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Coordinated regulation of life and death by RB
Author: B. N. Chau
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"� 2003 Nature Publishing Group The retinoblastoma-susceptibility gene (RB) is a proto- typical tumour suppressor. Germline mutation in the RB gene causes the highly penetrant hereditary retinoblastoma, which results from the bi-allelic loss of RB in embryonic retinoblasts. Consistent with its tumour-suppressor function, RB inhibits cell prolifera- tion. The mechanism by which RB inhibits cell growth has been elucidated. Through its interaction with the E2F family of transcription factors, RB represses genes that are required for DNA synthesis 1 . RB-mediated repression of transcription occurs at two levels. First, RB blocks the interaction between E2F and other transcrip- tion co-activators 1 . Second, RB recruits chromatin- modifying enzymes to further repress transcription 1?3 . The inactivation of RB, therefore, is a prerequisite for cell proliferation. RB is known to be inactivated by four mechanisms (FIG. 1). First, genetic inactivation of RB is observed in retinoblastoma, although the frequency of RB mutation is low among sporadic cancers. Second, viral oncopro- teins, such as T antigen, E1A and E7, sequester RB from its physiological partners, thereby disrupting its function 1 . Third, RB can be inactivated by phosphoryla- tion, which is catalysed by the cyclin-dependent protein kinases (CDKs) during cell-cycle progression. Finally, RB is degraded by caspases in response to apoptotic stimuli. In all cases, the inactivation of RB can confer a prolifera- tive advantage, due to the derepression of E2F-regulated genes 1 . Interestingly, RB mutation, RB sequestration by viral proteins and RB degradation by caspases have each been associated with increased levels of apoptosis. Recent studies in mice have shed new light on the mechanisms by which RB regulates cell death, and indicate its direct role in inhibiting apoptosis. Apoptosis in Rb-null mice Studies with Rb-null mice more than a decade ago first revealed the role of Rb in apoptosis regulation. Phenotypes of these mice include excessive apoptosis of cells in the nervous systems, lens and skeletal muscles 4?6 . Breeding of Rb-heterozygous mice with other geneti- cally engineered strains has identified suppressors of the ectopic apoptosis phenotype, indicating that these gene products interact with Rb in apoptosis regulation (TABLE 1; FIG. 2). Mutation of the mouse p53 gene (Trp53 ?/? ) diminished apoptosis in the lens and the cen- tral nervous system (CNS) of Rb-null embryos 7,8 , and mutation of E2f1 also suppressed apoptosis of the Rb- nullCNS neurons and the lens epithelial cells 9,10 .A com- mon target gene for transcriptional regulation by E2f1 and p53 is apoptosis protease activating factor-1 (Apaf1). Apaf1 is the key component of the apoptosome ? a downstream effector of mitochondria-dependent apoptosis 11 . E2F1 and p53 binding sites have been mapped in the promoter of Apaf1 (REF. 12). CNS neurons of Apaf1-null embryos are resistant to apoptosis induced by adenovirus-expressing p53 (REF. 13). Consistent with these observations, Apaf1 expression is COORDINATED REGULATION OF LIFE AND DEATH BY RB B. Nelson Chau and Jean Y. J. Wang Recent studies have shown that RB can inhibit apoptosis, independently of its ability to block cell proliferation. This poses the question of how cells choose to grow or to die when RB becomes inactivated. RB is phosphorylated following mitogenic stimulation, but it is degraded in response to death stimuli. Most sporadic cancers also inactivate RB by phosphorylation, rather than losing RB entirely ? possibly to exploit the survival advantage conferred by RB under stress. Drawing from the different mechanisms of RB inactivation, we propose two models for ways in which cells use RB to make the choice of life versus death. B. Nelson Chau and Jean Y. J. Wang are at the Division of Biological Sciences and the Cancer Center, University of California, San Diego, La Jolla, California 92093-0322, USA. Correspondence to J. Y. J. W. e-mail: jywang@ucsd.edu doi:10.1038/nrc993 130 | FEBRUARY 2003 | VOLUME 3 www.nature.com/reviews/cancer REVIEWS � 2003 Nature Publishing Group NATURE REVIEWS | CANCER VOLUME 3 | FEBRUARY 2003 | 131 REVIEWS deletion of E2f3 protected the Rb-null CNS and PNS neurons from apoptosis 10,15 . So, the deregulation of E2f3 in Rb-null neurons is an important contributing factor to apoptosis. These observations also indicate that the transcription function of E2f1 and E2f3 might be programmed differently in developing neurons and cultured fibroblasts. Apoptosis of the Rb-null PNS neurons can also be suppressed by mutation of the caspase-3 gene (Casp3) 17 . In the apoptosis signalling pathway, Apaf1 functions upstream of Casp3 (REF. 11). In the developing nervous systems of Rb-null embryos, Apaf1-knockout rescued apoptosis in the CNS, and Casp3-knockout res- cued apoptosis in the PNS 14,17 . These observations have several implications. First, death of Rb-null neurons requires the apoptotic machinery, because it can be pre- vented by the loss of Apaf1 or Casp3. Second, in the absence of Rb, CNS neurons undergo apoptosis through E2f1- and p53-dependent transcription of Apaf1. The upregulation of Apaf1 is necessary, but probably not sufficient, to kill cells, as external stress signals might also be required. Third, apoptosis of Rb- null CNS neurons can occur in the absence of Casp3 (REF. 17), so other caspases might be involved in killing CNS neurons. Mutation of a single caspase in mice has been shown to cause compensatory activation of other caspases 18 ? the observed death of Rb ?/? Casp3 ?/? CNS neurons could be the result of such adaptation. Recent studies have identified E2F-binding sites in the promoters of several caspase genes, including Casp3 (REF. 19). In the PNS of Rb-null embryos, deregulation of E2F3 might lead to increased Casp3 expression, and therefore sensitize these neurons to apoptosis (FIG. 2). Apoptosis of the Rb-null skeletal muscles was observed in neonatal mice of two different genetic back- grounds 20,21 . One group of mice was created by disrup- tion of both the Rb and Id2 genes 20 . Id2 is a helix?loop?helix transcription regulator that can bind to Rb 22 . Mutation of Id2 rescued most of the defects of Rb-null embryos 20 . Nevertheless, these mice died at birth with a severe reduction of muscle mass 20 . It is not yet clear how Id2 deficiency rescues the Rb-nullembry- onic lethality. However, it is clear that Id2 knockout does not suppress the death of Rb-null muscles. The second strain of mice was created through the transgenic expression of a wild-type Rb minigene (which consists of a genomic fragment that spans 1.3 kb of the mouse Rb promoter, the first exon and the intron fused to exons 2 to 27 of the mouse Rb cDNA) in Rb-null embryos 23 . The Rb minigene was expressed in neurons but not in muscle cells, and rescued the ectopic neuronal apoptotic phenotype, but skeletal muscles of these mice still underwent apoptosis 21 . Mutation of Trp53 or E2f1 did not rescue the muscle apoptotic phenotype in these mice 21 . Mutation of Cdkn1a (which encodes Cip1, also known as p21), a Cdk inhibitor, increased levels of apoptosis in Rb-null muscle cells 21 . Taken together, observations made in the various strains of Rb-null mice indicate that Rb can suppress apoptosis induced by different pathways during development (FIG. 2). deregulated in Rb-null CNS neurons 12 , and the pro- apoptotic phenotype of Rb-null CNS is suppressed by the mutation of Apaf1 (REF. 14). Therefore, in the absence of Rb, deregulated E2F1 and p53 contribute to the activation of mitochondria-dependent apoptosis in the CNS (FIG. 2). Interestingly, elimination of E2f1, Trp53 or Apaf1 did not reverse the apoptotic phenotype of Rb-null neurons in the peripheral nervous system (PNS) 7,9,14 . However, mutation of the related transcription factor, E2f3, did suppress PNS apoptosis in Rb-null embryos 10,15 . In cultured embryonic fibroblasts, dele- tion of E2f1, but not E2f3, prevented Myc-induced apoptosis 16 . Nevertheless, heterozygous or homozygous Summary ? Loss of RB sensitizes cells to apoptosis. ? Ectopic apoptosis of Rb-null neurons is not a default outcome of inappropriate S-phase entry. ? RB can be inactivated by phosphorylation and degradation. ? RB degradation is required for tumour necrosis factor type I receptor-induced apoptosis. ? Most sporadic human cancers inactivate RB function by exploiting pathways that regulate RB phosphorylation. ? Loss of RB can only contribute to tumour development under conditions in which apoptosis response is compromised. RB RB X X Y Y Z Z Z Z Z Genetic mutation RB X Y Z RB X Y Degradation RB RB X X Y Y Z Viral inactivation E1A RB RB X X Y Y Z Phosphorylation P P P P P a b c d Figure 1 | Mechanisms of RB inactivation. Retinoblastoma (RB) contains multiple protein binding sites and functions as a molecular scaffold to promote the assembly of transcription complexes. Disassembly of these complexes is mediated by RB inactivation by means of four known mechanisms. a | The RB gene is mutated (dashed line), causing release of its associated factors. RB mutations have been detected in retinoblastoma and a small fraction of sporadic tumours. b | RB is sequestered by viral oncoproteins, such as E1A, which prevent it from binding other factors. c | Phosphorylation (P) of RB by CDK?cyclin complexes during cell-cycle progression disrupts its ability to assemble transcriptional complexes. d | RB is degraded by a caspase-dependent proteolytic pathway during apoptosis. Except for RB phosphorylation (c), the other three mechanisms of RB inactivation (a, b and d) have been associated with sensitization to apoptosis. � 2003 Nature Publishing Group 132 | FEBRUARY 2003 | VOLUME 3 www.nature.com/reviews/cancer REVIEWS REVIEWS model is based on the ability of RB to block cell-cycle progression and to facilitate differentiation. In the absence of RB, cells cannot cease proliferation despite the stimulation by differentiation signals. Continuation of DNA synthesis during differentiation results in a conflict that triggers apoptosis. This model Pros and cons of the ?conflict? model The apoptosis phenotype of Rb-null embryos is corre- lated with the ectopic S-phase entry of Rb-null neu- rons and muscles 4?6,24 . This correlation has led to the hypothesis that apoptosis results from a ?conflict? in the Rb-null progenitor cells (FIG. 3a). The conflict Table 1 | Summary of Rb-mutant mouse strains Genotype Lethality Tumour spectrum Major phenotypes References Rb ?/? E13.5 ND Increased ectopic S-phase entry and massive 4,5,6 apoptosis in neurons; defects in terminal differentiation of muscle and erythrocytes Rb +/? Shortens lifespan Pituitary ND 59 Rb +/? Trp53 ?/? Shortens lifespan Pituitary, thyroid Trp53 dosage-dependent survival (lifespan 62,63 pancreatic islet, shortens as Trp53 gene dosage decreases) soft-tissue sarcoma Rb +/? E2f1 ?/? Lengthens lifespan Pituitary, lung, Decreased incidence of pituitary tumours, 64 compared to that adenocarcinoma, but increases the spectrum of tumours (similar to of Rb +/? thyroid the kinds of tumour that develop in E2f1 ?/? mice) Rb ?/? Trp53 ?/? E13.5 ND Reduced apoptosis in CNS but not in PNS; 7,8 increased ectopic S-phase entry is observed in both CNS and PNS Rb ?/? E2f1 ?/? E17 ND Reduced ectopic S-phase entry and apoptosis 9 in CNS and lens tissue; upregulation of p53 activity was abolished in Rb ?/? brains; skeletal muscle mass and the number of muscle fibres were significantly reduced; defects of lung development were observed Rb ?/? E2f2 ?/? E13.5 ND Partial reduction of ectopic S-phase entry in both 10 CNS and lens tissue; no reduction of apoptosis in these tissues was observed Rb ?/? E2f3 ?/? E17.5 ND Reduced ectopic S-phase entry and apoptosis 10,15 in CNS, PNS and lens tissue; two classes of phenotype were observed in Rb ?/? E2f3 +/? embryos (one class showed complete suppression of both ectopic entry and apoptosis in CNS, whereas the other class showed only suppression of apoptosis) Rb ?/? Id2 ?/? At birth ND Embryos survived to term; normal neurogenesis 20 and haematopoesis; normal apoptosis in CNS but moderate increase of apoptosis in PNS (however, mice died at birth due to severe defects in muscle differentiation ? greatly reduced number of muscle fibres) Rb ?/? Apaf1 ?/? E13.5 ND Complete suppression of apoptosis in CNS and 14 lens tissue; partial reduction of apoptosis in PNS Rb ?/? Casp3 ?/? E13.5 ND Rescue of apoptosis in PNS (trigeminal ganglia, 17 dorsal root ganglia); Casp3 deficiency did not rescue the CNS neurons from apoptosis in Rb-null embryos Rb ?/? Die minutes ND No suppression of ectopic S-phase entry or mitosis 25 (telencephalon) after birth was observed in Rb-null cells; the number of (respiratory tunel-positive cells was suppressed compared to defects) Rb-null embryos; Rb-null cells in telencephalon area express early neuronal markers; slight increase in telencephalon area was observed, indicating enhanced neurogenesis Phosphor- Normal lifespan Mammary Suppression of ductal growth in pubescent stage 61 ylation-resistant adenocarcinoma (5?7 weeks of age); development of multiple Rb (mammary suppression of hyperplastic nodules in the gland) mammary gland; apoptosis during postlactational involution Rb MI/MI Normal lifespan None Protection of male mice from lipopolysaccharide- 29 induced mortality; MI cells are resistant to apoptosis induced by type I TNFR but remain sensitive to DNA-damage-induced apoptosis CNS, central nervous system; ND, not detected; None, no tumour found; PNS, peripheral nervous system. � 2003 Nature Publishing Group NATURE REVIEWS | CANCER VOLUME 3 | FEBRUARY 2003 | 133 REVIEWS An alternative model proposes that Rb directly inhibits stress-induced apoptosis (FIG. 3b). This model proposes that Rb has an anti-apoptotic function in addition to its well-established antiproliferative func- tion. Recent experiments have added credence to the idea that Rb directly inhibits apoptosis. The ability of Rb to repress E2F1-dependent gene expression and the ability of E2F1 to induce apoptosis are well estab- lished 1,16 . Thymocytes from E2f1-null mice are resis- tant to activation-induced apoptosis 27 . Fibroblasts from E2f1-null mice are resistant to Myc-induced apoptosis 16 . Helin and colleagues have identified a number of genes that are upregulated by a condition- ally activated E2F1 (REF. 28). As expected, E2F1 stimu- lated the expression of cell-cycle genes ? for example, the G1/S cyclins. Interestingly, E2F1 also induced the expression of genes that encode the cell-death machin- ery, including several caspases and Apaf1 (REF. 28). E2F- binding sites have been defined in the Apaf1 promoter 12 . Recently, in silico analysis of the human genome has identified E2F-binding sites in promoters of several caspase genes 19 . These results support the conclusion that E2F1 is a bona fide pro-apoptotic transcription factor. The fact that RB can repress tran- scription from E2F1-occupied promoters makes it a negative regulator of apoptosis. RB attenuates apoptotic signalling The ability of Rb to directly inhibit apoptosis is also supported by the phenotype of a strain of mice that express a caspase-resistant Rb 29 . As discussed above, RB can be degraded through the action of caspase 30 . Caspases function in transducing death signals and executing cell killing. Since Rb-null cells are hypersen- sitive to apoptosis induced by a variety of agents, Rb degradation could contribute to cell death. RB con- tains several consensus sites for caspase cleavage 30?32 . The main caspase cleavage site in RB is located at the extreme carboxyl terminus. Mutation of this carboxy- terminal cleavage site creates a degradation-resistant form of RB, called RB-MI (mutated at the ICE-site) 33 . The ectopic expression of RB-MI attenuated apopto- sis that was induced by tumour necrosis factor-? (TNF-?) in fibroblasts 33 and by potassium depriva- tion in cultured cerebellar granular neurons 34 . These results indicate that RB is an important substrate of caspase, and that RB degradation contributes to the induction of apoptosis. To investigate the physiological significance of RB degradation in apoptosis, our laboratory has replaced the wild-type Rb allele with the Rb-MI allele in the mouse germline 29 . The Rb-MI mice are healthy and fertile with a normal lifespan. When challenged with bacterial endotoxin, the Rb-MI mice showed tis- sue-specific resistance to apoptosis 29 . In cells of the intestine, the presence of a single Rb-MI allele was sufficient to protect against apoptosis, showing that preservation of Rb can inhibit cell death. On the other hand, splenocytes of heterozygous or homozy- gous Rb-MI mice were not protected from apoptosis during endotoxic shock 29 . proposes that RB does not directly protect progeni- tor cells from apoptosis. Instead, the increased apop- tosis of Rb-null cells is the default outcome of ectopic S phase. Recent results from conditional Rb-knockout mice have shown that, in the developing CNS, ectopic S-phase entry does not lead to apoptosis by default 25,26 . By combining a conditional Rb knockout with neu- ron-specific Rb expression, Slack and colleagues targeted the inactivation of Rb in the developing telen- cephalon 25 . These mice survived through term and were born with the expected Mendelian ratio. However, they died within 20 minutes of birth, due to unexplained respiratory defects. The Rb-inactivated neurons showed ectopic S-phase entry and mitosis, but the inappropriate proliferation did not lead to apoptosis 25 . Jacks and colleagues have made a similar observation with an independently derived condi- tional knockout of Rb 26 . At present, it is not under- stood why the general knockout of Rb causes neurons to undergo apoptosis. It has been proposed that hypoxic stress, possibly caused by the developmental defects of Rb-null red blood cells, might contribute to the apoptosis of Rb-null neurons 26 . In a modified ?con- flict? model, ectopic proliferation, combined with stress generated in the Rb-null background, causes Rb-null progenitor cells to undergo apoptosis (FIG. 3b). CNS PNS Skeletal muscle Rb Rb Rb E2f1 E2f3 E2f3 p53 ? Apaf1 Apoptosis Caspase-3 Stress Stress Figure 2 | Genetic inactivation of Rb sensitizes developing neurons and skeletal muscle to apoptosis. The Rb ?/? mouse embryos undergo excessive apoptosis in the central nervous system (CNS), peripheral nervous system (PNS) and skeletal muscle. Neuron-specific disruption of Rb does not lead to apoptosis 25 , so additional factors are required to induce apoptosis of neurons of Rb-null embryos. In the CNS of Rb-null embryos, disruption of the genes E2f1 (REF. 9), E2f3 (REF. 15), Trp53 (REF. 8) or Apaf1 (REF. 14) can suppress excessive apoptosis. The Apaf1 gene is regulated by E2f1 and p53 (REF. 12). In the PNS of Rb-null embryos, loss of E2f3 (REF. 15) or caspase-3 (REF. 17), but not E2f1, p53 or Apaf1, prevents excessive apoptosis. The Casp3 gene is regulated by E2f 19 . The excessive apoptosis that occurs in Rb-null skeletal muscle is not suppressed by E2f1 or Trp53 gene disruption 21 . These results indicate that Rb can suppress apoptosis that is induced by several pathways during mouse development. � 2003 Nature Publishing Group 134 | FEBRUARY 2003 | VOLUME 3 www.nature.com/reviews/cancer REVIEWS The concept that caspases cleave specific substrates to amplify the apoptotic signal is supported by recent find- ings that cleavage of RIP (receptor interacting protein, a dead domain serine/threonine kinase) and IKK? (??B kinase-?) can each contribute to TNF-?-induced apop- tosis 35,36 . Preservation of RIP or IKK?, through expres- sion of non-cleavable variants of each protein, can protect cells from apoptosis by maintaining the activity of nuclear factor of ?B (NF-?B) 35,36 . It is interesting to note that RB-MI did not interfere with cytochrome c release or caspase-3 activation when these events were induced by DNA damage 29 . Despite the presence of intact RB-MI, DNA-damage signals induced apoptosis without any detectable delay 29 . The signal-specific protection from apoptosis argues that RB-MI does not function as a general inhibitor of caspases. Instead, these results show that the transduction and/or amplification of apoptotic signals from DNA damage can proceed without RB degradation. DNA damage preferentially kills prolifer- ating cells, which inactivate RB by phosphorylation. As RB-MI can be phosphorylated, its presence might be inconsequential in DNA-damage-induced apoptosis. As discussed below, either phosphorylation or degra- dation of RB might sensitize cells to apoptosis (FIG. 4). Alternatively, DNA damage and TNF-? might induce cytochrome c release and caspase-3 activation through distinct mechanisms, whereas RB-MI specifically inhibits the TNF-?-induced death pathway. RB inhibits pro-apoptotic factors other than E2F1 As summarized in TABLE 1 and FIG. 2, apoptosis in Rb- null embryos is not always suppressed by E2f1 gene dis- ruption. This might be explained by the fact that E2F1 is only one of a hundred or so cellular proteins that have been shown to interact with RB 37 . Several of the reported RB-binding proteins have a pro-apoptotic function. The nuclear c-ABL tyrosine kinase can induce apoptosis 38 , and RB directly binds to it and inhibits its kinase activity 39 . The JNK kinase is involved in stress- induced apoptosis, and this function seems to be inhib- ited by RB 40 . The amino-terminal region of RB contains a binding site for p84N5, which, when overproduced, has pro-apoptotic activity that is inhibited by RB 41,42 .It was reported that the large subunit of replication factor C can promote survival following DNA damage, and that this protection is dependent on its interaction with RB 43 . Taken together, these findings support the concept that RB regulates the activities of several proteins, including those that promote apoptosis. The inhibition of E2f1 by Rb contributes to the repression of Apaf1 and Casp3 expression, and there- fore suppresses apoptosis during mouse embryonic development. The regulated expression of Apaf1 and Casp3, however, is not likely to fully control the apop- totic response to TNF-?. In fact, TNF-?-induced apoptosis is enhanced by cycloheximide and actino- mycin D, which inhibit gene expression. This raises the interesting possibility that RB could inhibit apoptosis through mechanisms other than the regulation of gene expression. It is conceivable that RB participates in the The tissue-specific protection could be explained by the finding that Rb-MI is resistant to degradation induced by the type I receptor for tumour necrosis fac- tor (TNFRI) 29 , but activation of TNFRI plus TNFRII signalling causes Rb-MI degradation 29 . TNFRI is widely expressed, whereas the type II receptor is preferentially expressed in the haematopoietic lineage, which would explain why splenocytes were not protected from apop- tosis by Rb-MI expression. It will be of interest to con- struct a non-degradable form of RB, by mutating all of its caspase cleavage sites, to see whether it could protect cells from both TNFRI- and TNFRII-induced degrada- tion. So, preservation of Rb protein, in the form of Rb- MI, can interfere with TNFRI-induced apoptosis and can exert tissue-specific protection of apoptosis in vivo ? these results further support the concept that RB inhibits apoptosis. But how does RB-MI function? First of all, Rb-MI protects against TNF-?-induced apoptosis through a mechanism other than suppressing cell-cycle progres- sion. When treated with TNF-? and cycloheximide to induce apoptosis, DNA synthesis was equally inhibited in both Rb-WT and Rb-MI fibroblasts. Whereas both cells types arrested in G1, Rb-WT fibroblasts underwent apoptosis and Rb-MI cells remained viable. Further experiments with Rb-MI fibroblasts showed that this protein interferes with cytochrome c release and cas- pase-3 activation to protect against apoptosis 29 .This observation indicates that degradation of RB, through caspase, contributes to further activation of caspase. So, elimination of RB is required for caspase to transduce and/or amplify the apoptotic signal from TNF-? (BOX 1). Rb Stress Rb Stress a b Proliferation Apoptosis Differentiation Proliferation Apoptosis Differentiation Figure 3 | Two explanations for the apoptosis phenotype of Rb-null cells. During development, progenitor cells can choose among three fates: proliferation, differentiation or apoptosis. a | The ?conflict? model proposes that apoptosis is the default fate as Rb-null cells undergo unscheduled proliferation ? either in the presence or absence of cellular stress. The conflict model considers suppression of apoptosis to be a secondary effect to the antiproliferative function of Rb. b | The alternative model proposes that suppression of apoptosis is a primary function of Rb, independent of its antiproliferative activity. Rb also promotes differentiation in both these models. � 2003 Nature Publishing Group NATURE REVIEWS | CANCER VOLUME 3 | FEBRUARY 2003 | 135 REVIEWS apoptosis genes, on the other hand, is regulated through RB degradation. This model would predict that a fraction of the RB/E2F repression complexes are stable and functional in proliferating cells. Indeed, RB/E2F complexes have been detected in nuclear extracts from S-phase cells 44 . It remains to be determined whether these S-phase RB/E2F com- plexes occupy promoters of apoptosis genes. This model would also predict that RB/E2F complexes at some promoters to be resistant to CDK-mediated phosphorylation. Whether or how such phosphoryla- tion-resistant RB complexes are assembled would be an interesting problem to solve. An alternative model, illustrated in FIG. 4b as the ?context-dependent? regulation, can also explain how cells choose to proliferate or to die following RB inac- tivation. In this model, the different mechanisms of RB inactivation do not determine the cell fate. Rather, the activities of parallel pathways, in combination with RB inactivation, determine the biological out- come. FIGURE 4b shows a scenario in which mitogenic stimulation activates pro-survival factors, such as the transcription factor NF-?B 45 and the protein kinase AKT 46 . These survival signals antagonize the induc- tion of pro-apoptotic genes caused by the inactiva- tion of RB, thereby ensuring cell proliferation instead of apoptosis. nuclear sequestration of factors that stimulate cytochrome c release. Caspase-mediated degradation of RB could release such factors to the cytoplasm, leading to the transduction and/or amplification of apoptotic signals. To grow or to die? Because the inactivation of RB can stimulate prolifer- ation and apoptosis, and because the RB/E2F complex regulates the expression of S phase and apoptotic genes, the question arises as to how the decision to proliferate or to die is controlled. In response to mito- genic signals, cells inactivate RB by phosphorylation. Phosphorylation of RB is required for mitogens to stimulate DNA synthesis. In response to death-recep- tor signals, cells inactivate RB by caspase-dependent degradation. Degradation of RB, as we have shown, is required for TNFRI to induce apoptosis 29 . Because mitogenic and apoptotic signals inactivate RB through different molecular mechanisms, it is con- ceivable that RB/E2F promoter complexes could be differentially regulated. This model of ?promoter-specific? regulation of RB/E2F transcription complexes is illustrated in FIG. 4a. The RB/E2F complex at the promoters of S-phase genes is regulated through RB phosphoryla- tion. The RB/E2F complex at the promoters of Box 1 | Caspase-mediated amplification of apoptotic signal Tumor necrosis factor (TNF) is a cytokine that stimulates inflammation and apoptosis. Previous work has established the function of two types of caspases in TNF-induced apoptosis. The ?initiator? caspases ? for example, caspase-8 ? are directly activated by the type I TNF receptor (TNFRI). The ?executioner? caspases ? for example, caspase-3 ? are activated by the initiator caspases through mitochondria-dependent and -independent pathways, such as through cytochrome c (Cyto c) release. The previous model of direct connections between the initiator and executioner caspases would predict a rapid apoptosis following TNFRI activation. However, a rapid death would be incompatible with the physiological function of TNF, which is to regulate the inflammatory response to pathogens and stress. Recently, we have found that the cleavage of RB is required for TNFRI to activate the executioner caspases ? that is, caspase-3. The nuclear location of RB and the timing of its degradation indicate that RB is cleaved by caspases that are in-between the initiators and the executioners. We therefore propose the term ?amplifier? to describe the caspase that cleaves RB. The amplifier caspases are not defined by their molecular identities, but rather by their function in apoptotic signal transduction. For example, caspase-3, which cleaves RB 29 and ??B kinase-? (IKK?) 36 , can function as an amplifier and as an executioner. The caspase-resistant form of RB, which is called ?RB-MI?, can interfere with cytochrome c release from the mitochondria to diminish the processing of caspase-3 (REF. 29). The amplifier caspases might also cleave the pro- apoptotic proteins receptor interacting protein (RIP) 35 and IKK? 36 . TNF activates nuclear factor of ?B (NF-?B) through RIP and IKK? to stimulate inflammation. NF-?B also inhibits apoptosis through the upregulation of IAPs that block caspases. By cleaving RIP and IKK?,the amplifier caspases can extinguish inflammation and promote apoptosis. Caspase-resistant RB, RIP or IKK? could each interfere with the function of the amplifier caspases to diminish the apoptosis response to TNF. TNF TNFRI Initiator caspases Amplifier caspases RB Executioner caspases NF-?B RIP IKK? Cyto c IAP Apoptosis � 2003 Nature Publishing Group 136 | FEBRUARY 2003 | VOLUME 3 www.nature.com/reviews/cancer REVIEWS RB is a conditional tumour suppressor The direct regulation of apoptosis by RB has impor- tant implications for tumour development. Because RB has a central role in the inhibition of cell prolifer- ation, tumour development involves the inactivation of RB. However, as RB also has a crucial role in the inhibition of apoptosis, developing tumours need to preserve RB to antagonize apoptotic signalling. Previous work has shown that the ?RB pathway? is tar- geted for inactivation in more than 80% of sporadic human cancers 47 . Interestingly, the RB gene itself is rarely mutated in sporadic human cancers. Rather, tumour cells target the pathway that regulates the phosphorylation of RB. In particular, sporadic cancer cells repress the expression of CDKN2A (which encodes INK4A, also known as p16) which inhibits CDK4/6 (REF. 48) through gene deletion or promoter hypermethylation 49?51 . The increased expression of D-type cyclins that activate CDK4/6 is another strat- egy developed by tumour cells to inactivate RB. Cyclin D1 overproduction is observed in human mammary carcinomas 52 , and cyclin-D1-null mice are resistant to mammary tumour development induced by the murine mammary tumour virus 53 .Activated CDK4/6?cyclin-D complexes not only phosphorylate and inactivate RB, but also phosphorylate and inacti- vate the RB-related proteins p107 and p130, which share redundant function with RB in the regulation of E2Fs 44 . Moreover, this phosphorylation is reversible. So, targeting the phosphorylation pathway of RB provides tumour cells not only with a growth advantage, but also with the option to re-activate RB for protection against apoptotic stimuli. In keeping with the idea that RB inactivation needs to be coupled with apoptosis defects during tumour development, DNA tumour viruses have developed oncoproteins that inactivate both RB and p53. The best example occurs in adenovirus, which expresses E1A to inactivate RB, and E1B to inactivate p53. Expression of E1A alone causes apoptosis, which is suppressed by E1B 54 . These results are surprisingly similar to the suppression of apoptosis in Rb-null CNS neurons by the knockout of Trp53 (REFS 7,8).Moreover, somatic inactivation of the mouse Rb gene in the cere- bellar external granular layer cells of Trp53-null mice causes medulloblastoma 55 . These observations indicate that Trp53 inactivation can provide conditions that allow subsequent Rb loss to promote tumour growth. But what about human retinoblastoma? Hereditary and sporadic retinoblastoma results from the bi-allelic inactivation of RB, but does not acquire p53 mutation. There are two possible mechanisms by which retinoblas- toma cells avoid apoptosis. In the first mechanism, RB only inhibits proliferation, but does not regulate apopto- sis, in retinoblasts. In the second mechanism, in RB-null retinoblasts, apoptosis is prevented through epigenetic mechanisms, such as DNA methylation or gene amplifi- cation. Recent studies of retinoblastoma tumours have provided evidence for chromosomal aberrations outside the RB locus 56,57 . It will be of interest to determine if these additional aberrations provide survival signals for the Many studies have shown the importance of sur- vival signalling in cell proliferation, supporting this model. The coordinated induction of proliferative and apoptotic genetic programmes after RB inactivation would also explain the preferential sensitization of proliferating cells to apoptosis. This model would pre- dict that caspase-dependent inactivation of RB could contribute to cell proliferation if sufficient survival signals existed to antagonize the death function of cas- pases. The two models depicted in FIG. 4 are not mutu- ally exclusive and could be used simultaneously to integrate the myriad of signals that affect the decision of life and death. RB P P P P P CDK CDK DP E2F RB RB P P P P P Caspase DP E2F RB RB Caspase DP E2F RB RB a Promoter-specific regulation b Context-dependent regulation S-phase genes (e.g. cyclin A) S-phase genes (e.g. cyclin A) Apoptosis genes (e.g. caspase) Apoptosis genes (e.g. caspase) Survival signals (e.g. AKT, NF-?B) Figure 4 | Two models for the differential regulation of proliferation versus apoptosis. a | The ?promoter- specific regulation? model proposes that RB phosphorylation (P) by cyclin-dependent kinase (CDK) releases RB from the promoters of S-phase genes, allowing E2F (composed of E2F and DP heterodimer) to stimulate their expression. Phosphorylation does not disrupt the RB/E2F complexes that are assembled at the promoters of apoptosis genes. Instead, RB degradation, through the action of caspase, is required to disrupt the transcription repression complexes at the promoters of pro-apoptosis genes. b | The ?context-dependent regulation? model proposes that RB phosphorylation or degradation is equally effective at disrupting RB-mediated transcriptional repression of S-phase genes and pro-apoptosis genes. Following mitogenic stimulation and RB inactivation, cells do not undergo apoptosis because the expression of pro- apoptosis genes is counteracted by the activation of survival factors such as AKT and NF-?B. These two models are not mutually exclusive. In other words, promoter- specific regulation could be used in combination with parallel survival factors to control the decision of proliferation versus apoptosis. � 2003 Nature Publishing Group NATURE REVIEWS | CANCER VOLUME 3 | FEBRUARY 2003 | 137 REVIEWS mutation, viral oncoprotein sequestration or caspase- dependent degradation is, in each case, associated with the induction of apoptosis. Knockout of the Rb gene in mice sensitizes neurons and myocytes to apoptosis dur- ing development (FIG. 2). Through the creation of Rb- conditional knockouts, researchers have been able to dissociate Rb?s effects on apoptosis and proliferation (TABLE 1). The transcription factor E2F, which interacts with RB, has been shown to regulate genes involved in apoptosis, including Apaf1, Casp3, 7, 8 and 9 (REFS 12,19). Moreover, a degradation-resistant RB-MI can interfere with TNF-?-induced cytochrome c release and caspase activation (TABLE 1; BOX 1). So, RB is an inhibitor of cell death as well as an inhibitor of cell growth (FIG. 3b). We propose two models to explain how cells choose to proliferate or to die following the inactiva- tion of RB. Each model makes a previously untested prediction. The ?promoter-specific regulation? model predicts RB-assembled transcription complexes to remain on the promoters of apoptosis genes during S phase (FIG. 4). The ?context-dependent regulation? model predicts that caspases stimulate cell prolifera- tion when sufficient survival signals are present (FIG. 4). Experiments to test predictions of these two models are feasible, and results would advance our under- standing of how cells make the decision of life or death under complex physiological conditions. The studies of RB have been focused on its regulation of transcription. However, RB does interact with a number of proteins that do not seem to participate in transcription regulation 37 . We found that the caspase- resistant Rb-MI can attenuate the transduction of death signal under conditions when transcription is irrele- vant 29 . So, RB might inhibit apoptosis by mechanisms other than suppression of apoptosis gene expression. Whether RB inhibits apoptosis by post-transcriptional mechanisms remains to be determined. development of retinoblastoma. Unlike humans, the Rb +/? mice develop pituitary tumours with high pene- trance 4?6,58,59 . By examining a large number of pituitary glands of Rb +/? mice, Lee and colleagues found that tissue apoptosis preceded tumour growth 58 . Retinoblastoma can develop in chimeric mice generated from p107 ?/? , Rb ?/? embryonic stem cells 60 . Increased apoptosis in the retina was also observed in these mice prior to tumour formation 60 . The anti-apoptosis mechanisms that drive the formation of Rb-null pituitary tumours and retinoblastoma are unknown at present. In sporadic human cancers, the rare mutation of the RB gene, the increased phosphorylation of the RB pro- tein and the combined presence of both RB and p53 mutations support the concept that RB is a conditional tumour suppressor. As RB can inhibit cell death, it should in theory also function as a conditional tumour promoter. This concept is supported by observations made in mice that express phosphorylation-resistant (constitutively active) variants of Rb in the mammary epithelial cells. One-third of these mice developed hyper- plastic nodules, and ~7% of these mice developed ade- nocarcinomas 61 . In addition, we have recently observed that the Rb-MI allele, although having no effect on tumour incident by itself, can aggravate tumour devel- opment in Trp53-null mice (B.N.C. and J.Y.J.W., unpub- lished observations). The idea that RB can promote tumour development under specified circumstances might sound preposterous in light of our current under- standing of RB function. However, given the important role of apoptosis in guarding against tumorigenesis, the inhibition of apoptosis by RB might be exploited in some cancer cells to promote malignant transformation. Future directions The ability of RB to inhibit apoptosis is now supported by many lines of evidence. Loss of RB through genetic 1. Nevins, J. R. The Rb/E2F pathway and cancer. Hum. Mol. Genet. 10, 699?703 (2001). 2. Harbour, J. W. & Dean, D. C. Rb function in cell-cycle regulation and apoptosis. Nature Cell Biol. 2, E65?E67 (2000). 3. Nielsen, S. J. et al. Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561?565 (2001). 4. Clarke, A. R. et al. Requirement for a functional Rb-1 gene in murine development. Nature 359, 328?330 (1992). 5. Jacks, T. et al. Effects of an Rb mutation in the mouse. Nature 359, 295?300 (1992). 6. Lee, E. Y. et al. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359, 288?294 (1992). 7. Morgenbesser, S. 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It was shown that Apaf1 mRNA and protein were inappropriately upregulated in the Rb- null embryos. 13. Fortin, A. et al. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death. J. Cell Biol. 155, 207?216 (2001). By infecting Apaf1-null and wild-type neurons with an adenoviral vector that expresses 53, this study showed that Apaf1 is required for p53 to induce apoptosis in vivo. 14. Guo, Z., Yikang, S., Yoshida, H., Mak, T. W. & Zacksenhaus, E. Inactivation of the retinoblastoma tumor suppressor induces apoptosis protease-activating factor-1 dependent and independent apoptotic pathways during embryogenesis. Cancer Res. 61, 8395?8400 (2001). 15. Ziebold, U., Reza, T., Caron, A. & Lees, J. A. E2F3 contributes both to the inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev. 15, 386?391 (2001). In this study, Rb?/? 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Using in silico methods, this study identified E2F binding sites in the promoters of several caspase genes, including CASP3, 7, 8 and 9. When the expression of these caspases was examined during cell-cycle progression, it was found that some, but not all, of these caspases were induced as cells enter S phase. 20. Lasorella, A., Noseda, M., Beyna, M., Yokota, Y. & Iavarone, A. Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407, 592?598 (2000). 21. Jiang, Z. et al. E2F1 and p53 are dispensable, whereas p21(Waf1/Cip1) cooperates with Rb to restrict endoreduplication and apoptosis during skeletal myogenesis. Dev. Biol. 227, 8?41 (2000). This study showed that, unlike the developing nervous systems, E2f1 and p53 were not required for the ectopic apoptosis phenotype of Rb-null skeletal muscle. 22. Iavarone, A., Garg, P., Lasorella, A., Hsu, J. & Israel, M. A. The helix?loop?helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein. Genes Dev. 8, 1270?1284 (1994). � 2003 Nature Publishing Group 138 | FEBRUARY 2003 | VOLUME 3 www.nature.com/reviews/cancer REVIEWS 23. Zacksenhaus, E. et al. pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis. Genes Dev. 10, 3051?3064 (1996). 24. Wang, J., Guo, K., Wills, K. N. & Walsh, K. Rb functions to inhibit apoptosis during myocyte differentiation. Cancer Res. 57, 351?354 (1997). 25. Ferguson, K. L. et al. Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and abnormal cortical development. EMBO J. 21, 3337?3346 (2002). By examining conditional Rb-knockout mice, this study showed that ectopic apoptosis could be uncoupled from the ectopic S-phase entry in the Rb- null neurons. 26. MacPherson, D. et al. Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system. Mol. Cell. Biol. (in the press). By experimenting with an independently derived strain of conditional Rb-knockout mice, this study reported observations similar to those in the study cited in reference 25. The authors conclude that hypoxia induced by the developmental defect of Rb- null erythrocytes contributes to the ectopic apoptosis phenotype of neurons in Rb-null embryos. So, the ectopic apoptosis phenotypes in Rb-null embryos is a non-cell-autonomous event. 27. Field, S. J. et al. E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85, 549?561 (1996). 28. Muller, H. et al. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev. 15, 267?285 (2001). 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Regulation of cell death by the Abl tyrosine kinase. Oncogene 19, 5643?5650 (2000). 39. Welch, P. J. & Wang, J. Y. A C-terminal protein-binding domain in the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle. Cell 75, 779?790 (1993). 40. Shim, J. et al. Rb protein down-regulates the stress- activated signals through inhibiting c-Jun N-terminal kinase/stress-activated protein kinase. J. Biol. Chem. 275, 14107?14111 (2000). 41. Doostzadeh-Cizeron, J., Evans, R., Yin, S. & Goodrich, D. W. Apoptosis induced by the nuclear death domain protein p84N5 is inhibited by association with Rb protein. Mol. Biol. Cell 10, 3251?3261 (1999). 42. Doostzadeh-Cizeron, J., Yin, S. & Goodrich, D. W. Apoptosis induced by the nuclear death domain protein p84N5 is associated with caspase-6 and NF-?B activation. J. Biol. Chem. 275, 25336?25341 (2000). 43. Pennaneach, V. et al. The large subunit of replication factor C promotes cell survival after DNA damage in an LxCxE motif- and Rb-dependent manner. Mol. Cell 7, 715?727 (2001). 44. Schwarz, J. K. et al. Interactions of the p107 and Rb proteins with E2F during the cell proliferation response. EMBO J. 12, 1013?1020 (1993). 45. Barkett, M. & Gilmore, T. D. Control of apoptosis by Rel/NF- ?B transcription factors. Oncogene 18, 6910?6924 (1999). 46. Datta, S. R., Brunet, A. & Greenberg, M. E. Cellular survival: a play in three Akts. Genes Dev. 13, 2905?2927 (1999). 47. Sherr, C. J. Cancer cell cycles. Science 274, 1672?1677 (1996). 48. Sherr, C. J. & Roberts, J. M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501?1512 (1999). 49. Nobori, T. et al. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368, 753?756 (1994). 50. Merlo, A. et al. 5? CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nature Med. 1, 686?692 (1995). 51. Baylin, S. B., Herman, J. G., Graff, J. R., Vertino, P. M. & Issa, J. P. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res. 72, 141?196 (1998). 52. Zukerberg, L. R. et al. Cyclin D1 (PRAD1) protein expression in breast cancer: approximately one-third of infiltrating mammary carcinomas show overexpression of the cyclin D1 oncogene. Mod. Pathol. 8, 560?567 (1995). 53. Yu, Q., Geng, Y. & Sicinski, P. Specific protection against breast cancers by cyclin D1 ablation. Nature 411, 1017?1021 (2001). 54. White, E. Regulation of p53-dependent apoptosis by E1A and E1B. Curr. Top. Microbiol. Immunol. 199, 34?58 (1995). 55. Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J. & Berns, A. Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev. 14, 994?1004 (2000). In this study, tissue-specific knockout of Rb was introduced into the Trp53-knockout background. This led to the formation of medulloblastoma in mice. 56. Mairal, A. et al. Detection of chromosome imbalances in retinoblastoma by parallel karyotype and CGH analyses. Genes Chromosom. Cancer 28, 370?379 (2000). 57. Chen, D., Gallie, B. L. & Squire, J. A. Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridization. Cancer Genet. Cytogenet. 129, 57?63 (2001). 58. Lee, E. Y. et al. Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation. Genes Dev. 8, 2008?2021 (1994). 59. Hu, N. et al. Heterozygous Rb-1 delta 20/+mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance. Oncogene 9, 1021?1027 (1994). 60. Robanus-Maandag, E. et al. p107 is a suppressor of retinoblastoma development in pRb-deficient mice. Genes Dev. 12, 1599?1609 (1998). 61. Jiang, Z. & Zacksenhaus, E. Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression, precocious differentiation, and adenocarcinoma. J. Cell Biol. 156, 185?198 (2002). This study examined several lines of transgenic mice that express a phosphorylation-resistant (constitutively active) Rb in the mouse mammary epithelium. Some of these mice developed adenocarcinomas, indicating that constitutive Rb activity facilitates tumour development in vivo. This could be because the constitutively active form of Rb protects mammary epithelial cells from stress- induced apoptosis. 62. Harvey, M., Vogel, H., Lee, E. Y., Bradley, A. & Donehower, L. A. Mice deficient in both p53 and Rb develop tumors primarily of endocrine origin. Cancer Res. 55, 1146?1151 (1995). 63. Williams, B. O. et al. Cooperative tumorigenic effects of germline mutations in Rb and p53. Nature Genet. 7, 480?484 (1994). 64. Yamasaki, L. et al. Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(+/-)mice. Nature Genet. 18, 360?364 (1998). Acknowledgements We are grateful to the Wang lab members for stimulating discus- sion and critical reading of the manuscript throughout its prepara- tion. B.N.C is supported by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation. This work is sup- ported by a National Cancer Institute grant awarded to J.Y.J.W. Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nih.gov/LocusLink/ ABL | AKT | Apaf1 | Casp3 | CDK4 | CDK6 | Cdkn1a | CDKN2A | cyclin D1 | E2f1 | E2F1 | E2f3 | Id2 | IKK? | NF-?B | Rb | RB | RIP | TNF-? | TNFRI | TNFRII | Trp53 OMIM: http://www.ncbi.nlm.nih.gov/Omim/ hereditary retinoblastoma FURTHER INFORMATION CancerGeneticsWeb: http://www.cancerindex.org/geneweb/RB1.htm Mouse Knockout and Mutation database: http://research.bmn.com/mkmd Access to this interactive links box is free online. "
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