Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1).
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Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1). (+info)
Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse.
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Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in spermatogonial proliferation, in particular during the G(1)/S phase transition. Cyclin D(2) is first expressed at the start of spermatogenesis when gonocytes produce A(1) spermatogonia. In the adult testis, cyclin D(2) is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A(al) spermatogonia differentiate into A(1) spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D(2) during spermatogenesis, cyclin D(2) expression was studied in vitamin A-deficient testis. Cyclin D(2) was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A(1) spermatogonia by administration of retinoic acid. Overall, cyclin D(2) seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A(1) spermatogonia. Cyclin D(3) is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D(3) expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D(3) may control G(1)/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D(1) and D(3) seem to be involved in cell cycle regulation, whereas cyclin D(2) likely has a role in spermatogonial differentiation. (+info)
The Arabidopsis D-type cyclins CycD2 and CycD3 both interact in vivo with the PSTAIRE cyclin-dependent kinase Cdc2a but are differentially controlled.
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D-type cyclins (CycD) play key roles in linking the Arabidopsis cell cycle to extracellular and developmental signals, but little is known of their regulation at the post-transcriptional level or of their cyclin-dependent kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we demonstrate that the CDK partner of these Arabidopsis cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has shown that transcript levels of CycD2 and CycD3 are regulated in response to sucrose levels and that both their mRNA levels and kinase activity are induced with different kinetics during the G(1) phase of cells reentering the division cycle from quiescence. Here we analyze the protein levels and kinase activity of CycD2 and CycD3. We show that CycD3 protein and kinase activity parallel the abundance of its mRNA and that CycD3 protein is rapidly lost from cells in stationary phase or following sucrose removal. In contrast to both CycD3 and the regulation of its own mRNA levels, CycD2 protein is present at constant levels. CycD2 kinase activity is regulated by sequestration of CycD2 protein in a form inaccessible to immunoprecipitation and probably not complexed to Cdc2a. (+info)
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0).
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An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G(1) fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G(0)/G(1) and S/G(2)/M. Interestingly, the G(0) cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle-associated change in in vivo stem cell homing, the cultured G(0)/G(1) and S/G(2)/M CD34(+) CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor beta(1) that increased the G(0)/G(1) fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle-associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo-manipulated grafts. (Blood. 2000;96:4185-4193) (+info)
Isolation of three distinct CycD3 genes expressed during fruit development in tomato.
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Tomato (Lycopersicon esculentum Mill.) is an important fruit crop world-wide and a model for studying fruit development. As determined using flow cytometry, fruit growth was characterized by high cell division activity in tomato during the first week after anthesis and followed by endoreduplications (DNA replication without cell divisions). D-type cyclins are considered to be important parts of the signal transduction for stimulation of DNA replication and cell division. To study the function of D cyclins in fruit development, full-length cDNA clones for three D cyclin genes were isolated from young tomato fruit. They were classified as D3 cyclins by sequence similarities and a phylogenetic analysis and named as LeCycD3;1, LeCycD3;2 and LeCycD3;3. The deduced amino acid sequences for LeCycD3;1-3 contained a retinoblastoma-binding motif and a PEST-destruction motif. Pollination and fertilization were followed by a high increase in the transcript levels of LeCycD3;1-3 in young fruit. Using in situ hybridization, high expression of LeCycD3;3 was detected in the vascular tissue of young fruit suggesting a role in vascular development. The D3 cyclins are probably involved in transducing the signals leading to fruit growth by cell divisions. Distinct differences were detected in their temporal and spatial expression patterns suggesting that they play different roles in fruit development as well as in the development of other plant organs. (+info)
An enhancer trap line associated with a D-class cyclin gene in Arabidopsis.
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In yeast and animals, cyclins have been demonstrated to be important regulators of cell cycle progression. In recent years, a large number of A-, B-, and D-class cyclins have been isolated from a variety of plant species. One class of cyclins, the D-class cyclins, is important for progression through G1 phase of the cell cycle. In Arabidopsis, four D-class cyclins have been isolated and characterized (CYCLIN-D1;1, CYCLIN-D2;1, CYCLIN-D3;1, and CYCLIN-D4;1). In this report we describe the characterization of a fifth D-class cyclin gene, CYCLIN-D3;2 (CYCD3;2), from Arabidopsis. An enhancer trap line, line 5580, contains a T-DNA insertion in CYCD3;2. Enhancer trap line 5580 exhibits expression in young vegetative and floral primordia. In line 5580, T-DNA is inserted in the first exon of the CYCD3;2 gene; in homozygous 5580 plants CYCD3;2 RNA is not detectable. Even though CYCD3;2 gene function is eliminated, homozygous 5580 plants do not exhibit an obvious growth or developmental phenotype. Via in situ hybridization we demonstrate that CYCD3;2 RNA is expressed in developing vegetative and floral primordia. In addition, CYCD3;2 is also capable of rescuing a yeast strain that is deficient in G1 cyclin activity. (+info)
Role of cyclin D3 in the biology of herpes simplex virus 1 ICPO.
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Earlier reports from this laboratory have shown that the promiscuous transactivator infected-cell protein 0 (ICP0) binds and stabilizes cyclin D3, that the binding site maps to aspartic acid 199 (D199), and that replacement of D199 with alanine abolishes binding and reduces the capacity of the mutant virus to replicate in quiescent cells or to cause mortality in mice infected by a peripheral site. The objective of this report was to investigate the role of cyclin D3 in the biology of ICP0. We report the following results. (i) Wild-type ICP0 activates cyclin D-dependent kinase 4 (cdk4) and stabilizes cyclin D1 although ICP0 does not interact with this cyclin. (ii) The D199A mutant virus (R7914) does not activate cdk4 or stabilize cyclin D1, and neither the wild-type nor the mutant virus activates cdk2. (iii) Early in infection of human embryonic lung (HEL) fibroblasts both wild-type and D199A mutant ICP0s colocalize with PML, and in these cells the ND10 nuclear structures are dispersed. Whereas wild-type ICP0 is transported to the cytoplasm between 3 and 9 h. after infection, ICPO containing the D199A substitution remains quantitatively in the nucleus. (iv) To examine the interaction of ICP0 with cyclin D3, we used a previously described mutant carrying a wild-type ICP0 but expressing cyclin D3 (R7801) and in addition constructed a virus (R7916) that was identical except that it carried the D199A-substituted ICP0. Early in infection with R7801, ICP0 colocalized with cyclin D3 in structures similar to those containing PML. At 3 h after infection, ICP0 was translocated to the cytoplasm whereas cyclin D3 remained in the nucleus. The translocation of ICP0 to the cytoplasm was accelerated in cells expressing cyclin D3 compared with that of ICP0 expressed by wild-type virus. In contrast, ICP0 carrying the D199A substitution remained in the nucleus and did not colocalize with cyclin D3. These studies suggest the following conclusions. (i) ICP0 brings to the vicinity of ND10 cyclin D3 and, in consequence, an activated cdk4. The metabolic events occurring at or near that structure and involving cyclin D3 cause the translocation of ICP0 to the cytoplasm. (ii) In the absence of the cyclin D3 binding site in ICP0, cyclin D3 is not brought to ND10, cyclin D is not stabilized, and the function responsible for the translocation of ICP0 is not expressed, and in quiescent HEL fibroblasts the yields of virus are reduced. (+info)
De novo cortical cell division triggered by the phytopathogen Rhodococcus fascians in tobacco.
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Plant growth, development, and morphology can be affected by several environmental stimuli and by specific interactions with phytopathogens. In many cases, plants respond to pathogenic stimuli by adapting their hormone levels. Here, the interaction between the phytopathogen Rhodococcus fascians and one of its host plants, tobacco, was analyzed phenotypically and molecularly. To elucidate the basis of the cell division modulation and shoot primordia initiation caused by R. fascians, tobacco plants were infected at leaf axils and shoot apices. Adventitious meristems that gave rise to multiple-shoot primordia (leafy galls) were formed. The use of a transgenic line carrying the mitotic CycB1 promoter fused to the reporter gene coding for beta-glucuronidase from Escherichia coli (uidA), revealed that stem cortical cells were stimulated to divide in an initial phase of the leafy gall ontogenesis. Local cytokinin and auxin levels throughout the infection process as well as modulation of expression of the cell cycle regulator gene Nicta;CycD3;2 are discussed. (+info)