VIGAS

Are the same molecules involved in arresting proliferation during differentiation and in maintaining the post-mitotic arrest? Most of the studies, such as gene deletion, only address the question of whether those genes are im portant for terminal differentiation to occur. As such, the use of viral proteins that induce re-entry into the cell cycle has been an im portant tool for investigating the stability of the post-mitotic state. Two of these proteins are the SV40 large T antigen and the adenoviral protein E l A.

Large T may promote grow th through a variety of interactions, such as binding to pocket-proteins, p53 and the transcriptional co-activator p300 (reviewed in Ali and DeCaprio, 2001). It induces DNA synthesis and mitosis in mouse myotubes (Endo and Nadal-Ginard, 1989; Endo and Nadal-Ginard, 1998; lujvidin et al., 1990).

The adenoviral protein ElA induces DNA synthesis and mitosis in differentiated skeletal myotubes (Crescenzi et al., 1995), adipocytes (Crescenzi et al., 1995), and cortical neurons (Suda et al., 1994), and DNA synthesis w ithout subsequent mitosis in cardiomyocytes (Kirshenbaum and Schneider, 1995; Liu and Kitsis, 1996). E lA also interacts w ith a variety of ceU cycle regulatory

proteins, such as pocket proteins (Moran, 1993), p300 (Eckner et aL, 1994), p27 (Mai et aL, 1996) and p21 (Keblusek et aL, 1999).

Different m utants of ElA have been used to investigate the interactions that are im portant for its function. A n E lA m utant (ElA.RG2) tiiat is defective for p300 binding is still efficient in inducing DNA synthesis in myotubes (Table 1.3.1.; (Mai et aL, 2000). Thus p300 may not play an essential role in the maintenance of the post-mitotic state of fully differentiated multinucleated myotubes. In contrast, the abihty to induce ceU cycle re-entry is lost in an ElA m utant (E1A.928; Table 1.3.1.) which is unable to bind to pocket proteins, and in an ElA m utant unable to bind to p21 and p300 (ElA.dl2-36; Table 1.3.1 ; Mai et aL, 2000).

Table 1.3.1 Binding properties of ElA mutants and their ability to induce DNA synthesis in differentiated muscle cells. +, positive for binding to a cellular protein; -, negative for binding to a cellular protein. From (Mai et aL, 2000).

Mutant Binding BrdU-positive C2C12 myotubes p300 pRb p21 w t ElA + + + + E1A.928 + - + - E1A.RG2 - + + + ElA.dl2-36 - + - -

The role of pocket proteins in maintaining the post-mitotic arrest of multinucleated myotubes, particularly pRb, has been further substantiated w ith

other experiments. During myogenesis pRb expression is upregulated and remains high, whereas that of pl0 7 is normally downregulated (Schneider et al., 1994). In Kb-/- myotubes, pl0 7 expression remains high. The expression of p i 07 and of pl30 in Kb - /- myotubes is not sufficient to m aintain a stable cell cycle arrest. Upon serum stimulation, p l0 7 is downregulated and these cells re-enter the cell cycle (Schneider et al., 1994). CeU cycle re-entry was also observed in Rb-/-

embryonic fibroblasts driven to differentiate into myocytes by ectopic MyoD expression (Novitch et al., 1996). Myofibers of mgRb:Rb-/- mice contain abundant large nuclei that have undergone several rounds of endorepHcation without mitosis, indicating that there are defects in the post mitotic arrest (Zacksenhaus et al., 1996). The function of pRb in maintaining the post-mitotic arrest might be broader than just inhibiting the function of E2Fs. In fact endorepUcaüon persists in the myofibres of mgRb.Rb-/- ; E2F-/- mice (Jiang et al., 2000). AdditionaUy, overexpression of E2F4 or E2F1 does not induce ceU cycle re-entry per se in these cells (Pajalunga et al., 1999; Puri et al., 1998).

The GDI p21 is expressed at high levels in differentiating skeletal myotubes (Franklin and Xiong, 1996; Phelps et al., 1998). When an E lA m utant E1A.928 that binds p21 but not pRb is expressed in differentiated myotubes there is no DNA synthesis (Mai et al., 2000). In this m utant cyclin E-CDK2 is activated and pRb is phosphorylated (Mai et al., 2000). The fact that expression of this m utant does not lead to DNA synthesis might be due to a requirem ent for phosphorylation of other pRb sites. Other GDIs may also have a role in maintaining post mitotic arrest. The single knockout mice for p21, p l8, p27, or p57 do not present muscle phenotypes (reviewed in Zhang, 1999), but the p21, p57 double m utant mice show endorepHcation in the nuclei of multinucleated myotubes (Zhang et al., 1999b).

The molecular regulation of the post-mitotic state m ight thus depend on the function of several proteins. Another example is the case of neurons. 15% of cortical neurons from adult mice transfected w ith ElA are induced to enter S

phase in vivo (Suda et al., 1994). It is not clear whether ElA is acting through the E2F-pRb pathw ay to induce S phase. Although E2F1 overexpression can induce S phase re-entry in cortical neurons from adult mice in vivo and in cultured rat sensory neurons (Smith et al., 2000; Suda et al., 1994), a m utant version of ElA that is able to inactivate pocket proteins, but not p300, does not induce DNA synthesis in cultured cortical neurons (Slack et al., 1998). The interaction of this E lA m utant w ith CDls was not described (Slack et al., 1998). Further studies are necessary to clarify the importance of pocket proteins in the maintenance of the differentiated state in neurons.

GDIs may play an im portant role in maintaining the post-mitotic arrest in neurons. Ectopic entry into the cell cycle was observed, both in lens fibre cells in the p27 and p57 double-m utant mice (Zhang et al., 1998) and in neurons from the central nervous system in the p i 9 and p27 double-mutant mice (Zindy et al., 1999).

Although much remains to be learned about the precise mechanisms that m aintain cells in a post-mitotic state, there is evidence favouring redundancy in the set of molecules that are involved. It becomes clearer that the maintenance of the stability of the post-mitotic state carmot be ascribed to one single type of molecule. Thus, different players, such as pocket proteins and GDIs, and positive cell cycle regulators, such as E2F, may each play an im portant role.