Interactive Fly, Drosophila

Retinoblastoma-family protein


Table of contents

Retinoblastoma mutation and cell cycle regulation

The regulation of cell-cycle entry has been investigated in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, mutations in cyd-1 cyclin D and cdk-4 Cdk4/6 were identified. Recent results have indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, the data presented here indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, double mutants were constructed that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduces the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, since the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. lin-35 Rb and cki-1, a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. It is suggested that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals (Boxem, 2001).

A synthetic-lethal screen in Caenorhabditis elegans is describes that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans have been identified. Unlike the original SynMuv screens, this approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. This screen has identiifed fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. Genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23, have been uncovered. It is proposed that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels (Fay, 2002).

fzr-1 cooperates with lin-35 to control cell proliferation. A relatively small number of genes have been described that cause widespread hyperproliferation in C. elegans. These include the putative SCF components cul-1 and lin-23 , the CIP/KIP family member cki-1, and the CBP/p300 homolog cbp-1 . In the cases examined thus far, a hyperproliferation phenotype is observed following inactivation of a single gene product. Loss of cell proliferation control can also result from a synthetic genetic interaction. Although single mutants of lin-35 and fzr-1 show only subtle or low-penetrance phenotypes, lin-35; fzr-1 double mutants showed extensive tissue hyperproliferation affecting a wide range of cell types. Thus, uncontrolled proliferation in C. elegans can in essence follow the same genetic pattern as multistep carcinogenesis in mammals. Namely, proliferation control is abolished through the sequential loss of genes that function to restrain cell cycle progression (Fay, 2002).

Given these findings, and the large body of evidence implicating Rb in human cancers, it seems reasonable to suggest that this technical approach may facilitate the study of multistep carcinogenesis using C. elegans. Along these lines, it will be interesting to determine whether the human homolog of fzr-1, hCDH1, can function as a tumor-suppressor gene, and if so, whether it does so in cooperation with Rb (Fay, 2002).

Inactivation of fzr-1 function using RNAi injection leads to sterility and aberrancies in germ cell proliferation. Although the specific cause of this phenotype has not been determined, previous studies would implicate defects in either the execution of G1 arrest or in late-stage mitotic events such as cytokinesis. Embryonic lethality is also observed when fzr-1 is inactivated using RNAi injection in a lin-35 mutant background. The cause for this lethality is presently unknown. These embryos do not show obvious hallmarks associated with either excess cellular proliferation or grossly elevated levels of apoptosis. Although additional work will be necessary to determine the nature of this embryonic requirement, a role during embryonic development is consistent with the expression patterns observed for both fzr-1 and lin-35. The lack of an apparent hyperproliferation phenotype in embryos likely reflects significant differences in the means by which embryonic and postembryonic cell cycles are regulated. For example, cyclin D, an upstream regulator of Rb, has been shown to be required exclusively for the execution of postembryonic division cycles in C. elegans (Fay, 2002).

Work carried out over the past several years has produced an explosion in the number of identified SynMuv genes. Although certain functional classifications, such as transcriptional repressors, may accurately describe some of the SynMuv genes, others clearly defy straightforward categorization. This fact alone suggests that SynMuv genes most likely do not all act through the same mechanisms or pathways (Fay, 2002).

Both lin-36 and efl-1(RNAi) can phenocopy the effect of lin-35 LOF in an fzr-1 mutant background. However other class B genes, including lin-53, hda-1, and chd-4, did not show genetic interactions with fzr-1, nor did the class A gene lin-15a. These experiments are complicated by the fact that lin-53, hda-1, and chd-4 encode for essential genes, and RNAi leads to a highly penetrant sterile or lethal phenotype within several generations. Nevertheless, no evidence was seen for hyperproliferation in either the affected or unaffected classes of RNAi-treated animals. This suggests that neither a weak nor a severe reduction in the function of these genes is capable of producing a synthetic hyperproliferation phenotype with fzr-1. In addition, no evidence was found for an interaction in lin-53(n833); fzr-1 double-mutant animals. Although n833 results in only a partial loss of LIN-53, this allele does lead to a highly penetrant Muv phenotype in conjunction with class A SynMuv mutations (Fay, 2002).

Rb and its family members p107 and p130 have been shown in multiple systems to modulate transcription through direct interactions with a variety of transcriptional regulators. The majority of work indicates that Rb serves primarily as a transcriptional repressor, acting through a number of mechanisms including the recruitment of chromatin-modifying enzymes and the steric interference of transactivation domains. Acting as transcriptional corepressors with E2F, Rb and its family members regulate the expression of many key genes required for entry and progression through S-phase, including cyclin E and cyclin A. Consistent with these reports, a significant increase in the levels of ribonucleotide reductase mRNA, an E2F-regulated gene, is seen in lin-35 mutant animals (Fay, 2002).

In Drosophila, loss of fzr function leads to reentry into the cell cycle following embryonic cycle 16, thereby bypassing the normal G1 arrest. This ectopic division cycle is correlated with excess levels of cyclin A, which when overexpressed during G1 can lead to ectopic entry into S-phase. Interestingly, mutations in the Drosophila Rb homolog rbf , as well as in the CDK inhibitor decapo, show cell cycle defects similar to those of fzr mutants, suggesting complementary roles in G1/S-phase regulation. However, it is noted that conclusions regarding fzr functions were inferred from the analysis of a large deletion that removed several genes in addition to fzr. Therefore, fzr-1(ku298) is the first reported mutation in metazoans that specifically reduces CDH1/HCT1/fzr activity (Fay, 2002).

An analysis of distal-tip cell (DTC) hyperproduction in strains that overexpress either cyclin A or cyclin E mRNA supports the model that lin-35 and fzr-1 are likely to coregulate cyclin levels during G1. In addition, the E2F homolog efl-1 synergizes with fzr-1, adding further credence to this model. The ability of both cyclin A and cyclin E to induce extra DTCs in fzr-1 mutants could indicate that these cyclins may be functionally interchangeable and that sufficient levels of either cyclin A or cyclin E, or possibly both in combination, can work to override G1 arrest (Fay, 2002).

By screening ~3500 haploid genomes, seven synthetic with lin-35/Rb (Slr) mutations were uncovered that show synthetic lethality or inviability with mutations in lin-35 were uncovered. Other than fzr-1 and lin-23, no Slr mutations have been identified that produce an obvious synthetic hyperproliferation phenotype with lin-35. The means used to uncover the genetic interaction between lin-35 and fzr-1 will be of general use for those wishing to assign functions to genes lacking known biological roles or to identify novel functions for genes with previously characterized activities. In addition, this genetic approach serves to identify functional copartners through the isolation and cloning of the affected second-site mutations. Importantly, this method in no way depends on prior knowledge of the synthetic double-mutant phenotype, thereby permitting a nonbiased search for genetic modifiers of any gene of interest (Fay, 2002).

Given the inevitable saturation of the genome for mutations that cause easily detectable phenotypes, the ability to identify synthetic mutations will become increasingly important. Large-scale analyses carried out in yeast and C. elegans suggest that a large percentage of genes in higher organisms may fail to show easily discernable phenotypes when mutated. It is likely that the vast majority of these no-phenotype genes may nevertheless confer a weak selective advantage to the organism, thus accounting for their presence in the genome. At the same time, it can be argued that many of these genes fail to show mutational effects owing to genetic redundancy. Importantly, these two explanations are in no way mutually exclusive. By devising methods to experimentally address this latter issue, biological roles may be assignable to many genes that would normally not be amenable to straightforward functional analyses (Fay, 2002).

A key pathway that controls both cell division and differentiation in animal cells is mediated by the retinoblastoma (RB) family of tumor suppressors, which gate the passage of cells from G1 to S and through S phase. The role(s) of the RB pathway in plants are not yet clearly defined, nor has there been any evidence for its presence in unicellular organisms. An RB homolog encoded by the mat3 gene in Chlamydomonas reinhardtii, a unicellular green alga in the land plant lineage, has been identified. Chlamydomonas cells normally grow to many times their original size during a prolonged G1 and then undergo multiple alternating rounds of S phase and mitosis to produce daughter cells of uniform size. mat3 mutants produce small daughter cells and show defects in two size-dependent cell cycle controls: they initiate the cell cycle at a below-normal size, and they undergo extra rounds of S phase/mitosis. Unlike mammalian RB mutants, mat3 mutants do not have a shortened G1, do not enter S phase prematurely, and can exit the cell cycle and differentiate normally, indicating that the RB pathway in Chlamydomonas has a different role from that in animals (Umen, 2001).

The synthetic multivulva (synMuv) genes define two functionally redundant pathways that antagonize RTK/Ras signaling during C. elegans vulval induction. The synMuv gene lin-35 encodes a protein similar to the mammalian tumor suppressor pRB and has been proposed to act as a transcriptional repressor. Studies using mammalian cells have shown that pRB can prevent cell cycle progression by inhibiting DP/E2F-mediated transcriptional activation. C. elegans genes that encode proteins similar to DP or E2F have been identified. Loss-of-function mutations in two of these genes, dpl-1 DP and efl-1 E2F, cause the same vulval abnormalities as do lin-35 Rb loss-of-function mutations. It is proposed that rather than being inhibited by lin-35 Rb, dpl-1 DP and efl-1 E2F act with lin-35 Rb in transcriptional repression to antagonize RTK/Ras signaling during vulval development (Ceol, 2001).

Considerable evidence indicates that mammalian DP and E2F proteins can promote the entry of cells into S phase, thereby stimulating cell cycle progression. Observations of dpl-1(n3316 RNAi) animals, which are presumably deficient in both the zygotic and maternal contribution of DPL-1, suggest that DPL-1 activity is not essential in every cell for cell cycle progression. Since dpl-1 is the only predicted C. elegans DP family member, and since DP protein function is thought to be necessary for DP/E2F heterodimer function, DP/E2F activity in C. elegans may not be generally required for cell cycle progression. However, dpl-1 and efl-1 may be required to promote S phase entry in some cell types. Specifically, the Pn.a neuroblasts of dpl-1(n3316 RNAi) animals do not complete their divisions and sometimes generate large polyploid descendants. It is concluded that dpl-1 and possibly efl-1 may act in but are not essential for the cell cycle in C. elegans. It is possibile, however, that undetectable levels of dpl-1 activity and DPL-1 protein may be present in dpl-1(n3316 RNAi) animals and may fulfill a broader requirement for dpl-1 in promoting cell cycle progression. Loss-of-function mutations in dpl-1 and efl-1, like loss-of-function mutations in lin-35 Rb, cause cell-fate transformations that result in supernumerary cell divisions in the P(3,4,8).p lineages. The extra cell divisions in these mutants are consistent with the possibility that a DPL-1/EFL-1/LIN-35 protein complex normally inhibits cell cycle progression in P(3,4,8).p. Thus, while dpl-1 and efl-1 promote cell division in some cell types, e.g., in Pn.a descendants, dpl-1 and efl-1 may prevent cell division in other cell types, e.g., in P(3,4,8).p descendants. While it is tempting to speculate that dpl-1 and efl-1 are cell cycle regulators in C. elegans, it is not yet known if the effects of these genes on cell division are caused by the direct regulation of the cell cycle machinery or are caused by the regulation of cell fate-determining factors that subsequently impinge on the cell cycle machinery (Ceol, 2001).

In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 (and possibly cyclin E-dependent Cdk2) positively regulate the G1- to S-phase transition by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Ectopic expression of cyclin E (but not cyclin D1) can override G1 arrest imposed by either the p16INK4a Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. The cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells either downstream of or parallel to phosphorylation of pRb and independent of E2F activation. A lack of E2F-mediated transactivation can be compensated for by hyperactivation of this cyclin E-controlled event (Lukas, 1997).

p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppresses the activity of E2F-responsive promoters (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) are unaffected. Moreover, the simian virus 40 early promoter is rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis. E2F-1 (which binds pRb), but not E2F-4 (which does not), reverses both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppresses growth and E2F activity in cells lacking a functional pRb. p21 protein (wild type but not mutant) specifically disrupts an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for either the inhibition of growth or E2F-dependent transcription (Dimri, 1997).

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. It is suggested that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. These data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis (Alevizopoulos, 1997).

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, the functional properties of three identified RB mutations were examined in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein "pocket" binding activity, and do not suppress colony growth of RB(-) cells. In contrast, two low-penetrant alleles (661W and "deletion of codon 480") retain the ability to localize to the nucleus, show normal cdk-mediated hyperphosphorylation in vivo, exhibit a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that is intermediate between classic mutants (null) and wild-type RB, and has absent E2F1 binding in vitro. A third, low-penetrant allele, "deletion of RB exon 4," shows minimal hyperphosphorylation in vivo but demonstrates detectable E2F1 binding in vitro. Each low-penetrant RB mutant retains the ability to suppress colony growth of RB(-) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression (Otterson, 1997).

To assess biological roles of the retinoblastoma protein (RB), four independent transgenic mouse lines expressing human RB with different deletions in the N-terminal region (RBdeltaN) were generated and compared with mice expressing identically regulated, full-length RB. Expression of both RB and RBdeltaN causes developmental growth retardation, but the wild-type protein is more potent. In contrast to wild-type RB, the RBdeltaN proteins are unable to rescue Rb-/- mice completely from embryonic lethality. Embryos survive until gestational day 18.5 but display defects in the terminal differentiation of erythrocytes, neurons, and skeletal muscle. In Rb+/- mice, expression of the RBdeltaN transgenes fails to prevent pituitary melanotroph tumors but delays tumor formation or progression. These results strongly suggest that N-terminal regions are crucial for embryonic and postnatal development, tumor suppression, and the functional integrity of the entire RB protein. These transgenic mice provide models that may begin to explain human families with low-penetrance retinoblastoma and mutations in N-terminal regions of RB (Riley, 1997).

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells are observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlates with the presence of functional pRb. G2 arrest is more prominent in pRb-negative cells. The arrest distribution does not correlate with the p53 status, and the proliferating-cell nuclear antigen (PCNA) binding activity of p21 does not appear to be involved, since p27, which lacks a PCNA binding domain, produces a similar arrest profile. In addition, DNA endoreduplication occurs in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in cells arrested in G2 by p21. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. The tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway (Niculescu, 1998).

The predominant G2 arrest observed in pRb-negative cells and limited G2 arrest observed in pRb-positive cells is most likely due to p21-mediated inhibition of cyclin A-Cdk2. The dynamics of arrest in pRb-negative cells probably reflects the inefficiency of inhibition of cyclin E-Cdk2. It is unlikely that inhibition of cyclin B1-associated kinase by p21 contributes to G2 arrest, since there is no evidence for significant amounts of cyclin B1 associated with p21. Nevertheless, it has been observed that in p21-induced G2-arrested cells, cyclin B1-Cdk2 is inhibited by phosphorylation of Cdc2. Although the mechanism for this indirect inhibition by p21 is not yet known, a similar phenomenon has been observed in Xenopus egg extracts, where inhibition of Cdk2 leads to inhibitory phosphorylation of Cdc2 and concomitant G2 arrest. pRb is necessary to block endoreduplication. After arrest in G2, a significant subpopulation of pRb-negative cells responding to p21 or p27 expression undergo cycles of endoreduplicative DNA replication. Although a significant fraction of pRb-positive cells arrests in G2, endoreduplication is never observed. Two conclusions can be drawn from these observations: (1) p21 can arrest cells in G2 in a physiological environment that is permissive for entering the S phase without an intervening mitosis. This would seem to be a violation of the normal safeguards that prevent cell cycle events from occurring out of order. (2) Initiation of the S phase, however, whether from G1 or G2, requires neutralization of the inhibitory functions of pRb. This cannot happen in the presence of both pRb and p21. The incomplete inhibition of cyclin E- and/or cyclin A-associated kinase activities may allow sufficient activity for initiation of replication but not sufficient to proceed to mitosis. The appropriate balance may not be met in every cell, since many apparently do not undergo endoreduplication. Licensing of origins may not be efficient, since endoreduplicative replication appears to proceed slowly. But the fact that pRb appears to be capable of blocking endoreduplication in G2-arrested cells suggests a role in enforcing the order of cell cycle events. Whatever critical functions downstream of pRb are required for replication after passage through G1 appear to be required again if replication is to occur from G2. The regulation of pRb phosphorylation, in fact, may be an important G2 function of p21 in response to DNA damage (Niculescu, 1998).

Humans Hemizygous for the retinoblastoma gene RB are strongly predisposed to retinoblastoma. In the mouse, however, Rb hemizygosity leaves the retina normal, whereas in Rb-/- chimeras pRb-deficient retinoblasts undergo apoptosis. To test whether concomitant inactivation of the Rb-related gene p107 is required to unleash the oncogenic potential of pRb deficiency in the mouse retina, both Rb and p107 were inactivated by homologous recombination in embryonic stem cells and chimeric mice were generated. Retinoblastomas are found in five out of seven adult pRb/p107-deficient chimeras. The retinal tumors show amacrine cell differentiation, and therefore originate from cells committed to the inner but not the outer nuclear layer. Retinal lesions are already observed at embryonic day 17.5. At this stage, the primitive nuclear layer exhibits severe dysplasia, including rosette-like arrangements, and apoptosis. These findings provide formal proof for the role of loss of Rb in retinoblastoma development in the mouse and the first in vivo evidence that p107 can exert a tumor suppressor function (Robanus-Maandag, 1998).

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity are detected, but the expression of cyclin A is inhibited. The ectopic expression of cyclin E restored cyclin A expression and drives the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB can not complete DNA replication. Microinjection of cells that have passed through the G1 restriction point with plasmids expressing PSM-RB also leads to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. Taken together, these results indicate that unphosphorylated RB can inhibit DNA synthesis through a mechanism that is independent of the inhibition of the known G1/S cdk-cyclin activities. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication (Knudsen, 1998).

The commitment of cells to replicate and divide correlates with the activation of cyclin-dependent kinases and the inactivation of Rb, the product of the retinoblastoma tumor suppressor gene. Rb is a target of the cyclin-dependent kinases and, when phosphorylated, is inactivated. Biochemical studies exploring the nature of the relationship between cyclin-dependent kinase inhibitors and Rb have supported the hypothesis that these proteins are on a linear pathway regulating commitment. This relationship has been studied genetically by examining the phenotype of Rb+/-p27-/- mice. Tumors arise from the intermediate lobe cells of the pituitary gland in p27-/- mice, as well as in Rb+/- mice after loss of the remaining wild-type allele of Rb. Using these mouse models, the genetic interaction between Rb and p27 was examined. The development of pituitary tumors in Rb+/- mice correlates with a reduction in p27 mRNA and protein expression. To determine whether the loss of p27 is an indirect consequence of tumor formation or a contributing factor to the development of this tumor, the phenotype of Rb+/-p27-/- mice was analyzed. These mice develop pituitary adenocarcinoma with loss of the remaining wild-type allele of Rb and a high-grade thyroid C cell carcinoma that is more aggressive than the disease in either Rb+/- or p27-/- mice. Importantly, both pituitary and thyroid tumors are detected earlier in the Rb+/-p27-/- mice. It is therefore proposed that Rb and p27 cooperate to suppress tumor development by integrating different regulatory signals (Park, 1999).

The retinoblastoma protein, pRB, and the closely related proteins p107 and p130 are important regulators of the mammalian cell cycle. Biochemical and genetic studies have demonstrated overlapping as well as distinct functions for the three proteins in cell cycle control and mouse development. However, the role of the pRB family as a whole in the regulation of cell proliferation, cell death, or cell differentiation is not known. Embryonic stem (ES) cells and other cell types mutant for all three genes were generated. Triple knock-out mouse embryonic fibroblasts (TKO MEFs) have a shorter cell cycle than wild-type, single, or double knock-out control cells. TKO cells are resistant to G1 arrest following DNA damage, despite retaining functional p53 activity. They are also insensitive to G1 arrest signals following contact inhibition or serum starvation. Finally, TKO MEFs do not undergo senescence in culture and do possess some characteristics of transformed cells. These results confirm the essential role of the Rb family in the control of the G1/S transition; they place the three Rb family members downstream of multiple cell cycle control pathways, and strengthen the link between loss of cell cycle control and tumorigenesis (Sage, 2000).

Mouse embryonic fibroblasts mutant for the three pRB family members are unable to arrest in G1 following various inhibitory signals, such as confluence, low serum, detachment from the substratum, DNA damage, or normal or premature senescence. All of these signals use different transduction pathways to induce cell cycle arrest, but eventually, each is thought to converge on the cell cycle inhibitors of the INK4 and/or CIP/KIP families. For example, it is known that the senescence program involves increased levels of both p16INK4A and p21CIP1 and that low-serum conditions inhibit cyclin D1- and cyclin E-dependent kinases activities via p16INK4A and p21CIP1/p27KIP1, respectively. Absence of the pRB family eliminates key targets of these inhibitors and makes cells refractory to G1 arrest. Preliminary observations indicate that TKO cells are also resistant to G1 arrest induced by overexpression of p27KIP1 and p19ARF cell cycle inhibitors. It will be interesting to determine whether any conditions that normally induce G1 arrest would operate in cells lacking pRB family function (Sage, 2000).

It has been postulated that cells in which G1 control is compromised may undergo inappropriate S-phase entry, accumulate mutations, and undergo cellular transformation. Absence of pRB family function and consequent loss of G1 control leads to cellular immortalization directly. 3T3 assays, low-density plating experiments, and absence of senescence induced by oncogenic Ras have been used previously to demonstrate the immortalized properties of Ink4a-/-, ARF-/-, and p53-/- MEFs. Thus, the still-to-be-defined signal transduction pathway that leads from excessive mitogenic signaling or prolonged passage in culture to increased levels of p16INK4A, p19ARF, p53, and p21CIP1 converges on the pRB family. Also, in cells lacking just pRB or just p107 and p130, the senescence programs can effectively result in permanent G0/G1 arrest; in cells lacking all three proteins, such programs are ineffective (Sage, 2000).

Inactivation of the pRB family is not sufficient for full cellular transformation. For example, TKO MEFs are not tumorigenic in nude mice, and their ability to form colonies in soft agar or to form foci at confluency is reduced compared to fully transformed cells. This suggests that loss of G1 control leads to immortalization but is not sufficient for full transformation and that additional mutations are required for this effect. TKO cells are more susceptible than control cells to cell death under specific conditions; also, loss of p53 occurs in some late-passage TKO cultures. Thus, antiapoptotic mutations might cooperate with pRB family mutations in transformation. In addition, oncogenic Ras causes increased tumorigenicity of TKO cells, suggesting that increased mitogenic signaling can promote transformation in cells lacking G1 control. It must be noted, however, that Ras-transformed p53-/- MEFs or NIH3T3 cells form larger colonies in soft agar and tumors in nude mice more rapidly than Ras-transformed TKO cells. Therefore, mutations in other cellular pathways may be required for full transformation (Sage, 2000).

The analysis of TKO and control MEFs underscores the degree of functional overlap between the three pRB family members and indicates that this overlap is present in many aspects of cell cycle regulation. Depending on the assay, Rb-/- or p107-/-;p130-/- cells were either indistinguishable from wild-type cells (e.g., colony formation in soft agar or Ras-induced senescence) or they show an intermediate response between wild-type and TKO MEFs (e.g., G1 length or response to DNA-damaging agents). Therefore, the degree to which different pRB proteins can functionally compensate for one another may vary depending on the cellular context. The phenotypic differences between Rb-/- and TKO cells highlight the importance of p107 and p130 as cell cycle regulators, at least in cells with compromised pRB function. It is also possible that in certain conditions, p107 or p130 are more functionally related to pRB than they are to each other, and therefore, one might expect additional intermediate phenotypes in Rb-/-;p107-/- or Rb-/-;p130-/- cells. Indeed, Rb-/-;p107-/- MEFs are immortal (Sage, 2000).

The availability of cells carrying combined mutations for the pRB family members and the knowledge of the upstream and downstream regulatory pathways that affect them allow for a better understanding of the similarities and differences between the three family members. However, several points still remain unclear. For example, ectopic expression of p16INK4A does not induce G1 arrest in Rb-/- cells. This result is difficult to reconcile with the current understanding of functional compensation within the Rb family. Perhaps the residual cell cycle regulation provided by p107/p130 is not sufficient to confer growth arrest in this setting. In addition, it has recently been shown that p16INK4A overexpression does not arrest p107-/-;p130-/- cells. It is, therefore, possible that in some circumstances a certain threshold level of activation of the pRB family function is necessary for proper cell cycle arrest and that the level of this threshold may vary depending on the cellular context (Sage, 2000).

The E2F family of transcription factors can be divided in two groups, with E2F-1, E2F-2, and E2F-3 serving as activators of transcription and E2F-4 and E2F-5 being involved primarily in transcriptional repression through recruitment of pRB family proteins. pRB, p107, and p130 interact differentially with the E2F family proteins: pRB binds preferentially to the E2F-1-4, and p107 and p130 interact more specifically with E2F-4. These differences suggest that the pRB family members control two types of downstream pathways to arrest cells, perhaps targeting two different sets of genes. However, the target genes of the E2F family have not been well characterized, and little information exists concerning the specificity of each member of this family. Availability of E2F mutant cells in combination with Rb family mutations will permit investigation of these issues (Sage, 2000).

These observations with TKO cells raise the question of the importance of p107/p130 mutations in human cancer. Mutations in these genes have been found only rarely and in a small subset of tumor types. In contrast, many human tumor types exhibit structural mutations in RB itself. Thus, with respect to human tumorigenesis, loss of pRB function alone would appear to confer a selective advantage. This situation may resemble the p16INK4A-induced growth arrest in MEFs, where Rb mutation is sufficient to confer insensitivity. In other circumstances, however, loss of pRB family function as a whole may lead to some additional proliferation advantage. This may coincide with mutations in genes that regulate pRB, p107, and p130, such as INK4A or CDK4. In support of this possibility, some human tumors have been described as having RB mutation and CDK4 amplification or mutations in both RB and P16INK4A. Further mutational analysis in human cancer as well as the study of the tumorigenic potential of mouse cells carrying compound mutations in the pRB family will help to resolve this issue (Sage, 2000).

The retinoblastoma suppressor pRB belongs to the family of so-called pocket proteins, which also includes p107 and p130. These proteins may functionally overlap in cell cycle control and tumor suppression. An isogenic set has been generated of embryonic stem (ES) cell lines carrying single or compound loss-of-function mutations in the Rb gene family, including a cell line completely devoid of all three pocket proteins. None of the knockout combinations affected the growth characteristics of ES cells; however, concomitant ablation of all three pocket proteins strongly impairs their differentiation capacity. For the generated genotypes, primary mouse embryonic fibroblasts (MEFs) also were obtained. While inactivation of Rb alone does not alleviate the senescence response of MEFs, pRB/p107-deficient MEFs, after having adapted to in vitro culturing, continue to proliferate at a modest rate. Additional ablation of p130 renders MEFs completely insensitive to senescence-inducing signals and strongly increases their proliferation rate. Although triple-knockout MEFs retain anchorage dependence, they lack proper G1 control and show increased cell turnover under growth-inhibiting conditions (Dannenberg, 2000).

The hSNF5/INI1 gene encodes a member of the SWI/SNF chromatin remodelling complexes. The gene has been identified as a tumour suppressor gene mutated in sporadic and hereditary Malignant Rhabdoid Tumours (MRT). However, the role of hSNF5/INI1 loss-of-function in tumour development is still unknown. This study shows that the ectopic expression of wild-type hSNF5/INI1, but not that of truncated versions, leads to a cell cycle arrest by inhibiting the entry into S phase of MRT cells. This G1 arrest is associated with down-regulation of a subset of E2F targets, including cyclin A, E2F1 and CDC6. This arrest can be reverted by coexpression of cyclin D1, cyclin E or viral E1A, whereas it cannot be counteracted by pRB-binding deficient E1A mutants. Moreover, hSNF5/INI1 is not able to arrest cells lacking a functional pRB. These observations suggest that the hSNF5/INI1-induced G1 arrest is dependent upon the presence of a functional pRB. However, the observation that a constitutively active pRB can efficiently arrest MRT cells indicates that hSNF5/INI1 is dispensable for pRB function. Altogether, these data show that hSNF5/INI1 is a potent regulator of the entry into S phase, an effect that may account for its tumour suppressor role (Versteege, 2002).

Cancer cells arise from normal cells through the acquisition of a series of mutations in oncogenes and tumor suppressor genes. Mouse models of human cancer often rely on germline alterations that activate or inactivate genes of interest. One limitation of this approach is that germline mutations might have effects other than somatic mutations, owing to developmental compensation. To model sporadic cancers associated with inactivation of the retinoblastoma (RB) tumor suppressor gene in humans, a conditional allele of the mouse Rb gene was produced. Acute loss of Rb in primary quiescent cells is sufficient for cell cycle entry and has phenotypic consequences different from germline loss of Rb function. This difference is explained in part by functional compensation by the Rb-related gene p107. Acute loss of Rb in senescent cells leads to reversal of the cellular senescence program. Thus, the use of conditional knockout strategies might refine understanding of gene function and help to model human cancer more accurately (Sage, 2003).

Null mutations in lin-35, the Caenorhabditis elegans ortholog of the mammalian Rb protein, cause no obvious morphological defects. Using a genetic approach to identify genes that may function redundantly with lin-35, a mutation in the C. elegans psa-1 gene was isolated. lin-35; psa-1 double mutants display severe developmental defects leading to early larval arrest and adult sterility. The psa-1 gene encodes a C. elegans homolog of yeast SWI3, a critical component of the SWI/SNF complex, and has been shown to regulate asymmetric cell divisions during C. elegans development. Strong genetic interactions are observed between psa-1 and lin-35 as well as a subset of the class B synMuv genes that include lin-37 and lin-9. Loss-of-function mutations in lin-35, lin-37, and lin-9 strongly enhanced the defects of asymmetric T cell division associated with a psa-1 mutation. These results suggest that LIN-35/Rb and a certain class B synMuv proteins collaborate with the SWI/SNF protein complex to regulate the T cell division as well as other events essential for larval growth (Cui, 2004).

The retinoblastoma gene product, pRb, plays a crucial role in cell cycle regulation, differentiation and inhibition of oncogenic transformation. pRb and its closely related family members p107 and p130 perform exclusive and overlapping functions during mouse development. The embryonic lethality of Rb-null animals restricts the phenotypic analysis of these mice to mid-gestation embryogenesis. The Cre/loxP system was used to study the function of Rb in adult mouse stratified epithelium. RbF19/F19;K14cre mice display hyperplasia and hyperkeratosis in the epidermis with increased proliferation and aberrant expression of differentiation markers. In vitro, pRb is essential for the maintainance of the postmitotic state of terminally differentiated keratinocytes, preventing cell cycle re-entry. However, p107 compensates for the effects of Rb loss; the phenotypic abnormalities of RbF19/F19;K14cre keratinocytes in vivo and in vitro become more severe with the concurrent loss of p107 alleles. p107 alone appears to be dispensable for all these phenotypic changes; the presence of a single Rb allele in a p107-null background rescues all these alterations. Luciferase reporter experiments indicate that these phenotypic alterations might be mediated by increased E2F activity. These findings support a model in which pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis (Ruiz, 2004).

Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. The retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, since p21-null mice exhibit an unaltered inner ear phenotype. By contrast, Rb inactivation leads to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells express various cell type-specific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranges from near-normal to severe disorganization. Apoptosis contributes to the mutant phenotype, but does not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation leads to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. These findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia (Mantela, 2005).

These data show that the CKI p21 is expressed in the differentiating cochlear and vestibular HCs, and that the expression is induced at the initiation of HC differentiation. In the auditory sensory epithelium, p21 expression is initiated at E14.5, at the stage when Math1 expression is first detected. It is possible that p21 induction in HCs is regulated by Math1, by analogy to the positive role of bHLH proteins such as Myod1 and myogenin in skeletal myogenesis. Thereafter, p21 together with other CKI(s) might have an active role in keeping pRb in a hypophosphorylated form. Thus, negative regulation at the level of both pRb and CKIs seems to be responsible for the maintenance of HC quiescence. No phenotypic alterations or aberrant mitoses were found in the inner ears of developing or adult p21-/- mice. Interestingly, in addition to p21, another CKI, p19, has been shown to be expressed in the late-embryonic organ of Corti, but its inactivation does not result in developmental abnormalities. Thus, functional redundancy may exist between p21 and p19 in developing cochlear HCs. In addition, developing vestibular HCs express p21, but do not show phenotypic changes following targeted gene disruption, most probably owing to functional compensation. The identity of the CKI that may cooperate with p21 in vestibular HCs remains to be identified, since p19 expression and the consequences of p19 inactivation have not been reported in vestibular organs (Mantela, 2005).

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5. However, the actual cause of the embryonic lethality has not been fully investigated. This study shows that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. Two complementary techniques, tetraploid aggregation and conditional knockout strategies, were used to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals are virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development (Wu, 2003).

Table of contents

Retinoblastoma-family protein: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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