Glyphosate-based pesticides affect cell cycle regulation

Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.     

Comparison of Singlet and Doublet Paramecium tetraurelia: DNA Content,Protein Content and the Cell Cycle*

SYNOPSIS Doublet Paramecium tetraurelia contain either a single macronucleus which is substantially larger than that in a singlet cell, or 2 smaller macronuclei. Doublets have approximately twice the DNA content and twice the total protein content of singlets. The cell cycle of doublets is 164% as long as that of singlets, but the relative position of the macronuclear DNA synthesis period within the cell cycle is the same as in singlets. The DNA content of doublets is regulated so that differences in the number of macronuclei do not produce corresponding changes in DNA content; bimacronucleate doublets have only 27% more DNA than unimacronucleate doublets.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

周期蛋白和周期蛋白依赖性激酶及相关激酶抑制剂在细胞周期进程中的调控机制研究进展

在细胞发育过程中,细胞周期起着至关重要的作用。细胞周期进程主要受细胞周期蛋白依赖性激酶(cyclin dependent kinase, CDK)、周期蛋白和内源性CDK抑制剂(cyclin-dependent kinase inhibitors,CKI)调控。其中,CDK是主要的细胞周期调节因子,可与周期蛋白结合形成周期蛋白-CDK复合物,从而使数百种底物磷酸化,调控分裂间期和有丝分裂进程。各类细胞周期蛋白的活性异常,可引起不受控制的癌细胞增殖,导致癌症的发生与发展。因此,了解CDK的活性变化情况、周期蛋白-CDK的组装以及CKI的作用,将有助于了解细胞周期进程中潜在的调控过程,为癌症与疾病的治疗和CKI治疗药物的研发提供基础。本文关注了CDK激活和灭活的关键事件,并总结了周期蛋白-CDK在特定时期及位置的调控过程,以及相关CKI治疗药物在癌症及疾病中的研究进展,最后简单阐述了细胞周期进程研究面临的问题和存在的挑战,以期为后续细胞周期进程的深入研究提供参考和思路。     

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.     

PP2A function toward mitotic kinases and substrates during the cell cycle

To maintain cellular homeostasis against the demands of the extracellular environment, a precise regulation of kinases and phosphatases is essential. In cell cycle regulation mechanisms, activation of the cyclin-dependent kinase (CDK1) and cyclin B complex (CDK1:cyclin B) causes a remarkable change in protein phosphorylation. Activation of CDK1:cyclin B is regulated by two auto-amplification loops-CDK1:cyclin B activates Cdc25, its own activating phosphatase, and inhibits Wee1, its own inhibiting kinase. Recent biological evidence has revealed that the inhibition of its counteracting phosphatase activity also occurs, and it is parallel to CDK1:cyclin B activation during mitosis. Phosphatase regulation of mitotic kinases and their substrates is essential to ensure that the progression of the cell cycle is ordered. Outlining how the mutual control of kinases and phosphatases governs the localization and timing of cell division will give us a new understanding about cell cycle regulation. [BMB Reports 2013; 46(6): 289-294]     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

Genome-wide analysis of core cell cycle genes in the unicellular green alga Ostreococcus tauri

The cell cycle has been extensively studied in various organisms, and the recent access to an overwhelming amount of genomic data has given birth to a new integrated approach called comparative genomics. Comparing the cell cycle across species shows that its regulation is evolutionarily conserved; the best-known example is the pivotal role of cyclin-dependent kinases in all the eukaryotic lineages hitherto investigated. Interestingly, the molecular network associated with the activity of the CDK-cyclin complexes is also evolutionarily conserved, thus, defining a core cell cycle set of genes together with lineage-specific adaptations. In this paper, we describe the core cell cycle genes of Ostreococcus tauri, the smallest free-living eukaryotic cell having a minimal cellular organization with a nucleus, a single chloroplast, and only one mitochondrion. This unicellular marine green alga, which has diverged at the base of the green lineage, shows the minimal yet complete set of core cell cycle genes described to date. It has only one homolog of CDKA, CDKB, CDKD, cyclin A, cyclin B, cyclin D, cyclin H, Cks, Rb, E2F, DP, DEL, Cdc25, and Wee1. We have also added the APC and SCF E3 ligases to the core cell cycle gene set. We discuss the potential of genome-wide analysis in the identification of divergent orthologs of cell cycle genes in different lineages by mining the genomes of evolutionarily important and strategic organisms.     

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 μM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 μM AG126 (an inhibitor of ERK1/2), but not by 25 μM SP600125 (an inhibitor of JNK1/2) or 5 μM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1→S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1→S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.     

Early effects of floral induction on cell division in the shoot apex of Silene

The changes in cell number, the relative proportions of interphase nuclei with different amounts of DNA, mitotic index and labelling index have been investigated in the shoot apex of Silene coeli-rosa L. (a long-day plant) during the first long day of photoinduction, and compared with the corresponding changes in plants in short days. 3 h after the start of induction the proportion of nuclei in the G2 phase of the cell cycle had increased, the mitotic index tended to be higher, and the labelling index was lower than in plants in short days. 8–9 h later the values for plants in the long day had become similar to those for plants in short days. No evidence was obtained for a synchronisation of cells in one phase of the cell cycle as a result of photoinduction. The results obtained were consistent with a temporary shortening of the cell cycle in the induced apices over the first long day which resulted in a greater increase in cell number by the end of the first day of photoinduction than in plants in short days.Abbreviations LD long day - SD short day     

Regulation of Multiple Fission and Cell-Cycle-Dependent Gene Expression by CDKA1 and the Rb-E2F Pathway in Chlamydomonas

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Arsenic trioxide inhibits the growth of A498 renal cell carcinoma cells via cell cycle arrest or apoptosis

Previously, we showed that arsenic trioxide potently inhibited the growth of myeloma cells and head and neck cancer cells. Here, we demonstrate that arsenic trioxide inhibited the proliferation of all the renal cell carcinoma cell lines (ACHN, A498, Caki-2, Cos-7, and Renca) except only one cell line (Caki-1) with IC(50) of about 2.5-10 microM. Arsenic trioxide induced a G(1) or a G(2)-M phase arrest in these cells. When we examined the effects of this drug on A498 cells, arsenic trioxide (2.5 microM) decreased the levels of CDK2, CDK6, cyclin D1, cyclin E, and cyclin A proteins. Although p21 protein was not increased by arsenic trioxide, this drug markedly enhanced the binding of p21 with CDK2. In addition, the activities of CDK2- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Arsenic trioxide (10 microM) also induced apoptosis in A498 cells. Apoptotic process of A498 cells was associated with the changes of Bcl-(XL), caspase-9, caspase-3, and caspase-7 proteins as well as mitochondria transmembrane potential (Deltapsi(m)) loss. Taken together, these results demonstrate that arsenic trioxide inhibits the growth of renal cell carcinoma cells via cell cycle arrest or apoptosis.     

The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2–7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2–7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2–7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.     

Structural basis for the ORC1‐Cyclin A association

Progression of cell cycle is regulated by sequential expression of cyclins, which associate with distinct cyclin kinases to drive the transition between different cell cycle phases. The complex of Cyclin A with cyclin‐dependent kinase 2 (CDK2) controls the DNA replication activity through phosphorylation of a set of chromatin factors, which critically influences the S phase transition. It has been shown that the direct interaction between the Cyclin A‐CDK2 complex and origin recognition complex subunit 1 (ORC1) mediates the localization of ORC1 to centrosomes, where ORC1 inhibits cyclin E‐mediated centrosome reduplication. However, the molecular basis underlying the specific recognition between ORC1 and cyclins remains elusive. Here we report the crystal structure of Cyclin A‐CDK2 complex bound to a peptide derived from ORC1 at 2.54 å resolution. The structure revealed that the ORC1 peptide interacts with a hydrophobic groove, termed cyclin binding groove (CBG), of Cyclin A via a KXL motif. Distinct from other identified CBG‐binding sequences, an arginine residue flanking the KXL motif of ORC1 inserts into a neighboring acidic pocket, contributing to the strong ORC1‐Cyclin A association. Furthermore, structural and sequence analysis of cyclins reveals divergence on the ORC1‐binding sites, which may underpin their differential ORC1‐binding activities. This study provides a structural basis of the specific ORC1‐cyclins recognition, with implication in development of novel inhibitors against the cyclin/CDK complexes.     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

EFFECT OF IRRADIANCE ON GROWTH AND REPRODUCTIVE PROCESSES DURING THE CELL CYCLE IN SCENEDESMUS ARMATUS (CHLOROPHYTA)1

Synchronized populations of the chlorococcal alga Scenedesmus armatus (Chod.) Chod. were grown under five irradiance levels. During the cell cycles of these populations, reproductive processes such as DNA replication, nuclear division, protoplast fission, and daughter cell release and growth processes such as RNA and protein accumulation were followed. The amount of RNA and proteins increased stepwise with a short time interval between individual steps during which the rate of RNA and protein accumulation decreased. At each of the steps, the amount of RNA and protein approximately doubled and the number of steps increased with irradiance. At the end of each of the growth steps, a commitment to trigger the sequence of reproductive events (DNA replication, nuclear division, protoplast fission) was attained. After attaining the commitment point, the cells were able to trigger and terminate the whole reproductive sequence without any further growth, that is, even in the dark when the external supply of energy was cut off. With increasing irradiance, the number of commitment points attained during one cell cycle increased from one to four. Consequently, one to four sequences of the reproductive steps were triggered, and each of them ended by doubling the reproductive structures, which resulted in the formation of 2, 4, 8, or 16 daughter cells. The length of the precommitment periods shortened with increasing irradiance as the result of an increasing rate in growth. The length of postcommitment periods showed light independence and remained constant at the range of irradiances at which the number of growth steps and, consequently, the number of sequences of reproductive events did not change. At higher irradiances, the number of sequences of reproductive events increased, which caused a prolongation of postcommitment periods. The length of the cell cycle varied as a result of this distinct effect of irradiance on pre- and postcommitment periods.