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. We have generated an isogenic set 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 impaired their differentiation capacity. For the generated genotypes, primary mouse embryonic fibroblasts (MEFs) also were obtained. While inactivation of Rb alone did not alleviate the senescence response of MEFs, pRB/p107-deficient MEFs, after having adapted to in vitro culturing, continued to proliferate at modest rate. Additional ablation of p130 rendered MEFs completely insensitive to senescence-inducing signals and strongly increased their proliferation rate. Although triple-knockout MEFs retained anchorage dependence, they lacked proper G 1 control and showed increased cell turnover under growth-inhibiting conditions.

The retinoblastoma gene family consists of three genes: RB, p107, and p130. While loss of pRB causes retinoblastoma in humans and pituitary gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107 and p130. To test this hypothesis, we have generated chimeric mice from embryonic stem cells carrying compound loss-of-function mutations in the Rb gene family. We found that Rb/p107-and Rb/p130-deficient mice were highly cancer prone. We conclude that in a variety of tissues tumor development by loss of pRB is suppressed by its homologs p107 and p130. The redundancy of the retinoblastoma proteins in vivo is reflected by the behavior of Rb-family-defective mouse embryonic fibroblasts in vitro.[Keywords: Retinoblastoma; cancer; mouse model; pocket proteins] Supplemental material is available at http://www.genesdev.org. Inactivation of the retinoblastoma suppressor pRB has been found in many human cancers, including hereditary retinoblastoma and sporadic breast, bladder, prostate, and small cell lung carcinoma (Friend et al. 1986(Friend et al. , 1987Harbour et al. 1988;Lee et al. 1988;T'Ang et al. 1988;Bookstein et al. 1990). pRB controls the G 1 /S transition of the cell cycle by modulating the activity of E2F transcription factors. Hypophosphorylated pRB binds to E2Fs and forms complexes harboring chromatin remodeling proteins like histone deacetylases, SWI/SNF and histone methyltransferases, which actively repress genes that are essential for cell cycle regulation, DNA replication, DNA repair, G 2 /M checkpoints and differentiation (Harbour and Dean 2000;Muller et al. 2001;Ishida et al. 2001;Kalma et al. 2001;Rayman et al. 2002;Ren et al. 2002). Upon sequential phosphorylation by Cyclin D/CDK4 and Cyclin E/CDK2 kinases, pRB undergoes a conformational change leading to the release of E2Fs. Ink4A function has been found in melanoma, pancreatic, and bladder carcinomas, amplification of Cyclin D1 in breast, oesophagus, and head-andneck cancer, and CDK4 amplification or mutational activation in melanoma (for review, see Sherr 1996).RB is a member of the retinoblastoma gene family that encodes the so-called pocket proteins pRB, p107, and p130 (Ewen et al. 1991;Hannon et al. 1993;Li et al. 1993;Chow and Dean 1996). All three can repress transcription from E2F-responsive promoters (Zamanian and La 1993;Bremner et al. 1995;Starostik et al. 1996) and are regulated by cell-cycle dependent phosphorylation. (Graña et al. 1998;Lundberg and Weinberg 1998;Canhoto et al. 2000;Hansen et al. 2001). Over-expression of each of the pocket proteins results in growth suppression, although not every (tumor) cell type is equally sensitive to each family member (Zhu et al. 1993;Claudio et al. 1994;Beijersbergen et al. 1995). Whereas pRB predominantly binds E2F1, E2F2, and E2F3, p107 and p130 specifically bind E2F4 and E2F5 (Dyson 1998;Trimarchi and Lees 2002) and each of these complexes appears at different stages of the cell cycle: p130/E2F4 is mainly found in G 0 , pRB/E2F in G 0 and G 1 , and p107/E2F in S-phase (Dyson ...

Newborns are highly susceptible to infectious diseases, which may be due to impaired immune responses. This study aims to characterize the ontogeny of neonatal TLR-based innate immunity during the first month of life.Cellularity and Toll-like receptor (TLR) agonist-induced cytokine production were compared between cord blood obtained from healthy neonates born after uncomplicated gestation and delivery (n=18), neonatal venous blood obtained at the age of one month (n=96), and adult venous blood (n=17). Cord blood TLR agonist-induced production of the Th1-polarizing cytokines IL-12p70 and IFN-α was generally impaired, but for TLR3, 7 and 9 agonists, rapidly increased to adult levels during the first month of life. In contrast, TLR4 demonstrated a slower maturation, with low LPS-induced IL-12p70 production and high IL-10 production up until the age of one month. Polarization in neonatal cytokine responses to LPS could contribute to neonatal susceptibility to severe bacterial infection.

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed.

Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix.We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.