ABSTRACT
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.
Subject(s)
CDC2-CDC28 Kinases , Cell Cycle Proteins , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Liver Regeneration/physiology , Liver/enzymology , Microtubule-Associated Proteins/metabolism , Proto-Oncogene Proteins , Tumor Suppressor Proteins , Animals , Cell Nucleus/enzymology , Cyclin D1/metabolism , Cyclin E/metabolism , Cyclin G , Cyclin G1 , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase Inhibitor p27 , Cytoplasm/enzymology , Enzyme Inhibitors/metabolism , Male , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Sprague-Dawley , S Phase/physiologyABSTRACT
In tissue culture systems, p21 and p27 inhibit cyclin-dependent kinase (CDK) activity and cell cycle progression in response to numerous stimuli, but little is known about their involvement in cell growth in vivo. We examined the modulation of CDK activity by these proteins after 70% partial hepatectomy (PH), an in vivo model of synchronous hepatocyte cell cycle progression. After PH in BALB/c mice, p21 was induced during the prereplicative (G1) phase and was maximally expressed after peak hepatocyte DNA synthesis. p27 was present in quiescent liver and was minimally induced after PH. p21 and p27 immunoprecipitated with CDK2, CDK4, and cyclin D1 in the regenerating liver. The activity of CDK2-, CDK4- and cyclin D1-associated kinases was upregulated after PH, and maximal activity of these enzyme complexes corresponded to peak DNA synthesis. Immunodepletion experiments suggested that p27 plays a role in downregulating CDK2 activity before and after peak DNA synthesis. Compared to cogenic wild-type mice, p21-/- mice demonstrated evidence of markedly accelerated hepatocyte progression through G1 phase after PH: DNA synthesis, upregulation of cyclin A and PCNA, induction of cyclin D1- and CDK2-associated kinase activity, and appearance of a phosphorylated retinoblastoma protein (Rb) species occurred earlier in the p21-/- mice. These results suggest that p21 and p27 modulate CDK activity in the regenerating liver, and that p21 regulates the rate of progression through G1 phase of the cell cycle in vivo.
Subject(s)
CDC2-CDC28 Kinases , Cell Cycle Proteins , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclins/metabolism , Enzyme Inhibitors/metabolism , Liver Regeneration/physiology , Liver/metabolism , Microtubule-Associated Proteins/metabolism , Proto-Oncogene Proteins , Tumor Suppressor Proteins , Animals , Cell Cycle , Cyclin A/metabolism , Cyclin D1/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/metabolism , Cyclins/genetics , Female , Gene Deletion , Liver/cytology , Male , Mice , Mice, Inbred BALB C , Phosphorylation , Proliferating Cell Nuclear Antigen/metabolism , Protein Serine-Threonine Kinases/metabolism , Retinoblastoma Protein/metabolism , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.