Shigeki Jinno

Functionalized Fullerene as an Artificial Vector for Transfection

Materials. Dulbecco modified Eagle medium (DMEM) was purchased from Sigma, fetal bovine serum (FBS) from Equitech-Bio. The green fluorescent protein (GFP) vector plasmid (pGreen LANTERN-1) was purchased from Life Technologies and was propagated by the lysozyme-Triton procedure. Vector DNA was purified by the CsCl equilibrium density gradient centrifugation and phenol extractions in the presence of 1% sodium dodecyl sulfate (SDS). Final concentration of plasmid was determined by absorbance at 260 nm. Organic solvents were distilled and, when necessary, dried over molecular sieves 4Å before use.

Bone morphogenetic protein 2-induced osteoblast differentiation requires Smad-mediated down-regulation of Cdk6.

ABSTRACT Because a temporal arrest in the G1 phase of the cell cycle is thought to be a prerequisite for cell differentiation, we investigated cell cycle factors that critically influence the differentiation of mouse osteoblastic MC3T3-E1 cells induced by bone morphogenetic protein 2 (BMP-2), a potent inducer of osteoblast differentiation. Of the G1 cell cycle factors examined, the expression of cyclin-dependent kinase 6 (Cdk6) was found to be strongly down-regulated by BMP-2/Smads signaling, mainly via transcriptional repression. The enforced expression of Cdk6 blocked BMP-2-induced osteoblast differentiation to various degrees, depending on the level of its overexpression. However, neither BMP-2 treatment nor Cdk6 overexpression significantly affected cell proliferation, suggesting that the inhibitory effect of Cdk6 on cell differentiation was exerted by a mechanism that is largely independent of its cell cycle regulation. These results indicate that Cdk6 is a critical regulator of BMP-2-induced osteoblast differentiation and that its Smads-mediated down-regulation is essential for efficient osteoblast differentiation.

Isolation of a Mammalian Homologue of a Fission Yeast Differentiation Regulator

ABSTRACT In the fission yeast Schizosaccharomyces pombe thenrd1 + gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombetemperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have clonedROD1, a functional homologue ofnrd1 +, from rat and human cDNA libraries. Likenrd1 +, ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast,ROD1 behaves in a way that is functionally similar tonrd1 +, being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1 + for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

Cell cycle start from quiescence controlled by tyrosine phosphorylation of Cdk4

In mammals Cdk4 (or Cdk6 in some cell types) is required for starting the cell cycle. Recently we showed that Cdk4 is regulated by tyrosine phosphorylation and dephosphorylation, and that this regulation is required for a DNA damage-induced G1 arrest. We report here that a generic anti-phosphotyrosine antibody can detect tyrosine-phosphorylated Cdk4 and that as revealed by immunoblot detection and kinase assay, this regulation is employed for DNA damage-responsive checkpoint control during cell cycle start from quiescence. In rat fibroblasts traversing G1 or arrested in G1 by deprivation of anchorage, Cdk4 does not undergo tyrosine phosphorylation. Tyrosine phosphorylation occurs only during cells arrest in quiescence and dephosphorylation during their cell cycle start. Ultraviolet irradiation blocks dephosphorylation and concomitant activation of Cdk4, thereby preventing the start of cell cycling. Thus, unlike tyrosine phosphorylation of Cdc2, which controls phase transition in the regular cell cycle, tyrosine phosphorylation of Cdk4 is employed for controlling cell cycle start from quiescence in a rat fibroblast.

Cdk6-cyclin D3 complex evades inhibition by inhibitor proteins and uniquely controls cell's proliferation competence

Mammalian cells require a cyclin D-dependent kinase for the cell cycle start, yet many mesenchymal cells express three seemingly redundant D cyclins and similarly, seemingly redundant Cdk4 and Cdk6 as their kinase partners. We have found that the Cdk6-cyclin D3 complex is unique among the D cyclin and kinase combinations in the ability to promote the cell cycle start. In an anchorage-minus G1-arrested rat fibroblast, only Cdk6-D3 retains kinase activity due mainly to its ability to evade inhibition by p27KIP1 and p21CIP1 with a resemblance to viral cyclin-bound Cdk6. Rodent fibroblasts engineered to overexpress both Cdk6 and cyclin D3 highly resist serum starvation- or cell–cell contact-imposed G1-arrest. In BALB/c 3T3 cells, D3 is constitutively expressed, but Cdk6 is markedly induced with concomitant activation upon stimulation with a growth-promoting factor. These results suggest a role for the Cdk6-D3 complex in regulating cells proliferation ability in response to external stimuli.

Mammalian Rcd1 is a novel transcriptional cofactor that mediates retinoic acid-induced cell differentiation

Rcd1, initially identified as a factor essential for the commitment to nitrogen starvation‐invoked differentiation in fission yeast, is one of the most conserved proteins found across eukaryotes, and its mammalian homolog is expressed in a variety of differentiating tissues. Here we show that mammalian Rcd1 is a novel transcriptional cofactor and is critically involved in the commitment step in the retinoic acid‐induced differentiation of F9 mouse teratocarcinoma cells, at least in part, via forming complexes with retinoic acid receptor and activation transcription factor‐2 (ATF‐2). In addition, antisense oligonucleotide treatment of embryonic mouse lung explants suggests that Rcd1 also plays a role in retinoic acid‐controlled lung development.

Cell cycle control in fission yeast and mammals: identification of new regulatory mechanisms.

Publisher Summary This chapter discusses the recent rapid progress in understanding of the molecular mechanisms controlling the G1 and G2 phases of the cell cycle in fission yeast and mammals, focusing on the newly identified control genes and highly conserved control mechanisms between these two apparently remote organisms. The cells of the fission yeast, Schizosaccharomyces pombe, are rod shaped, grow in the longitudinal direction, and divide by septation and medial fission. The chapter describes the cell cycle starts control of fission yeast and mammals. The mitotic start control of fission yeast and mammals have also been described in the chapter. Cell cycle control is one of the most complex and fundamental cellular regulatory processes that eukaryotes possess. Cell cycle control is one of the most complex and fundamental cellular regulatory processes that eukaryotes possess. Once cells have committed to start the cell cycle, they are unable to differentiate until they return to G1. The next few years will be the period during which rapid progress continues to be made and will witness the discovery of new factors and new mechanisms and the resolution of some of these questions.

Overexpression of Cdk6-cyclin D3 highly sensitizes cells to physical and chemical transformation.

Virtually all mammalian cells express two seemingly redundant cyclin–D-dependent kinases (Cdk4 and Cdk6) and three partner cyclins (D1, D2 and D3) essential for the G1–S transition, with predominant expression of Cdk4 and D1 in mesenchymal cells and Cdk6 and D3 in hematopoietic cells. We recently found two novel functions for Cdk6 executed in fibroblasts although unlike Cdk4 it is dispensable for their proliferation. In the rat fibroblast NRK-49F cells, oncogenic stimulation recruits Cdk6 to participate in a step of the cell cycle start that seems to be critical for anchorage-independent S-phase onset. Among the kinase-D-type cyclin combinations, the Cdk6–cyclin-D3 complex has a unique ability to evade inhibition by cyclin-dependent kinase inhibitors and thereby control the cells proliferative competence under growth-suppressive conditions. We describe here that 2–5-fold overexpression of both Cdk6 and D3 enhances by 5×103–106-fold the susceptibility of the BALB/c3T3 and C3H10T1/2 mouse fibroblast lines to ultraviolet irradiation- as well as 3-methylcholanthrene-induced transformation. This result suggests that deregulated expression of Cdk6 and cyclinD3 may predispose cells to malignant transformation, supporting the recent finding that cyclin D3 activated by chromosomal rearrangement is the causative gene of non-Hodgkin B lymphoma, in which Cdk6 is the major partner kinase.

Cdc6 requires anchorage for its expression

Fibroblasts need anchorage to extracellular matrix to transit from G1 to S phase, but no longer after oncogenic transformation. Here we report that Cdc6 protein essential for the activation of replication origins requires anchorage or oncogenic stimulation for its execution. Upon anchorage loss, Cdc6 expression is shut off both transcriptionally and post-transcriptionally in a rat fibroblast despite enforced activation of E2F-dependent promoters. However, stimulation of this cell with oncogenic growth factors suppresses this shutoff and concurrently activates Cdk2 and Cdk6/4, thereby overriding the anchorage requirement for the G1-S transition and consequently enabling cells to perform anchorage-independent S phase entry. Analysis with enforced expression of Cdc6 indicates that the G1 cyclin-dependent kinases and Cdc6 constitute major cell cycle targets for the restriction of the G1-S transition by anchorage loss.

Cdc6 protein obstructs apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules

Abstract Cdc6 is the bifunctional AAA+ ATPase that assembles pre-replicative complexes (preRC) on origins of replication and activates p21CIP1- or p27KIP1-bound Cdk2. During the G1-S transition, the Cdc6 gene essential for chromosomal replication is activated by the E2F transcriptional factor. Paradoxically, Apaf-1 encoding the central component of the apoptosome is activated also at the same time and by E2F. Consequently, genes for antipodal life and death are regulated in the same manner by the same transcriptional factor. Here we report a striking solution to this paradox. Besides performing preRC assembly and Cdk2 activation, Cdc6 obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules. This function depended on its own ATPase but not cyclin-binding motif. In proliferating rodent fibroblasts, Cdc6 continued to block apoptosome assembly induced by non-cytochrome c or some other mechanism, suppressing seemingly unintended apoptosis when promoting cell proliferation. Thus Cdc6 is a trifunctional AAA+ ATPase all working for life.