Progression of cells into S phase is controlled by the retinoblastoma protein (pRB) and relies on the functional inactivation of this tumour suppressor in late G1 via protein phosphorylation. We provide evidence here that, besides controlling entry of cells into S phase, pRB can operate to inhibit S phase completion. Dierential arrays of phosphorylation appear to regulate these dierent events, suggesting that cycle progression at these two stages of the cell cycle may be achieved via activation of distinct downstream pRB eector pathways. In agreement with this hypothesis, pRB's ability to prevent S phase entry, but not its ability to inhibit S phase completion, correlates with repression of E2F-regulated promoters. Furthermore, ectopic expression of E2F or the E2F-regulated cyclin E gene promote S phase entry in cells expressing phosphorylation-defective pRB but neither is sucient to trigger completion of S phase. Our ®ndings raise the possibility that pRB, in addition to its well-established role in controlling a checkpoint in late G1, could be involved in the control of a further checkpoint operating during S phase and that implementation of this checkpoint relies on an as yet unidenti®ed pRB eector distinct from E2F.Keywords: retinoblastoma protein; pRB; E2F; phosphorylation; checkpoint control; cell cycle IntroductionThe growth-controlling activity of the tumour suppressor, retinoblastoma protein (pRB), relies on its capacity to reversibly associate with E2F and other cellular transcription factors (Herwig and Strauss, 1997;Weinberg, 1995). The phosphorylation state of pRB determines its ability to bind to these factors (reviewed in Bandara et al., 1993;Taya, 1997). Evidence to this eect was ®rst provided through analysis of pRB's association with SV40 T antigen (Sterner et al., 1998;Templeton et al., 1991;Whyte et al., 1989). Only un-or poorly phosphorylated pRB associated with this viral oncoprotein and this has since been similarly demonstrated for many of the cellular pRB ligands.Phosphorylation of pRB, leading to its inactivation, is executed by members of the cyclin-dependent family of protein kinases (ConnellCrowley et al., 1997;Johnson et al., 1995;Lees et al., 1991;Lin et al., 1991;Mittnacht et al., 1994;Sterner et al., 1998;Templeton et al., 1991;Whyte et al., 1989;. In growing cells, the phosphorylation state of pRB changes periodically and in accordance with the cellular division cycle. Un-or poorly phosphorylated pRB, distinguished from its highly phosphorylated variant by a faster migration in SDS gels and its tight association with the cell nucleus, exists in cells only during the early phases of G1 (Buchkovich et al., 1989;Chen et al., 1989;DeCaprio et al., 1989;Mittnacht et al., 1991Mittnacht et al., , 1994. This form of pRB is no longer detectable upon passage of cells into S phase. Instead, highly phosphorylated pRB accumulates and prevails throughout the remainder of the cell cycle until the cells enter the next G1 phase, when pRB is converted into an un-or poorly phosphorylated form by d...

Translocations of the retinoic acid receptor alpha (RARA) locus with the PLZF or PML genes lead to expression of oncogenic PLZF-RARα or PML-RARα fusion proteins, respectively. These fusion oncoproteins constitutively repress RARα target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARα have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (POZ) and DNA binding (zinc-finger) domains. Additionally, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction between PML or E2F1 with Rb, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. The interaction between PLZF and Rb is further underlined by chromatin immunoprecipitation analysis of PLZF binding to genomic DNA, which shows that PLZF associates with genes controlling cell proliferation known to be regulated by Rb and E2F (for example cdc6). Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to have roles in stem cell biology and, taken together, these data provide a plausible scenario in which interactions between PLZF and Rb function in stem cell commitment or maintenance and self-renewal. The oncogenic PLZF-RARα fusion also interacts with Rb, suggesting that deregulation of Rb function may be a factor in the molecular pathogenesis of PLZF-RARα associated acute promyelocytic leukemia.

Hepatocellular carcinoma (HCC) is a one of the most prevalent cancers worldwide, especially in the Asia Pacific region. At present, the five-year survival of individuals with HCC is low, mainly due to the late presentation of the disease, and limited therapeutic options. The major risk factors for HCC in the Asia Pacific region include hepatitis B and C viral infection. Removing or reducing these risk factors, such as by immunizing against the hepatitis B virus, may reduce the incidence of hepatitis B-associated HCC in the distant future. However, immunization does not decrease the risk of HCC in individuals who are currently infected with HBV worldwide highlighting the continued importance of examining strategies for the treatment of HCC. Current treatment strategies include surgical resection, liver transplantation, chemotherapy, transcatheter arterial chemoembolism and percutaneous injection. Except for surgical resection and liver transplantation, which represent the most viable treatment options, most of the other present treatments are mainly for palliation. Hence novel treatment modalities continue to be investigated and several of these are currently in clinical trials. One of the more promising novel approaches for HCC treatment is gene therapy. Potential promising gene therapeutic approaches for the treatment of HCC include augmentation of tumor suppressor genes, inhibition of abnormally over-expressed oncogenes as well as specifically inducing death of cancer cells either via "suicide" gene therapy, conditional replicative adenovirus strategy, immunomodulation or inhibiting tumor angiogenesis. Nonetheless, successful implementation of these gene therapeutic approaches is dependent on overcoming current practical and technical hurdles underscoring the need for a better understanding of the basic aspects of gene therapy.

The estrogen receptor (ER) is a ligand-dependent transcription factor that is involved in diverse aspects of growth and development. Both ER and cyclin D1 are important regulators of