Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2.

Cyclin-dependent kinase 1 (CDK1) plays a crucial role in establishing metaphase and has also been shown to prevent DNA re-replication. Cyclins B1 and B2 are two known activators of CDK1 operating during mitosis in human cells. Little is known about the specific roles of each of these cyclins in CDK1 activation, but cyclin B2 is thought to play a minor role and to be unable to replace cyclin B1 for mitosis completion. In our study, we found that severe reduction by separate RNA interference of either cyclin B1 or cyclin B2 protein levels results in little or no alteration of the cell cycle and, more specifically, of mitosis progression. In contrast, simultaneous depletion of both B-type cyclins leads to massive accumulation of 4N cells, mitotic failure, premature mitosis exit and DNA re-replication. These defects can be corrected by the ectopic expression of a cyclin B2 resistant to the short hairpin RNA. Altogether, these data show that, in cycling human cells, cyclin B2 can compensate for the downregulation of cyclin B1 during mitosis. They also clearly implicate cyclins B1 and B2 as crucial activators of CDK1 in its biological function of DNA re-replication prevention.

Synthesis, crystal structure, and characterization of the first Nb(3) triangular cluster compound bonded to fluorine ligands: association of Nb(3)I(i)F(i)(3)F(a)(8)L(a) units and Nb(IV)L(6) Octahedra with L=O and F.

The synthesis and the crystal structure of the first compound containing Nb(3) triangular clusters bonded to fluorine ligands are presented in this work. The structure of Nb(3)IF(7)L(NbL(2))(0.25) with L = O and F, determined by single-crystal X-ray diffraction, is based on a Nb(3)I(i)F(i)(3)F(a)(8)L(a) unit and a NbL(6) octahedron (tetragonal, space group I4/m, a = 13.8638(3) A, c = 8.9183(2) A, V = 1714.14(7) A(3), Z = 8). Two crystallographic positions (noted L5 and L6) are randomly occupied by fluorine and oxygen with two different F:O occupancies. These L ligands build an octahedral site for a single niobium atom, located between the units. The four L5 ligands of the NbL(6) octahedron are shared with four Nb(3) cluster units, while the two other L6 ligands are terminal. The Nb(3) cluster is face-capped by one iodine and edge-bridged by three fluorine ligands. Two of the three niobium atoms constituting the cluster are bonded to three additional apical fluorine ligands, while the third one is bonded to two fluorines and one L5 ligand. The Nb(3) cluster is linked to six adjacent ones via all the apical fluorine ligands. The developed formula of the unit is therefore Nb(3)I(i)F(i)(3)F(a)(-)(a)(8/2)L(a) according to the Schäfer and Schnering notation. The oxidation state of the single niobium and the random distribution of fluorine and oxygen on the two L sites will be discussed on the basis of structural analysis, the bond valence method, and IR and EPR measurements. The structural results will be compared to those of previously reported niobium compounds containing NbF(6) or Nb(F,O)(6) octahedra.

Membrane-anchored cyclin A2 triggers Cdc2 activation in Xenopus oocyte.

In Xenopus oocyte, the formation of complexes between neosynthesized cyclins and Cdc2 contributes to Cdc2 kinase activation that triggers meiotic divisions. It has been proposed that cytoplasmic membranes could be involved in this process. To investigate this possibility, we have injected in the oocyte two undegradable human cyclin A2 mutants anchored to the endoplasmic reticulum (ER) membrane. They encode fusion proteins between the truncated cyclin A2-Delta152 and a viral or cellular ER-targeting domain. We show that both mutants are fully functional as mitotic cyclins when expressed in Xenopus oocytes, bind Cdc2 and activate M-phase promoting factor.

NS1- and minute virus of mice-induced cell cycle arrest: involvement of p53 and p21(cip1).

The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G(1), S, and G(2), well reproducing the arrest in S and G(2) observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21(cip1) associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21(cip1). By comparison, MVMp infection induced the accumulation of both p53 and p21(cip1). We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G(2) by using p53 wild-type (+/+) and null (-/-) cells. Furthermore, only the G(2) arrest was abrogated in p21(cip1) null (-/-) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21(cip1) independent, whereas the arrest in G(2) depends on both p53 and its downstream effector p21(cip1). They also suggest that induction of p21(cip1) by the viral protein NS1 arrests cells in G(2) through inhibition of cyclin A-dependent kinase activity.

Early development of mouse embryos null mutant for the cyclin A2 gene occurs in the absence of maternally derived cyclin A2 gene products.

Progression through the mammalian cell cycle is regulated by the sequential activation and inactivation of the cyclin-dependent kinases. In adult cells, cyclin A2-dependent kinases are required for entry into S and M phases, completion of S phase, and centrosome duplication. However, mouse embryos lacking the cyclin A2 gene nonetheless complete preimplantation development, but die soon after implantation. In this report, we investigated whether a contribution of maternal cyclin A2 mRNA and protein to early embryonic cell cycles might explain these conflicting observations. Our data show that a maternal stock of cyclin A2 mRNA is present in the oocyte and persists after fertilization until the second mitotic cell cycle, when it is degraded to undetectable levels coincident with transcriptional activation of the zygotic genome. A portion of maternally derived cyclin A2 protein is stable during the first mitosis and persists in the cytoplasm, but is completely degraded at the second mitosis. The ability of cyclin A2-null mutants to develop normally from the four-cell to the postimplantation stage in the absence of detectable cyclin A2 gene product indicates therefore that cyclin A2 is dispensable for cellular progression during the preimplantation nongrowth period of mouse embryo development.

p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line.

In vivo transfer of wild-type (wt) p53 gene via a recombinant adenovirus has been proposed to induce apoptosis and increase radiosensitivity in several human carcinoma models. In the context of combining p53 gene transfer and irradiation, we investigated the consequences of adenoviral-mediated wtp53 gene transfer on the cell cycle and radiosensitivity of a human head and neck squamous cell carcinoma line (SCC97) with a p53 mutated phenotype. We showed that ectopic expression of wtp53 in SCC97 cells resulted in a prolonged G1 arrest, associated with an increased expression of the cyclin-dependent kinase inhibitor WAF1/p21 target gene. A transient arrest in G2 but not in G1 was observed after irradiation. This G2 arrest was permanent when exponentially growing cells were transduced by Ad5CMV-p53 (RPR/INGN201) immediately after irradiation with 5 or 10 Gy. Moreover, levels of cyclins A2 and B1, which are known to regulate the G2/M transition, dramatically decreased as cells arrived in G2, whereas maximal levels of expression were observed in the absence of wtp53. In conclusion, adenoviral mediated transfer of wtp53 in irradiated SCC97 cells, which are mutated for p53, appeared to increase WAF1/p21 expression and decrease levels of the mitotic cyclins A2 and B1. These observations suggest that the G2 arrest resulted from a p53-dependent premature inactivation of the mitosis promoting factor.

cAMP-dependent positive control of cyclin A2 expression during G1/S transition in primary hepatocytes.

cAMP positively and negatively regulates hepatocyte proliferation but its molecular targets are still unknown. Cyclin A2 is a major regulator of the cell cycle progression and its synthesis is required for progression to S phase. We have investigated whether cyclin A2 and cyclin A2-associated kinase might be one of the targets for the cAMP transduction pathway during progression of hepatocytes through G1 and G1/S. We show that stimulation of primary cultured hepatocytes by glucagon differentially modulated the expression of G1/S cyclins. Glucagon indeed upregulated cyclin A2 and cyclin A2-associated kinase while cyclin E-associated kinase was unmodified. In conclusion, our study identifies cyclin A2 as an important effector of the cAMP transduction network during hepatocyte proliferation.

Resonant cavity gas-phase polarimeter.

A high-sensitivity polarimeter is demonstrated for application to gas-phase chirality measurement. This device is based on the physics of the eigenstates of a Fabry-Perot cavity, permitting improvement in the sensitivity with respect to the usual polarimeters. Typical measurements of rotations of 50 (±1) × 10(-)(5)° induced by the optical activity of (R)-(+)-limonene and (S)-(-)-limonene in the vapor phase are shown. A noise level corresponding to a rotation of 10(-)(6)° is experimentally demonstrated. Application to the polarimetric monitoring of an enantiomer mixing racemization of limonene in the gas phase is also presented.

Delayed early embryonic lethality following disruption of the murine cyclin A2 gene.

In higher eukaryotes, cell cycle progression is controlled by cyclin dependent kinases (Cdks) complexed with cyclins. A-type cyclins are involved at both G1/S and G2/M transitions of the cell cycle. Cyclin A2 activates cdc2 (Cdk1) on passage into mitosis and Cdk2 at the G1/S transition. Antisense constructs, or antibodies directed against cyclin A2 block cultured mammalian cells at both of these transitions. In contrast, overexpression of cyclin A2 appears to advance S phase entry and confer anchorage-independent growth, and can lead to apoptosis. A second A-type cyclin, cyclin A1 has been described recently which, in the mouse, is expressed in germ cells but not somatic tissues. To address the possible redundancy between different cyclins in vivo and also the control of early embryonic cell cycles, we undertook the targeted deletion of the murine cyclin A2 gene. The homozygous null mutant is embryonically lethal, demonstrating that the cyclin A2 gene is essential. Surprisingly, homozygous null mutant embryos develop normally until post-implantation, around day 5.5 p.c. This observation may be explained by the persistence of a maternal pool of cyclin A2 protein until at least the blastocyst stage, or an unexpected role for cyclin A1 during early embryo development.

ATF/CREB site mediated transcriptional activation and p53 dependent repression of the cyclin A promoter.

Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated through tight control of its promoter. We have previously shown that the ATF/CREB site, present in the cyclin A promoter, mediates transcriptional regulation by cAMP responsive element binding proteins. The main goal of the present study was to investigate whether this site is involved in transcriptional regulation of the gene. We have constructed stable NIH-3T3 cell lines that express the luciferase reporter gene under the control of normal or mutated versions of the cyclin A promoter. We show that the ATF/CREB is required to achieve maximal levels of transcription from the cyclin A promoter starting in late G1. We also show that down-regulation of the cyclin A promoter by p53 does not implicate a direct binding of p53 to its cognate consensus sequence but occurs probably by interference with trans-activating factors. This result suggests that p53 can interfere with transcription of the cyclin A gene, in the absence of a TATA sequence in the promoter.

Proliferation and differentiation of a human hepatoblastoma transplanted in the Nude mouse.

A pure epithelial human hepatoblastoma was directly transplanted to athymic Nude mice to provide a model system to study proliferation and differentiation of these tumoral cells. The first transplantation selected the embryonal component of this tumor, while subsequent passages selected in addition neuroendocrine and mesenchymal cells that evolved into osteoid and bony trabeculae. The embryonal character of this hepatoblastoma was further demonstrated by the expression of glutamine synthetase mRNA and a fetal pattern of mRNAs encoding insulin-like growth factor II. However, alphafetoprotein mRNA was detectable in neither the original nor the transplanted tumors. Finally, although p53 mRNA levels were increased, no mutation was detected in the p53 gene.

Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM.

Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S.

The PML growth-suppressor has an altered expression in human oncogenesis.

Altered sub-nuclear localisation of the nuclear body-associated PML protein in acute promyelocytic leukaemia, has been proposed to contribute to leukaemogenesis. We have recently shown that PML is a primary target gene of interferons. Here, it is shown that PML has growth suppressive properties and displays an altered expression pattern during human oncogenesis. PML is widely expressed in cell-lines and is cell-cycle regulated. Overexpression of the protein induces a sharp reduction in growth rates in vitro and in vivo. In contrast with cell-lines, in normal tissues (including those that rapidly proliferate) only a few cells have detectable PML levels. However, these can be upregulated by soluble factors (e.g. IFN, estrogens). Human epithelial tumors show a gradual increase of PML levels as the lesion progresses from benign dysplasia to carcinoma. A similar induction is found in the surrounding stroma and vessels, which likely results from paracrine interactions. Strikingly, when malignant cells turn invasive, they loose PML expression, while expression is conserved in the stromal compartment. These observations point to the existence of a consistent deregulation in the expression of the PML growth-suppressor during human oncogenesis.

Cyclin A: function and expression during cell proliferation.

Cyclin A is a key regulatory protein which, in mammalian cells, is involved in both S phase and the G2/M transition of the cell cycle through its association with distinct cdks. Several lines of evidence have also implicated cyclin A in carcinogenesis. Our review concentrates on the role of cyclin A in S phase, in the S/G2 transition and in human carcinogenesis; it will also discuss the transcriptional regulation of cyclin A gene.

Localization of cyclin A at the sites of cellular DNA replication.

Cyclin A is a nuclear protein which is part of a kinase complex with either p34cdc2 or p33cdk2. Cyclin A is required in higher eukaryotic cells at the G1/S and the G2/M transitions. To examine the relationship between cyclin A and DNA replication, we simultaneously labeled exponentially growing HeLa cells for the distribution of cyclin A and proliferating cell nuclear antigen (PCNA). We have now demonstrated, by means of immunoelectron microscopy, that cyclin A is located at the sites of DNA replication visualized by both BrdU and PCNA labeling. Thus cyclin A may play a significant role in the phosphorylation of proteins at or near the sites of DNA replication.