Temporal distribution of CDK4, CDK6, D-type cyclins,and p27 in developing mouse oocytes

Various molecular interactions not operating in other cell types are most likely required for mammalian oocytes to develop into fully competent eggs. This study seeks to initiate analyses of the potential oocyte-specific functions of regulators of G1/S progression-CDK4, CDK6, D-type cyclins, and p27-by first determining their expression patterns in growing and maturing mouse oocytes and in mouse embryos early after fertilization. Western blot and immunofluorescence analyses on isolated oocytes were employed to evaluate both their levels and their localization. The data show that 1). mouse oocytes contain significant amounts of all studied regulators; 2). their amounts and localization undergo dramatic changes as the oocytes grow, meiotically mature, and transit into embryogenesis; and 3). some regulators (CDK4, CDK6, cyclin D2, and p27) appear in unusual, most likely posttranslationally modified, forms. These data distinguish G1/S regulators as the potential players in molecular processes that are important for oocytes to function normally.     

Bone growth retardation in mouse embryos expressing human collagenase 1

Cellular growth and differentiation are readouts of multiple signaling pathways from the intercellular and/or extracellular milieu. The extracellular matrix through the activation of cellular receptors transmits these signals. Therefore, extracellular matrix proteolysis could affect cell fate in a variety of biological events. However, the biological consequence of inadequate extracellular matrix degradation in vivo is not clear. We developed a mouse model expressing human collagenase (matrix metalloproteinase-1, MMP-1) under the control of Col2a1 promoter. The mice showed significant growth retardation during embryogenesis and a loss of the demarcation of zonal structure and columnar array of the cartilage. Immunological examination revealed increased degradation of type II collagen and upregulation of fibronectin and alpha(5)-integrin subunit in the transgenic cartilage. The resting zone and proliferating zone of the growth plate cartilage exhibited a simultaneous increase in bromodeoxyuridine (BrdU)-incorporated proliferating cells and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling-positive apoptotic cells, respectively. Chondrocyte differentiation was not disturbed in the transgenic mice as evidenced by normal expression of the Ihh and type X collagen expression. These data demonstrate that type II collagen proteolysis is an important determinant for the skeletal outgrowth through modulation of chondrocyte survival and cartilagenous growth.     

Measurement of metabolic turnover in single mouse embryos

The method for estimation of metabolic turnover by a single embryo at each stage of preimplantation development is based on the incubation of an embryo in the presence of labelled substrate at high specific activity in a miniaturized incubation chamber and the subsequent radioassay of metabolic products. Preliminary tests indicated that the treatment did not affect embryos adversely. Estimates of glycolysis, substrate oxidation and incorporation by mouse embryos throughout the whole of the preimplantation period of development were made. This technique could prove particularly useful for investigating substrate requirements and metabolic turnover in species for which few embryos are available for study.     

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.     

利用CRISPR/Cas9技术产生肌肉特异表达Cas9的小鼠胚胎

目的通过CRISPR/Cas9技术获得肌肉特异性表达Cas9蛋白小鼠胚胎,为建立肌肉特异表达Cas9小鼠动物模型奠定基础。方法设计小鼠Rosa26位点sgRNA并通过体外酶切验证活性,同时使用同源重组构建肌肉特异性同源打靶载体;通过显微注射将Rosa26sgRNA与Cas9蛋白注射到小鼠胚胎,通过PCR及测序检测胚胎的编辑情况,同时移植到假孕母鼠体内,待其生产;将同源打靶载体与Rosa26sgRNA和Cas9一起注射到小鼠胚胎,通过PCR检测整合情况。结果体外酶切实验表明,体外转录的sgRNA与Cas9蛋白联合可对靶位点产生编辑作用;成功构建了肌肉特异性同源打靶载体Donor-SP-px459;通过原核注射获得Rosa26基因编辑胚胎,经移植获得Rosa26基因编辑小鼠;注射同源打靶载体后,成功获得肌肉特异表达Cas9蛋白的基因打靶小鼠胚胎。结论利用CRISPR/Cas9技术,成功获得Rosa26基因编辑胚胎和小鼠,并获得了肌肉特异性表达Cas9蛋白小鼠胚胎,为利用基因打靶构建肌肉特异表达Cas9的小鼠动物模型奠定基础。     

Fascaplysin-inspired diindolyls as selective inhibitors of CDK4/cyclin D1

We present the design, synthesis and biological activity of a new series of substituted 3-(2-(1H-indol-1-yl)ethyl)-1H-indoles and 1,2-di(1H-indol-1-yl)alkanes as selective inhibitors of CDK4/cyclin D1. The compounds were designed to explore the relationship between the connection mode of the indolyl moieties and their CDK inhibitory activities. We found all the above-mentioned designed compounds to be selective inhibitors of CDK4/cyclin D1 compared to the closely related CDK2/cyclin A, with IC₅₀ for the best compounds 10m and 13a being 39 and 37 μm, respectively.     

The Arabidopsis D-type cyclin CYCD4 controls cell division in the stomatal lineage of the hypocotyl epidermis

Cyclin D (CYCD) plays an important role in cell cycle progression and reentry in response to external signals. Here, we demonstrate that Arabidopsis thaliana CYCD4 is associated with specific cell divisions in the hypocotyl. We observed that cycd4 T-DNA insertion mutants had a reduced number of nonprotruding cells and stomata in the hypocotyl epidermis. Conversely, CYCD4 overexpression enhanced cell division in nonprotruding cell files in the upper region of the hypocotyls, where stomata are usually formed in wild-type plants. The overproliferative cells were of stomatal lineage, which is marked by the expression of the TOO MANY MOUTHS gene, but unlike the meristemoids, most of them were not triangular. Although the phytohormone gibberellin promoted stomatal differentiation in the hypocotyl, inhibition of gibberellin biosynthesis did not prevent CYCD4 from inducing cell division. These results suggested that CYCD4 has a specialized function in the proliferation of stomatal lineage progenitors rather than in stomatal differentiation. We propose that CYCD4 controls cell division in the initial step of stomata formation in the hypocotyl.     

Impaired intermediate formation in mouse embryos expressing reduced levels of Tbx6

Intermediate mesoderm (IM) is the strip of tissue lying between the paraxial mesoderm (PAM) and the lateral plate mesoderm that gives rise to the kidneys and gonads. Chick fate mapping studies suggest that IM is specified shortly after cells leave the primitive streak and that these cells do not require external signals to express IM‐specific genes. Surgical manipulations of the chick embryo, however, revealed that PAM‐specific signals are required for IM differentiation into pronephros—the first kidney. Here, we use a genetic approach in mice to examine the dependency of IM on proper PAM formation. In Tbx6 null mutant embryos, which form 7–9 improperly patterned anterior somites, IM formation is severely compromised, while in Tbx6 hypomorphic embryos, where somites form but are improperly patterned along the axis, the impact to IM formation is lessened. These results suggest that IM and its derivatives, the kidneys and the gonads, are directly or indirectly dependent on proper PAM formation. This has implications for humans harboring Tbx6 mutations which are known to have somite‐derived defects including congenital scoliosis.     

Structural basis for CDK6 activation by a virus-encoded cyclin

Cyclin from herpesvirus saimiri (Vcyclin) preferentially forms complexes with cyclin-dependent kinase 6 (CDK6) from primate host cells. These complexes show higher kinase activity than host cell CDKs in complex with cellular cyclins and are resistant to cyclin-dependent inhibitory proteins (CDKIs). The crystal structure of human CDK6--Vcyclin in an active state was determined to 3.1 A resolution to better understand the structural basis of CDK6 activation by viral cyclins. The unphosphorylated CDK6 in complex with Vcyclin has many features characteristic of cyclinA-activated, phosphorylated CDK2. There are, however, differences in the conformation at the tip of the T-loop and its interactions with Vcyclin. Residues in the N-terminal extension of Vcyclin wrap around the tip of the CDK6 T-loop and form a short beta-sheet with the T-loop backbone. These interactions lead to a 20% larger buried surface in the CDK6--Vcyclin interface than in the CDK2--cyclinA complex and are probably largely responsible for the specificity of Vcyclin for CDK6 and resistance of the complex to inhibition by INK-type CDKIs.     

Targeting cyclin D3/CDK6 activity for treatment of Parkinson's disease

At present, treatment for Parkinson's disease (PD) is only symptomatic; therefore, it is important to identify new targets tackling the molecular causes of the disease. We previously found that lymphoblasts from sporadic PD patients display increased activity of the cyclin D3/CDK6/pRb pathway and higher proliferation than control cells. These features were considered systemic manifestations of the disease, as aberrant activation of the cell cycle is involved in neuronal apoptosis. The main goal of this work was to elucidate whether the inhibition of cyclin D3/CDK6‐associated kinase activity could be useful in PD treatment. For this purpose, we investigated the effects of two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic (SAHA) acid and sodium butyrate (NaB), and the m‐TOR inhibitor rapamycin on cell viability and cyclin D3/CDK6 activity. Moreover, the potential neuroprotective action of these drugs was evaluated in 6‐hydroxy‐dopamine (6‐OHDA) treated dopaminergic SH‐SY5Y cells and primary rat mesencephalic cultures. Here, we report that both compounds normalized the proliferative activity of PD lymphoblasts and reduced the 6‐OHDA‐induced cell death in neuronal cells by preventing the over‐activation of the cyclin D3/CDK6/pRb cascade. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it is plausible that they may serve as novel therapeutic drugs for PD.

     

Increased D-type cyclin expression together with decreased cdc2 activity confers megakaryocytic differentiation of a human thrombopoietin-dependent hematopoietic cell line

At the late phase of megakaryocytopoiesis, megakaryocytes undergo endomitosis, which is characterized by DNA replication without cell division. Although a number of cell cycle regulatory molecules have been identified, the precise roles of these molecules in megakaryocytic endomitosis are largely unknown. In a human interleukin-3-dependent cell line transfected with the thrombopoietin (TPO) receptor c-mpl (F-36P-mpl), either treatment with TPO or the overexpression of activated ras (Ha-Ras(G12V)) induced megakaryocytic maturation with polyploid formation. We found that TPO stimulation or Ha-Ras(G12V) expression led to up-regulation of cyclin D1, cyclin D2, and cyclin D3 expression. In addition, expression levels of cyclin A and cyclin B were reduced during the total course of both TPO- and Ha-Ras(G12V)-induced megakaryocytic differentiation, thereby leading to decreased cdc2 kinase activity. Neither the induced expression of cyclin D1, cyclin D2, or cyclin D3 nor the expression of a dominant negative form of cdc2 alone could induce megakaryocytic differentiation of F-36P-mpl cells. In contrast, overexpression of dominant negative cdc2 together with cyclin D1, cyclin D2, or cyclin D3 facilitated megakaryocytic differentiation in the absence of TPO. These results suggest that both D-type cyclin expression and decreased cdc2 kinase activity may participate in megakaryocytic differentiation.     

DNA polymerase activity in preimplantation mouse embryos.

DNA polymerase activity was measured in mouse embryos at stages before implantation to determine whether it increases in proportion to the amount of DNA synthesis, as it does in populations of differentiated mammalian cells, or remains constant, as it does in early sea urchin embryos. Total enzyme activity was found to be relatively unchanged following fertilization and in the first few cleavage stages. However, between the 12- and 120-cell (blastocyst) stage, the amount of activity increased by several-fold. These results indicate that the relationship between amount of DNA polymerase activity and DNA synthesis in mouse embryos exhibits two phases: in the early cleavage phase it is similar to that in sea urchin embryos, whereas, in the blastocyst phase, it is similar to that in differentiated mammalian cells.     

CDK5 is present in sea urchin and starfish eggs and embryos and can interact with p35, cyclin E and cyclin B3

While most cyclin‐dependent kinases (CDKs) are involved in cell cycle control, CDK5 is mostly known for crucial functions in neurogenesis. However, we cloned sea urchin CDK5 from a two‐cell stage cDNA library and found that the protein is present in eggs and embryos, up to the pluteus stage, but without associated kinase activity. To investigate the potential for nonneuronal roles, we screened a starfish cDNA library with the yeast two‐hybrid system, for possible CDK5 partners. Interactions with clones expressing part of cyclin B3 and cyclin E proteins were found and the full‐length cyclins were cloned. These interactions were verified in vitro but not in extracts of starfish oocytes and embryos, at any stages, despite the presence of detectable amounts of CDK5, cyclin B3, and cyclin E. We then looked for p35, the CDK5‐specific activator, and cloned the sea urchin ortholog. A sea urchin‐specific anomaly in the amino acid sequence is the absence of N‐terminal myristoylation signal, but nucleotide environment analysis suggests a much higher probability of translation initiation on the second methionine(Met44), that is associated with a conserved myristoylation signal. p35 was found to associate with CDK5 and, when bacterially produced, to confer protein kinase activity to CDK5 immunoprecipitated from sea urchin eggs and embryos. However, p35 mRNA expression was found to begin only at the end of the blastula stage, and the protein was undetectable at any embryonic stage, suggesting a neuronal role beginning in late larval stages. Mol. Reprod. Dev. 77: 449–461, 2010. © 2010 Wiley‐Liss, Inc.     

2-Substituted paullones: CDK1/cyclin B-inhibiting property and in vitro antiproliferative activity

9-Trifluoromethyl-paullones with a carbon chain in the 2-position were synthesized by palladium-catalyzed coupling reactions of a 2-iodoprecursor with terminal alkenes or alkynes, respectively. The introduction of a 2-cyanoethyl substituent led to a significant enhancement of CDK1/cyclin B inhibiting property and in vitro antiproliferative activity.     

A fine analysis of glucose-phosphate-isomerase patterns in single preimplantation mouse embryos

Mouse embryos were derived from eggs heterozygous for alleles of the dimeric enzyme glucose phosphate isomerase (Gpi-1a/Gpi-1b) that had been fertilized with sperm carrying a third allele (Gpi-1c). This particular combination makes it possible to study the activity of the paternally derived as well as the maternally derived genes, the persistence of oocyte-coded enzyme throughout early development and the possible simultaneous expression of both the paternally derived allele and the maternal message. The different isozymes present in single embryos were separated by electrophoresis. The results show that the oocyte-coded glucose phosphate isomerase is gradually replaced by embryo-coded enzyme. Expression of the paternally derived allele was first detected at the morula stage, during which the translation of the maternally derived message seemed to be either exhausted or below the detection limit of our system. Some oocyte-coded enzyme persisted until after implantation.     

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.