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CDK inhibitors, cell cycle arrest and differentiation (part 1/2) Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, overlapping chromosomal deletions were identified that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants reveals that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. These findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses (Fukuyama, 2003).
Mice lacking the imprinted Cdk inhibitor p57(KIP2) have altered cell proliferation and differentiation,
leading to abdominal muscle defects, cleft palate, endochondral bone ossification defects with
incomplete differentiation of hypertrophic chondrocytes, renal medullary dysplasia, adrenal cortical
hyperplasia and cytomegaly, and lens cell hyperproliferation and apoptosis. Many of these phenotypes
are also seen in patients with Beckwith-Wiedemann syndrome, a pleiotropic hereditary disorder
characterized by overgrowth and predisposition to cancer, suggesting that loss of p57(KIP2) expression
may play a role in the condition (Zhang, 1997).
C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage (Hong, 1998).
To begin an analysis of cki-1 promoter
activity a green fluorescent protein reporter under the control of cki-1 regulatory sequences was created. The resulting transcriptional fusion was
transformed into worms and attached to a chromosome. The term cki-1::GFP is used in reference
to this construct. Embryonic expression of cki-1::GFP begins at the comma
stage in the pharyngeal primordium as pharyngeal muscles
begin terminal differentiation. cki-1::GFP
is highly expressed in most cells during late embryogenesis,
when cells are either differentiating or undergoing cell cycle
arrest prior to hatching. At hatching, and in L1 animals
maintained in the absence of food, cki-1::GFP is detected in
specific identified cells. The expression in these cells
fades after feeding, when cell division resumes. Throughout
postembryonic development, cki-1::GFP is expressed with a
temporal and cell type-specific pattern that corresponds to
developmental patterns of cell cycle progression. Expression is also strong in many postmitotic
neurons and muscle cells. cki-1::GFP expression tends to be
stronger in newly differentiated cells and then gradually
decreases. cki-1::GFP is also expressed in dauer larvae (Hong, 1998).
In larvae that hatch in the absence of food, cells are arrested in
G1, and elevated levels of cki-1::GFP expression persist until
animals begin feeding and cells reenter the cell cycle. In
starved cki-1(RNAi) L1 larvae, certain postembryonic blast
cells escape the starvation-induced G1 arrest and enter the cell
cycle prematurely. Perhaps the transition between embryonic
and larval development coincides with a reduction in
maternally supplied growth factors, causing an increase in cki-1
expression and consequent cell cycle arrest. It should
be noted that cells in cki-1(RNAi) starved L1 larvae undergo
only limited rounds of division, suggesting additional
constraints, besides the CKI-1 level, on cell cycle progression
in the absence of food. The observations implicating cki-1 in a larval cell division
checkpoint are analogous to the stimulation of p27KIP levels
observed after serum withdrawal in fibroblasts, and the reduced dependence on growth factors observed
in p27-/- mammalian cells. These results suggest a general role for CKIs in
mediating growth factor responses (Hong, 1998).
Although developmental control of the cell cycle is universal,
few genetic pathways have been directly implicated in
controlling the developmental signals that influence cell cycle
progression. In C. elegans, a genetic pathway that includes gon-2 mediates feeding-dependent
postembryonic cell cycling in somatic gonadal lineages. gon-2
encodes a TRP channel protein that may mediate the
transduction of signals for gonadal growth (R. Biron, Y. Sun,
D. Church and E. Lambie, personal communication to Hong, 1998),
resulting directly or indirectly in cki-1 down-regulation. cki-1 appears to be activated in dauer larvae to affect cell cycle arrest, as one consequence of a complex signaling
cascade that includes daf-7 (TGF-beta), daf-2
(insulin-related receptor), and daf-12 (nuclear hormone receptor). The current experiments do not distinguish between direct or indirect regulation of cki-1 by specific components of the dauer regulatory pathway. Interestingly, the possible role of cki-1 as a downstream effector of TGF-beta (daf-7) signaling is analogous to the role of p27KIP as a mediator of TGF-beta-induced epithelial cell division arrest. However, daf-7 does not activate (but rather antagonizes) dauer arrest, suggesting that the effect of TGF-beta/daf-7 on downstream CKIs may be cell-type specific (Hong, 1998).
A pathway of heterochronic genes, including lin-14 and
lin-28 and their repressor, lin-4, controls the timing of G1 progression and
developmental competence in VPCs. The heterchronic gene lin-14 controls the temporal sequence of developmental events in the Caenorhabditis elegans
postembryonic cell lineage. It encodes a nuclear protein that normally is present in most somatic cells of late embryos and L1
larvae but is absent at later stages. Mutations in the heterochronic gene lin-28 of C. elegans cause precocious development where diverse events specific to
the second larval stage are skipped. lin-28 encodes a cytoplasmic protein with a cold shock domain and retroviral-type (CCHC) zinc finger motifs, consistent with a role for LIN-28 in posttranscriptional regulation. The 3'UTR of lin-28 contains a conserved element that is complementary to the 22 nt regulatory RNA product of lin-4 and that resembles
seven such elements in the 3'UTR of the heterochronic gene lin-14. Both lin-4 activity and the lin-4-complementary element (LCE) are necessary for stage-specific regulation of lin-28.
The observation that cki-1::GFP expression is reduced
specifically in the VPCs of lin-14(0) animals strongly
suggests that cki-1 is activated in VPCs by lin-14. lin-14 and
lin-28 expression are mutually interdependent, so it cannot be said whether cki-1 is a direct target of either (or both) of lin-14 or lin-28. Since the cki-1(RNAi)
phenotype results in precocious VPC division, but is not identical to the lin-14(0) VPC defect, it is conclude that lin-14 acts via cki-1 to keep VPCs in G1 during the first half of
the L2 stage, but acts independently of cki-1 to maintain VPC G1 through the end of L2. Further molecular and biochemical analyses are required to determine precisely how
cki-1 is targeted for regulation by the heterochronic pathway in VPCs (Hong, 1998).
Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and
differentiation in several tissues. Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdk activity to
activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, it is shown that the Cdk inhibitors p27(KIP1) and p57(KIP2)
function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27(KIP1) and
p57(KIP2) are critical terminal effectors of signal transduction pathways that control cell differentiation (Zhang, 1998).
The phenotypes observed in p27/p57 mutant lenses are reminiscent of those seen in Rb-deficient lenses, consistent with the biochemical roles of cyclin-dependent kinase inhibitors CKIs as activators of Rb.
Because hypophosphorylated Rb plays a critical role in differentiation, it is likely that the inability of lens
fiber cells to differentiate in p27/p57 mutants reflects increased Rb phosphorylation and inhibition of its
differentiation-promoting function. However, two significant differences exist between the phenotypes of
the Rb versus p27/p57 double mutants. The first difference has to do with the extent of overproliferation as assessed by BrdU
incorporation: this appears to be significantly greater in p27/p57 mutants than in Rb mutants. This may
reflect the fact that these two CKIs function not only upstream of Rb by blocking cyclin D/Cdk4 activity
but also function downstream of Rb by blocking cyclin E/Cdk2-mediated S-phase entry. Alternatively,
the increase in Cdk activity due to CKI loss may result in inactivation of additional Rb-family members
such as p130 and p107, thereby producing a more severe proliferation defect than Rb loss alone. Thus,
proliferation of lens fiber cells lacking Rb may be limited because of the action of p27 and p57 on
Cdks. The second major difference is that the rates of apoptosis in CKI-deficient lenses are much lower
than those in Rb-deficient lenses and are similar to the rates seen in Rb/p53 double mutant lenses. Rb is
required to establish the transcriptional program that brings about differentiation of multiple cell types but
has also been shown to inhibit apoptosis during myoblast differentiation and in
other situations. Thus, low rates of apoptosis in p27/p57-mutant
lenses may reflect an antiapoptotic role for Rb. If the absence of p27 and p57 result in the inactivation of
Rb to such an extent that it phenocopies the differentiation defect of the Rb null mutant lenses, why the
difference in apoptosis rates? There are several plausible explanations for this difference: (1) Rb could
have an antiapoptotic function that is not regulated by Cdk phosphorylation and therefore would not be
altered by CKI loss. (2) Even in the absence of the CKIs, there may be residual Rb activity such that
apoptosis-inhibiting functions of Rb are largely intact. Even in the absence of CKIs, there is likely to be
residual regulation of Rb if Cdk activity is still cyclical. In contrast, an Rb null mutant cell would
constituively derepress all Rb-regulated genes such as E2F1, an apoptosis-inducing gene and might display a more severe
phenotype for this reason. (3) It is also possible that CKI mutant cells have higher Cdk activity levels
and these act to prematurely inactivate E2F1 function, thereby balancing the apoptotic-inducing consequences of inactivating Rb. The fact that the
apoptosis rates of the p27/p57 double mutants are similar to the rates observed in the Rb/p53 double
mutant mice is consistent with interfering with E2F1 function because
apoptosis caused by Rb loss is partially mediated by E2F1 and E2F1-mediated
apoptosis is p53-dependent. CKIs are the ultimate effectors of signal transduction pathways intended to bring about cell cycle arrest:
the patterns of expression during embryonic development suggest that particular CKIs play important roles
in terminal differentiation in a tissue-specific manner. However, the fact that mice lacking single CKIs
display surprisingly few developmental phenotypes has brought into question the essential nature of CKIs
for cell cycle arrest and differentiation. It has been shown here that two CKIs, p57 and p27, cooperate
to control proliferation and differentiation in multiple tissues and reiterate the critical importance of CKIs to
cell cycle control during development (Zhang, 1998).
Cell-cycle arrest is thought to be required for differentiation of muscle cells. However, the molecules
controlling cell-cycle exit and the differentiation step(s) dependent on cell-cycle arrest are poorly
understood. Two Cdk inhibitors, p21(CIP1) and p57(KIP2), redundantly control
differentiation of skeletal muscle and alveoli in the lungs. Mice lacking both p21 and p57 fail to form
myotubes, display increased proliferation and apoptotic rates of myoblasts, and display endoreplication
in residual myotubes. This point of arrest during muscle development is identical to that of mice lacking
the myogenic transcription factor myogenin, indicating a role for cell-cycle exit in myogenin function.
Expression of myogenin, p21, and p57 is parallel but independent, and in response to differentiation
signals, these proteins are coordinately regulated to trigger both cell-cycle exit and a dependent
muscle-specific program of gene expression to initiate myoblast terminal differentiation and muscle
formation (Zhang, 1999).
Interphasic nuclear organization has a key function in genome biology. p21WAF-1,
by influencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a
major role as a nuclear organizer. Transfection of U937 tumor cells with p21WAF-1 results in
expression of the HUMSIAH (human seven in absentia homolog), Rb, and Rbr-2 genes, and
strong suppression of the malignant phenotype. This gene is activated by wild-type p53 at the early onset of programmed cell death, during physiological apoptosis and tumor suppression. p21WAF-1 drastically modifies the compartmentalization
of the nuclear genome. DNase I genome exposure and fluorescence in situ hybridization show,
respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of
chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide
potentially significant perspectives for the understanding of tumor suppression (Linares-Cruz, 1998).
In the fly, Cyclin E is required for induction of S-phase in a process that does not require transcription. Ectopic cyclin E can bypass the S-phase requirement for E2F in epidermal cells arrested in G1 at stage 17. A similar function for cyclin E is found in Xenopus, where the p21 cyclin-dependent kinase inhibitor prevents DNA replication. This inhibition can be restored by addition of cyclin E to p21-arrested extracts (Strausfeld, 1994).
In the retina and the cortex, there is a well-defined histogenetic order of laminar fates, but investigations into cell cycle aspects of fate decision in these tissues
have led to a variety of conclusions about when fate is determined during the cell cycle. For example, the laminar
fate of cortical progenitor cells must be acquired by S phase of final cell division. Retinal
progenitors lose their competence to respond to an amacrine inhibitory factor, and motor neuron precursors lose their competence to respond to Sonic
Hedgehog, once they enter M phase in their final cell division. The mechanisms that relate the cell
cycle to such determination events are unknown. p27Xic1, a member of the Cip/Kip family of Cdk inhibitors, besides its known function of inhibiting cell division, induces Müller glia from retinoblasts. This novel gliogenic function of p27Xic1 is mediated by part of the N-terminal domain near (but distinct from) the region that inhibits cyclin-dependent kinases. Cotransfections with dominant-negative and constitutively active Delta and Notch constructs indicate that the gliogenic effects of p27Xic1 work within the context of an active Notch pathway (Ohnuma, 1999).
How does p27Xic1 induce the Müller cells, the last cell type to be differentiated, in a normal differentiation scheme? An intrinsic timer model is proposed. The cell fate decision to become Müller cells is influenced by endogenous expression of p27Xic1, which gradually increases during retinal cell development, competing with other differentiation factors, so that the last cells to be born generally have the most p27Xic1. If the Müller cell determining activity of p27Xic1, expressed at high levels, overcomes the activity of other determinants, these cells will become committed to a Müller fate. Such a situation is reached early in p27Xic1 lipofected retinas, resulting in an increase in the ratio of Müller glia. A similar model has been proposed to explain the function of p27Kip1 on oligodendrocyte differentiation. It is particularly revealing that the influence of p27Xic1 on Müller cells, the last cell type in the retina, is gated by the activity of the Delta/Notch pathway, which inhibits the determination of early cells types. The Notch pathway, the cell cycle, and cell determination are thus interconnected by the functions of p27Xic1 (Ohnuma, 1999).
The cyclin-dependent kinase inhibitor protein p27
Kip1 is necessary for the timing of cell cycle withdrawal, which precedes
terminal differentiation in oligodendrocytes of the optic nerve. Although p27
Kip1 is widely expressed in the developing central nervous system, it is not known whether this protein has a similar role in neuronal differentiation. To address this
issue, the expression and function of p27Kip1 was examined
in the developing retina, a well-characterized part of the
central nervous system. p27Kip1 is expressed in a pattern coincident with the onset of differentiation of most retinal cell types. In vitro analyses show that p27Kip1 accumulation in retinal cells correlates with cell cycle withdrawal and
differentiation, and when overexpressed, p27Kip1 inhibits proliferation of the progenitor cells. Furthermore, the histogenesis
of photoreceptors and Muller glia is extended in the retina of p27Kip1-deficient mice. Finally, the adult retinal
dysplasia in p27Kip1-deficient mice was examined with cell-type-specific markers. Contrary to previous suggestions that the dysplasia is
caused by excess production of photoreceptors, it is suggested that the dysplasia is due to the displacement of reactive Muller
glia into the layer of photoreceptor outer segments. These results demonstrate that (1) p27Kip1 is part of the molecular mechanism that controls the decision of multipotent central nervous system progenitors to withdraw from the cell cycle, and (2) postmitotic Muller glia have a novel and intrinsic requirement for p27Kip1 in maintaining their differentiated state (Levine, 2000).
A cell-intrinsic timer helps control when rodent oligodendrocyte precursor cells (OPCs) exit the cell cycle and terminally
differentiate when cultured in platelet-derived growth factor (PDGF) and thyroid hormone (TH). There is evidence that the cyclin-dependent kinase inhibitor (CKI) p27/Kip1 (p27) is a component of this TH-regulated timer, since it increases as OPCs proliferate and is required for the timer to operate accurately. Evidence is provided that another CKI, p18/INK (p18), may also be a component of the timer: it increases as OPCs proliferate, and its overexpression in OPCs accelerates the timer, causing the cells to differentiate prematurely. The overexpression of p27 accelerates the timer, and the increases in both p27 and p18 that occur in proliferating OPCs are controlled posttranscriptionally. By contrast, the overexpression of either p18 or p27 in OPCs proliferating in PDGF and the absence of TH greatly slows the cell cycle but fails to accelerate the spontaneous differentiation that normally occurs independently of TH (Tokumoto, 2002).
Cells deprived of serum mitogens will either undergo immediate cell cycle arrest or complete
mitosis and arrest in the next cell cycle. The transition from mitogen dependence to mitogen
independence occurs at the so-called restriction point, in mid-to late G1 phase of the cell cycle. Murine Balb/c-3T3 fibroblasts deprived of serum mitogens accumulate the
cyclin-dependent kinase inhibitor p27Kip1. This is correlated with inactivation of
essential G1 cyclin-cdk complexes and with cell cycle arrest in G1. The ability of specific
mitogens to allow transit through the restriction point parallels their ability to down-regulate
p27. Antisense inhibition of p27 expression prevents cell cycle arrest in response to mitogen
depletion. Therefore, p27 is an essential component of the pathway that connects mitogenic
signals to the cell cycle at the restriction point (Coats, 1996).
Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in the contact inhibition of the
growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell-cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent
growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, these cells were transfected with an
exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells results in tighter adhesion of multicellular
spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin
expression also results in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase
inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also show increased levels of p27
bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in
three-dimensional culture simultaneously prevents adhesion and stimulates proliferation of E-cadherin transfectants as well as a
panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in
E-cadherin-dependent growth inhibition, E-cadherin-positive cells were engineered to express inducible p27. By forcing expression
of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on
proliferation can be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a
major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27 (St. Croix, 1998).
Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21Cip1 arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21Cip1 independent of cell cycle arrest. Rapamycin induces p21Cip1 binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21Cip1 (Huang, 2003).
Continued: CDK inhibitors, cell cycle arrest and differentiation part 2/2
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