Because of the simplicity of the Drosophila E2F/Rb protein families, significant insights into the biological functions of the E2F/Rb proteins have been derived from studies of this model system. Analysis of the phenotype of flies with mutations of de2fl showed that dE2Fl plays a critical role in the expression of a set of S phase genes such as RNR2 and PCNA, which are two replication factors regulated by E2F (Duronio et al, 1995). However, in de2fl mutants, although the rate of BrdU incorporation was significantly reduced, it was not completely blocked (Royzman et al, 1997). This effect of de2fl mutation on BrdU incorporation is in contrast to that of one of its targets, the cyclin E mutant, which showed a complete block to BrdU incorporation (Royzman et al, 1997). Therefore, during normal development dE2Fl is limiting for the expression of replication factors but is not limiting for the expression of cell cycle regulators such as cyclin E. Unexpectedly, reducing the gene dosage of rbf or de2f2, or dDp all substantially suppress the phenotype of de2fl null mutants (Du, 2000; Frolov et al, 2001), suggesting that the phenotype of de2fl mutant is at least in part due to the presence of an RBF/dE2F2 repressor complex.
Interestingly, not all of the E2F target genes are coordinately deregulated by rbf mutation. For example, the epidermal cells of the developing embryo normally arrest at G1 after the 16th cell division and have low levels of the E2F targets PCNA, RNR2, and cyclin E expression. In embryos devoid of RBF, although all the epidermal cells initially still arrest in Gl, they exhibit strongly deregulated PCNA and RNR2 expression. In contrast, cyclin E is not immediately upregulated, but later a subset of epidermal cells accumulate cyclin E and enter S phase (Du and Dyson, 1999). The initial Gl cell cycle arrest of the epidermal cells of the developing embryos requires the cdk inhibitor dacapo (de Nooij et al., 1996; Lane et al., 1996), and sufficient cyclin E expression in the absence of RBF can overcome this arrest. RBF therefore functions to maintain the Gl arrest by inhibiting the expression of key cell cycle regulators such as cyclin E (Du and Dyson, 1999).
In summary, it appears that while E2F activity is not absolutely required for S phase entry, lack of E2F activation, either due to mutation of de2fl or due to overexpression of RBF, slows S phase progression (Royzman et al, 1997; Xin et al, 2002). On the other hand, inappropriate activation of E2F, either by ectopic expression of dE2Fl or by mutation of rbf, does lead to ectopic S phase entry and increased apoptosis (Du and Dyson, 1999; Du et al, 1996b). These effects of E2F on the cell cycle are likely due to the fact that the expression of the key regulator of S phase, cyclin E, does not absolutely require E2F but ectopic expression of E2F does activate cyclin E.
Biochemical studies using mammalian tissue culture systems have provided significant insight into the different E2F/Rb complexes that function in different phases of the cell cycle. Although simplified models suggest that the Rb/E2F complexes present during quiescence or GO are replaced with free E2Fs at the Gl/S transition, the actual composition of these complexes appears to vary with the cell cycle (reviewed in (Bracken et al, 2004; Classon and Harlow, 2002). In quiescent cells, pl30 is the main pocket protein complexed to inhibitory E2Fs; however, in cycling cells it is replaced by pl07/E2F 4 in G0/G1 phases and by Rb/E2F 1-3 complexes in S phase. Although mouse embryonic fibroblasts (MEFs) deficient in any one of the inhibitory E2Fs or in both pi07 and pi30 proliferate normally, they show defects in response to growth inhibitory signals. While pocket protein members may compensate for one another to some extent, Rb appears to be particularly important for senescence, and MEFs that are acutely inactivated for Rb are able to re-enter the cell cycle even from a quiescent state (Sage et al, 2003).
Given this important role of E2F and Rb in the cell cycle, it is not surprising that alterations in the Rb pathway are found in most tumors. Although mutation of Rb itself does not necessarily occur, inactivation may occur through overexpression of cyclin D or through loss of cdk inhibitors such as pi6, both leading to phosphorylation of Rb. In addition to adenovirus El a,
E2F can also be released from E2F/Rb complexes by viral oncogenes such as the SV40 large T antigen or by the human papilloma virus E7 protein. The strains of HPV that are associated with cervical cancer encode E7 proteins with the highest affinity and the ability to degrade Rb (Munger, 2002).
In addition to the cell cycle, Rb and E2F are involved in functions such as apoptosis, differentiation, and development (reviewed in Bracken et al, 2004; Classon and Harlow, 2002; Liu et al., 2004). Besides altering proliferation, loss of Rb may contribute to cancer by affecting differentiation. It was initially believed that loss of Rb would indirectly contribute to loss of differentiation because cells would be unable to terminally exit the cell cycle. However, screens of E2F-dependent genes have demonstrated that tissue-specific genes are among the targets of Rb/E2F. The importance of E2F and pocket protein activity has been shown in confluent (non-dividing) MEFs that are induced to differentiate into adipocytes. Under these conditions, the loss of E2F 4 results in spontaneous differentiation even though the cell cycle is not affected, suggesting that E2F 4 suppresses differentiation independently of proliferation (Landsberg et al., 2003). Moreover, mouse models of pocket protein or E2F family members show that loss of various E2Fs is associated with specific developmental abnormalities. For example, in some hematopoetic cell types Rb null cells cannot reach the final stage of differentiation and, the mice are prone to myeloproliferative disorders. It has been speculated that this is due to the increase in the pool of precursor cells that can give rise to tumors (Spike et al, 2004).
In addition to their roles in the cell cycle and development, Rb and E2F have also been found to affect apoptosis. Overexpression of E2F 1 has been shown to induce apoptosis, and E2F 1-/- thymocytes are defective in undergoing apoptosis (Field et al, 1996). Whether this ability to induce apoptosis is restricted to E2F 1 or is shared by other activating E2Fs is still unclear. E2F targets related to apoptosis include APAF1, caspases 3 and 7, and p73. Although some apoptosis appears to be independent of p53, E2F can activate the p53 pathway through upregulation of Arf and Pin-1, both of which contribute to p53 stabilization. There is also evidence that Rb itself may contribute to inhibiting apoptosis by stabilizing the anti-apoptotic protein Bcl-xL (reviewed in Liu et al, 2004). The ability of Rb and E2F to affect apoptosis would be expected to inhibit tumor formation by leading to apoptosis of cells with excessive E2F activity. Consistent with a role for E2F activity in tumor suppression, E2F 1-/- mice are surprisingly tumor prone (Cloud et al, 2002), although they show a different tumor spectrum from that of Rb+/- mice. Tumor incidence of E2F 1-/- mice can be further increased by removing E2F 2, but not E2F 3, suggesting that tumor suppression is a characteristic only of E2Fs 1 and 2.
Besides apoptosis, mouse models confirm many of the additional biological functions ascribed to Rb and E2F (reviewed in Classon and Harlow, 2002; Liu et al, 2004). The best characterized of the pocket protein knockout animals are Rb deficient mice. Rb heterozygous mice show a similar phenotype to heterozygous humans except that instead of developing retinoblastomas, Rb+/- mice develop pituitary and thyroid tumors (Jacks et al, 1992). The incidence of these tumors can be decreased by eliminating E2F 1, suggesting that the ability of Rb to suppress tumors is due to its inhibition of activating E2Fs. Rb null mice die at embryonic day 13.5 and show ectopic S phases, extensive neuronal apoptosis, and defects in the differentiation of muscle and red blood cells, among other tissues. However, many of these defects appear not to be cell autonomous but may instead be due to hypoxia since the Rb-/- placenta and red blood cell development are abnormal (Wu et al, 2003). In contrast, pi07 or pi30 null animals appear to develop normally, although pl07-/-;pl30-/- animals have defects in chondrocytes leading to bone abnormalities (Cobrinik et al, 1996), suggesting some essential, tissue-specific roles for these proteins. The ability of E2F 1-/- or E2F 3-/- to decrease the extent of neuronal apoptosis in Rb-/-embryos has suggested that it is excessive E2F activity that leads to apoptosis; however, restoration of Rb in the placenta also rescued apoptosis in the central nervous system, so it is not clear whether the apoptotic rescue by E2Fs 1 and 3 is cell autonomous or whether loss of the E2Fs corrected the placental phenotypes.
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