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The Albert Einstein Cancer Center (R.G.P., C.A., A.T.R., R.J.L.), Department of Developmental and Molecular Biology and Department of Medicine, Department of Anatomy and Structural Biology (J.E.S.), Albert Einstein College of Medicine, Bronx, New York 10461; and Center for Molecular Medicine (A.A.), and Division of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington, Connecticut 06030
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Abstract |
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 Top Abstract I. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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The normal mammalian cell cycle consists of several temporally distinct
phases (Fig. 1
). One current model of
the cell cycle envisages transitions between different cell cycle
states by passage through checkpoints (1, 2, 3, 5, 8) (Fig. 1). Examples
of these states are the initiation and completion of DNA replication
(S) phase and of cell division or mitosis (M). Between these phases are
gaps (G). One important checkpoint in mammalian cells is the
restriction point in late G1, also known as START in yeast.
This is the point at which the cell commits itself irrevocably to
another round of DNA replication. Passage through the restriction point
is promoted by a group of G1 cyclins, which include in
mid-G1, the D type cyclins, and in late G1,
cyclin E. These cyclins can heterodimerize with specific catalytic
subunits, the cyclin-dependent kinases (Cdks), to form holoenzymes.
Some substrates of these holoenzymes, which are inactivated upon
phosphorylation, are the retinoblastoma tumor suppressor protein, pRB
(retinoblastoma protein) (Fig. 2B) and
the related proteins, p130 and p107. It is thought that phosphorylation
and inactivation of pRB leads to progression through the restriction
point. The ability of the cyclin/Cdk holoenzymes to phosphorylate pRB
is inhibited by a family of small molecular weight proteins, known as
cyclin-dependent kinase inhibitors (CKIs) (Fig. 2A).
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II. Regulation of the Cell Cycle G1 Phase |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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The human cyclin D1 gene was cloned as an endocrine tumor oncogene in
human parathyroid adenomas. During structural analysis of the PTH gene,
a pericentromeric inversion was observed in chromosome 11 (22) (Fig. 3A). Cloning and sequencing of the
chromosomal breakpoint in these tumors revealed the presence of an
overexpressed gene, downstream of the PTH gene promoter (22). The PRAD1
cDNA (parathyroid adenoma 1) revealed structural homology to a class of
proteins previously identified in yeast known as cyclins (23). Cyclins
were known to regulate the first Gap phase (G1) in
nonmammalian cells. The PRAD1 gene product was thus the first putative
mammalian G1 cyclin cloned. Furthermore, the PRAD1 cDNA was
capable of rescuing a yeast mutant in its G1 CLN cyclins,
which suggested the PRAD1 gene may encode a functional cyclin now
called a D-type cyclin (24). Independently, the murine homolog of PRAD1
was cloned as a colony-stimulating factor-1 (CSF-1)-responsive gene
product (25).
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The 35-kDa cyclin D1 protein is encoded by 5 exons in the structurally
similar human and mouse genes (26). The cyclin D1 protein shares
structural homology to the other cyclins. The amino terminus of cyclin
D1 contains a motif Leu-X-Cys-X-Glu (where X represents any amino
acid). This motif, which is shared by the viral oncoproteins E1A,
simian virus (SV) 40 large T antigen, and papillomavirus E7, is
involved in binding the pRB pocket domain (Fig. 3B
). A region located
carboxy terminal to the pRB-binding domain is known as the "cyclin
box" because it is conserved between the known cyclins. The acidic
rich carboxy terminus of cyclin D1 inhibits myogenic helix loop helix
(HLH) protein function (27). An alternate splice form of cyclin D1
encodes a protein with an altered carboxy-terminal domain (CTD) (28).
The cyclin D1 protein is quite unstable, with a half-life of less than
20 min, with degradation occurring through ubiquitin
proteosome-mediated degradation (29). In quiescent cells, cyclin D1
protein levels are low. However, nuclear abundance increases as cells
progress through G1 phase (11). As cells pass into S phase,
cyclin D1 moves from the nucleus to the cytoplasm. Exclusion of cyclin
D1 from the nucleus is required for progression into S phase in human
fibroblasts (11). Proliferating cell nuclear antigen (PCNA), which is
required for DNA polymerase 
activity, binds cyclin D1 in the
nucleus. As cells enter S phase, cyclin D1 protein no longer binds PCNA
and is extruded from the nucleus (30).
Two other structurally related cyclins, D2 and D3, are also capable of heterodimerizing with Cdk4/6 and phosphorylating pRB in vitro. The amino acid identity between the human D-type cyclins is 53–63%. Cyclin D2 was mapped to chromosome 12 at 12p13 and cyclin D3 was mapped to 6p21. It is currently thought that each of the D-type cyclins subserve multiple functions, some of which are shared, such as phosphorylation of the pRB protein, and some of which are distinct. The mRNA distribution and expression profiles differ between the D-type cyclins. Steady state cyclin D2 mRNA levels, for example, peaked in late G1 (31) and for cyclin D3 peaked in S phase rather than early in G1 as seen for cyclin D1. Unlike the relatively ubiquitous distribution of cyclin D1 expression, the expression of cyclin D2 is somewhat more restricted with mRNA expressed abundantly in T lymphocytes and gonadal cells, although several different transformed cell lines also express cyclin D2 (25, 31, 32, 33).
Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with a G1 function in mammalian cells. mRNA levels for cyclin E peak later in G1 phase than cyclin D1. Mammalian cells express several isoforms of cyclin E protein. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase (34). The cyclin E-Cdk2 complex is maximally active at G1/S, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. The destruction of cyclin E is linked to ubiquitin-mediated degradation. Cyclin E phosphorylation is coupled to cyclin E turnover via site-specific phosphorylation, which acts as a signal for ubiquitination and proteasome processing (35, 36).
Cyclin D1 and cyclin E appear to subserve at least partially overlapping functions. pRB is phosphorylated at an overlapping subset of sites by these two distinct kinases (37). Several different scenarios may explain why two different kinases subserve this function. Phosphorylation of pRB at distinct sites may alter the ability of pRB to interact with a distinct subset of transcription factors or substrates (38). Alternatively, phosphorylation at distinct sites by both kinases may alter the epitopes of pRB required for full phosphorylation. It is clear, however, that the effect of cyclin D1 to promote cell cycle progression in cultured cells requires pRB, while the effect of cyclin E occurs independently of pRB (19, 39, 40). Thus, fibroblasts engineered to constitutively overexpress cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle (40, 41). Under certain circumstances, in cultured Rat-1 cells, cyclin D1, but not cyclin E, induced pRB phosphorylation, suggesting cyclin D1 and cyclin E promote G1 phase progression through different mechanisms (42). However, recent studies in NIH3T3 cells demonstrated that cyclin E can phosphorylate pRB and located a pRB-binding motif VxCxE (43). Mutation of the pRB-binding motif abolished the ability of cyclin E to promote S phase entry in NIH3T3 cells (43). It is clear that, in addition to pRB, alternate substrates for cyclin D/Cdk exist (below). Histone H1 is also phosphorylated well by cyclin E/Cdk2. Because cyclin E enhancement of G1 phase progression occurs independently of pRB (14, 40), a search has begun for alternate cyclin E/Cdk substrates. Recent studies have identified one new substrate for cyclin E/Cdk2, known as the NPAT protein (nuclear protein mapped to the ATM locus) (44). It is thought that phosphorylation of NPAT by cyclin E/Cdk2 may promote S phase entry.
Cyclin A plays an essential role in the progression through the cell cycle as a regulatory component of the Cdk2 and cdc2 kinases (1, 45). The mRNA and protein of cyclin A accumulate at the end of the G1 phase. Microinjection of immunoneutralizing antibodies to cyclin A or antisense expression vectors indicate a requirement for cyclin A in DNA replication (46). Complexed to Cdk2 in cooperation with cyclin E, cyclin A has been shown to trigger S phase entry of G1 nuclei from HeLa cells (47). Cyclin A has been shown to promote S phase entry by associating with transcription factors and by regulating target genes involved in cell growth regulation.
In normal human fibroblasts, cyclin A/Cdk2 forms a quaternary complex with p21Cip1 and PCNA and cyclin A forms associations with p107 and E2F transcription factors. Cyclin A/Cdk2 binds to the amino terminus of the E2F-1–3 transcription factors. The E2F multigene family contains six members, and preferential binding of cyclin A/Cdk2 to E2F-1–3 likely alters the repertoire of E2F transcriptional target genes. The E2F proteins bind to DNA with an heterodimeric binding partner from the DP protein family and activate gene transcription through the E2F enhancer sequence. This DNA-binding site was initially identified as an adenovirus AdE2a enhancer sequence and has since been identified in the promoter of a number of genes induced during G1/S phase transition. E2F-1 is phosphorylated by cyclin A/Cdk2, down-regulating its ability to bind DNA and activate transcription (48, 49, 50). The region of E2F-1 phosphorylated by cyclin A/Cdk2 is conserved among E2F proteins (E2F-1, E2F-2, -E2F-3), implying each of the protein’s binding activity may be regulated in this manner. E2F-1 mutants defective in cyclin A binding cause apoptosis and deregulated cell cycle progression, indicating the importance of E2F-1 phosphorylation-dependent inactivation (49, 50).
A number of other cyclins and Cdks have been identified and are touched on only briefly herein. The abundance of cyclin F fluctuates during cell cycle progression like cyclin A, peaking in G2 (51). Overexpression of cyclin F increases the proportion of cells in G2 phase (51). The expression of cyclin G (52) is induced upon nerve injury in the motor neurons during the early phase of the nerve regeneration process (53). A neuron-specific cyclin p35 has been described that is expressed in postmitotic neurons of the central nervous system (CNS) (54, 55). The heterodimeric partner for p35 is Cdk5 (55, 56). Mice lacking p35 develop abnormal lamination likely due to abnormal neuronal migration (57). The related cyclin p39 is also required for neurite outgrowth in a cultured hippocampal cell line model (58). Cyclin H and Cdk7 form a Cdk-activating kinase (CAK), which regulates activities of the cyclin/Cdk complexes and activity of the RNA Pol II CTD. Unlike many of the other cyclins the abundance of CAK does not change during cell cycle progression, although its activity and substrates may (discussed below). Mitotic entry is signaled by the accumulation of cyclin B-cdc2 (59). Although accumulating in S and G2 phases, the activity of cyclin B/cdc2 is inhibited by phosphorylation on Tyr-15 and Thr-14 by Wee1/Mik1/Myt1-related protein kinases. The Cdc25c phosphatase is stimulated to dephosphorylate T14/Y15 and activate Cdc2. Destruction of the B-type cyclins through a ubiquitin-dependent proteolysis is required for progression past anaphase (60, 61).
Thus, specific cyclins convey cell-type dependent developmental functions and regulate specific steps in progression through the cell cycle of dividing cells. Recent attention has been drawn to aberrations in function of these proteins in tumorigenesis. The normal function of these cyclins in the mature animal in nondividing cells, however, remains to be fully understood.
B. The Cdk-inhibitory proteins
Activity of the holoenzyme containing cyclin D1 (cyclin D1/Cdk4
and cyclin D1/Cdk6) referred to here as cyclin D1 kinase
(CD1K), is modulated in vitro by the
Cdk-regulatory proteins (62), which are divided into two broad
categories (Fig. 2). The first family, the Ink4s (p16Ink4a,
p15Ink4b, p18Ink4c, and p19Ink4d),
inhibit specifically Cdk4 or Cdk6 and contain a set of highly conserved
ankyrin ring motifs (Fig. 2A). The second group include the Cip/KIP
family (p21Cip1, p27Kip1, and
p57Kip2), which share partial structural homology and
possess the ability to inhibit cyclin/Cdk complexes. All CKIs can cause
G1 arrest when overexpressed in transfected cells (reviewed
in Ref. 2). The expression and subcellular distribution of the CKIs is
regulated by hormones in a complex and cell-type specific manner (Fig. 2).
Overexpression of Ink4 proteins in vitro dissociates the
CD1K complexes and overexpression of p16Ink4a
is associated with reduced levels of cyclin D1-holoenzyme complexes
(Fig. 2B). The p16 (INK4a/ARF) locus encodes two alternate transcripts,
the p16Ink4a and the p19ARF (alternate reading
frame). Overexpression of either of these transcripts can induce cell
cycle arrest and block transformation (63, 64, 65). Transgenic mice
homozygously deleted of the p16 locus spontaneously developed a variety
of malignancies including lymphomas and fibrosarcomas (66), and
deletion of the p19ARF gene resulted in animals with a
similar phenotype (67). Unlike p16Ink4a, however, the cell
cycle arrest induced by p19ARF is p53 dependent.
p19ARF directly binds MDM2 and stabilizes p53 (65, 68). The
human equivalent to p19ARF, p14ARF, also binds
MDM2, resulting in stabilization of both p53 and MDM2 (69) (Fig. 2B).
Consistent with a negative feedback loop, p53 in turn inhibits
p14ARF (69) and p19ARF expression (70). Unlike
p53, however, p19ARF is not involved in the DNA damage
response (69). Together these studies suggest that the alternate
reading frames of the CdkN2A locus function to inhibit cell
cycle progression through two different mechanisms (71). The
evolutionary pressure to sustain the presence of these two tumor
suppressor genes, possibly as the result of gene duplication, in such
close proximity and given their vulnerability to co-deletion, remains
an area of considerable speculation. The possibility that each
transcript plays a role in hormonal signal transduction specificity has
yet to be explored.
The second class of inhibitors, the p21 family, includes
p21Cip1 (72, 73), p27Kip1 (74, 75), and
p57Kip2 (76, 77, 78). Although intially referred to as CKIs,
with biochemical properties supporting this notion (79), strong
evidence is accumulating that p21Cip1 and
p27Kip1 function as assembly factors in vitro
(80) and in vivo (81) to enhance cyclin D-dependent
activity. The p21 family members have a conserved region near the amino
terminus that is necessary and sufficient for binding to and inhibiting
Cdk2 (82, 83, 84). At certain concentrations, however, p21Cip1
does not inhibit cdc2 or Cdk2 kinase activity (85). Thus although
p21Cip1 inhibits Cdk4 and Cdk6 kinase activity, with an
inhibition constant (Ki) of 0.5–15 nM,
p21Cip1 is a poor inhibitor of cdc2/cyclin B in
vitro with a Ki of 400 nM (86). In
subsequent studies, functional subdomains of p21Cip1 were
defined with fine deletional analysis. For example, the binding of
p21Cip1 to cyclin E/Cdk2 and cyclin A/Cdk2 was shown to
involve both a Cdk2-binding domain and either an amino-terminal or
carboxy-terminal cyclin-binding domain whereas binding by cyclin D1
involved only the amino-terminal cyclin-binding domain (87) (Fig. 2A).
The carboxy-terminal region of p21Cip1 allows it to
associate with PCNA, a processivity subunit of the DNA polymerase
holoenzyme (82, 83, 84, 88, 89). Because the binding of p21Cip1
to PCNA inhibits the processivity of polymerization, but does not
affect excision repair, it was suggested that p21Cip1 may
serve to coordinate DNA replication with cell cycle progression (90).
The p21-/- embryonic fibroblasts derived from mice homozygously deleted of the p21Cip1 gene (91) exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed in p53-/- cells. In addition, p21-/- cells are significantly deficient in their ability to arrest in G1 in response to DNA damage and nucleotide pool perturbation. Although the p21 family of proteins inhibit the activity of Cdk2-containing complexes, recent studies demonstrated that the p21 family may also function under some circumstances as assembly factors to promote the association of Cdk4 with D-type cyclins (80, 92). The p21 family proteins enhance activity of the cyclin D1-kinase (CD1K) complex at low concentrations, while inhibiting CD1K complex at high concentrations.
The p27Kip1 protein was initially characterized as a protein homologous to the tumor suppressor p21Cip1. When overexpressed in fibroblasts, cell cycle progression was delayed and antisense p27Kip1 experiments resulted in mitogen-independent G1 phase progression, indicating a critical role for p27Kip1 in the establishment or maintenance of cellular quiescence (93, 94). The abundance of p27Kip1 is regulated primarily at a posttranslational level, although translational control also contributes. Thus, p27Kip1 mRNA levels remain relatively unchanged during the cell cycle transition; however, the addition of mitogens reduces p27Kip1 protein levels. For example, in quiescent 3T3 cells p27Kip1 protein levels decrease after mitogenic stimulation (95, 96, 97). The growth factor-mediated reduction in p27Kip1 protein levels is mediated primarily through enhanced ubiquitin-mediated degradation (98). It will be of interest to determine whether progression through the G1/S phase of the cell cycle is associated with induced expression of proteolytic enzymes, such as members of the caspase family. p27Kip1 abundance was also implicated as an important mediator of the cytostatic effects of rapamycin and cAMP (75, 99, 100), although these implications were not borne out in all studies using transgenic mice models (below).
The cyclin/Cdk complex to which p27Kip1 is bound determines its functional activity. p27Kip1 is found associated with cyclin E in a variety of cell types during quiescence (95, 99). When bound to cyclin D1/Cdk4, p27Kip1 may not be inhibitory (74, 99, 101, 102), whereas cyclin E/Cdk2 activity is inhibited by p27Kip1. It is thought that the removal of p27Kip1 from the cyclin E/Cdk complex is an essential step for S-phase entry. Through binding cyclin D1/Cdk4, p27Kip1 is sequestered from cyclin E/Cdk2, reducing its inhibition by p27Kip1 (74, 99, 101, 102).
The degradation of p27Kip1 upon mitogen stimulation is dependent upon prior phosphorylation. Expression of cyclin E/Cdk2 in murine fibroblasts was found to induce phosphorylation of p27Kip1 on T187 (103). A mutant of p27Kip1 at amino acid T187, to alanine, created a p27Kip1 protein that caused a G1 block resistant to cyclin E overexpression and whose level of expression was not modulated by cyclin E. Phosphorylation of p27Kip1 by cyclin E/Cdk2, enhanced degradation of p27Kip1, thereby promoting G1-S phase transition. Thus, the cyclin/Cdk complexes promote cell cycle progression in mammalian cells by also enhancing degradation of the CKI.
Together these studies suggest that p27Kip1 interacts with cyclin E/Cdk2 in two distinct ways. First, through tightly binding to cyclin E/Cdk2, p27Kip1 inhibits the ability of the holoenzyme to phosphorylate target substrates, such as pRB, or the NPAT protein (44). Second, p27Kip1 is phosphorylated by cyclin E/Cdk2, leading to the release and subsequent degradation of p27Kip1. The ability of p27Kip1 to either block Cdk activity or serve as a substrate for the Cdk appears to be determined by the ambient concentration of ATP within the reaction. At low ATP concentrations (<50 mM) p27Kip1 is primarily a CKI, but at ATP concentrations approaching physiological levels (>1 mM), p27Kip1 is more likely to be a substrate.
The homozygous deletion of the p27Kip1 gene resulted in animals with organomegaly, intermediate lobe pituitary tumors, and testicular and ovarian cell hyperplasia (104, 105). Because p27Kip1 expression was induced upon oligodendrocyte differentiation, the role of p27Kip1 in this process was closely examined in mice homozygously deleted of the p27Kip1 gene [p27 knockout (KO)]. The oligodendrocytes derived from p27 KO mice underwent prolonged proliferation and delayed differentiation, suggesting a role for p27Kip1 in promoting oligodendrocyte proliferation (106). In view of previous studies demonstrating the induction of p27Kip1 by transforming growth factor-ß (TGFß) and rapamycin, signaling by these agents was assessed in the p27 KO mice. Surprisingly, initial analysis of p27Kip1 KO T cells suggested their proliferation rates in response to TGFß and rapamycin were similar to those of wild type, suggesting that p27Kip1 was not required for the cytostatic effect of these two agents (104, 105). In subsequent experiments, however, exponentially growing p27-/- fibroblasts were found to have an impaired antiproliferative response to rapamycin. The inhibition of DNA synthesis by rapamycin was approximately half of control p27+/+ fibroblasts (107). In addition, the p27-/- T lymphocytes were 15- to 30-fold more sensitive to the growth-inhibitory effect of rapamycin (107). The different results obtained by these investigators may be due to methodological differences, but together provide support for the role of p27 in rapamycin-dependent inhibition of cellular proliferation. The p27Kip1 gene product is not a significant source of specific selected inactivating mutations, however, raising suspicions that this locus may not encode a human tumor suppressor gene. The results of studies exposing p27Kip1 KO mice to carcinogenic agents or matings to transgenic tumor-prone animals may provide important insights into the potential tumor suppressor function of the p27Kip1 gene product.
p57Kip2 was also cloned as a protein related to p21Cip1 and p27Kip1. Overexpression of p57Kip2 was found to arrest cells in G1 (77). Both the amino-terminal cyclin/Cdk binding domain and the carboxy-terminal PCNA binding domains of p57Kip2 were required for full antimitogenic activity and inhibition of cellular transformation much like p21Cip1 (78). Unlike p21Cip1, however, p57Kip2 was not regulated by p53. Homozygous deletion of the murine p57Kip2 allele by one group of investigators resulted in animals with abnormal endochondral ossification (108), while other investigators observed increased prenatal growth, adrenal cortical hyperplasia, and cytomegaly (109), features found in patients with Beckwith-Wiedemann syndrome. The pituitary tumors, together with the testicular and ovarian cell hyperplasia in the p27Kip1 KO mice and the adrenal cortical hyperplasia and cytomegaly in the p57Kip2 KO mice, launched an excited search for a role of these proteins in the regulation of normal function in these endocrine tissues (below).
Since the original cloning of the CKI proteins, it has become clear that the abundance and activity of these proteins are regulated in a complex manner by hormonal stimuli. Frequently the overexpression of the protein may be sufficient to induce cell cycle arrest, but equally frequently the overexpression of the protein, as observed during differentiation, has been shown to be insufficient to recapitulate the differentiated phenotype induced by a particular hormone. Furthermore, in most circumstances, homozygous deletion of the CKIs has not affected the signaling pathway that induced the abundance of the CKI. Together, these types of findings have led to an understanding that the precise timing and orchestration of the alteration in expression of the CKI, together with changes in their subcellular distribution and the formation of multimeric complexes in the cell, may be critical for the induction of the differentiated phenotype. Thus, although the instruments have been identified, the mechanisms of orchestration are poorly understood and are critical for the hormonal induction of the cellular phenotype.
C. Regulation of transcription by cell cycle-control proteins
As noted above, hormonal signals regulate the abundance of cell
cycle-control proteins. The altered levels of the cyclins/Cdks in turn
regulate downstream target genes. These genetic effects are conveyed by
altering the activity of transcription factors and/or by regulating the
basal transcription apparatus. Transcription factors that coordinate
hormone-mediated signal transduction can be regulated by the cyclin/Cdk
complexes through several different mechanisms (Fig. 4). These effects can be thought of as
either regulating transactivation function (Fig. 4, A and B), by
altering DNA binding (Fig. 4, C and D), or by altering protein/protein
interaction (Fig. 4, E and F).
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). Another example of this mechanism is the inhibition of the
DNA binding affinity of the E2F/DP transcription factors by cyclin
A/Cdk2 (Fig. 4B). This phosphorylation-mediated inhibition of E2F/DP
protein binding may be important in attenuating the transcriptional
enhancer activity of these complexes after S phase entry. Because E2F-1
overexpression is capable of inducing apoptosis in many cell types, the
inhibition of E2F/DP activity is likely important in cell survival.
Cell cycle-regulatory proteins can also affect the transactivation
function of specific transcription factors. The transcription factor
B-Myb, for example, which regulates activity of gene expression through
the Myb-binding site (PyAACG/TG), is phosphorylated and activated by
cyclin A/Cdk2 (111) (Fig. 4C). Several nuclear receptors are also
phosphorylated and activated by the cyclin/Cdks, including the
glucocorticoid and estrogen receptors (Section III.C below).
The glucocorticoid receptor, for example, is phosphorylated by cyclin
E/Cdk2 (112). Cell cycle-control proteins can also regulate gene
expression by promoting transcription factor binding through inhibiting
repressors. The HLH protein Id2 resembles the basic HLH proteins that
govern gene expression through E box sequences (Fig. 4D). The Id family
of proteins lack basic DNA-binding domains and therefore function as
transdominant negative regulators through E box sequences.
Phosphorylation of Id2 by cyclin E/Cdk2 or cyclin A/Cdk2 leads to a
reduction in the transcriptional repressor function of Id2 and enhances
activity through the E box sequence (113). As noted above, the
transcriptional activity of the E2F-1 factor is inhibited by cyclin
A/Cdk2 binding and phosphorylation (48, 49, 50).
Cell cycle-regulatory proteins can also affect signaling by forming
direct protein-protein interactions with known transcription factors.
For example, cyclin D1 can bind the estrogen receptor (ER), enhancing
the activity of the ER through a synthetic estrogen response element
(114, 115). Cyclin D1 also binds a Myb-related protein, DMP1, and
modulates its activity (116). Although the nature of the association
remains to be fully defined, cyclin D1 also inhibits v-Myb
transcriptional activity independently of Cdk activity (117) and cyclin
D1 interacts with HLH proteins to inhibit their activity, at least in
part, independently of the Cdk domain of cyclin D1 (27). Finally, a
growing list of transcription factors are regulated in their activity
by either the pRB or pRB-related proteins. Activity of the Sp1
transcription factor is induced by the pRB protein (118). Although the
mechanisms by which pRB regulates the activity of these other
transcription factors remain to be fully determined, other
transcription factors capable of binding to and/or regulated by pRB
include Elf-1, E2Fs, PU.1, c-Myc, Sp-1, MyoD, ATF-2, NF-IL6, and UBF
(reviewed in Refs. 119, 120). For the time being, these interactions
are loosely classified as pRB dependent (Fig. 4E).
Components of the basal transcription apparatus are also regulated by the cyclin/Cdks (121). The RNA polymerase II large subunit contains an essential CTD. The CTD is phosphorylated on a fraction of the RNA polymerase II molecules in vivo and is phosphorylated by the general transcription factor TFIIH in vitro. TFIIH is composed of at least nine subunits, which include Cdk7, cyclin H, and MAT1 (p36). These three subunits (Cdk7, cyclin H, MAT1) form a ternary complex, Cdk-activating kinase (CAK). The kinase activity of the TFIIH complex is directed toward the RNA Pol II CTD (122, 123). CAK preferentially phosphorylates Cdk2, whereas TFIIH preferentially phosphorylates the CTD (124). CAK also phosphorylates the threonine residue of the Cdk when cyclin D1-Cdk has formed a dimeric complex, and CAK is required for full cyclin D1/Cdk4 activity (1, 125). Recent studies have demonstrated that the cyclin H/Cdk7 complex preferentially phosphorylated Cdk2, whereas the multimeric complex that included cyclin H/Cdk7 and MAT1(p36) preferentially phosphorylated the RNA Pol II CTD (126). Thus, the substrate specificity of cyclin H/Cdk7, which is abundant within the cell, is dictated by the presence of MAT1 (124, 126). The CTD is also phosphorylated by a complex known as P-TEFb which includes Cdk9 and cyclin T (which includes T1, T2a, and T2b) (127).
RNA Pol II and Pol III-dependent transcription is reduced during mitosis in part through cell cycle-regulatory proteins (128, 129). RNA Pol III activity is increased in pRB-deficient mice and pRB targets TFIIB. As pRB contains regions of homology to TATA-binding protein and BRF, components of TFIIB, it is thought that pRB disrupts TFIIB by mimicking these two components (130). Mitosis-specific exclusion of transcription factors from chromatin may also be important in cell cycle-specific gene regulation. Phosphorylation and inhibition of poly (A) polymerase by cyclin B/Cdc2 may contribute to the overall decrease in RNA and protein synthesis that occurs during mitosis (129, 131, 132).
Cell cycle-regulatory proteins also affect the high molecular weight coactivator proteins of the p300/CBP family. The p300 protein was noted to undergo phosphorylation during cell cycle transition and during cell cycle exit in myocyte differentiation (133). These high molecular weight coactivators, (p300/CBP), interact with a broad array of nuclear receptors and general transcription factors, including members of the AP-1 family, which are involved in transducing hormone signals (134, 135). The general effects of coactivators on gene transcription are mediated in part through modulating histone structure either through direct histone acetylation activity of the transcription coregulator (136) or through the ability of the coregulator to recruit other proteins with histone-regulatory function (137, 138). The p300/CBP family also regulate cell cycle-regulatory transcriptional targets including p21Cip1 and cyclin D1 (139, 140, 141). To date, relatively little is known about the hormonal regulation of these associations, although these interactions provide important evidence for cross-talk between the cyclins and the basal transcription apparatus.
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III. Endocrine Regulation of Cyclin and Cdk-Regulatory Proteins |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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As noted above, the murine homolog of PRAD1 was initially cloned as a CSF-1-responsive gene product (25). Many studies of CSF-1 signaling were performed in the murine macrophage cell line, BAC1.2F5. In these cells CSF-1 induces dimerization and autophosphorylation of the CSF-1 receptor, as well as tyrosine phosphorylation of the p85 subunit of PI3-kinase, c-Cbl, SHP-1, and Tyk2. CSF-1 induces the activity of STAT proteins, Raf-1, MEK, ERKs (extracellular signal-regulated kinases), and S6 kinase. Although it is clear that the expression of c-fos, c-myc, and cyclin D1 are enhanced by CSF-1, there is controversy as to whether the induction of c-fos and c-myc both lie downstream of a Ras-signaling pathway (145). Overexpression of oncogenically activated Raf-1 protein in BAC1.2F5 macrophages induced cellular proliferation and immediate early gene expression without induction of ERK activity, demonstrating that ERK signaling was not required for cellular proliferation (146).
The introduction of ligand-activated human c-Fms receptor can also induce proliferation when ectopically expressed in other cell types, including NIH-3T3 cells. The use of dominant negative expression vectors in NIH3T3 cells helped map an important role for a proliferative pathway involving Ras, Ets proteins, and c-Myc (147). This pathway, although necessary, was not sufficient for CSF-1 signaling to promote proliferation (147). In NIH3T3 cells overexpressing a mutant c-Fms receptor, Y809F, the defective mitogenic signaling and c-myc expression could be rescued by cyclin D1 overexpression (148). Studies by Barone and Courtneidge (149) suggested that the induction of DNA synthesis by CSF-1 in NIH3T3 cells required both a Ras-dependent pathway that induced Fos and a Ras-independent pathway that induced c-myc transcription, suggesting that Fos and c-myc may function in parallel pathways.
b. Platelet-derived growth factor (PDGF).
The presence of
mitogens in platelets was first suggested by Balk (150) who observed
that serum-supplemented culture medium supported the proliferation of
chicken fibroblasts better than plasma-supplemented medium. Each of the
three possible isoforms of this dimeric peptide, PDGF, made of A and B
chains, have been isolated from natural sources including human
platelets. The most prominent activity of PDGF is to stimulate
proliferation. Growth factors have been categorized as either
competence- or progression-inducing agents (151). Transient exposure of
BALB/c3T3 cells to PDGF or fibroblast growth factor (FGF) induces a
state of competence to undergo DNA synthesis, while insulin-like growth
factors (IGFs), epidermal growth factor (EGF), and other substances
that are present in plasma permit progression through the
G1 phase of the cell cycle (152). In BALB/c-3T3 cells, two
arbitrary control points were described in the G1 phase:
the V-point, typically occurring 6 h into G1 phase —
a point at which the cell fails to progress further without the
addition of essential nutrients including amino acids, and the W point,
which occurs later at the G1/S border (153).
In keeping with the known effect of PDGF early in G1 phase, PDGF treatment of a variety of cell types was associated with an induction of cyclin D1 abundance (17, 154, 155) and CD1K activity (17, 155). In primary tracheal myocytes, PDGF induced cyclin D1 expression with a sequential induction of pRB phosphorylation (17). The induction of S phase traversal was also blocked by immunoneutralizing antibodies to cyclin D1 in these cells (17). The induction of cyclin D1 by PDGF in CHO cells was dependent upon Ras (155).
PDGF directly affects the abundance and subcellular localization of the CKI, p27Kip1. PDGF has been shown to reduce p27Kip1 levels in fibroblasts (96); however, the relative abundance of p27Kip1 in a p27Kip1/cyclin D1/Cdk4 multiprotein complex increased with PDGF treatment of fibroblasts and was associated with increased pRB kinase activity, consistent with a model in which p27Kip1 binding to specific partners is important in determining pRB kinase activity (156). PDGF treatment of BALB/c-3T3 cells resulted in both a reduction in p27Kip1 protein synthesis (156) and an increase in p27Kip1 protein degradation. In PDGF-treated BALB/c-3T3 cells, p27Kip1 protein associated with cyclin D1 increased as the abundance of p27Kip1 bound to cyclin E complexes decreased. The induction of p27Kip1 degradation by PDGF in Chinese hamster embryo fibroblasts was blocked by dominant negative mutants of Ras and RhoA (155). Because the induction of cyclin D1 by PDGF was dependent only upon Ras, these findings suggest distinct Ras family members regulate the effect of PDGF on distinct components of the cell cycle-regulatory apparatus.
c. Basic FGF (bFGF).
bFGF was the first member of a family of
structurally related proteins, isolated as a biochemically distinct
mitogen for 3T3 fibroblasts from pituitary gland crude extracts (157).
Together with the 11 other members of the FGF family (aFGF, int-2, hst,
FGF-5, FGF-6, KGF, AIGF, GAF, FGF-10, FGF-11, and FGF-12), bFGF has
been shown to exert mitogenic and differentiation inducing potential in
a wide variety of cell types. The high-affinity FGF receptors are
single-chain transmembrane proteins of the Ig-like receptor
superfamily. The binding of ligand induces dimerization, tyrosine
transphosphorylation, and phosphorylation and activation of a broad
array of substrates including c-Src and the F-actin-binding protein
cortactin, which correlates with the induction of DNA synthesis (158).
The induction of SHC/Grb2/SOS complex formation with Ras and
subsequently ERK activation has been linked to the induction of DNA
synthesis primarily in cell types that are involved in tissue repair
and revascularization such as fibroblasts, chondrocytes, myoblasts,
smooth muscle, and endothelial cells (159).
FGF can induce either proliferation or differentiation depending upon the cell type. It was anticipated, therefore, that distinct changes in the abundance or composition of cell cycle-control proteins may mediate these distinct responses. To examine this possibility, a number of different groups analyzed cell cycle-control protein abundance in FGF-treated cells. In fibroblasts, FGF treatment is associated with the induction of DNA synthesis and the induction of cyclin D1 levels. In MCF7 cells, FGF induces ERK activity with an initial mitogenic and subsequently a cytostatic signal. Cyclin D1 protein levels are initially induced in bFGF-treated MCF7 cells, and then increasing p21Cip1 levels contribute to reduced pRB phosphorylation and the inhibition of cellular proliferation (160). Differentiation of skeletal myoblasts in culture is negatively regulated by certain growth factors, including bFGF. Cyclin D1 was induced in myoblasts by bFGF, and stable expression of cyclin D1 inhibited C2C12 myoblast differentiation. Cyclin D1 inhibited myoblast differentiation by certain growth factors (161) and directly inhibited myogenic HLH proteins through an acidic region in cyclin D1’s carboxy terminus (27). Forced expression of cyclin D1, but not cyclins A, B or E, inhibited the ability of MyoD to transactivate muscle-specific genes and correlated with phosphorylation of MyoD. There are also pRB-independent mechanisms by which cyclin D1 inhibits growth factor-mediated differentiation (162). Transfection of myoblasts with CKIs p21Cip1 and p16Ink4a augmented muscle-specific gene expression in cells maintained in high concentrations of serum, suggesting that an active cyclin-Cdk complex suppresses MyoD function in proliferating cells (163). The direct binding and inhibition of MyoD by Cdk4, together with the ability of cyclin D1 to promote nuclear translocation of Cdk4, may contribute to the inhibition of myocyte differentiation by cyclin D1 (164). The possibility that the carboxyl termiunus of cyclin D1, which was required for inhibition of MyoD (27), may contribute to inhibition of differentiation through distinct mechanisms, including recruitment of histone acetylase-regulatory proteins (below), remains to be examined.
d. IGFs.
As noted above, IGF-I has been classified as a
progression factor in the cell cycle. In density-arrested BALB/c-3T3
cells made competent with PDGF treatment, IGF-I with EGF induced
G1 phase progression and initiation of DNA synthesis.
Subnanogram amounts of IGF-I with EGF allow progression to the V-point
but not S phase. Once cells have progressed to the V-point within
G1, IGF-I alone is sufficient to promote progression into
S-phase (165). Transcription of new mRNAs do not appear to be required
for IGF-I to function as a progression factor, and IGF-I is known to
stimulate posttranslational modifications of several proteins (166, 167).
IGF-I and IGF-II were isolated from human plasma as peptides with insulin- and somatomedin-like properties (168). Signaling by IGF-I requires a tetrameric type II IGF receptor. The requirement for IGF and the IGF receptor in normal growth was well illustrated by studies using targeted disruption of the IGF-I receptor. These mice were growth retarded, their size being 30% of wild-type control (169, 170). The mouse embryo fibroblasts (MEFs) derived from these animals also showed defective mitogenic capabilities, failing to grow in medium supplemented with growth factors that sustained the growth of wild-type cells (171). In the knockout MEFs, all phases of the cell cycle were delayed (171, 172), and this abnormality was reverted with reintroduction of the wild-type receptor (171). The IGF-I receptor is also required for EGF receptor function to exert its mitogenic effect (173).
IGF-I induces DNA synthesis in a broad array of cell types, and intracellular signaling involves activation of Ras via a Grb2-mSOS pathway. There is also a requirement for a docking protein IRS-1 in IGF signaling, which in turn regulates the activity of SH2 domain-containing proteins such as PI3 kinase, Grb-2, Nck, and Syp (174, 175). The induction of cell cycle progression by IGF-I treatment correlated with the induction of cyclin D1 mRNA levels in T47D breast cancer cells (176), in quiescent WI-38 cells (177), and MG63 osteosarcoma cells (178). The induction of cyclin D1 mRNA was, at least in part, regulated at the level of transcription, and the human cyclin D1 promoter linked to a luciferase reporter gene was induced by IGF-I in cultured cells (179). Recent studies suggested that IGF-I may also regulate cyclin D1 levels in vivo. During development, cyclin D1 is expressed in proliferating cells of the developing CNS (179, 180) and retina (181, 182). Protein-calorie malnutrition inhibits neonatal cerebellar growth and development, in part, through a reduction in IGF-I. Developmental delay induced by malnutrition was associated with reduced cyclin D1 protein and CD1K activity assessed using glutathione-S-transferase-pRB as a synthetic substrate (179, 180). Nutritional replacement induced cyclin D1 protein levels and kinase activity in conjunction with a partial restoration of normal cerebellar development (179, 180).
As with PDGF, IGF also regulates the abundance of p27Kip1. The induction of p27Kip1 protein levels that occurs with growth factor withdrawal is likely mediated through reduced ubiquitin-mediated degradation (98). Although the reduction in p27 by interleukin-2 was blocked by rapamycin (183), rapamycin did not block insulin-mediated inhibition of p27Kip1 levels in Swiss 3T3 cells (184), suggesting a rapamycin-independent pathway is involved in insulin-mediated DNA synthesis (184). Using a surrogate in vivo model of IGF-I deficiency, nutritional deprivation-induced cerebellar developmental arrest was associated with a reduction in p27Kip1 levels from the same tissues in which CD1K activity was decreased (180). These findings are consistent with the growing evidence from in vitro experiments that p21-related proteins may function to enhance pRB kinase activity (80).
e. EGF.
EGF, like IGF, functions as a progression factor. EGF
is a potent mitogen for cells of ectodermal and mesodermal origin and
is the prototype of a large superfamily of ligands that signal through
a family of related receptors. EGF binding leads to receptor
aggregation, autophosphorylation and phosphorylation of intracellular
substrates including phospholipase C-
, MAP kinase, and the Ras
GTPase-activating protein (GAP). Binding of EGF to the EGF receptor
(EGFR) can also induce heterodimerization with other members of the
EGFR family, including c-ErbB-2 (185).
EGF binding to the EGFR induces expression of immediate early
genes such as c-jun, c-fos, and c-myc
(186). In fibroblast cells induced to proliferate by EGF, cyclin D1
levels were increased (154). The induction of cyclin D1 protein was
preceded by the induction of cyclin D1 promoter activity in JEG-3 and
CHO cells (187). Dominant negative mutants of Ras, mitogen-activated
protein kinase (MAPK), and Ets inhibited EGF induction of the cyclin D1
promoter, mapping a likely signal transduction pathway from mitogenic
signals directly to the cell cycle-regulatory apparatus (187). The
EGF-related ligand TGF
in esophageal and colon cancer cells (188)
and activating mutants of c-ErbB-2 in MCF7 breast cancer cells (R. J.
Lee, C. Albanese, G. Watanabe, G. K. I. Haines, P. M. Siegel, W. J.
Muller, M. C. Hung, and R. G. Pestell, submitted) were also shown to
regulate cyclin D1 abundance and promoter activity. Point mutation in
the context of the native cyclin D1 promoter demonstrated that the
induction of cyclin D1 by c-ErbB-2 involved the E2F- and Sp1-binding
sites (R. J. Lee, C. Albanese, G. Watanabe, G. K. I. Haines, P. M.
Siegel, W. J. Muller, M. C. Hung, and R. G. Pestell, submitted), and
deletion of both the E2F and Sp1 sequences reduced TGF induction
(188).
In PC12 cells, EGF stimulated cellular proliferation and increased the levels of several cell cycle progression factors including Cdk2, Cdk4, and cyclin B1 (190). p27Kip1 levels were reduced by EGF in NIH3T3 cells, and dominant negative mutants of Ras blocked the EGF-induced down-regulation of p27Kip1, consistent with dual roles of Ras in early G1 and late G1/S (191). As with PDGF, EGF-mediated induction of cyclin D1 and inhibition of p27Kip1 abundance appears to be a common finding in several different cell types.
f. Nerve growth factor (NGF).
The differentiation and survival
of CNS and peripheral nervous system (PNS) neurons is influenced by
neurotrophic factors including NGF, brain-derived neurotrophic factor
(BDNF), ciliary neurotrophic factors, and the fibroblast growth
factors. One of the better characterized neurotrophins, NGF, is
required for the differentiation and survival of sympathetic and
sensory neurons of the PNS. NGF binds to two transmembrane proteins,
p140trk and p75, on the cell surface (192). PC12 cells, which are
derived from a neural crest-derived pheochromocytoma cell line, respond
to NGF by withdrawing from the cell cycle, extending neurites and
acquiring features of sympathetic neurons. NGF induces a sustained
activation of the MAPK pathway and induces a pp90Rsk kinase
(193). Activation of the MAPK pathway by an activating Raf mutant in
NIH3T3 cells overexpressing the TrkA receptor is sufficient to induce
p21Cip1 and growth arrest (194).
The response of cell cycle-regulatory proteins to NGF suggests a concurrent induction of G1 cyclins, and CKIs may participate in coordinating the differentiation signal. NGF induces expression of cyclin D1 and the CKI p21Cip1 (195, 196, 197). In association with these increases in cyclin D1 and p21Cip1, the protein levels and associated activities of Cdk4, Cdk6, and cdc2 decreased. The induction of the cyclin D1 and p21Cip1 genes by NGF requires DNA sequences in their respective promoters that include Sp1-binding sites (198).
The induction of cyclin D1 is not sufficient for the induction of neurite outgrowth in PC12 cells, however, raising the possibility that the induction of cyclin D1 by NGF subserves a distinct function (196). Because the addition of Cdk chemical inhibitors to undifferentiated PC12 cells promoted cell death, it was suggested that cyclin D1 may convey a cell survival function (199). During neuronal apoptosis, however, CD1K activity was stimulated and cyclin D1 was required for the induction of apoptosis in R970B cells (200). The induction of apoptosis by cyclin D1 did not require p53 and was inhibited by Bcl-2 and the 21-kDa E1B protein, suggesting cyclin D1 inhibited cell survival (200). Expression of cyclin D1 in the HC11 breast cell line (201) or NIH3T3 cells (200) also induced apoptosis, and the induction of apoptosis in rat fibroblasts was observed with cyclin D1 but not with cyclin E (202). It remains possible, therefore, that cyclin D1 may either promote apoptosis or function as a sensor of impending cell death. It has been suggested that the induction of apoptosis by cyclin D1, particularly after ionizing radiation, represents a G1/S checkpoint function of cyclin D1 (30, 203) (for a review see Ref. 4). Under these circumstances, cyclin D1 is actually required for the cell cycle arrest, and a similar function was observed for cyclin D1 in p53-mediated cell cycle arrest (204). In part, this cell cycle-arrest function of cyclin D1 may be through its ability to bind PCNA and sequester its activity in the nucleus.
Recent studies have suggested a role for Cdk5 in neurite outgrowth, and overexpression of a Cdk5 dominant negative mutant blocked neurite extension in cultured cortical neuron cultures (205). The heterodimeric partners for Cdk5, p35, and the related p39 are required for normal neurite extension in a rat hippocampal cell line (58). The role of the cyclin/Cdk complexes in NGF signaling remains to be fully explored.
The abundance of other cell cycle-control proteins are altered during NGF treatment of PC12 cells, although their role in mediating the differentiated phenotype is not understood. One of the earlier changes induced in PC12 cells treated with NGF is a decrease in cyclin F, raising the possibility that cyclin F is also involved in NGF-mediated cell cycle events during the differentiation of PC12EY cells (206). In addition the DNA binding activity of the E2F/DP proteins, which bind DNA enhancer sequences in a number of different genes involved in promoting cell cycle progression, was reduced with NGF treatment (195).
2. Serine/threonine kinase receptors.
a. TGFß.
The TGFß family of cytokines regulates diverse
functions including differentiation and inhibition of cell growth
(207). In addition, TGFß can induce cellular proliferation in certain
cell types, such as subconjunctival and adult lung fibroblasts (208, 209). The proliferative effects are likely in part related to release
of paracrine growth factors such as connective tissue growth factor
(210). TGFß inhibits cellular growth in most epithelial cells and can
act at both early and late points during the prereplicative G1period (211, 212, 213). The diverse effects of TGFß are, in part,
regulated by the cell type, the state of cellular differentiation,
local paracrine context, and the relative abundance of the TGFß
receptors. The signaling pathway induced by TGFß is dependent upon
binding of ligand to the TGFß type II receptor, which forms a
heteromultimeric transphosphorylated complex with the TGFß1 receptor.
Studies of pathways activated by TGFß indicate important roles for members of a multigene family related to Drosophila Mad (mothers against decapentaplegia) in intracellular signaling. In addition the CKIs appear to play an important role in the cytostatic phenotype induced by TGFß. In the developing Drosophila eye (214), signaling mediated by the TGFß-related gene decapentaplegia (dpp) was required for the synchronization of the cell cycle. dpp May affect cell cycle synchronization by promoting cell cycle progression. This synchronization is critical for the precise assembly of the Drosophila eye. More than six mammalian proteins related to Drosophila Mad, Smads, have been cloned and are implicated in transducing TGFß signaling to the nucleus, with Smad3 and Smad4 functioning as DNA sequence-specific binding proteins (215, 216, 217, 218, 219).
The effect of TGFß on components of the cell cycle-regulatory
apparatus are quite complex (211). TGFß functions in part
through both transcriptional effects and posttranscriptional and
posttranslational mechanisms. For example, the inhibitory effect of
TGFß on CCL64 cells late in G1 occurred in the presence
of RNA synthesis inhibitors (220). Unifying themes are appearing,
however, indicating a critical role for the CKIs (p21Cip1,
p27Cip1 and p15Ink4b) in which the
transcriptional induction and subnuclear localization appear to mediate
their cell cycle inhibitor effects. In addition, TGFß has been shown
to inhibit expression of Cdk2, cyclin D2, and cyclin A and to reduce
pRB phosphorylation (221). TGFß inhibits growth in fibroblasts and
Mv1Lu cells in association with a delayed reduction in Cdk4 abundance
(222) while in several cell types it appears that the earliest and
critical steps in the cytostatic function of TGFß is the induction of
the Cdk4/6 inhibitor p15(Ink4b/MTS2). TGFß stabilizes
p15Ink4b protein, increases p15Ink4b-Cdk4
complexes, and inhibits cyclin D1/Cdk4 association in human mammary
epithelial cells (223). The recent studies from Massague’s laboratory
(92) have been particularly insightful in understanding TGFß
signaling to the CKIs with their recent finding that the subcellular
locations of p15Ink4b and p27Kip1 coordinate
their inhibitory interactions with Cdk4 and Cdk2. In lung epithelial
cells, treatment with TGFß led to an induction of
p15Ink4b bound to cyclin D1/Cdk4 and reduced binding of
cyclin D1/Cdk4 by p27Kip1. The displacement of
p27Kip1 led to its binding to cyclin E/Cdk2, which forms an
inhibitory complex. These studies suggested the subcellular
distribution, and the proteins to which p27Kip1 was bound
may be critical in mediating the cytostatic effect of TGFß (92) (Fig. 5). In cells lacking
p15Ink4b, however, TGFß is still capable of arresting
cells in G1 through a reduction in cyclin D1 and an
induction of p21Cip1 and p27Kip1 (224).
|
b. Activins.
Activins are members of the TGFß family encoded
by two closely related genes, activin-ßA and activin-ßB. Activins
inhibit cellular proliferation and induce differentiation of mesoderm,
erythroid precursors, and other cell types (229, 230, 231). Activin also
inhibited basal and stimulated proliferation in prostate cancer cell
lines (232). In plasmacytic cells, the induction of cell cycle arrest
by activin A was associated with an induction of p21Cip1,
but not the other CKIs (p27Kip1, p16Ink4a, or
p15Ink4b), and a suppression of cyclin D2 (233). Because
pRB phosphorylation was reduced in association with the induction of
p21Cip1 in activin A-treated cells, the cell cycle arrest
induced by activin was attributed to the induction of
21Cip1 expression.
B. Seven-transmembrane receptors
1. Rhodopsin family.
a. Angiotensin II (AII).
The octapeptide AII binds to specific
high-affinity receptors present in the adrenal cortex, liver epithelial
cells, and in vascular smooth muscle cells where it elicits a vast
array of biological effects. AII increases DNA synthesis, cell
proliferation, and steroidogenesis in cultured adrenal cortical cells,
both in cells derived from the adrenal fasciculata and
glomerulosa cell layer (234, 235). AII functions as a growth factor in
cardiac fibroblasts, myocytes, vascular smooth muscle cells, and
adrenal cells (236, 237, 238). Many of the known biological actions of AII,
including enhanced DNA synthesis, are mediated by stimulation of the
AT1 receptor (234, 235).
The AT1 receptor is a member of the G protein-coupled seven-transmembrane spanning receptor family (239, 240). Binding of AII to the AT1 receptor activates phospholipase C, which initiates a rapid release of inositol triphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate, causing intracellular calcium release (241). The intracellular transmission of signaling by Ca+2 and activation of cytosolic phospholipases involves, in part, sequential activation of Ras (242, 243, 244) and thereby protein kinases, including MAPK (240, 245). Previous studies showed that AII can stimulate phosphorylation of several intracellular signaling protein kinases at tyrosine residues including the ERKs in vascular cells (246, 247) and the related stress-activated protein kinases [SAPKs or Jun N-terminal kinases (JNKs)] in hepatic cells (248). In addition, AII stimulates tyrosine phosphorylation of p44/p56SHC (246), the Jak family proteins Jak2 and Tyk2 (249), and focal adhesion kinase (p125FAK) in vascular smooth muscle cells (246).
In the human adrenal cell line H295R, AII (10-6 M) stimulated G1 phase progression within 12 h, with a maximal effect after 72 h (250). This action was preceded by the induction of cyclin D1 mRNA, the presence of nuclear cyclin D1 protein, and CD1K activity. Acting through the AT1 receptor, AII induced cyclin D1 promoter activity 4-fold via an enhancer sequence at -954 bp. c-Fos and c-Jun bound the cyclin D1 -954 enhancer sequence, and the abundance of c-Fos within this complex was increased by AII treatment (250). AII induced ERK activity 7-fold, and dominant negative mutants of either Ras or ERK reduced AII-stimulated cyclin D1 promoter activity. These findings suggest AII may stimulate mitogenesis by increasing CD1K activity through a Ras/ERK/AP-1 pathway.
b. Parathyroid hormone (PTH).
Osteoblasts are the major direct
cellular target of PTH action in bone. The high-affinity receptors for
PTH generate a variety of intracellular signaling pathways, including
changes in intracellular cAMP, inositol triphosphate, diacyl glycerol,
Ca+2, membrane potential, and pH. The effect of PTH on
osteoblast proliferation and differentiation is complex, and PTH may
either inhibit or induce cellular proliferation depending upon the cell
system examined and upon whether the exposure to PTH is tonic or
intermittent. Acute exposure to high concentrations of PTH leads to an
inhibition of many osteoblastic functions. Continuous exposure to high
concentration of PTH in vivo results in a progressive bone
loss and osteopenia, although osteoblasts may increase in number. PTH
is known to inhibit proliferation of the UMR-106 osteoblast cell lines
and reduce the proportion of cells entering S-phase. Associated with
these changes, PTH increased p27Kip1, but not
p21Cip1 levels. This effect was mimicked by 8-bromo-cAMP,
but not by phorbol 12-myristate 13-acetate. The protein kinase A
inhibitor KT5720 abolished the PTH induction of p27Kip1.
PTH increased Cdk2-associated p27Kip1 without affecting the
levels of Cdk2 (251). Extracellular calcium inhibits parathyroid cell
proliferation. In a rat epithelial parathyroid cell line, PT-r, cyclin
D1 mRNA levels oscillate during the cell cycle, and increasing amounts
of calcium in the incubation medium reduced the expression of rat
cyclins D1 and D2 (252).
c. TSH.
TSH is a member of a dimeric family of
glycoprotein hormones that contain a common - and a unique
ß-subunit. The binding of TSH to its cognate G protein-coupled
receptor (Gs) initiates a cascade of intracellular signaling, including
the induction of cAMP, and in the human the activation of phospholipase
C (253). The cAMP pathway induces DNA synthesis with kinetics
comparable to the effect of TSH and is associated with the induction of
immediate early genes and PCNA (254). In the rat thyroid cancer cell
line FRTL5, TSH induces cellular proliferation and cell cycle
progression. A FRTL5 cell line containing a dominant negative mutant of
CREB (cAMP reponse element binding protein) had reduced
induction of DNA synthesis in response to TSH, providing supportive
evidence that the cAMP/CREB pathway was linked to DNA synthesis in
thyroid cells (255). The proliferative effects of TSH are associated
with an enhanced rate of G1 phase progression and induction
of cyclin D1 and cyclin E (256). The phosphatase inhibitor okadaic
acid, which inhibits protein phosphatase 2A (PP2A) and protein
phosphatase 1, stimulates the TSH-induced G1-S phase
transition in thyroid cells (257). In dog thyrocytes, microinjection of
human GST-p16 inhibited BrdU incorporation induced by TSH (18).
Cyclin D3 abundance increased in the nucleus of TSH-treated dog
thyrocytes, and microinjection of immunoneutralizing cyclin D3
antibodies blocked TSH-induced BrdU uptake, suggesting a specific role
for cyclin D3 in G1 phase entry induced by TSH (258).
d. FSH.
The binding of FSH to its seven membrane-spanning
receptor leads to induction of cAMP within the cell through
heterodimeric G protein coupling to adenyl cyclase (259). FSH induces
ovarian folliculogenesis and male spermatogenesis, and the mitogenic
effect of FSH is, in part, induced through cAMP (259, 260). Many
actions of FSH can be mimicked by the addition of cAMP to cultured
ovarian cells. Cyclin D2 was found in FSH-responsive Sertoli cells and
spermatagonia; furthermore, testicular germ cell tumors express very
high levels of cyclin D2 (261). cAMP treatment of granulosa cells
induced cyclin D2 mRNA with similar kinetics to the induction by FSH
(261, 262). Serum addition or induction of the protein kinase C (PKC)
pathway by mitogens did not induce cyclin D2 mRNA levels, suggesting
activation of the cAMP pathway by FSH was critical for the induction of
cyclin D2 mRNA. Homozygous deletion of the cyclin D2 gene resulted in
female mice with hypoplastic granulosa cells, unresponsive to FSH,
suggesting a critical role for cyclin D2 in FSH-induced cellular
proliferation in vivo (261). The male cyclin D2 KO mice
displayed hypoplastic testis, with normal differentiation and normal
serum testosterone levels, suggesting an important role for cyclin D2
in cellular proliferation required for the formation of a critical mass
of tissue during development.
C. Steroid hormones
1. Estrogens. Steroid hormones mediate diverse effects on
cellular proliferation in association with modulating activity of the
cyclins and the CKIs. The proliferative effects of estrogen on
responsive tissues, including breast and uterus, have been well
documented (for reviews see Refs. 263, 264, 265). The use of animals
homozygously deleted of the ER- gene (ER KO mice) confirmed the
requirement of the ER for several normal proliferative processes
including normal mammary gland ductal growth (266, 267), angiogenesis
(268), and spermatogenesis (269).
A primary mechanism of estrogen action is the ligand dependent steroid hormone receptor activation through specific response elements of target genes (270, 271). In addition, estradiol regulates the Ras-Raf-MAPK pathway with kinetics similar to those of polypeptide growth factors operating through membrane tyrosine kinase receptors (272). Peptide growth factors also modulate ER activity independently of ligand (273), and the ER is capable of interacting with a number of other transcription factors to coordinate expression of downstream target genes, including members of the AP-1 family (274) and Sp1. In part, the coordinate regulation of these downstream transcription factors is regulated by interaction with high molecular weight co-integrator proteins, including SRC-1, RIP-140, SNF2ß-BRG1 (275), TASF(II)130 (276), and TIFI (277) (reviewed in Ref. 278).
Recent studies have focused on the molecular mechanisms by which estrogens induce components of the cell cycle-regulatory apparatus to induce cellular proliferation. Estrogens stimulate cell cycle progression early in G1 phase in cultured breast epithelial cells (279, 280, 281). Cyclin D1 expression is reduced by antiestrogen treatment in T47-D cells (176). The induction of cellular proliferation by estrogen in breast cancer cell lines was found to correlate with increased expression of cyclin D1 protein levels and CD1K activity in MCF-7 and T-47D cells (282, 283, 284). In the experiments conducted by Altucci, the MCF-7 cells were released from cell cycle arrest by 24–48 h of treatment with the hydroxymethylglutyryl-coenzyme A reductase inhibitor, simvastatin, before the addition of estrogen. Under these experimental conditions, cyclin D1 levels were induced rapidly between 1–3 h, and the induction of cyclin D1 mRNA was blocked by actinomycin D, suggesting a role for RNA polymerase II. The cyclin D1 promoter was also induced by estrogens, indicating the induction by estrogen may be a direct transcriptional event (283).
The recent series of studies demonstrating the activation of the unliganded ER by EGF through the Ras-ERK pathway suggest additional mechanisms by which growth factors and estrogen may regulate cyclin D1 expression (285). The addition of estrogen to lovastatin-treated cells induced CD1K activity. The induction of DNA synthesis by estrogens was inhibited by the microinjection of cyclin D1-neutralizing antibodies but not by dominant negative mutants of the MAPK kinase pathway (18). These results suggest that the induction of cyclin D1 is necessary whereas the induction of ERK activity that occurs in response to estrogens in MCF7 cells is not directly responsible for the induction of S phase entry (18).
The CKIs are also important in estrogen-induced mitogenesis in breast
cancer cell lines (Fig. 6B).
Overexpression of p16Ink4a, for example, blocked the
estrogen-induced S phase entry in MCF7 cells (18), indicating a
critical role for the CKIs in estrogen-induced mitogenesis. The
mechanisms by which estrogen regulates CKI function can be
mechanistically considered as three distinct but functionally
interrelated effects. First, estrogen treatment induces alterations in
the subcellular localization of the CKI. Estrogens induce cyclin E/Cdk2
activity in association with an alteration in the relative distribution
of p21Cip1 from an inhibitory cyclin E/Cdk2 complex to the
cyclin D1/Cdk4 complex (286). Because p21Cip1 can inhibit
cyclin E/Cdk2 activity, but may, at specific stoichiometric ratios,
foster cyclin D1/Cdk4 activity, the net effect of the relocation of
p21Cip1 is to promote cell cycle progression and cellular
proliferation (286). Second, estrogen alters the nature of the
multimeric complexes formed between the cyclin/Cdk complexes. In
studies by Prall et al. (287), estrogens reduced the amount
of the CKIs p21Cip1 and p27Kip1 protein bound
to cyclin E/Cdk2 (Fig. 6B). Third, estrogen treatment results in the
formation of higher molecular weight complexes of cyclin E/Cdk2, which
lack p21Cip1 and p27Kip1. These high molecular
weight complexes contained increased Cdk2 phosphorylation at Thr 160.
In part, therefore, the estrogen-induced activation of cyclin E/Cdk2
appeared to involve both a reduction in the associated CKIs and
increased CAK activity. The use of gel filtration chromatography
was critical to demonstrating the alterations in the cyclin/Cdk/CKI
multimeric complexes. The nature of these high molecular weight
complexes is currently unknown; however, it is possible that these
complexes include ER coactivator proteins, such as SRC-1 or p300.
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Recent studies using mammary gland transplants from ER knockout mice
suggest that stromal cells play a role in the mitogenic effects of
estrogens (289). Mammary tissue from ER-deficient mice and wild-type
mice were used to produce tissue recombinants that were grown as
subrenal capsule grafts in intact female nude mice. In these
experiments ductal growth in the ovariectomized hosts treated with
estrogen required stromal ER but not epithelial ER (289).
Cyclin D1 binds directly to the ER and regulates estrogen-dependent
enhancer activity (114, 115). The transactivation function of the ER,
when fused to a GAL-4 DNA-binding domain, was enhanced by
overexpression of cyclin D1 (Fig. 6C). This activity was induced
further by the addition of estrogens, an effect that was mediated
through the EF domain of the ER (114, 115). Cyclin D1 activated the
ER-mediated transcription in the absence of ligand, and the induction
occurred independently of the Cdk- or pRB-binding domains of cyclin D1
(114, 115). Because ER status is often positive in postmenopausal
women, it has been proposed that the overexpression of cyclin D1,
frequently seen in ER-positive tumors, may function to promote ER
activation of target genes in the presence of low estrogen levels. In
analysis of the molecular mechanisms by which cyclin D1 enhances ER
activity, cyclin D1 was found to bind the ER coactivator SRC-1 through
a carboxy-terminal LLXXXL motif, with deletion of this region reducing
ER activation by 80% (290). Because the LXXL motif of SRC-1, which was
required for cyclin D1 binding (290), is a conserved motif among
several coactivators (291), these studies imply cyclin D1 may interact
with other coactivator proteins. These studies demonstrated that cyclin
D1 may stimulate estrogen enhancer activity and provided further
evidence for a model in which cyclin D1 interacts with proteins other
than the Cdks to mediate hormonal effects on target gene sequences.
Cyclin A overexpression also enhanced ER activity, and ER is
phosphorylated between amino acids 82 and 121 in vitro by
cyclin A, an effect that was inhibited by p27Kip1
overexpression (292) (Fig. 6D). Together, these results suggest that
the regulation of the Cdks by estrogens is under complex feedback loops
in which the transcriptional targets also serve as substrates. Most of
the studies described above have focused on the ER gene, although
recent studies have shown that the ERß gene mRNA abundance appears to
be regulated by the ER gene (293) and that the ER and ERß genes
can differentially regulate AP-1 activity (294).
The effects of estrogens in other cell types may also be under equally complex regulatory pathways. In the rat uterus, estrogen induces endometrial epithelial cell proliferation (295). The administration of 17ß-estradiol (E2) to ovariectomized rats induced Cdk4, -5, and -6, but not Cdk2 activation in the uterus, and increased expression of cyclin D1 and D3, cyclin A, and cyclin E mRNAs (295). p27Kip1 was also induced by E2 in the uterus (295). It will be of interest to determine whether the mechanisms regulating the effects of specific synthetic estrogens differ in different tissues.
2. Progestigens. The physiological functions of progesterone are mediated by two naturally occurring receptor isoforms, PRA and PRB, derived from a common gene. The progesterone receptor (PR), as with other nuclear receptors, contains DNA-binding, ligand-binding, and transactivation regions. Evidence for a normal role for progesterone in promoting mammary epithelial cell growth during puberty and pregnancy was supported by the augmentation of mammary gland growth by the administration of progesterone (296, 297), the inhibition of mammary gland tumor growth by antiprogestins (298), the positive correlation between proliferative potential and PR abundance, and the finding that PR mRNA was concentrated at the end bud cells and in the undifferentiated progenitors of the mammary gland duct wall (299). Transgenic mice homozygously deleted of both PR isoforms provided further support for progesterone’s critical role in mammary gland proliferation (300). In the PR KO mice, mammary gland development was severely limited with absence of end buds and failure of lobuloalveolar development. The animals were unable to ovulate and displayed uterine hyperplasia consistent with the loss of the progesterone decidual response.
In breast cancer cell lines, progestigens both stimulate and inhibit cell cycle progression (176, 265, 301, 302). The initial response is acceleration of cells from G1 to S and then, upon completion of the cell cycle, the progestin-treated cells arrest in G1 (176, 265, 301, 302). The transient induction of G1 phase by progestigens is accompanied by an induction of cyclin D1. However, the inhibition of cell cycle progression by antiprogestigen treatment in T47D cells is not accompanied by changes in cyclin D1 expression (176). The reduction in CD1K activity that occurs with antiprogestigens, such as RU486 or ORG 31710, is associated with an induction of p21Cip1 mRNA and protein within the cyclin D and cyclin E kinase complexes. RU486 has potent antiglucocorticoid activity whereas ORG 31710 is thought to be more selective for its antiprogestigen activity. Overexpression of cyclin D1 markedly reduced the antiproliferative effect of the antiprogestigens (303). Progesterone enhances the induction of cyclin D1 by estrogen in the murine mammary gland, and this induction was abolished in animals homozygously deleted of the PR (304), indicating the effect was likely mediated through the PR. The induction of p21Cip1 may also contribute to the differentiation and apoptosis-inducing properties of progestins that are observed after prolonged treatment, as p21Cip1 overexpression is capable of inducing differentiation and apoptosis in other cell types. c-Myc mRNA levels rose before the induction of cyclin D1 mRNA levels, raising the possibility that c-myc may be a positive regulator of cyclin D1 expression in the presence of progestin (176).
3. Retinoic acid. The highly pleiotropic effects of retinoic
acids in regulating proliferative and differentiation pathways in
mammalian cells (305) result in part through combinatorial interactions
of their receptors (306, 307). The three retinoic acid receptors
(RAR, -ß, and -) and retinoid X receptors (RXR, -ß, and
-) and their isoforms exhibit distinct patterns of expression.
All-trans- and 9-cis- retinoic acids are among
the physiological retinoids. RARs and RXRs act as ligand-inducible
transcription factors, with the homo- and heterodimers binding distinct
DNA sequences. Retinoids had been shown to affect cellular
proliferation at many different levels, and the predominant effect is
to inhibit cellular proliferation and induce differentiation (305, 308). The expression of receptors that bind growth factors was shown to
be directly inhibited by retinoids (309). The retinoids have also been
shown to directly affect expression of cell cycle-regulatory genes. The
p21Cip1 gene, for example, is a retinoic acid-responsive
target gene, and an RA response element in the promoter is required to
confer retinoic acid induction through RAR/RXR heterodimers (310). The
induction of monocytic differentiation by RA in U937 cells correlated
with the transcriptional activation of the p21Cip1 gene and
suggests a role for this cyclin/Cdk complex inhibitor in facilitating
this differentiation pathway. The RA-mediated inhibition of cyclin D1
protein in bronchial epithelial cells is regulated at the
posttranslational level, likely through increased ubiquitin-dependent
proteasome degradation. In these studies, calpain inhibitor I and
lactacystin each prevented the decrease in cyclin D1 protein expression
in the presence of RA treatment (311). Free E2F-1, which is induced by
phosphorylation of pRB, inhibited RAR transactivation function but
did not inhibit glucocorticoid receptor (GR) or thyroid hormone
receptor (TR) transactivation (312).
The RARs and RXRs also act as ligand-dependent trans-repressors of AP-1 (activator protein-1) (c-Jun/c-Fos) activity, and reciprocally AP-1 can inhibit transactivation by RARs and RXR (313, 314). Synthetic retinoids were identified that selectively enhanced the activation function of RARß and efficiently inhibited AP-1 activity. These activities were associated with an inhibition of anchorage-independent growth (315). These studies suggested that retinoid-induced growth inhibition may be related to AP-1 transrepression. The identification of high molecular weight coactivator proteins, CBP and p300, required for both nuclear receptor activation and the inhibition of AP-1 activity (316), raised the possibility that limiting abundance of coregulatory proteins may dictate the genomic response to the addition of a receptor ligand (reviewed in Ref. 317). Linking activity of these rate-limiting coregulatory proteins to the regulation of genes that encode rate-limiting steps in cellular proliferation may provide important insights into the antiproliferative effects of retinoids. In this context it will be of considerable interest to determine whether the RXR receptors, like the ER, are capable of binding to cyclin D1 and directly modulating cell cycle function.
4. Glucocorticoids. The glucocorticoid receptor is a member of the intracellular receptor superfamily of transcriptional regulatory proteins and contains separable transactivation, hormone-binding, and DNA-binding domains (270, 318). The activity of the GR is regulated in a cell cycle-dependent manner and glucocorticoids directly affect cellular proliferation and cell cycle progression. The action of glucocorticoids is cell cycle dependent. The inability of glucocorticoids to induce gene expression during the G2 phase of the mammalian cell cycle has long been known (319). In cultured rat hepatoma cells, glucocorticoids induce tyrosine amino transferase (TAT) activity in late G1 and S phases but not during G2/M and early G1 phases (319). Glucocorticoid induction of several other genes, including the metallothionein-1 gene (320), were subsequently shown to be inhibited during the G2 phase of the cell cycle (321). It was observed that G2-arrested cells contained a modified form of the GR with altered chromatographic properties (322), and it was observed that GRs that translocate to the nucleus in G2 in response to dexamethasone treatment were inefficiently retained and redistributed rapidly to the cytoplasm. The inefficient nuclear retention of the GR in G2-arrested cells was associated with altered phosphorylation of the GR, and it was proposed that dephosphorylation of the GR may be important in its nuclear retention (320).
The amino terminus contains several sites that undergo ligand-dependent
(323) and cell cycle-dependent phosphorylation, with basal GR
phosphorylation being higher in G2/M than in S phase (320, 324). Selective mutations of single or multiple phosphorylation sites
of the mouse (325) or the human GR (326) only reduced the
transcriptional activity modestly. The phosphorylation status of the
GR, however, may co-determine its subcellular location (327) and
interaction with other transcription factors or coactivators. Four
major sites within the rat GR are phosphorylated in the presence of
hormone: S224 and S232 are phosphorylated by the Cdks, whereas
the constitutively phosphorylated residues, T171 and S246, are
phosphorylated by members of the MAPK family (112) (Fig. 7). Cyclin E/Cdk2, cyclin A/Cdk2, and
cyclin B/cdc2 were each capable of phosphorylating the GR in
vitro. The transcriptional activity of the GR was reduced in yeast
strains deficient in particular G1 (CLN) and
G2 (CLB) genes, whereas strains of yeast bearing
deletions of MAPK genes, FUS3 and KSS1, enhanced
GR transactivation function. Together these studies suggest that
transactivation function of the GR may be enhanced by several cell
cycle-regulatory kinases.
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synthesis (331, 332), which binds NF-kB, keeping it in its inactive
form in the cytoplasm, and may thereby regulate CKI abundance
indirectly.
5. Vitamin D. In addition to a role in regulating calcium
homeostasis, immune responses, and cellular differentiation, vitamin D
and its metabolites play important roles in the regulation of cellular
proliferation and differentiation of normal and malignant cells
(reviewed in Ref. 333). 1,25(OH)2D3
generates biological responses through genomic and nongenomic pathways,
and the genomic responses involve homo- or heterodimerization with
other nuclear receptors including members of the RXR and T3
receptor family. The antiproliferative effects of
1,25(OH)2D3 and several vitamin D analogs
have been documented for human, mouse, or rat breast cancer lines
(334, 335, 336) and implantable breast tumor models (337, 338, 339),
keratinocytes (340, 341), and prostate cancer cell lines (342).
The inhibition of human and murine keratinocyte proliferation by vitamin D3 was associated with a decreased number of EGFRs, a rapid decrease in c-myc expression (343), an induction of TGFß (344), and a reduction in pRB phosphorylation (345).
The human promyeloid leukemia cell line HL-60 and a human myeloid
leukemia cell line U-937 have been well studied for the effect of
vitamin D3. In HL-60 cells, vitamin D3-induced
antiproliferation and differentiation is associated with a reduction in
c-myc and pRB mRNAs (346) and an induction of the
c-fms gene, which encodes the monocyte CSF receptor that
favors differentiation (347). The principal block to cell cycle
progression in 1,25(OH)2D3-treated human
cells occurs in G1. In HL-60 cells, the addition of
1,25(OH)2D3 induces late G1
arrest (348) associated with elevated levels of p27Kip1
protein, cyclin D1 (349), and p21Cip1 (350, 351) and a
reduction in pRB phosphorylation. p21Cip1 was
transcriptionally induced by 1,25(OH)2D3 in
a vitamin D receptor-dependent, but not p53-dependent manner. A vitamin
D response element in the p21Cip1 promoter was also
identified. The induction of p21Cip1 was an early event,
however, and not a uniform finding by all investigators, whereas the
induction of p27Kip1 protein occurred later, correlated
with the alteration in cellular phenotype (349, 352), and occurred at a
posttranslational level as mRNA abundance was unchanged (353). The main
target of the elevated p27Kip1 in this system is Cdk6
(352). The activity of Cdk2 is also down-regulated, and this is
associated with altered and reduced levels of cyclin E in the kinase
complex. The kinase activity of Cdk4 was elevated, in spite of an
almost complete G1 block. These data show that the
functions of Cdk4 and Cdk6 are not redundant and that Cdk6 and Cdk2
activities are regulated by 1,25(OH)2D3
(352).
Transient overexpression of p21Cip1 and/or the related CKI
p27Kip1 in U937 cells in the absence of
1,25(OH)2D3 results in the cell surface
expression of monocyte/macrophage-specific markers, suggesting that
these two CKIs were sufficient for at least a partial induction of the
differentiated phenotype (354). Overexpression of cyclin D1 induced
differentiation in the DAMI megakaryocytic cell line (355) and in the
HC11 breast cancer cell line (201), suggesting that the abundance of
the cyclin D1/p27Kip1 complex may play a more complex role
in differentiation.
6. Androgens and orphan nuclear receptors. In immature males, androgens stimulate prostate cellular growth (356). Castration is associated with a reduction in cellular proliferation and apoptosis in androgen-dependent epithelial cells (357). Androgen replacement to castrated animals induces prostate epithelial cell proliferation (356) concordant with the induction of a variety of immediate early and other genes (358). Rat ventral prostate undergoes proliferation upon androgen replacement to castrated rats, in association with an induction of G1 cyclins (cyclin D1, cyclin E, cyclin A), and a reduction of these cyclins upon androgen withdrawal-induced apoptosis, although the apoptosis was p53 independent (359). Cyclin D1 levels increased 9-fold, peaking at 48 h after androgen treatment (360). p27Kip1 levels increased after androgen treatment with an initial peak after 24 h of treatment. p21Cip1 levels increased after 72 h and continued to increase in association with a reduction in the proliferative changes in the prostatic epithelial cells (360). In the LNCaP-FGC prostate cancer cell line, androgens increased Cdk2 and Cdk4 expression and down-regulated p16Ink4a levels (361). In the PC3 prostate cancer cell line, which lacks AR, p16Ink4a levels were very low, raising the possibility that the reduction in p16Ink4a may contribute to the enhanced proliferation in prostate cells.
The growing family of orphan nuclear receptors is being delegated roles
more frequently in the regulation of cellular differentiation
and proliferation. The peroxisome proliferator-activated receptor
(PPAR) binds two distinct ligands, the synthetic antidiabetic
thiazolidinediones (362, 363) and the 15-deoxy
12,14 PGJ2
(362, 364). The induction of PPAR during adipocyte differentiation
occurs relatively early (365, 366), and retroviral overexpression of
PPAR in the presence of Pioglitazone (Pharmacia & Upjohn, Inc., Kalamazoo, MI), a synthetic
thiazolidinedione, is sufficient to induce growth arrest and adipocyte
differentiation (367). These changes were unassociated with changes in
p21Cip1 or p27Kip1 expression but were
associated with a reduction in binding of the E2F/DP proteins to an
AdE2 E2F site, and it was proposed that this reduction in binding may
play a role in withdrawal of cells from the cell cycle (367).
D. Intracellular second messengers
1. cAMP. cAMP has long been known to either inhibit (368) or
induce cellular proliferation depending upon the cell type (369). Cell
cycle progression in Xenopus egg extracts is accompanied by
fluctuations in cAMP levels and the activity of PKA (370). The
antiproliferative effect of cAMP has been correlated with the induction
of a protein phosphatase activity (371). The CREB and related
cAMP-responsive element modulator (CREM) proteins have specific
roles in regulating cell cycle changes. In the FRTL5 thyroid cell line,
cAMP induces cellular proliferation, and a dominant negative of CREB
caused G1 phase arrest (255). In pituitary cells, cAMP
serves as a mitogenic signal for somatotrophs. Pituitary expression of
a dominant negative mutant of CREB resulted in pituitary hypoplasia
specific for the somatotrophs, in conjunction with a dwarf phenotype
(372). Cyclin D1 expression is inhibited by cAMP (177). Treatment of
macrophages with cAMP analogs induces G1 phase arrest
(373), and cAMP blocked the mitogenic effect of CSF-1, arresting the
cells in mid-G1 (100). cAMP inhibited cyclin D1 and Cdk4
expression in CSF-1-treated macrophages in some studies (374, 375) but
not others (100). Kato et al. found that cAMP treatment of
macrophages was associated with the induction of a cyclin D1/Cdk4
complex that was poorly phosphorylated by the CAK. In the cAMP-treated
cells, p27Kip1 levels were elevated and immunodepletion of
p27Kip1 restored the ability of CAK to activate the cyclin
D1/Cdk4 complexes (376).
Recent studies have analyzed the importance of the changes in D-type cyclin expression observed in cAMP-treated cells. The reduction in cyclin D1 induced by cAMP appears to be critical for the inhibition of cellular proliferation in fibroblasts (377). TSH induced nuclear cyclin D3 expression in dog thyrocytes (258), and immunoneutralizing antibodies to cyclin D3 reduced G1-S phase progression induced by TSH. Microinjection of p16 blocked TSH-induced BrdU incorporation in FRTL5 cells, consistent with a role for cyclin D/Cdk activity in TSH-induced mitogenesis (18). In ovarian granulosa cells, cyclin D2 is induced by forskolin and by FSH through a cAMP-dependent pathway (261). Although p27Kip1 levels change in response to cAMP (376), as yet the requirement for p27Kip1 in the cytostatic effect remains to be formally established using either cells derived from animals homozygously deleted of the p27Kip1 gene or immunoneutralizing antibodies in cultured mammalian cells.
The cyclin A gene is also inhibited by cAMP in a cell cycle-dependent manner, and components of the molecular mechanisms governing the expression of cyclin A have been examined in several cell types. In human diploid fibroblasts (Hs 27), cyclin A gene expression at G1/S is stimulated by 8- bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which delayed S phase entry in Hs 27 primary embryo fibroblasts (378). The cyclin A gene CRE conveyed cAMP-induced promoter activation (378). The CRE bound CREB/CREM proteins in primary fibroblasts (378) and ATF-1 in growing bovine aortic endothelial cells (379). In CHO cells, inhibition of cellular proliferation by cAMP was associated with inhibition of cyclin A promoter activity (380). In AtT20 cells corticotroph cells, cAMP induces a cell cycle arrest at the G2/M phase associated with an inhibition of cyclin A expression (381). The CREM gene isoform ICER (inducible cAMP early repressor), when overexpressed in AtT20 cells, was capable of inhibiting G2/M phase progression and inhibited cyclin A expression. Thus, cyclin A expression is repressed in several different cell types in which cAMP is cytostatic, and the effects appear to be mediated through a consensus CRE site.
cAMP may also indirectly affect the cell cycle-regulatory apparatus through an alternate mechanism affecting cyclin degradation. cAMP also directly affects components of the ubiquitin pathway. Anaphase-promoting complex has ubiquitin ligase activity and is required for mitotic cyclin destruction and sister chromatid separation. The 20S cyclosome complex formation and proteolytic activity was inhibited by the cAMP/PKA pathway (382). Together these studies suggest that both the transcriptional repression and altered protein degradation may mediate the effects of cAMP on the cell cycle.
2. 12-O-tetradecanoylphorbol 13-acetate (TPA). The phorbol
ester, TPA, activates PKC and thereby induces the activities of many
different transcription factors including AP-1, NF-
B, p67TCF, and
related Ets protein family members (383). Although induction of MAPKs
and immediate early gene expression is a common finding in many
different cell types, the effect of TPA on cell cycle progression
varies considerably. Thus, TPA inhibits NIH 3T3 cell proliferation and
arrests growth of many leukemia cell lines such as the HL60 and K562
cells, but stimulates the proliferation of Swiss 3T3 cells (384). TPA
has been shown to stimulate or inhibit different subtypes of NIH3T3
cells (385). In both NIH3T3 cell sublines, TPA induced immediate early
gene expression. In the NIH 3T3 subline that proliferated in response
to TPA (P-3T3 cells), cyclin D1 levels were also induced. In the NIH3T3
subline inhibited by TPA, cyclin E kinase activity was reduced.
p21Cip1 and p27Kip1 levels were not
differentially affected in the two sublines, suggesting the
differential induction of cyclin D1 may be an important distinguishing
feature of the proliferative phenotype.
In K562 cells, p21Cip1 levels were induced by TPA in a p53-independent manner, associated with cell cycle arrest and the induction of cellular differentiation (351). TPA induction of p21Cip1 in K5632 cells was associated with growth arrest, and induction occurred through an AP-2 binding site (386). AP-2 has long been known as a TPA-inducible transcription factor (387); however, the ability of AP-2 to induce cell cycle arrest (386) raises important questions about its role in cell cycle regulation.
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IV. Cyclins and CKIs in Endocrine Tumors |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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A. Parathyroid adenomas
As noted above, the human cyclin D1 gene was cloned during
molecular investigation of parathyroid adenomas in which the PTH gene
was found in a pericentromeric inversion in chromosome 11 (22) (Fig. 3). Such clonally selected abnormalities involving the cyclin D1/PRAD1
gene provide overwhelming evidence for PRAD1's oncogenicity in this
tissue. This is confirmed by transgenic mice overexpressing cyclin D1
in parathyroid tissue, in which parathyroid growth and functional
abnormalities are seen (Y. Hosokawa, K. Yoshimoto, and A. Arnold,
unpublished). In parathyroid neoplasms, overexpression of the
wild-type PRAD1 sequence, rather than mutational activation, appears to
be the predominant mechanism by which PRAD1 exerts its oncogenic action
(390). Overexpression of cyclin D1 was found in 18% of parathyroid
neoplasms in one study (391). Cyclin D1 overexpression is a feature of
typical parathyroid adenomas (391). Hyperplasias had 3-fold more
p27Kip1-positive cells than parathyroid adenomas,
suggesting that p27Kip1 immunostaining may be useful in
distinguishing between these two conditions (392). Deletions of the
p16Ink4a and p15Ink4d genes occur uncommonly,
if ever, in parathyroid adenomas (393). In one study the large majority
of parathyroid carcinomas showed allelic losses on 13q that included
the RB gene, associated with significantly reduced or absent nuclear
staining for pRB, whereas none of the benign parathyroid adenomas
examined contained abnormal pRB staining (394). Frequent allelic loss
on 13q has been confirmed in aggressive parathyroid tumors (395), but
it remains to be determined whether pRB itself is a functionally
relevant target of such deletions.
B. Thyroid cancer
Oncogenes known to disrupt the cyclin D1/pRB axis have been shown
to induce thyroid tumors. When targeted to the thyroid gland by the
bovine thyroglobulin promoter, the SV40 large T antigen induced thyroid
adenocarcinoma (396), and the E7 protein induced papillary and
follicular thyroid cancer (397). p53 Is frequently mutated in
anaplastic thyroid carcinomas, and p21Cip1 is a downstream
effector of p53. It is conceivable that genetic defects of genes
downstream in the p53 pathway could also be oncogenic. In analysis of
thyroid tissues including adenomas and carcinomas by one group, normal
thyroid follicles and adenomas rarely were immunopositive for
p21Cip1; however 30 of 93 carcinomas studied were positive
for p21Cip1. There was no correlation between
p21Cip1 immunopositivity and p53 status. The incidence of
p21Cip1 status was unrelated to thyroid carcinoma subtype
when undifferentiated, poorly differentiated, and well differentiated
tumors were compared. There was also no correlation between
p21Cip1 status and clinical parameters (398). In other
studies p21Cip1 abnormalities were not found in thyroid
adenomas; however, 12% of the papillary carcinomas harbored
p21Cip1 mRNA deletions of exon 2, without evidence for gene
deletion (399) and therefore of uncertain pathogenetic importance. A
polymorphism in the p15Ink4b gene was noted to occur more
frequently in papillary and medullary thyroid cancer compared with
normal subjects, although the functional significance of this
polymorphism in the tumor suppressor function remains undetermined
(400). The role of other cell cycle-regulatory proteins such as cyclin
D1 and the other CKI as independent prognostic indicators in thyroid
cancer, as previously performed in breast and prostate cancer, remains
to be assessed.
C. Pituitary tumors
Homozygous deletion of the p27Kip1 gene resulted in
larger animals with organomegaly and intermediate lobe pituitary
tumors, suggesting the p27Kip1 product played a role in
inhibiting normal cellular growth, particularly in the pituitary (104, 105). Among 31 sporadic and 2 familial pituitary adenomas,
PCR-single-strand conformation polymorphism analysis detected three
polymorphic changes but no tumor-specific mutations of the p27Kip1gene (401). p27Kip1 levels were reduced
in neoplastic pituitary tissue (392), and Jin et al. (402)
found that p27Kip1 levels were normally high in pituitary
cells but protein levels were decreased in adenomas and carcinomas
(402).
Transgenic mice heterozygous for a pRB mutation developed pituitary corticotrope intermediate lobe tumors (403, 404). These tumors were in part dependent upon inhibition of E2F-1 function as mating of mice nullizygous for E2F-1 with the pRB mutant mice resulted in offspring with a reduced prevlance of pituitary tumors (405). In studies of human pituitary tumors, however, pRB mutations were not found (406, 407), and in aggressive tumors the loss of heterozygosity was found at sites telomeric and centromeric to the pRB locus (408). Functional inactivation of pRB through overexpression of G1cyclins in pituitary tumors remains to be formally examined. As abnormalities of either the pRB or p53 axis are observed almost uniformly in human tumors of all types (1), it was surprising that p53 mutations were not found in pituitary tumors (409). In transgenic mice homozygously deleted of the p18INK4c gene, hyperplasia of the intermediate and anterior pituitary gland was found as early as 4 weeks (410). Intermediate-lobe pituitary tumors staining for ACTH subsequently developed with nearly complete penetrance (410). Mating of the p18Ink4c-/- and the p27Kip1-/- mice resulted in an acceleration of the pituitary pathology consistent with the model that these two CKI work in distinct parallel pathways. Thus, to summarize, pituitary tumors in the mouse result from deletion of genes for specific CKI that are expressed well in the normal murine pituitary (p18Ink4c and p27Kip1), but are not found with deletion of other CKI (p16Ink4a, p21Cip1, p57Kip2). In all, the role of cyclin/CKIs in human pituitary tumors as prognostic markers or mechanistic abnormalities remains relatively poorly understood (411).
D. Adrenal tumors
As noted above, homozygous deletion of the p57Kip2
gene resulted in mice that developed adrenal cortical hyperplasia and
cytomegaly (109). Although the low frequency of p57Kip2
mutations in patients with Beckwith-Wiedemann syndrome in conjunction
with the frequent disruption of the K(v)LQT1 gene in patients with
chromosomal rearrangements suggested that Beckwith-Wiedemann syndrome
involves disruption of multiple independent 11p15.5 genes (412), these
results stimulated interest in the role of p57Kip2 in
adrenal tumorigenesis. Expression of p57Kip2 was restricted
in the mouse to the fetal adrenal. In normal human adrenals, however,
p57Kip2 mRNA (and H19 RNA) was abundantly expressed and was
found in adrenocortical adenomas from patients with Cushing’s or
Conn’s syndrome. In most adrenocortical carcinomas and virilizing
adrenal adenomas, very low levels of both p57Kip2 and H19
RNAs were observed (413). These findings remain consistent with a model
in which p57Kip2 may play a role in the inhibition of
abnormal adrenal cellular proliferation.
E. Ovarian and testicular cancer
Assessment of components of the CKI/E2F axis in ovarian and
testicular tumors revealed surprising results. Based on epithelial cell
tumor analysis, it was anticipated that CKI levels would be reduced and
E2F-1 levels may be increased. Surprisingly, p16Ink4a mRNA
and protein levels were significantly elevated in 28/32 ovarian tumors
(414). When p16Ink4a gene deletions and rearrangements were
sought in 21 ovarian and 42 testicular tumors, no abnormalities other
than polymorphisms were found (415). These findings were somewhat
counterintuitive, as overexpression of E2F-1 typically induces S-phase
progression and apoptosis. p21Cip1 levels were reduced,
however, in ovarian cancer (416). The homozygous deletion of the
p27Kip1 gene resulted in animals with testicular and
ovarian cell hyperplasia (104, 105), raising the possibility that
p27Kip1 may normally inhibit testicular and ovarian cell
proliferation. The testes of mice homozygously deleted of the E2F-1
gene underwent atrophy (417, 418), although the mechanism is currently
not defined. These findings suggest that the role of
p21Cip1 and p27Kip1 as ovarian and testicular
cell tumor suppressors warrants further investigation.
Homozygous deletion of cyclin D2 was associated with selective abnormalities of gonadal development (261). The association with tumor formation is much less frequent for cyclin D2 and cyclin D3 than with cyclin D1, and cyclins D2 and D3 have not yet proven to be human oncogenes. Cyclin D2 overexpression has been observed in a subset of gonadal tumors in which cyclin D1 levels were unaltered (261). Cyclin D2 levels, which were normally undetectable, were increased in male germ cell tumors (419). The levels of cyclin D2 were inversely correlated with the level of tumor differentiation. p21Cip1 levels were inversely correlated with cyclin D2 levels (419). Cyclin D2 was associated with Cdk6 and Cdk4 in the tumors (419). At present, among the D type cyclins, cyclin D2 overexpression appears to be linked specifically to a subset of gonadal tumors.
F. Mechanisms of oncogenic transformation by cyclin D1
The cyclin D1 gene maps to the region of chromosome band 11q13,
which has been frequently found in tumor-associated translocations.
Evidence from several laboratories has indicated that the PRAD1 is the
BCL-1 (B-cell lymphoma/leukemia 1) protooncogene (420, 421, 422) critical in
the pathogenesis of mantle cell lymphoma. The cyclin D1 part of the
11q13 chromosomal band was also found to be amplified in numerous other
types of malignancy including breast, squamous cell, esophageal
cancers, and a smaller number of bladder, hepatocellular, and non-small
cell lung cancers (9, 423). The association with tumor formation is
much less frequent for cyclin D2 and cyclin D3 than with cyclin D1.
Cyclin D2 overexpression has been observed in a subset of gonadal
tumors (261), and cyclins D2 and D3 have also been implicated in
chronic lymphocytic leukemia and malignant lymphoma. The relative
importance of structural differences in the D-type cyclin promoters to
their tumorigenic or developmental roles is an area of active
investigation.
Cyclin D1 mRNA is overexpressed in many different neoplasms including breast, esophageal, hepatic, squamous cell carcinoma of the head and neck, and melanoma. The prevalence of cyclin D1 overexpression is particularly high in mantle cell lymphomas and breast cancers. Some 30–45% of human breast cancers and 70–100% of breast tumor cell lines have increased cyclin D1 expression. Although in head and neck cancers, the increase in cyclin D1 abundance is usually associated with cyclin D1 gene amplification, gene amplification can explain only some of the overexpression found in breast cancers, suggesting that additional factors contribute to the increased abundance of cyclin D1 (1). Immunohistochemical analysis of cyclin D1 abundance provides independent prognostic information in squamous cell carcinoma of the head and neck (424) and pancreatic cancer (425), in which the overexpression of cyclin D1 is associated with poor prognosis. Some (426) but not all studies (427) suggest cyclin D1 abundance may provide useful prognostic information in breast cancer. Immunohistochemical analysis of cyclin D1 abundance may be useful in predicting relapse in patients with good-prognosis breast cancer (428). The recent development of cyclin D1 enzyme-linked immunosorbent assays of tumors may provide information with higher predictive value for patient management in breast and other cancers but remains to be fully evaluated.
Cyclin D1 alone is not capable of transforming primary cultured cells but is capable of cooperating in classical assays of transformation with other known oncogenes. Primary baby rat kidney cells (BRK) cells, like rat embryo fibroblasts, cannot be transformed by oncogenes such as E1A or Myc without a collaborating oncogene such as Ras. Although cyclin D1 alone did not transform BRK cells, it enhanced 3-fold the transformation efficiency of a mutant E1A that was incapable of binding pRB (429). The complementation of cyclin D1- transforming ability required binding to its catalytic partner, Cdk4, but did not require the pRB-binding domain (429).
The oncogenic capacity of cyclin D1 has been investigated in transgenic mice. Overexpression of cyclin D1 in the mammary glands of transgenic mice resulted in mammary gland tumor formation, (430) supporting cyclin D1’s role as a "driver oncogene" on the 11q13 amplicon in breast cancer. Coexpression of cyclin D1 and N-Myc or L-Myc under control of an Eµ-directed enhancer sequence in double transgenic mice revealed a strong cooperative effect in the rapid development of clonal pre-B and B cell lymphomas (431, 432). Transgenic mice in which the cyclin D1 cDNA was linked to the Epstein-Barr virus promoter developed squamous epithelial cell dysplasia in the tongue, esophagus, and forestomach, sites demonstrated to express the transgene (433). Similar types of transgenic experiments demonstrated that keratin promoter-directed overexpression of cyclin D1 in epithelial cells could induce epithelial cell hyperplasia (434).
Because of the clear evidence for the oncogenicity of cyclin D1 in vivo, recent studies have further examined the requirement for cyclin D1 in cellular transformation. In cultured fibroblasts, antisense cyclin D1 reduced Ras-induced transformation (435). When transgenic mice with skin tumor induced by Ras were mated with mice homozygously deleted of the cyclin D1 gene, the prevalence of skin tumors was significantly decreased, providing further supporting evidence for a critical role of cyclin D1 in Ras-induced transformation (436). Stable integration of an antisense cyclin D1 cDNA in a transplantable colonic cancer cell line inhibited tumor formation of the implanted cells in nude mice (437). These results provide encouragement to investigators seeking important targets for alternative molecular or pharmacological tumor therapy, as the cyclin D1/p16/pRB axis is frequently perturbed in tumors.
As noted above, a variety of oncogenes induce overexpression of cyclin D1. Several different protooncogenes induce cyclin D1 abundance and cyclin D1 promoter activity including activating mutants of Ras, pp60src, Rac, Neu, and Dbl and overexpression of SV40 small t antigen (187, 438, 439, 440). In some tumors, the mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF- 4E]) increases cyclin D1 protein levels likely at a posttranscriptional level (441, 442). The mechanism by which cyclin D1 overexpression induces cellular transformation is distinct from many other oncogenes. The cyclin D1 protein and coding sequence from tumors examined to date are normal, suggesting it is the overexpression of cyclin D1 per se that is responsible for the formation of tumors (7, 9). The weight of evidence supports the proposal that overexpression of wild-type cyclin D1 contributes to transformation, in large part, through phosphorylation and inactivation of the pRB protein (5), although this has not been well examined in complex in vivo systems. Additional mechanisms may well contribute to the transforming ability of cyclin D1 including the sequestration of CKI, such as p27Kip1, and perhaps the ability to bind other proteins through Cdk-independent mechanisms.
The coding sequence derived from some immortal cell lines had substitutions that would lead to amino acid changes, but the possibility that these amino acid changes may lead to activating mutations has not been directly assessed. An alternatively spliced form of cyclin D1 that lacks the carboxy terminus involved in targeting cyclin D1 for destruction has been described (28). The relatively long 3'-noncoding region of cyclin D1 is also occasionally truncated in tumor cell lines. Overexpression of this alternate splice form appears to differentially inhibit G0-G1 phase progression compared with the wild type in U87 MG cells (443). Cyclin D1 overexpression is frequently associated with gene amplification at other distinct loci, and it has been suggested that cyclin D1 overexpression may contribute to gene amplification and genomic instability that occurs during cellular transformation (444).
Indirect evidence for other mechanisms contributing to cellular transformation by cyclin D1, however, do exist. Cyclin D1 interacts directly with other proteins to affect gene transcription, which in turn may contribute to the transforming phenotype. For example, cyclin D1 binds the ER, enhancing estrogen-dependent enhancer activity (114, 115). As noted above, this effect is independent of the Cdk4 function of cyclin D1. Cyclin D1 also affects activity of V-Myb (117) and binds a Myb-related protein (116). Although a formal assessment has not yet been made, it is tempting to speculate that these protein-protein interactions may also contribute to cyclin D1’s tumorigenic properties.
G. CKIs in treatment of endocrine disease
Because major changes in the activity of the Cdks are found in the
majority of tumors, enzymatic screening has been used to uncover
chemical inhibitors for tumor therapy (445). The first generation of
these inhibitors have been studied in some detail and are being used in
clinical trials of tumor therapy. Although there are several different
theoretical approaches to identification of CKIs, the majority of these
drugs were identified as inhibitors of Cdk catalytic activity.
Molecular interaction-based screening is beginning to identify
inhibitory peptides (446), and the resolution by crystal structure of
the interfaces between the CKI and their target proteins provides a
logical basis for ongoing studies. Although the ability of these
compounds to preferentially inhibit Cdk activity compared with other
kinases such as PKC has been suggested, further studies need to be
performed using synthetic substrates including pRB in cultured cells.
The seven types of CKIs currently described in detail include 1)
staurosporine and UCN-01 (two microbial alkaloids); 2) butyrolactone;
3) flavopiridol and L868276 (derived from an Indian plant extract); 4)
9-hydroxy-ellipticine (derived from the plant Ochrosia
ellipticia); 5) suramin (a naturally occurring glycosaminoglycan);
6) olomoucine, roscovitine, and isopentenyladenine; and 7) peptides,
some of which are derived from the sequence of the naturally occurring
CKI described above (445). Flavoperidol was the first specific CKI to
undergo clinical trials on a spectrum of malignancies. Other than the
peptides, these inhibitors were all derived from natural sources. It is
hoped that the preferential activity of some of these inhibitors for
particular Cdks may allow selective use for tumor therapy.
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V. Transcription Factors Regulating the Cell Cycle |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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with the transcription factor-binding
sites designated. Transcription factors binding to the cyclin D1 (26),
cyclin A (447), and cyclin E promoters (448) were identified through a
combination of transient expression studies, mutational analyses, and
electrophoretic mobility shift assays. Specific components of the basal
transcription apparatus are required for normal cell cycle progression
and, in some cell types, have been shown to link directly to the
induction of cyclin gene expression. TAFII250 is a core
component of transcription factor IID (TFIID) and binds to a number of
other subunits including TATA-binding protein (TBP) and other
transcription-activating factors (TAFs). Mutant strains of
Saccharomyces cerevisiae, containing temperature-sensitive
mutants of yTAFII145, the yeast homolog of human
TAFII250, have been shown to arrest at the restrictive
temperature (449). The mutant hamster cell line, tsBN462, exhibits cell
cycle arrest at the restrictive temperature due to a point mutation in
the TAFII250/CCG1 gene (450). This cell line fails to
progress out of G0 into G1 and was found to
express reduced levels of cyclin D1, cyclin A, and cyclin E and
increased levels of p21Cip1 and p27Kip1 (450).
The arrest may be due to the reduction in cyclin D1 and cyclin A as
cyclin D1 was shown to be directly induced by TAFII250
(451). The cyclin A promoter was also a direct transcriptional target,
with induction by TAFII250 occurring through ATF- binding
sites consisting of ATF1, CREB1, and ATFa2 in HeLa cell extracts (451).
|
). Overexpression of either human or
Xenopus ERK cDNAs (187, 250) and specific activating
mutations of the ERK kinase (439, 452) induced cyclin D1 promoter
activity. The induction of ERK activity likely plays an important role
in the induction of cyclin D1 by oncogenic mutations of p21Ras (187),
Rac (438), members of the dbl family (440), and Neu (453). The relative
abundance of cyclin D1 is dependent upon the relative abundance of both
the MAPKs and their MAPK phosphatases such as mitogen-activated MKP-1
and PP2A, both of which reduce MAPK activity. MKP-1 is known to have
cell cycle-inhibitory activity, and overexpression of MKP-1 inhibited
cyclin D1 (439). Inhibition of the MAPK phosphatase PP2A by the SV40
small t antigen induced cyclin D1 levels and CD1K activity
(439). The domain of SV40 small t required for PP2A binding was
required for induction of cyclin D1. These results suggest that
relative balance of the MAPKs and their phosphatases determine the
abundance of cyclin D1.
Analysis of transcriptional regulation of the cyclin D1 gene has led to
the identification of distinct binding sites at the distal end of
oncogenic signaling pathways. This structure of the cyclin D1
regulatory region may allow for collaborative interactions between
oncogenic stimuli. c-Jun activation of the cyclin D1 promoter (187, 454) required an activator protein 1 (AP-1) (c-Fos/c-Jun) binding site
at -953 (187). This AP-1 binding site is the same region shown to be
involved in AII-induced mitogenesis (250) (Fig. 8B). Sequences at -58
bind CREB in trophoblastic cells (439), ATF-2 in breast cancer cells
(455) and chondrocytes (456), and Fos in primary fibroblasts (457). The
cyclin D1 gene was activated by ß-catenin (458, 459) through DNA
sequences shown to bind a lymphoid enhancer factor-1
(LEF-1)/ß-catenin heterodimer (458). Constitutively active STAT5
induced the cyclin D1 promoter through a STAT-binding site at -481
(460). pRB was shown to induce cyclin D1 expression, and Sp-1 and E2F
proteins bind to the proximal promoter modulating regulation by pRB and
the E2F transcription factors (Fig. 8B) (461). In several of these
analyses, cyclin D1 abundance was rate limiting in the proliferative or
transforming phenotype of the transcription factor examined (456, 457).
Thus, cyclin D1 levels were reduced in the chondrocytes of the
ATF-2-/- chondrocytes and the
c-fos/fosB-/- mouse embryo fibroblasts in
conjunction with reduced levels specifically of cyclin D1. The
abundance of cyclin D1 was also critical in interleukin-3 and
STAT5-dependent cell growth (460). It has been proposed therefore that
the relative abundance of the transcription factors binding to these
sites in the cyclin D1 promoter may contribute to the ability of a
particular cell type to respond to a given mitogenic signal.
Controversy surrounds the role of c-Myc in regulating cyclin D1 expression (462, 463), and the role of c-Myc in hormone signaling is not well understood. Cyclin D1 levels are frequently increased in pRB-overexpressing tumors, and overexpression of pRB induced cyclin D1 levels (464). c-Myc induces cyclin D1-kinase activity (462) or protein levels in some (465) but not other studies (463). Although in most circumstances, cyclin D1 abundance is regulated at the level of enhanced transcription, a contribution to cyclin D1 abundance at posttranscriptional level may mediate the effect of c-Myc on cyclin D1. The relative abundance of pRB and p53 in the cell may determine the effect of a particular signaling pathway on cyclin D1 abundance. Induction of p53 was associated with increased cyclin D1 abundance, and the induction occurred independently of the p21Cip1 status of the cell in one study (466). However other investigators have shown that p53 induction of cyclin D1 is through p21Cip1 (466). The requirement for cyclin D1 in p53-mediated arrest may reflect the ability of cyclin D1 to induce cell cycle arrest in specific cell types including normal human diploid fibroblasts (11, 30, 467). p300, A coactivator protein that regulates a diverse array of transcription factors, regulates cyclin D1 gene expression and associates with it in multiprotein complexes (Ref. 140 and C. Albanese and R. G. Pestell, submitted). The interaction between cyclin D1 and p300 may therefore be of considerable importance, allowing cyclin D1 to regulate a broad array of genes during cell cycle progression. Ongoing studies are attempting to unravel the contribution of these additional protein-protein interactions to the diverse role of cyclin D1 in cellular differentiation, proliferation, and transformation.
The human cyclin D2 and cyclin D3 promoter regions have been sequenced (468). The cyclin D2 promoter contains DNA sequences homologous to binding sites for AP-2, PUF, and Sp1 binding (468). The human cyclin D3 contains sequences resembling AP-2-, Sp-1-, and CTF-binding sites within the proximal -366 to -167-bp region involved in basal enhancer activity. The rat (469) and mouse (470) cyclin D3 promoters also contain Sp-1- and AP-2-like sequences in the proximal -350 bp, although the functional significance and the nature of the proteins binding to these sequences remain to be determined.
The human cyclin A gene contains a CRE that contributed to the timing of cyclin A expression in fibroblasts and was shown to bind CREB/CREM proteins in one study (378) and ATF-1 in another (380). The TGFß1-induced down-regulation of cyclin A promoter activity appeared to be mediated via the activating transcription factor (ATF) site (379). This effect occurred in association with reduced CREB phosphorylation bound to the proximal ATF/CRE-binding site (471). Because of the distinct temporal profile of induction of the cyclins at the transcriptional level and the distinct responses of these genes to different trophic stimuli, it is likely that focused chimeric promoter swaps will provide important information about the mechanisms governing these differential responses.
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VI. Conclusion |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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). The focus of these recent studies has been the
role of altered phosphorylation in regulating transcriptional activity
and gene expression. Attention is now turning to the role of
acetylation in the function of transcription factors and cell
cycle-regulatory proteins (227, 472, 473). An understanding of the role
of acetylation in hormone signal transduction will likely become
increasingly important. Historically the management of endocrine
disease has been directed at modification of the hormone secreted.
Ongoing studies of hormonal regulation of cell cycle proteins may lead
to the identification of common downstream targets responsible for
regulating hormonal effects. It will be of enormous value to
endocrinologists if targeted drug design can be used to treat
complications of endocrine disease through usurping these final common
pathways. In addition, the systematic analysis and identification of
cell cycle abnormalities in endocrine tumors may provide a rational
basis for alternate forms of tumor therapy. |
Note Added in Proof |
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TopAbstractI. IntroductionII. Regulation of the...III. Endocrine Regulation of...IV. Cyclins and CKIs...V. Transcription Factors...VI. ConclusionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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1 This work was supported in part by NIH Grants
1R29CA-70897–01, R01CA-75503, P50-HL-56399 (to R.G.P.). R.G.P. is a
recipient of the Irma T. Hirschl award and an award from the Susan G.
Komen Breast Cancer Foundation and Mortimer Harrison Foundation. Work
at the Albert Einstein College of Medicine was supported by Cancer
Center Core NIH Grant 5-P30-CA13330–26 (R.G.P.). A.T.R. was supported
by a P. F. Sabotka Postgraduate Scholarship from the University of
Western Australia. J.E.S. is an Established Scientist of the New York
City Affiliate of the American Heart Association and was supported
by Grant USAMRDC-2466. A.A. was supported by NIH Grant RO1
CA-55909. 
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S. J. Marx and W. F. Simonds Hereditary Hormone Excess: Genes, Molecular Pathways, and Syndromes Endocr. Rev., August 1, 2005; 26(5): 615 - 661. [Abstract] [Full Text] [PDF]
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C. Wang, S. Fan, Z. Li, M. Fu, M. Rao, Y. Ma, M. P. Lisanti, C. Albanese, B. S. Katzenellenbogen, P. J. Kushner, et al. Cyclin D1 Antagonizes BRCA1 Repression of Estrogen Receptor {alpha} Activity Cancer Res., August 1, 2005; 65(15): 6557 - 6567. [Abstract] [Full Text] [PDF]
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 D. N. Abrous, M. Koehl, and M. Le Moal Adult Neurogenesis: From Precursors to Network and Physiology Physiol Rev, April 1, 2005; 85(2): 523 - 569. [Abstract] [Full Text] [PDF]
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X. Zhang, J. P. Gaspard, Y. Mizukami, J. Li, F. Graeme-Cook, and D. C. Chung Overexpression of Cyclin D1 in Pancreatic {beta}-Cells In Vivo Results in Islet Hyperplasia Without Hypoglycemia Diabetes, March 1, 2005; 54(3): 712 - 719. [Abstract] [Full Text] [PDF]
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A. Chen and J. Xu Activation of PPAR{gamma} by curcumin inhibits Moser cell growth and mediates suppression of gene expression of cyclin D1 and EGFR Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G447 - G456. [Abstract] [Full Text] [PDF]
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L Hilakivi-Clarke, C Wang, M Kalil, R Riggins, and R G Pestell Nutritional modulation of the cell cycle and breast cancer Endocr. Relat. Cancer, December 1, 2004; 11(4): 603 - 622. [Abstract] [Full Text] [PDF]
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M. Fu, C. Wang, Z. Li, T. Sakamaki, and R. G. Pestell Minireview: Cyclin D1: Normal and Abnormal Functions Endocrinology, December 1, 2004; 145(12): 5439 - 5447. [Abstract] [Full Text] [PDF]
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S. Benzeno, G. Narla, J. Allina, G. Z. Cheng, H. L. Reeves, M. S. Banck, J. A. Odin, J. A. Diehl, D. Germain, and S. L. Friedman Cyclin-Dependent Kinase Inhibition by the KLF6 Tumor Suppressor Protein through Interaction with Cyclin D1 Cancer Res., June 1, 2004; 64(11): 3885 - 3891. [Abstract] [Full Text] [PDF]
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C. Sharma, A. Pradeep, R. G. Pestell, and B. Rana Peroxisome Proliferator-activated Receptor {gamma} Activation Modulates Cyclin D1 Transcription via {beta}-Catenin-independent and cAMP-response Element-binding Protein-dependent Pathways in Mouse Hepatocytes J. Biol. Chem., April 23, 2004; 279(17): 16927 - 16938. [Abstract] [Full Text] [PDF]
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P. Rouet-Benzineb, T. Aparicio, S. Guilmeau, C. Pouzet, V. Descatoire, M. Buyse, and A. Bado Leptin Counteracts Sodium Butyrate-induced Apoptosis in Human Colon Cancer HT-29 Cells via NF-{kappa}B Signaling J. Biol. Chem., April 16, 2004; 279(16): 16495 - 16502. [Abstract] [Full Text] [PDF]
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A. Sunters, D. P. Thomas, W. A. Yeudall, and A. E. Grigoriadis Accelerated Cell Cycle Progression in Osteoblasts Overexpressing the c-fos Proto-oncogene: INDUCTION OF CYCLIN A AND ENHANCED CDK2 ACTIVITY J. Biol. Chem., March 12, 2004; 279(11): 9882 - 9891. [Abstract] [Full Text] [PDF]
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K. H. H. Wong, H. D. Wintch, and M. R. Capecchi Hoxa11 Regulates Stromal Cell Death and Proliferation during Neonatal Uterine Development Mol. Endocrinol., January 1, 2004; 18(1): 184 - 193. [Abstract] [Full Text] [PDF]
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I. Cozar-Castellano, K. K. Takane, R. Bottino, A.N. Balamurugan, and A. F. Stewart Induction of {beta}-Cell Proliferation and Retinoblastoma Protein Phosphorylation in Rat and Human Islets Using Adenovirus-Mediated Transfer of Cyclin-Dependent Kinase-4 and Cyclin D1 Diabetes, January 1, 2004; 53(1): 149 - 159. [Abstract] [Full Text] [PDF]
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C. R. Kahl and A. R. Means Regulation of Cell Cycle Progression by Calcium/Calmodulin-Dependent Pathways Endocr. Rev., December 1, 2003; 24(6): 719 - 736. [Abstract] [Full Text] [PDF]
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C. Wang, N. Pattabiraman, J. N. Zhou, M. Fu, T. Sakamaki, C. Albanese, Z. Li, K. Wu, J. Hulit, P. Neumeister, et al. Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor {gamma} Expression and Transactivation Mol. Cell. Biol., September 1, 2003; 23(17): 6159 - 6173. [Abstract] [Full Text] [PDF]
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W. H. Walker Molecular Mechanisms Controlling Sertoli Cell Proliferation and Differentiation Endocrinology, September 1, 2003; 144(9): 3719 - 3721. [Full Text] [PDF]
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H. K. Kinyamu and T. K. Archer Estrogen Receptor-Dependent Proteasomal Degradation of the Glucocorticoid Receptor Is Coupled to an Increase in Mdm2 Protein Expression Mol. Cell. Biol., August 15, 2003; 23(16): 5867 - 5881. [Abstract] [Full Text] [PDF]
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S. Paternot, K. Coulonval, J. E. Dumont, and P. P. Roger Cyclic AMP-dependent Phosphorylation of Cyclin D3-bound CDK4 Determines the Passage through the Cell Cycle Restriction Point in Thyroid Epithelial Cells J. Biol. Chem., July 11, 2003; 278(29): 26533 - 26540. [Abstract] [Full Text] [PDF]
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 H. Huang, S. B. Petkova, A. W. Cohen, B. Bouzahzah, J. Chan, J.-n. Zhou, |
