Transient expression of cyclin D1 is sufficient to promote hepatocyte replication and liver growth in vivo.

Cyclin D1 regulates mitogen-dependent progression through G(1) phase in cultured cells, and its overexpression in malignant cells is thought to contribute to autonomous proliferation in vivo. However, previous studies in cell lines have not demonstrated that cyclin D1 is sufficient to trigger cell replication. In this study, we found that transient transfection of adult hepatocytes with cyclin D1 stimulated assembly of active cyclin D1/cdk4 complexes, robust hepatocyte proliferation, and liver growth in the intact animal. After several days, hepatocyte proliferation was inhibited despite the persistence of high levels of cyclin D1 and cyclin E, suggesting that endogenous antiproliferative mechanisms were induced. Our data suggest that this antiproliferative response includes the marked up-regulation of p21, which in turn inhibits cyclin D1/cdk4 and cyclin E/cdk2 complexes. This study offers further evidence that cyclin D1 plays a pivotal role in the regulation of hepatocyte proliferation in the liver. Furthermore, this model may offer a unique system to study the normal cellular response to cyclin D1 expression in vivo.

Induction of hepatocyte proliferation and liver hyperplasia by the targeted expression of cyclin E and skp2.

Cells in culture become competent to replicate in the absence of growth factor after progressing beyond the late G1 restriction point, suggesting that a set of genes expressed during G1 phase is sufficient to trigger completion of the cell cycle. However, this has not been demonstrated in an in vivo system. In this study, we examined whether transfection of genes associated with the G1/S transition could trigger hepatocyte replication. Co-transfection of cyclin E and skp2 synergistically promoted cell cycle progression in cultured primary hepatocytes in the absence of mitogen or in the presence of growth inhibitors. Furthermore, transfection of hepatocytes in vivo with cyclin E and skp2 promoted abundant hepatocyte replication and hyperplasia of the liver. These studies confirm that transfection with a small number of genes can trigger proliferation of quiescent hepatocytes in vivo, and suggest that therapies to enhance liver regeneration by targeting cell cycle control genes may be feasible.

Regulation of G(1) cyclin-dependent kinases in the liver: role of nuclear localization and p27 sequestration.

Recent studies suggest that cyclin D1 mediates progression of hepatocytes through G(1) phase of the cell cycle. The present study further examines the regulation of cyclin D1-dependent kinase activity and the interplay between cyclin D1 and other G(1) phase regulatory proteins during liver regeneration. After 70% partial hepatectomy in rats, there was upregulation of kinase activity associated with cyclins (A, D1, D3, and E), cyclin-dependent kinases (Cdk2 and Cdk4), and Cdk-inhibitory proteins (p27, p107, and p130). Although cyclin D1/Cdk4 complexes were more abundant in the cytoplasmic fraction after partial hepatectomy, kinase activity was detected primarily in the nuclear fraction. Cytoplasmic cyclin D1/Cdk4 complexes were activated by recombinant cyclin H/Cdk7. Because endogenous Cdk7 activity was found in the nucleus, this suggests that activation of cyclin D1/Cdk4 requires nuclear importation and subsequent phosphorylation by cyclin H/Cdk7. Recombinant cyclin E/Cdk2 was inhibited by extracts from quiescent liver, and cyclin D1 could titrate out this inhibitory activity. Induction of cyclin D1 was accompanied by increased abundance of cyclin D1/p27 complexes, and most p27 was sequestered by cyclin D1 after partial hepatectomy. Thus cyclin D1 appears to play two roles during G(1) phase progression in the regenerating liver: it forms a nuclear kinase complex, and it promotes activation of Cdk2 by sequestering inhibitory proteins such as p27. These experiments underscore the complexity of cyclin/Cdk regulatory networks in the regenerating liver.

Recombination between North American strains of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), a recently discovered arterivirus swine pathogen, was shown to undergo homologous recombination. Co-infection of MA-104 cells with two culture-adapted North American PRRSV strains resulted in recombinant viral particles containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Recombinant products persisted in culture for at least three passages, indicating continuous formation of recombinant viruses, growth of recombinant viruses in competition with parental viruses, or both. The frequency of recombination was estimated from <2% up to 10% in the 1182 b fragment analyzed, which is similar to recombination frequencies observed in coronaviruses. An apparent example of natural ORF 5 recombination between naturally occurring wild type viruses was also found, indicating that recombination is likely an important genetic mechanism contributing to PRRSV evolution.

Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.

Subgenomic RNA7 is transcribed with different leader-body junction sites in PRRSV (strain VR2332) infection of CL2621 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV), like all members of the order Nidoviridae, is expressed in the infected cell as a nested set of subgenomic (sg) RNAs with a common 5'-leader sequence. We have determined that the 5'-leader sequence for the US prototype strain (VR2332, Collins, et al., 1992) is distinct from the European prototype strain [Lelystad (LV); Wensvoort, et al., 1991, Meulenberg et al., 1993a], yet these two strains use almost the same sequence for downstream sites of 5'-leader-body junction formation. Analysis of VR2332 genomic sequence identified several potential 5'-leader-body junction sequences upstream of open reading frame (ORF) 7, coding for the nucleocapsid protein, that could be used for generation of VR2332 sgRNA7 transcripts. Sequence determinations of RT-PCR-generated cDNA clones of sgRNA7 identified two species of RNA7 transcripts in infected cells, one utilizing a leader-body junction sequence (AUAACC) 123 nucleotides upstream of the AUG start site and one utilizing a sequence (UAAACC) 9 nucleotides upstream of the AUG start site for ORF7 translation.