Regulation of cell cycle progression by retinoblastomasusceptibilitygene product

When the normal rb gene is transferred into retinoblastoma cells and/or into cells which carry the inactivated rb gene, there is suppression of growth and tumorigenicity (Huang et al., 1988; Bookstein et al., 1990b); therefore, it has been regarded as a tumour suppressor gene (Figure 9). The function of tumour suppression appears to be mainly due to its ability to act as a negative regulator of cell cycle progression. It has been shown that the product of this gene is a 110 kDa (pllOrt>) phosphoprotein. This protein is differentially phosphorylated in relation to the cell cycle (Buchkovich et al, 1989; Chen et al., 1989). The unphosphorylated rb protein is found predominantly in the Gi phase of the cell cycle (Pardee, 1989). Stimulation of resting cells by mitogens is associated with phosphorylation of the rb protein and transition of cells from G, to S phase of the cell cycle (Chen et al., 1989). Relatively higher levels of phosphorylated rb are found in the logarithmic phase of growth of cells compared with those arrested in the G, phase (Xu et al., 1989). The unphosphorylated form has therefore been regarded as a suppressor of cell proliferation. The hyperphosphorylated status of rb protein is maintained throughout the remainder of the cell cycle and it is dephosphorylated upon the exit of the cells from mitosis. The cell cycle-dependent phosphorylation of the rb protein is mediated by cyclin D and CDK4/6 (Dowdy et al., 1993; Ewen et al., 1993a). The expression of cyclin E also can lead to rb phosphorylation (Hinds et al., 1992) and it has been suggested that cyclin E/CDK2 complexes may mediate this process.

The sequestration of unphosphorylated rb protein will allow the cells to transit into the S-phase. It is known that functional rb protein forms stable complexes with transforming oncoproteins such as those encoded by the simian virus 40 (SV40) and human papilloma viruses (Templeton et al, 1991). HPV E7 binds to rb protein and such binding is necessary for E7 protein to immortalise and transform cells (Dyson et al., 1989; Munger et al, 1989; Gage et al, 1990). The SV40 T-antigen is known to bind preferentially to the underphosphorylated rb protein (Ludlow et al., 1989). Although these studies suggest a close correlation between phosphorylation and the transition of cells from G, into the S-phase of the cell cycle, phosphorylation might be a progressive event and this may be reflected in the molecular heterogeneity found in rb proteins (Xu et al., 1989). The process of rb phosphorylation may be an incremental process beginning in late G,, several hours before transition into the S-phase

Figure 9. A schematic representation of cooperative control of cell cycle progression by p53 and rb proteins. The phosphorylation of rb is a major requirement for progression of the cell cycle beyond the restriction point. p53 may suppress rb phosphorylation by inducing the expression of CDK inhibitors. The phosphorylation of rb releases transcription factors such as E2F (see Figure 10) from their complexes with rb, and this allows the transcription of genes required for entry of cells into the S-phase. Viral oncoproteins may tether underphosphorylated rb and allow GrS transition of the cells. The figure also presents a postulate which implicates mdm2 with the function of both p53 and rb.

Figure 9. A schematic representation of cooperative control of cell cycle progression by p53 and rb proteins. The phosphorylation of rb is a major requirement for progression of the cell cycle beyond the restriction point. p53 may suppress rb phosphorylation by inducing the expression of CDK inhibitors. The phosphorylation of rb releases transcription factors such as E2F (see Figure 10) from their complexes with rb, and this allows the transcription of genes required for entry of cells into the S-phase. Viral oncoproteins may tether underphosphorylated rb and allow GrS transition of the cells. The figure also presents a postulate which implicates mdm2 with the function of both p53 and rb.

(Mittnacht et al, 1994). Furthermore, underphosphorylated rb binds to other cellular proteins such as the E2F transcription factor (Chellappan et al., 1991; Shirodkar et al., 1992) and cyclins D1 and D3 (Dowdy et al., 1993; Ewen et al., 1993a).

The rb protein needs to control transcription of genes which are essential for the progression of the cell through the cell cycle and this appears to be effected by rb interaction with the E2F transcription factors (Figure 10). The latter comprise a family of transcription factors. Five members of this family have been identified, namely E2F1, E2F2, etc. (Lam and La Thangue, 1994; Hijmans et al., 1995; Sardet et al, 1995). These transcription factors show sequence-specific binding as homodimers to DNA and this can be enhanced when E2Fs form heterodimers with the DPI family of transcription factors (Bandara et al, 1993,1994; Helin et al, 1993; Krek et al, 1993; Wu et al, 1995). Other rb-related proteins deemed as members of the rb family of proteins also interact with E2F in a manner similar to rb itself (Vairo et al, 1995).

E2F-1

E2F-1

E2F-1/rb

Phosphorylation

Phosphorylation

Transcription of E2F-1 responsive genes

Cyclin/CDK

Cell cycle progression

P16/INK4 p21 Waf1/cip1

Figure 10. The significance of the complex formation between rb protein and the transcription factor E2F-1 is depicted here. Phosphorylation of rb results in the release of E2F-1 from the complex and this transcription factor mediates the transcription of E2F-1 responsive genes necessary for the progression of the cell cycle. As shown also in Figure 9, a p53-mediated control mechanism might be the inhibition of cyclin-dependent kinases by pl6/ink4 and wafl/cipl.

Johnson DG et al. (1993) found that if E2F-1 protein is introduced into a quiescent cell it enters the S-phase. But interaction between rb and E2F proteins produces a down-regulation of E2F-dependent transcription (Lam and La Thangue, 1994; Wu CL et al, 1995). E2F binding occurs predominantly to the underphosphorylated form of the rb protein (Chellappan et al., 1991; Helin et al., 1992; Kaelin et al., 1992; Shan et al., 1992). The phosphorylation of rb releases it from its association with E2F, thus allowing E2F-dependent gene transcription. E2F-1 is itself differentially regulated. In the late G]-early S phase, E2F-1 is bound by cyclin A/cdk2. The latter also phosphorylates the DPI in the DP1/E2F complex. As a consequence E2F-1 loses its DNA-binding ability and is essentially negatively regulated (Dynlacht et al., 1994; Krek et al., 1994). This is believed to be the pathway by which the rb protein exerts a negative regulatory control on cell cycle progression (Figure 10).

The cell cycle regulatory function of rb may involve other transcription factors, such as the Ets family members which are involved in cell cycle progression in activated T cells. The Ets related transcription Elf-1 has been found to bind rb protein by a sequence motif which is related to rb-binding motifs carried by viral oncoproteins (Wang CY et al., 1993). As with E2F, phosphorylation of rb results in the release of Elf-1. The rb protein is also known to bind to other transcription factors such as the ATF2, causing transcription of ATF2 target genes (Kim et al, 1992).

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