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RB and cell cycle progression

Abstract

The Rb protein is a tumor suppressor, which plays a pivotal role in the negative control of the cell cycle and in tumor progression. It has been shown that Rb protein (pRb) is responsible for a major G1 checkpoint, blocking S-phase entry and cell growth. The retinoblastoma family includes three members, Rb/p105, p107 and Rb2/p130, collectively referred to as ‘pocket proteins’. The pRb protein represses gene transcription, required for transition from G1 to S phase, by directly binding to the transactivation domain of E2F and by binding to the promoter of these genes as a complex with E2F. pRb represses transcription also by remodeling chromatin structure through interaction with proteins such as hBRM, BRG1, HDAC1 and SUV39H1, which are involved in nucleosome remodeling, histone acetylation/deacetylation and methylation, respectively. Loss of pRb functions may induce cell cycle deregulation and so lead to a malignant phenotype. Gene inactivation of pRB through chromosomal mutations is one of the principal reasons for retinoblastoma tumor development. Functional inactivation of pRb by viral oncoprotein binding is also shown in many neoplasias such as cervical cancer, mesothelioma and AIDS-related Burkitt's lymphoma.

Introduction

The Rb gene is an archetypal tumor suppressor gene that was first identified in a malignant tumor of the retina known as retinoblastoma.

Retinoblastoma is a sporadic or hereditary pediatric neoplasm arising from retinal cells, and Knudson (1971) hypothesized that the tumor phenotype is not apparent unless both copies of the gene are damaged. The cloning of the retinoblastoma Rb gene and the identification of biallelic Rb mutations in retinoblastoma tumors confirm the hypothesis that such gene product exerts the action of a tumor suppressor. Several human tumors show mutations and deletions of the Rb gene, and inherited allelic loss of Rb confers increased susceptibility to cancer formation (Dunn et al., 1988). The Rb gene is functionally inactivated in most human neoplasms either by direct mutation/deletion, such as in retinoblastoma, osteosarcoma and small-cell lung carcinoma, or indirectly through altered expression/activity of upstream regulators (Liu et al., 2004). Nevertheless, the Rb protein plays a pivotal role in the negative control of the cell cycle and in tumor progression.

It has been shown that Rb protein (pRb) is responsible for a major G1 checkpoint (restriction point) blocking S-phase entry and cell growth, promoting terminal differentiation by inducing both cell cycle exit and tissue-specific gene expression (Weinberg, 1995).

The Rb gene family includes three members, Rb/p105, p107 and Rb2/p130, collectively referred to as ‘pocket proteins’. The term ‘pocket proteins’ derives from the conserved binding pocket region through which pRb, p107 and Rb2/p130 bind viral oncoproteins and cellular factors such as the E2F family of transcription factors. Further studies have shown that overexpression of all three pocket proteins in cells can induce growth arrest in the G1 phase of the cell cycle (Dyson, 1998). These studies helped define the role of the Rb gene family in regulating transition between cell proliferation and terminal differentiation. How pRb family members control cell cycle proliferation is not completely understood. Moreover, the interaction between the pRb family proteins and the E2F family transcription factors plays a central role in governing cell cycle progression and DNA replication by controlling the expression of cell cycle E2F-dependent genes (Macaluso et al., 2005). In addition, pRb recruits chromatin remodeling factors such as histone deacetylase 1 (HDAC1) (Luo et al., 1998), SWI/SNF factors (Harbour and Dean, 2000; De Luca et al., 1997), Polycomb group proteins (Dahiya et al., 2001) or methyltransferase (Nielsen et al., 2001) that act on the nearby surrounding nucleosome structure.

The pRb protein has also a central role in various differentiation processes, including eye, lens, brain, peripheral nervous system, epidermis, melanocytes, hair cells, muscle and liver. For example, pRb is necessary for the completion of the muscle differentiation program and for myogenic helix–loop–helix-dependent transcription (reviewed by De Falco, in this issue). The most convincing evidence of the importance of pRb in cellular differentiation comes from studies of Rb knockout mice, where the disruptions of the Rb gene cause death by day 14 of gestation, associated with defects in the development of the hematopoietic system and central nervous system (Lee et al., 1992).

Rb family proteins

The Rb/p105 and Rb2/p130 map to human chromosomal areas 13q14 and 16p12.2, respectively (Baldi et al., 1996), in which mutations have been found in several human neoplasias. The p107 maps to chromosomal area 20q11.2, which is not frequently found to be altered in human neoplasia, although there is a recent report (Ichimura et al., 2000) of deletion or functional inactivation for p107 in human tumors. The Rb family proteins share large regions of homology, especially in a bipartite region, the so-called ‘pocket domain’ (Claudio et al., 2002). Rb2/p130 and p107 are more related to each other, with about 50% amino-acid identity, than they are to Rb/p105 (30–35% identity). The Rb family proteins show a peculiar steric conformation owing to the pocket region, which is responsible for many of the protein–protein interactions in the homeostasis of the cell cycle (Paggi et al., 1996). The pocket region is characterized structurally by two conserved functional domains (A and B) separated by a spacer (S), which substantially differs among the three Rb proteins, Rb/p105, Rb2/p130 and p107. Members of the E2F family of transcription factors, which are cellular Rb-binding proteins, interact with the A and B pocket domains (Puri et al., 1999), as well as proteins that possess the LXCXE peptide motif, such as oncoproteins from several DNA tumor viruses (Lee et al., 1998), the D-type cyclins (Weinberg, 1995) and HDAC. Rb proteins can bind E2F and several other LXCXE-containing proteins at the same time because the two binding sites for E2F peptide and LXCXE peptide are close but distinct from each other (Lee et al., 1998; Gallo and Giordano, 2005).

Rb family members are post-translation regulated proteins and their gradual phosphorylation leads to their functional inactivation; Rb protein phosphorylation is a cell cycle-dependent phenomenon and cyclin/cyclin-dependent kinase (cdk) complexes are responsible for this regulation. In the early G1 phase, D-type cyclins interact with the A/B pocket of the Rb gene products via their LXCXE motif; in particular, they couple with the kinase cdk4 or cdk6 and phosphorylate pRb/105 and Rb2/p130 (Dowdy et al., 1993). In the middle to late G1 phase, cyclins E and A form complexes with cdk2, which specifically target Rb/p105, Rb2/p130 and p107 (De Luca et al., 1997). This step is crucial for the inactivation of members of the pRb family and the release of E2F factors; however, it is still not evident how phosphorylation could induce a conformational change to allow physical dissociation of E2F factors from Rb family proteins. Rb proteins can be phosphorylated both on A/B domains of the pocket region and on the C-terminal domain. The main hypothesis is that the phosphorylation at the carboxy terminus of pRb by cyclin D/cdk4 (or ckd6) causes a conformational modification, which displaces the HDAC bound to pRb through its LXCXE motif. This facilitates the phosphorylation of the A/B pocket region by cyclin E (A)/cdk2, leading to the release of E2F from pRb/p105.

Pocket protein phosphorylation is often reversed by dephosphorylation, which causes transient inactivation. Pocket protein dephosphorylation is known to take place in the period from anaphase to G1 and to depend upon protein phosphatase 1 (Ludlow et al., 1993); dephosphorylation occurs in response to growth inhibitory signals. In some situations, phosphorylation can cause permanent inactivation, for example, by binding skp2 (Tedesco et al., 2002) and leading to degradation. High levels of Cdk activity may result in phosphorylation of pRb-Ser567, which exposes the Rb protein to a proteolytic cleavage site and degradation by an unidentified protease (Ma et al., 2003).

The C-terminal region of pRb proteins also plays an important role in orchestrating the activity of these proteins. The carboxy-terminal region differs in length between Rb/p105 and the other two proteins Rb2/p130 and p170, which are instead very similar in the amino-acid sequence. As described above, analysis of structural similarities among the Rb family members discloses that p107 and Rb2/p130 are more strictly related to each other than to Rb. The carboxy-terminal region is quite important as it contains the nuclear localization signal (NLS) and domains responsible for HADC1 and cyclin/cdk complex binding. The NLS controls the transport of Rb proteins from the cytoplasm to the nucleus; it consists of two cluster basic residues separated by a stretch of amino acids including proline residues (Dingwall et al., 1991). Nuclear localization signal works as a carrier also for E2F4 and E2F5 proteins, both of which do not present the NLS.

It has been demonstrated that HDAC1 needs both the LXCXE-binding motif of the pocket region and the C-terminus region in order to form a stable complex (Stiegler et al., 1998) with Rb proteins. The binding sites for E2F and HDAC1 are distinct from each other (Lee et al., 1998), so we can hypothesize that Rb works as a link between HDAC1 and E2F proteins when associated to E2F-responsive promoters.

Rb and cell cycle

Two of the most important proteins involved in the cell cycle machinery are cyclin-dependent kinases and cyclins. A variety of cyclin/cdk complexes are in fact able to guide the cdks to appropriate substrates and activate their catalytic activity (Figure 1). Cyclin/cdk complexes are formed during distinct phases of the cell cycle, and are specifically involved in the phosphorylation of a distinct set of target proteins. The cyclin/cdk complexes orchestrate the advance of the cell through different phases of its growth cycle. Mammalian G1 cyclins D and E mediate progression through G1/S phases. Three D-type cyclins exist (cyclin D1, D2 and D3), which are expressed differently in various cell lineages, with most cells expressing cyclin D3 and either D1 or D2. Two types of cyclin E (E1 and E2) exist, which show overlapping expression patterns in mouse tissues and can be co-overexpressed in human tumors (Geng et al., 2001). Mitotic cyclins A and B mediate progression through the S/G2/M phases. Cyclin A1 is expressed in meiosis and early embryogenesis, whereas cyclin A2 is found in proliferating somatic cells. Cyclin B2 probably plays a role in Golgi remodeling during mitosis (Jackman et al., 1995), whereas cyclin B1 controls other functions of this cyclin type. Until now, at least 16 cyclins have been discovered, for many of which binding partners and functions have yet to be identified.

Figure 1
figure1

Rb and cell cycle machinery. Rb and Rb-p represent the unphosphorylated and the phosphorylated forms of the retinoblastoma protein. In G0 and early G1, Rb physically associates with E2F factors and blocks their transactivation domain. In late G1, Rb-p releases E2F, allowing the expression of genes that encode products necessary for S-phase progression.

D-type cyclins are short-lived proteins whose synthesis and assembly with Cdk4 or Cdk6 in G1 is dependent on mitogenic signaling (Hitomi and Stacey, 1999). Cyclin D/Cdk activity goes on through the first and subsequent cycles as long as mitogenic stimulation continues. Cyclin E protein levels peak at the G1/S progression, followed by an increase in cyclin A levels in the S phase. Both cyclin E and A interact with and activate Cdk2, whereas cyclin A can also bind Cdk1 (Lees et al. 1993). At the G2/M boundary, cyclin B levels increase, resulting in activation of its partner, Cdk1. This fluctuation in cyclin expression and the resultant oscillation in Cdk activity form the basis of a coordinated cell cycle progression.

The cell responds to mitogenic stimuli and decides to advance through the various phases of the cell cycle only during a limited phase of its cycle. In fact, the cell needs stimulation only during the first two-thirds of its G1 phase where it may decide to continue its advance and complete its cell cycle. This point is termed ‘restriction point’ (R point); it is a central event in normal cellular proliferation control (Lundberg et al., 1999). It has been demonstrated that pRb is the molecular device that serves as the R point switch. pRb is hypophosphorylated in resting G0 cells, is increasingly phosphorylated during progression through G1 and is maintained in a hyperphosphorylated state until late mitosis (Weinberg, 1995; Claudio et al., 1996, Claudio et al., 2002). pRb phosphorylation seems to be related to mitogenic signals, which converge on the cell cycle machinery, represented by the cyclin D1/cdk4 (cdk6) complex in the early and mid-G1, and composed of cyclin E/cdk2 in late G1.

Like pRb, p107 is phosphorylated by the cyclin D1–cdk4/6 complex activity, whereas Rb2/p130 phosphorylation depends on the cyclin D3–cdk4 complex activity. All of the three pocket proteins are phosphorylated by the cyclin E/cdk2 complex, and Rb2/p130 and p107, but not Rb/p105, are phosphorylated by cyclin A–cdk2. Pocket proteins may also regulate the earlier transition from G0 to G1 (Cobrinik, 2005). In G0, the RNA content is at low levels and hypophosphorylated pocket proteins contribute to the G0 state by repressing ribosomal RNA and tRNA gene expression. Ren and Rollins (2004) recently found that the cyclin C/cdk3 complex is able to increase the RNA content while mediating G0 exit by phosphorylating pRb in the G0 state. As expression of cyclin C precedes that of the cyclin D, in keeping with these considerations, the pRb phosphorylation by cyclin C–Cdk3 mediates the G0–G1 transition.

When in its actively growth-suppressing hypophosphorylated state, pRb physically associates with E2F factors and blocks their ability to activate expression of genes that encode products necessary for S-phase progression.

The Rb proteins repress gene transcription, required for transition from G1 to S phase, by directly binding to the transactivation domain of E2F and by binding to the promoter of these genes as a complex with E2F (Adams et al., 1995). Pocket proteins regulate G1–S transition also through E2F-independent mechanisms: (1) p107 and p130 bind and inhibit the cyclin E/cdk2 and cyclin A/cdk2 kinases and (2) pRb inhibits cdk activity and G1–S progression by increasing the expression of p27 and stabilizing the p27 protein by binding the Skp2 protein and interfering with the Skp2–p27 complex, thus avoiding p27 ubiquitination. Progression of the cells through G1 and S phases requires inactivation of Rb protein phosphorylation. The phosphorylation status of these proteins is regulated by Cdk inhibitor (CKI) binding to the cyclin/cdk complexes (Sherr et al., 1995). The CKI inhibitors p15, p16 and p17 specifically act on cyclin D kinase activity, whereas p21, p27 and p57 act on all other cyclin/cdk complexes. It has been reported that both cyclin D1 overexpression and related CKI inhibitor alterations produce persistent hyperphosphorylation of pRb, resulting in cell cycle arrest.

Extracellular physiological signals induce the phosphorylation of Rb protein, affecting the decision to transit the R point. Mitogens directly induce rapid expression of cyclin D, which begins to inactivate pRb through its ability to associate with its previously cited cdk4 (cdk6) partners (Matsushime et al., 1991). Each type of cyclin D could receive different upstream signals that converge on it, so that we can say that the control of cell proliferation in various cell types, known to be exercised by different mitogens, is modulated via expression of a distinct D-type cyclin gene, which is under the control of a distinct transcriptional promoter. The common point is that all these different signals converge to a common target, which is pRb.

There is no evidence that mitogenic stimuli directly modulate the levels of cyclin E or cyclin/cdk2 complex. Extracellular signals can negatively affect the cyclin/cdk machinery as well. Serum starvation leads to a collapse of cyclin D levels and activity and to an increase of specific CKI (Resnitzky et al., 1994).

Rb activity in cell cycle machinery can also be regulated at the transcriptional and protein levels by the transcription factor ICBP90 (Hopnefer, 2000). ICBP90 (inverted CCAAT box binding protein of 90 kDa) is a transcriptional regulator of the topoisomerase II alpha gene and it has also two consensus binding sites for pRb in its primary sequence. The promoter of the Rb gene contains several putative binding sites for ICBP90. Overexpression of ICBP90 induces downregulation of pRb expression in lung fibroblasts, as a result of mRNA decrease, and increases both the S- and G2/M-phase cell fractions of fibroblasts and the topoisomerase IIalpha expressions. DNA chromatin immunoprecipitation experiments show that ICBP90 binds to the Rb gene promoter under its methylated status. These results show that ICBP90 regulates Rb at the protein and gene transcription levels, thus favoring the entry into the S phase of the cells, and we can hypothesize that ICBP90 overexpression, found in cancer cells, could be involved in the altered checkpoint controls occurring in carcinogenesis (Jeanblanc, 2005).

How does Rb control cell proliferation?

How Rb proteins control cell proliferation is not completely understood. It has been established that RB protein associates with a wide variety of transcription factors and chromatin-associated complexes.

Rb protein binding inhibits the transcriptional activation ability of E2F factors, masking the transcriptional activation domain and, in some cases, converting E2F factors to transcription repressors. The importance of the Rb–E2F interaction in cell growth control is demonstrated by the finding that all naturally occurring Rb mutants isolated from human tumors lack the ability to bind and negatively regulate E2F (Qian et al., 1992).

Rb proteins are thought to inhibit expression of E2F-regulated genes in two ways (Dyson et al., 2002): by directly binding and blocking the activation domain of E2F proteins or by active repression through the recruitment of HDAC, SWI/SNF factors, Polycomb group proteins (Dahiya et al., 2001) or methyltransferase (Vandel et al., 2001).

Binding to E2F transcription factors

The E2F family of transcription factors consists of at least seven E2F family members, E2F1, E2F2, E2F3, E2F4, E2F5, 2A7E and E2F7.

A functional E2F transcription factor consists of a heterodimer containing an E2F polypeptide and a DP polypeptide (Helin et al., 1993). Each of the E2F polypeptides can heterodimerize with either DP1 or DP2 (DP3) (Ormondroyd et al., 1995), although the E2F7 transcription factor binds DNA in a DP-independent manner.

There are functional and structural differences within the E2F family. E2F1, E2F2 and E2F3 are transcriptional activators, which interact with pRB. E2F4 and E2F5 are transcriptional repressors and mostly bind Rb2/p130 and p107 (Gaubatz et al., 2000). 2A7E seems not to have a pocket protein interaction domain; it interacts with Polycomb proteins and represses transcription (Morkel et al., 1997). The recently identified E2F7 and E2F8 are also believed to repress specific promoters (Di Stefano et al., 2003). The DP heterodimerization partner does not seem to determine the binding specificity of E2F factors for the pocket proteins. However, DP factors do function in stabilizing the interaction between E2F factors and the Rb proteins (Helin et al., 1993).

During the cell proliferation response, there is a different expression of the various E2Fs, presenting an increase in E2F3 in early-G1 to mid-G1 and with E2F1 and E2F2 increasing at the G1/S boundary (Johnson et al., 1998). E2F4 and E2F5 are expressed throughout the cell cycle, but in G0 and early G1, they are bound to the nucleus by p107 and Rb2/p130, forming transcriptional repressor complexes. p107 and Rb2/p130 recruit E2F4 into the nuclei of the cell, inducing the repression of S-phase genes transcription and cell proliferation. Meanwhile, pRb is believed to bind E2F1–3 either at or sequestered away from E2F-responding promoters (De La Luna et al., 1996).

After stimulation, quiescent cells enter the cell cycle, and in the early-G1 phase, pRb is phosphorylated and consequently E2F1–3 transcriptional factors are released, whereas only in mid-G1 to late G1, we can observe the loss of Rb2/p130 complexes. Therefore, in the late G1 phase, Rb proteins are phosphorylated and dissociate from E2Fs; E2F4 and E2F5 move to the cytoplasm and E2F1–3 bind to the E2F-responding promoters in sites that often are different from the binding site of E2F repressor proteins (Cinti et al., 2000). At the G1/S-phase transition, complexes containing p107 in association with E2F4 can still be detected (Mudryj, 1991). These E2F4–p107 complexes also contain cyclin E or A and cdk2 (Shirodkar, 1992). E2F–p130 complexes have also been shown to accumulate, as some cell types, including myoblasts and melanocytes, undergo terminal differentiation (Shin et al., 1995).

pRb and the related proteins p107 and Rb2/p130 do not have a fully redundant function in regulating E2F genes transcription; instead they show a great redundancy at the cell cycle level. For example, in pRb−/− fibroblasts, there is a compensatory increase in p107 protein levels during the arrest in G0/G1 phase (Sage et al., 2003). The mechanism of this antiproliferative effect of p107 is unknown – p107 could substitute pRb in the regulation of E2F1–3 or it may repress some E2F-responsive genes that are controlled by pRb (Lee et al., 2002). Furthermore, pRb can also bind E2F4 in vitro but fails to regulate the E2F-responsive promoter in p107−/− and p130−/− cells. It has been shown that the pRb–E2F4 complex, in p107−/− cells, fails to bind the same promoter sites that are usually linked by the p107–E2F4 complex in wild-type cells (Rayman et al., 2002).

However, pRb is involved in the repression of a limited set of E2F genes in G0 and G1 cells, and recently its role in cyclin E gene repression has been defined. The cyclin E promoter is in fact deregulated in cells that have lost pRb, even though the p107/p130–E2F4 complex is still available (Herrera et al., 1996). pRb mediates the repression of cyclin E promoter by the recruitment of HDAC and histone methyltransferase on a particular E2F-SP1 site that is exclusively linked by the pRb–E2F1–3 complexes. Cyclin E is derepressed not only by the loss of pRb but also by the loss of E2F1–3 proteins, so it is clear that E2F1–3 proteins act in this case as repressors instead of activators (Wu et al., 2001). p107 or Rb2/p130 can substitute pRb only if they are present in a sufficient amount to bind E2F1–3 proteins (Lee et al., 2002).

pRb can inhibit proliferation through an E2F-independent mechanism. For example, pRb can induce the formation of promyelocytic leukemia (PML) nuclear bodies (Fang et al., 2002), increase p27 expression (see above), suppress Ras signaling (Lee et al., 2002) and cooperate with hLin-9, a protein that is involved, similar to pRb, in suppressing transformation but in an E2F- independent manner (Gagrica et al., 2004).

RB/E2F repressor complexes can also be regulated in a Cdk-independent manner. For example, transforming growth factor-β recruits E2F4/p107 and E2F5/p107 by the c-myc promoter irrespective of cell phase, and it is still stable in the presence of high activity of Cdk (Chen et al., 2004). In human cells, it has been noticed that p107 and p130 are removed from cell cycle-regulated gene promoters during S phase, but are still located at the promoters of apoptotic genes (Young et al., 2004). The regulation of RB/E2F complex in a Cdk-independent manner is not yet well understood.

Binding to proteins that modify chromatin structure

Repression by Rb proteins requires the conserved A and B domains of the pocket protein and appears to involve the association of other proteins in addition to E2F factors. Rb proteins repress transcription by remodeling chromatin structure through interaction with proteins such as hBRM, BRG1, HDAC1 and SUV39H1 (Shao et al., 1995), which are respectively involved in nucleosome remodeling, histone acetylation/deacetylation and methylation.

hBRM and BRG1 are mammalian homologs of components of the yeast chromatin remodeling complex SNF2/SWI2 (Strober et al., 1996). Both BRG1 and hBRM have been shown to associate with pRB and they are implicated in pRb-mediated repression. A physical interaction between pRb and BRG1 is not required for pRB-induced growth arrest and transcriptional repression of E2F target genes (Kang et al., 2004).

Histone deacetylase 1 is an HDAC that has recently been shown to be recruited to E2F complexes by pRb and to function in repressing cyclin E gene expression (Magnaghi-Jaulin et al., 1998). Histones are generally hyperacetylated at the promoters of actively transcribed genes but are hypoacetylated at silenced genes. Histone deacetylase is generally recruited on gene promoters by many transcriptional regulators. pRB associates with HDAC activity in vivo and binds to class I HDACs (HDAC1–HDAC3) in vitro. pRB-mediated repression is enhanced by HDAC1 in transient transfection experiments, and to confirm this association between pRb and HDAC, it has been shown that inhibition of HDAC activity with trichostatin A interferes with pRB-mediated repression at a subset of E2F-regulated promoters (Zhang et al., 2000). Histone deacetylase is thus involved in modulating the repressive activity of pRb on E2F gene promoters. The recruitment of HDAC may be indirect and accompanied by recruitment of other binding proteins such as RBP1, corepressors and chromatin remodeling proteins (Lai et al., 1999). Rayman et al. (2002) have shown that in Rb−/− cells, HDAC is on E2F gene promoters; this allowed us to hypothesize that pRb is not strictly necessary in E2F transcriptional repression, but that other mechanisms could be involved. Histone acetyltransferase proteins (CCBP, p300, Tip6, etc.) participate in the activation of E2F proteins (Condorelli and Giordano, 1997); they interact with the activation domain of E2F proteins and stimulate their activation.

SUV39H1 is a methyltransferase that specifically methylates K9 of histone H3 and cooperates with pRb in the repression of the promoter of E2F-responding genes. In cells lacking pRB, there is no association of me-K9-H3 in these promoters (Rea et al., 2000); in particular, these results have been developed by studying cyclin E, one of the best E2F target genes modulated by pRb. Both pRb and SUV39H1 are directly involved in repressing cyclin E transcription, as in both pRb−/− and SUV39H1−/− double knockout cells, cyclin E is overexpressed. Rb proteins are also able to prevent the assembly of pre-initiation complexes, inhibiting the activity of transcription factors recruited on E2F gene promoters (Ross et al., 1999).

The type of repression that Rb proteins exert on cell growth depends on the type of corepressor proteins that bind E2F target genes. For example, different types of transcriptional repression can operate at the same promoter in a different cellular state or in a different cell phenotype (Dimova and Dyson, 2005).

The pRb/E2F complex can also promote stable repression independently of cell cycle position or by being resistant to cell cycle progression. Unfortunately, the mechanisms through which pRb is involved in processes that permanently repress E2F-dependent transcription are not well known until now. For example, E2F4 can be found at E2F-regulated promoters in S phase, but it is unclear whether it acts as an activator. Senescent cells, for example, show a higher level of H3K9 methylation in E2F-responsive promoters in comparison to quiescent cells (Narita et al., 2003). Rb proteins mediate E2F-responsive genes both in a quiescent and senescent/differentiated state, the former being characterized by low levels of H3K9 methylation, and the latter by high levels of H3K9 methylation. The identity of histone methyltransferase that is recruited on the promoter can also modulate the transition between a quiescent cell and a senescent/differentiated cell.

Rb and cancer

Loss of pocket protein functions may induce cell cycle deregulation and lead to a malignant phenotype. Rb protein can be functionally inactivated by phosphorylation, mutations or viral oncoprotein binding. As a direct consequence, E2F transcription factors are liberated by control of Rb protein and induce deregulation of the cell cycle.

Loss of heterozygosity of tumor suppressor genes is observed in a variety of human tumors, including retinoblastomas, osteosarcomas and small-cell lung carcinomas. In retinoblastoma, a malignant childhood tumor, pRb is functionally inactivated by mutations or partial deletions (Bignon et al., 1993). Bilateral familial retinoblastomas show germline mutation in pRb, and tumors develop when both the alleles are mutated (pRb−/−). These data are supported by studies on animals in which heterozygous mutation of pRb (pRb+/−) is not sufficient for the development of the tumor, whereas pRb+/− and p107−/− animals develop retinal dysplasia, showing that p107 is able to prevent this type of tumor in pRb−/− mice and so it acts as a tumor suppressor (Lee et al., 1996). Gene inactivation through chromosomal mutations is one of the principal reasons for retinoblastoma loss of function in cancer.

Other insults can act on pRb altering cell cycle growth. For example, in tumor cells, pRB/E2F interaction is broken by pRb binding to DNA tumor virus oncoprotein such as papilloma virus E7 and displacing E2F transcription factors. Functional inactivation by viral oncoprotein binding is observed in many neoplasias such as cervical cancer, mesothelioma and AIDS-related Burkitt's lymphoma. For example, in AIDS-related Burkitt's lymphoma, Rb2/p130 and p107 are expressed at high levels, suggesting that these proteins could be functionally inactivated during HIV infection (Cinti et al., 2000; De Falco et al., 2003; Lazzi et al., 2002; Cinti and Giordano, 2000).

In cervical cancer, the inactivation of pRb occurs through the binding of the E7 oncoprotein, which is associated with over 90% of these cancer cases (Kim et al., 2005; Cobrinik et al., 1993).

Inappropriate pRb phosphorylation due to rearrangement and overexpression of cyclin D1 also contributes to the development of several types of human tumors, including parathyroid adenomas, B-cell lymphomas and squamous cell carcinomas (Hunter et al., 1994). The overproduction of cyclin D1 and its partner cdk4 leads to a constitutive phosphorylation of Rb proteins and to a deregulation of E2F transcriptional activity. It has also been reported that deregulation of other G1 cyclins (D2, D3 and E) is associated with tumorigenesis (Keyomarsi et al., 1995).

The finding that the cyclin D/cdk inhibitor, p16/INK4A, is lost in several different tumor types (for example, breast carcinoma, hepatocellular carcinoma, etc.) suggests that the altered activity of cyclin/cdk complexes in G1 phase can be oncogenic (Hunter et al., 1994). p16/INK4 controls Rb activity by binding cdk4/6 and inhibiting the action of cyclin D; in this way, p16/INK4 promotes the inhibition of pRb phosphorylation and the formation of the pRb/E2F repressor complex. Loss of function of the kinase inhibitor enhances cyclin D/cdk4 kinase activity, which promotes hyperphosphorylation of pRb that is no more able to regulate cell cycle checkpoints.

For example, most of pRb-positive lung cancer cell lines are p16/INK4-negative. This would indicate that the pathogenesis of some lung cancers occurs by the absence of p16 inhibitor, which functions to keep pRb hypophosphorylated (Caputi et al., 2005). The main role of pRb in lung cancer is also evaluated by the notion that in small lung cancer cells, the frequency of deletion or mutation of pRb is 90% (Slagia et al., 1998).

In nasopharyngeal carcinoma, no pRb rearrangements have been found, whereas Rb2/p130 results in a drastic reduction in the expression level of both nasopharyngeal cell line and in primary tumors owing to a mutation in the C-terminal domain of the oncosuppressor protein (Claudio et al., 2000).

We can summarize that a breakdown of cell cycle, owing to products of oncogenes (cyclins, cdks and oncovirus) and tumor suppressor genes (Rb proteins and CKI) whose functions converge on alteration of E2F genes, leads to a cancer phenotype.

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Acknowledgements

This work is supported in part by grants from NIH and Sbarro Health Research Organization. We thank Simone C, De Falco G and Bagella L for their critical comments.

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Correspondence to A Giordano.

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Giacinti, C., Giordano, A. RB and cell cycle progression. Oncogene 25, 5220–5227 (2006). https://doi.org/10.1038/sj.onc.1209615

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Keywords

  • Rb
  • cell cycle
  • E2F
  • cancer
  • Rb2/p130
  • tumor suppressor

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