliferation rates without affecting individual cell size or overall cell-cycle phasing. Likewise, although inhibiting the Hh pathway by overexpressing Ptc decreases growth and decelerates cell proliferation rates, it does not affect individual cell size or cell cycle phasing (Duman-Scheel, 2002).

The ability of Cyclin D-Cdk4 to mediate induction of growth by Hh was examined in several ways. First, the ability of Cyclin D-Cdk4 to rescue the inhibitory effects of Ptc on growth was examined. The average size of clones expressing Ptc and Cyclin D-Cdk4 is significantly larger than the size of Ptc overexpression clones and is comparable to the size of clones expressing CyclinD-Cdk4 alone. Thus, overexpression of CyclinD-Cdk4 can suppress the inhibition of growth by Ptc. Furthermore, although clones of cells lacking Cdk4 (the only apparent kinase subunit for Drosophila Cyclin D) grow more slowly than wild-type cells, overexpression of Ptc does not significantly reduce the size of clones induced in a Cdk4 mutant background. Also, overexpression of Ci cannot stimulate growth in a Cdk4 mutant background. These results indicate that Cyclin D-Cdk4 is the principal growth-regulating target of Hh signaling (Duman-Scheel, 2002).

The investigation demonstrates that Hh signaling has a distinct ability to promote cellular growth, which is mediated by Cyclin D. In addition, Hh signaling can induce proliferation during development by promoting expression of Cyclin D and Cyclin E. This study reveals a direct connection between Hh signaling and induction of Cyclin E expression, which is accomplished through binding of Ci to the Cyclin E promoter. Upregulation of murine cyclin D1, D2 and E in response to Hh signaling has been observed. It is therefore likely that the mechanism for Cyclin E induction by Hh described here is conserved in mammals. Furthermore, because both overexpression of Ptc-1 or mutation of cyclin D1 produces a small mouse phenotype, it is likely that the ability of Hh to promote cellular growth through upregulation of D-type cyclins is also conserved in mice. Thus, constitutive Hh signaling (which promotes deregulated expression of G1/S cyclins that have been associated with diverse forms of human cancer) would promote both cell proliferation and growth in tumors. In contrast, during development, cell growth and proliferation must be carefully regulated and coordinated with the processes of cell patterning and differentiation. These same processes are also regulated by Hh signaling. This delicate balance is probably maintained by tight control of the temporal and spatial expression patterns of Hh targets and the molecules that regulate them (Duman-Scheel, 2002).

INI1/hSNF5 is a component of the ATP-dependent chromatin remodeling hSWI/SNF complex and a tumor suppressor gene of aggressive pediatric atypical teratoid and malignant rhabdoid tumors (AT/RT). To understand the molecular mechanisms underlying its tumor suppressor function, the effect has been studied of reintroduction of INI1/hSNF5 into AT/RT-derived cell lines such as MON that carry biallelic deletions of the INI1/hSNF5 locus. Expression of INI1/hSNF5 causes G(0)-G(1) arrest and flat cell formation in these cells. In addition, INI1/hSNF5 represses transcription of cyclin D1 gene in MON, in a histone deacetylase (HDAC)-dependent manner. Chromatin immunoprecipitation studies reveal that INI1/hSNF5 is directly recruited to the cyclin D1 promoter and that its binding correlates with recruitment of HDAC1 and deacetylation of histones at the promoter. Analysis of INI1/hSNF5 truncations indicates that cyclin D1 repression and flat cell formation are tightly correlated. Coexpression of cyclin D1 from a heterologous promoter in MON is sufficient to eliminate the INI1-mediated flat cell formation and cell cycle arrest. Furthermore, cyclin D1 was overexpressed in AT/RT tumors. These data suggest that one of the mechanisms by which INI1/hSNF5 exerts its tumor suppressor function is by mediating the cell cycle arrest due to the direct recruitment of HDAC activity to the cyclin D1 promoter thereby causing its repression and G(0)-G(1) arrest. Repression of cyclin D1 gene expression may serve as a useful strategy to treat AT/RT (Zhang, 2002).

Activation of cyclin-dependent kinases

Activation of the cyclin-dependent kinase to promote cell cycle progression requires their association with cyclins as well as phosphorylation of a threonine residue. This phosphorylation is carried out by the Cdk-activating kinase (CAK). Purification of CAK from mammals, starfish, and Xenopus has identified it as a heterotrimeric complex composed of a catalytic subunit, p40MO15/cdk7, a regulatory subunit, cyclin H, and an assembly factor, MAT1. CAK phosphorylates not only c34cdc2 but also other Cdks, including p33cdk2 and cdk4, which function earlier in the cell cycle. Additionally, the CAK subunits are components of TFIIH, a basal transcription factor involved in the initiation of transcription, phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II and DNA repair. The cloning of the CAK from S. cerevisiae raises the possibility that the the predominant CAK in vertebrate cell extracts, may not function as a physiological CAK. S. cerevisiae CAK is active as a monomer and is not a component of the basal transcription factor (Kaldis, 1996 and references).

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D-CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity is well tolerated and has no overt effects on the cell cycle (Cheng, 1999).

Nuclear localization of Cyclin D

M-phase promoting factor or maturation promoting factor, a key regulator of the G2-->M transition of the cell cycle, is a complex of cdc2 and a B-type cyclin. Xenopus cyclin B1 contains five sites for Ser phosphorylation, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the underlying mechanism remains unclear. Phosphorylation of cyclin B1 is not required for cdc2 binding or cdc2 kinase activity. However, when all of the Ser phosphorylation sites in the CRS are mutated to Ala, thereby abolishing phosphorylation, the mutant cyclin B1Ala is inactivated; activity can be enhanced by mutation of these residues to Glu in order to mimic phosphoserine, suggesting that phosphorylation of cyclin B1 is required for its biological activity. Biological activity can be restored to cyclin B1Ala by appending either a nuclear localization signal (NLS), or a second CRS domain with the Ser phosphorylation sites mutated to Glu, while fusion of a second CRS domain with the Ser phosphorylation sites mutated to Ala inactivates wild-type cyclin B1. Nuclear histone H1 kinase activity is detected in association with cyclin B1Ala targeted to the nucleus by a wild-type NLS, but not by a mutant NLS. These results demonstrate that nuclear translocation mediates the biological activity of cyclin B1 and suggest that phosphorylation within the CRS domain of cyclin B1 plays a regulatory role in this process. Given the similar in vitro substrate specificity of cyclin-dependent kinases, this investigation provides direct evidence for the hypothesis that the control of subcellular localization of cyclins plays a key role in regulating the biological activity of cyclin-dependent kinase-cyclin complexes (Li, 1997).

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of a linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. When it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhances their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase (Diehl, 1997b).

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts. About 20% of the total cellular levels of cyclin D1 is found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein are found in the middle to late G1 phase of the cell cycle. Both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions are reactive to an anti-phosphotyrosine antibody, while only the latter is detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduces cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels is reduced by about 60%, as compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induces a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increase by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway (Scovassi, 1997).

GSK-3beta-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3beta could inhibit nuclear import. GSK-3beta-dependent phosphorylation is shown in this study to promote cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3beta, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division (Alt, 2000).

Transit of proteins between nuclear and cytoplasmic compartments occurs via nuclear pores. For cytoplasmic to nuclear translocation through the nuclear pore, proteins must first interact with soluble, cytoplasmic import factors (importins), followed by the docking of this complex to the cytoplasmic face of the nuclear pore. The mechanisms governing nuclear export are analogous, with nuclear export substrates binding to soluble, nuclear export factors (exportins). CRM1 is one such exportin that binds to leucine-rich nuclear export signals (NES). CRM1 likely mediates nuclear export of several cell cycle regulatory proteins, including cyclin B1, CDC25, and p27Kip1 , and the data presented here demonstrate that cyclin D1 should be added to this list (Alt, 2000).

The binding of CRM1 to a leucine-rich NES within the cargo is an early step in the nuclear export process. Because the exact spacing of the leucine/hydrophobic residues within each substrate is subject to variation, it can be difficult to define an NES signal on this basis alone. Examination of cyclin D1 reveals two sequences that closely resemble characterized NES signals. The first is located within the 'cyclin box' from residues 87-94 (RFLSLEPL), and the second is located at the C terminus beginning with Thr-286 (TPTDVRDVDI). Deletion of either of these sequences alone is not sufficient to abolish CRM1-dependent nuclear-to-cytoplasmic relocalization of cyclin D1. The possibility remains that cyclin D1 may have more than one functional NES, as has been demonstrated for the adenomatous polyposis coli (APC) tumor suppressor. Accordingly, removal of any one NES would not be sufficient to abolish CRM1-mediated nuclear export. Alternately, D1-CRM1 binding may be mediated by a novel type of interaction. Further investigation of cyclin D1-CRM1 binding is clearly warranted (Alt, 2000).

The capacity of the cell to direct nuclear import of cyclin D1 during G1 phase and nuclear export during S phase suggests that movement of cyclin D1 must be considered bi-directional. Consequently, cyclin D1 subcellular distribution is in fact an equilibrium established between nuclear import and nuclear export. The observed overexpression of nuclear cyclin D1 in a variety of cancers suggests that its overexpression provides cells with a distinct growth advantage. The development of mammary hyperplasia and cancer in mice bearing a cyclin D1 transgene is consistent with this hypothesis. Yet, cell lines engineered to overexpress wild-type cyclin D1 remain growth factor dependent and fail to exhibit overt signs of cell transformation. Thus, cyclin D1 overexpression by itself is not sufficient to promote uncontrolled cell growth. In contrast, the constitutively nuclear cyclin D1-T286A protein promotes cell transformation. This single-point mutation alters two known properties of cyclin D1: (1) D1-T286A is resistant to polyubiquitination, resulting in an extended half-life relative to wild-type cyclin D1; (2) this mutation inhibits the nuclear export of cyclin D1 during S phase. The steady-state levels of wild-type versus mutant cyclin D1 are comparable in the respective cell lines, resulting in roughly equivalent levels of D-type cyclin kinase activity. Therefore, increased protein stability is likely not the primary contributor to cell transformation by D1-T286A. Rather, the oncogenicity of D1-T286A correlates with its constitutive nuclear localization (Alt, 2000).

These observations highlight the possibility that the tolerance of cells to wild-type cyclin D1 may reflect their ability to 'restrain' cyclin D1 through regulation of its subcellular localization. In fact, the subcellular localization of overexpressed wild-type cyclin D1 mirrors that of endogenous cyclin D1, being nuclear in G1 and cytoplasmic during S phase. This is significant given that the critical functions of cyclin D1 are nuclear.

What then is the mechanism whereby D1-T286A promotes cell transformation? The cyclin D1/CDK4 complex is thought to regulate cell cycle progression via its propensity to promote Rb phosphorylation. However, the presence of D1-T286A/CDK4 complexes in the nucleus during S phase suggests that it could function downstream of Rb. One possibility is that the inability of a cell to eliminate nuclear cyclin D1 during S phase perturbs the fidelity of DNA synthesis, perhaps the result of phosphorylation of proteins that are normally substrates of the CDK2 kinase. High-fidelity DNA replication depends on appropriate timing and activation of the DNA replication machinery. The cyclin E/CDK2 and cyclin A/CDK2 kinases play an active role in regulating and activating DNA synthesis. Phosphorylation of these substrates by the D1-T286A/CDK4 kinase could result in the premature or inappropriate activation of these CDK2 substrates. Implicit to this model is the possibility that D1-T286A-dependent transformation occurs downstream of Rb phosphorylation and thus may be Rb independent. The capacity of cyclin D1-dependent kinase to promote S-phase entry independent of Rb phosphorylation is consistent with this notion. Alternately, the constitutively nuclear D1-T286A/CDK4 complex may perturb the normal temporal and spatial course of the Rb phosphorylation/dephosphorylation cycle. The growth-suppressive activity of Rb is intimately associated with its ability to bind to and inactivate a large number of growth-promoting proteins, including members of the E2F family of transcription factors, Myc family proteins, and even proteins that directly participate in DNA replication such as MCM7. Phosphorylation of Rb triggers the release of these bound proteins, thereby permitting activation of their growth-promoting functions. Dephosphorylation of Rb during mitosis is an important step in the re-activation of Rb and likely serves to reset the cell cycle clock for the ensuing G1 interval. Perturbation of either phosphorylation or dephosphorylation of Rb through overexpression of D1-T286A would likely perturb the critical temporal and spatial timing of Rb-dependent growth control, resulting in neoplastic growth (Alt, 2000).

Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. These Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allow for the sustained activation of MEK, and this effect is mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with myosin light chain kinase inhibitor ML-7 leads to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase leads to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocks sustained ERK signaling, but only Rho kinase inhibition allows for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), are not affected by inhibition of MLCK or Rho kinase. Overall, these results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1 (Roovers, 2003a).

The Rho-Rho kinase pathway controls cyclin D1 expression by preventing its early G1 phase induction in response to Rac and/or Cdc42, thus increasing its dependence on ERK signaling and actin stress fiber formation. The Rho kinase effector LIM kinase (see Drosophila LIM-kinase1) is responsible for this effect. Surprisingly, inhibition of Rac-dependent cyclin D1 expression by LIM kinase is independent of both cofilin phosphorylation and actin polymerization. Instead, specific mutation of its nuclear localization and export sequences show that LIM kinase acts in the nucleus to suppress Rac/Cdc42-dependent cyclin D1 expression. These results therefore describe an unexpected role for LIM kinase that requires nuclear translocation. The effect of nuclear LIM kinase on cyclin D1 expression ultimately regulates the duration of G1 phase and the degree to which G1 phase progression depends on actin stress fiber formation and imposition of cellular tension (Roovers, 2003b).

G1 phase progression in mammalian cells is mediated by the activities of cyclin D-cdk4 (or cdk6) and cyclin E-cdk2. The activation of these enzymes is regulated by a complex interplay of signaling pathways that reflect conditions in the extracellular environment. For example, the induction of cyclin D1, typically the rate-limiting step in the activation of cdk4/6, involves cooperative signaling by receptor tyrosine kinases (RTKs; receptors for many mitogenic growth factors), integrins (receptors for extracellular matrix proteins), and the actin cytoskeleton. At least in most fibroblasts, cyclin D1 is induced in mid-G1 phase (9 hr after mitogen stimulation of quiescent cells), and this mid-G1 phase induction requires sustained (5-6 hr) ERK activity. RTKs, integrins, and actin stress fibers are jointly required to sustain the ERK signal long enough to induce cyclin D1 (Roovers, 2003b).

Cyclin D1 can also be induced by Rac and/or Cdc42 in an ERK-independent manner. Rac/Cdc42-dependent induction of cyclin D1 requires RTK and integrin signaling, but it is independent of stress fiber formation. In fact, if cyclin D1 is induced by Rac/Cdc42, then all of G1 phase progression in fibroblasts can occur in the absence of stress fibers and the consequent imposition of cellular tension. Rac/Cdc42 signaling also results in an early G1 phase induction of cyclin D1 (3 hr after mitogenic stimulation of quiescent cells), and this premature induction leads to a correspondingly early activation of cdk4 and cdk2, as well as a several hour decrease in the duration of G1 phase as cells leave quiescence. Thus, the choice of signaling pathways used to induce cyclin D1 (sustained ERK versus Rac/Cdc42) has at least two distinct consequences for cell cycle progression through G1 phase (Roovers, 2003b).

Rho kinase determines whether cyclin D1 is induced by sustained ERK or Rac/Cdc42. Rho kinase is required for sustained ERK signaling because it promotes stress fiber formation and integrin clustering/signaling in growth factor-treated cells. Rho kinase also suppresses Rac/Cdc42-dependent cyclin D1 induction downstream of GTP-loading. This inhibitory effect of Rho kinase on Rac/Cdc42 signaling maintains the mid-G1 phase, ERK-dependent induction of cyclin D1 that is typically seen in fibroblastic cells (Roovers, 2003b).

Rho kinase is best known as a regulator of actin stress fibers through its stimulatory effects on contractility and actin polymerization. Rho kinase promotes contractility by inhibiting myosin light chain (MLC) phosphatase and by direct phosphorylation of MLC itself. Rho kinase promotes actin polymerization by activating LIM kinase (LIMK) and phosphatidylinositol 4-phosphate 5-kinase. Its effect on LIMK is the best understood: Rho kinase activates LIMK1 and LIMK2 by phosphorylating T508 and T505, respectively, which in turn catalyze the inactivating phosphoryation of cofilin on S3. Although exceptions exist, cofilin typically promotes actin depolymerization, so its inactivation by the Rho kinase-LIMK pathway stimulates actin polymerization. The combined effects of Rho kinase on MLC and LIMK phosphorylation result in stress fiber formation. Note, however, that mDia (a Rho kinase-independent effector of Rho) and PAK (an effector of Rac and Cdc42) also contribute to actin polymerization. Besides regulating the kinetics of ERK activation, the polymerization of actin that is associated with stress fiber formation can directly regulate gene expression because a subset of SRF-dependent genes is strongly stimulated by the consequent depletion of the g-actin pool (Roovers, 2003 and references thereinb).

In contrast to its well-characterized effects on stress fiber formation, the mechanism by which Rho kinase suppresses Rac/Cdc42 signaling to cyclin D1 remains completely unexplored. LIMK is the effector that suppresses Rac/Cdc42 signaling to cyclin D1. Surprisingly, the suppressive effect of LIMK on Rac/Cdc42-mediated cyclin D1 induction is independent of cofilin (its only characterized substrate) and actin polymerization (its only characterized effect). Moreover, specific mutation of its nuclear localization and export sequences show that LIM kinase acts in the nucleus to suppress Rac/Cdc42-dependent expression of cyclin D1. Thus, in addition to identifying the Rho kinase effector that suppresses Rac/Cdc42-signaling to cyclin D1, these studies reveal a specific role for LIMK in the nucleus (Roovers, 2003b).


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.