Cdc2 Gene Research Articles

Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands

BackgroundG-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay.ResultsWhen G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex.ConclusionDele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.

Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest

SummaryDuring mitosis, cells must segregate the replicated copies of their genome to their daughter cells with extremely high fidelity. Segregation errors lead to an abnormal chromosome number (aneuploidy), which typically results in disease or cell death [1]. Chromosome segregation and anaphase onset are initiated through the action of the multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C [2]). The APC/C is inhibited by the spindle checkpoint in the presence of kinetochore attachment defects [3, 4]. Here we demonstrate that two non-essential APC/C subunits (Apc14 and Apc15) regulate association of spindle checkpoint proteins, in the form of the mitotic checkpoint complex (MCC), with the APC/C. apc14Δ mutants display increased MCC association with the APC/C and are unable to silence the checkpoint efficiently. Conversely, apc15Δ mutants display reduced association between the MCC and APC/C, are defective in poly-ubiquitination of Cdc20, and are checkpoint defective. In vitro reconstitution studies have shown that human MCC-APC/C can contain two molecules of Cdc20 [5, 6, 7]. Using a yeast strain expressing two Cdc20 genes with different epitope tags, we show by co-immunoprecipitation that this is true in vivo. MCC binding to the second molecule of Cdc20 is mediated via the C-terminal KEN box in Mad3. Somewhat surprisingly, complexes containing both molecules of Cdc20 accumulate in apc15Δ cells, and the implications of this observation are discussed.

Effect of potential role of p53 on embryo development arrest induced by H2O2 in mouse

During mammalian embryo development in vitro, mechanism of embryonic development arrest caused by oxidative stress has not been clear so far. The tumor suppressor protein p53 controls cell cycle and programmed cell death by regulating relevant signal pathway. Recent researches revealed that the concentration and distribution of p53 are closely related with reactive oxygen species (ROS). The main objective of this experiment was to explore the role of p53 on embryonic development arrest caused by oxidative stress. Results showed that embryo arrest at two-four-cell stage was significantly increased in the presence of 50μM H2O2 (39.01±2.74 vs. 77.20±5.34%, p<0.05). Supplementation of N-acetyl-L-cysteine (NAC) obviously reduced the ratio of development arrest (39.01±2.74 vs. 71.18±5.34%, p<0.05), which was accompanied by an increase in ROS level, and H2O2 treatment sharply increased messenger RNA (mRNA) expression and protein levels of p53 and p53-ser15. Further increased transcription of GADD45a and p21, a downstream of p53, has an especially significant effect on the mRNA expression of GADD45a. However, expressions of cdc2 were reduced by H2O2. In addition, using Pifithrin-α (PFT-α), the suppresser of p53, the result showed that GADD45a and p21 were significantly downregulated, but the cell cycle gene cdc2 was significantly upregulated, while the protein level of p53 and p53-ser15 was significantly decreased. Taken together, these results demonstrate that ROS could activate p53 and regulate p53 target genes to influence early embryo development in in vitro culture.

CDC20siRNA and paclitaxel co-loaded nanometric liposomes of a nipecotic acid-derived cationic amphiphile inhibit xenografted neuroblastoma.

Despite significant recent progress in the area of translational genomics of neuroblastoma, the overall survival rates for children with high-risk NB continue to be not more than 5 years due to tumor relapse and/or drug-resistant tumors. Herein we report on the development of a neuroblastoma targeting nanometric (130-150 nm) circulation stable liposomal system prepared from a novel nipecotic acid-derived cationic amphiphile (NACA). The size ranges of liposomes (130-150 nm) were confirmed by both dynamic light scattering and transmission electron microscopy. The findings in the gel electrophoresis assay revealed that siRNAs encapsulated within the liposomes of NACA (with 90% entrapment efficiency) are protected from attack by RNase. Cellular uptake experiments using FAM-siRNA loaded liposomes of NACA showed the liposomal entry in human neuroblastoma cells (IMR-32) to be mediated via the GABAA receptor. CDC20siRNA-loaded liposomes of NACA caused significantly higher CDC20 gene silencing efficiency in IMR-32 cells compared to CDC20 gene knockdown efficiency mediated by CDC20siRNA-loaded control non-targeting liposomes (NTL). The findings in the annexin-V binding based flow cytometric apoptosis assay and MTT-based cellular cytotoxicity assay support the notion that pronounced (80%) neuroblastoma cell death upon treatment with CDC20siRNA & PTX co-loaded liposomes of NACA presumably originates from enhanced apoptosis of cells. Importantly, intravenously administered CDC20siRNA & PTX co-loaded liposomes of NACA significantly inhibited growth of xenografted human neuroblastoma in athymic nude mice. The presently disclosed strategy of co-delivering potent anticancer siRNA and small molecule chemotherapeutics using liposomes of NACA opens a new door for combating the dreaded disease of neuroblastoma.

Abstract A21: Epigenetic regulation of cell cycle progression at the G2/M transition and mitosis in high-risk leukemia

Abstract High-risk acute lymphoblastic leukemia (ALL) is a clinical challenge due to drug resistance and poor prognosis. A characteristic molecular defect of most high-risk ALL is the deletion or inactivating mutation of one allele of the IKZF1 (Ikaros) tumor suppressor. Ikaros encodes a DNA-binding protein that regulates transcription of its target genes via chromatin remodeling. The mechanisms through which Ikaros regulates cellular proliferation in high-risk leukemia, are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that are critical in the control of G2/M transition (CDC2) and mitotic progression (ANAPC1 and ANAPC7) in leukemia. Gain- and loss-of-function experiments demonstrate that Ikaros represses the transcription of CDC2, ANAPC1 and ANAPC7. Overexpression of Ikaros in leukemia also results in cell cycle arrest. We studied the mechanism through which Ikaros represses CDC2, ANAPC1 ad ANAPC7. The use of serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes demonstrated that Ikaros can repress transcription of its target genes by two different mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and loss of H3K9ac (for ANAPC1 and CDC2); and 2) via an HDAC1-independent mechanism which is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac (for ANAPC7). In high-risk ALL that is characterized by deletion of one Ikaros allele, the function of Ikaros as a transcriptional regulator is impaired due to reduced binding to promoters of Ikaros target genes. We showed previously that Ikaros DNA-binding affinity is regulated via direct phosphorylation by pro-oncogenic Casein Kinase II (CK2). CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, which further reduces Ikaros function in this disease. In vivo CK2 inhibition with the CK2 specific inhibitor, CX-4945, results in a strong therapeutic effect in primary high-risk ALL xenografts. Analysis of primary high-risk B-ALL (that have deletion of one Ikaros allele) showed that treatment with CX-4945, restored Ikaros function as a transcriptional regulator of CDC2, ANAPC1 and ANAPC7, and was associated with cell cycle arrest. Epigenetic analysis of promoters of CDC2, ANAPC1 and ANAPC7 genes revealed that restoration of Ikaros binding to the promoters of these genes is associated with epigenetic alterations that are consistent with Ikaros overexpression and formation of repressive heterochromatin. In conclusion, our results reveal that: 1) Ikaros functions as a tumor suppressor by repressing transcription of genes that are critical for G/M transition (CDC2) and mitotic progression (ANAPC1 and ANAPC7); 2) Ikaros represses transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator of CDC2, ANAPC1 and ANAPC7 in high-risk leukemia. These results provide novel insights into the control of cell cycle progression in high-risk leukemia and the mechanisms by which CK2 inhibitors exert their therapeutic effects. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. Citation Format: Chunhua Song, Chandrika Gowda, Yali Ding, Kimberly J. Payne, Sinisa Dovat. Epigenetic regulation of cell cycle progression at the G2/M transition and mitosis in high-risk leukemia. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A21.

Cell division cycle 2 participates in eyestalk ablation-induced ovarian maturation of Procambarus clarkii

Eyestalk ablation is commonly used to promote ovarian development and maturation in captive economic crustaceans. Understanding the molecular mechanism of eyestalk ablation in Procambarus clarkii is essential in finding alternative measures to trigger ovarian maturation on this species without undesirable effects. The cell division cycle 2 (Cdc2) is a catalytic subunit of the maturation-promoting factor, which is a central factor for inducing oocyte meiotic maturation. This study analyzed the Cdc2 gene expression patterns and the progesterone level in the P. clarkii ovary during its eyestalk ablation-induced maturation course with or without synthetic pcGnRH injection. The RNA interference technique was used to elucidate the role of Cdc2 in the eyestalk ablation mechanism. A combination of the present and previous results revealed that the progesterone-mediated oocyte maturation pathway could exist in the ovary. Furthermore, this pathway could play a crucial role in the eyestalk ablation-induced ovarian maturation in P. clarkii. Statement of relevanceThe results of this study enable the utilization of the Cdc2 function to control oocyte maturation in Procambarus clarkii.

Low Gene Dosage ofCdc42Is Not Associated with Protein Dysfunction in Patients with Colorectal Cancer

High incidence of Rho Cdc42-GTPase overexpression has been found in Colorectal Cancer (CRC) samples, suggesting its potential role in tumor development. However, no conclusive studies have shown the lack of mutations and/or copy number of Cdc42 gene in this type of samples. To understand mutation/deletion and copy number status of Cdc42 gene, CRC patients were evaluated for both parameters. More than Cdc42 mutants, single-nucleotide variants were found. Analysis of regions flanking the Cdc42 gene showed allelic imbalance; 58.7% were loss of heterozygosity (LOH) positive and 14.8% presented microsatellite instability. The highest LOH percentage was located between microsatellite markers D1S199 and D1S2674, where the Cdc42 gene is located. No association between gender, age, and tumor stage was found. LOH validation through gene dosage analysis showed most CRC patients with allelic imbalance also presented a low gene dosage of Cdc42, although equal amounts of Cdc42 mRNA were detected in all samples. Although changes in Cdc42 expression were not found in any condition, Cdc42 activation was different between high and normal gene dosage samples, but not between samples with normal and low copy number. Low dosage of Cdc42 was also not related to changes in methylation status at the Cdc42 promoter region. Results suggest that low copy of Cdc42 gene is not associated with Cdc42 protein dysfunction in CRC patients.

The Urtica dioica extract enhances sensitivity of paclitaxel drug to MDA-MB-468 breast cancer cells.

Due to the chemo resistant nature of cancer cells and adverse effects of current therapies, researchers are looking for the most efficient therapeutic approach which has the lowest side effects and the highest toxicity on cancer cells. The aim of the present study was to investigate the synergic effect of Urtica dioica extract in combination with paclitaxel on cell death and invasion of human breast cancer MDA-MB-468 cell line. To determine the cytotoxic effects of Urtica dioica extract with paclitaxel, MTT assay was performed. The scratch test was exploited to assess the effects of Urtica dioica, Paclitaxel alone and combination on migration of cancer cells. The expression levels of snail-1, ZEB1, ZEB2, twist, Cdc2, cyclin B1 and Wee1 genes were quantified using qRT-PCR and western blot performed for snail-1expression. The effects of plant extract, Paclitaxel alone and combination on different phases of cell cycle was analyzed using flow cytometry. Results of MTT assay showed that Urtica dioica significantly destroyed cancer cells. Interestingly, Concurrent use of Urtica dioica extract with paclitaxel resulted in decreased IC50 dose of paclitaxel. Moreover, findings of scratch assay exhibited the inhibitory effects of Urtica dioica, Paclitaxel alone and combination on migration of MDA-MB-468 cell line. Our findings also demonstrated that the extract substantially decreased the Snail-1 and related gene expression. Ultimately, Cell cycle arrest occurred at G2/M phase post-treatment by deregulating Cdc2 and wee1. Our results demonstrated that the dichloromethane extract of Urtica dioica inhibit cell growth and migration. Also, Urtica dioica extract substantially increased sensitivity of breast cancer cells to paclitaxel. Therefore, it can be used as a potential candidate for treatment of breast cancer with paclitaxel.

Synchronizing Progression of Schizosaccharomyces pombe Cells from G2 through Repeated Rounds of Mitosis and S Phase with cdc25-22 Arrest Release.

Transient inactivation of the cdc25(+) gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures.

A comprehensive genome-wide analysis of melanoma Breslow thickness identifies interaction between CDC42 and SCIN genetic variants.

Breslow thickness (BT) is a major prognostic factor of cutaneous melanoma (CM), the most fatal skin cancer. The genetic component of BT has only been explored by candidate gene studies with inconsistent results. Our objective was to uncover the genetic factors underlying BT using an hypothesis-free genome-wide approach. Our analysis strategy integrated a genome-wide association study (GWAS) of single nucleotide polymorphisms (SNPs) for BT followed by pathway analysis of GWAS outcomes using the gene-set enrichment analysis (GSEA) method and epistasis analysis within BT-associated pathways. This strategy was applied to two large CM datasets with Hapmap3-imputed SNP data: the French MELARISK study for discovery (966 cases) and the MD Anderson Cancer Center study (1,546 cases) for replication. While no marginal effect of individual SNPs was revealed through GWAS, three pathways, defined by gene ontology (GO) categories were significantly enriched in genes associated with BT (false discovery rate ≤5% in both studies): hormone activity, cytokine activity and myeloid cell differentiation. Epistasis analysis, within each significant GO, identified a statistically significant interaction between CDC42 and SCIN SNPs (pmeta-int =2.2 × 10(-6) , which met the overall multiple-testing corrected threshold of 2.5 × 10(-6) ). These two SNPs (and proxies) are strongly associated with CDC42 and SCIN gene expression levels and map to regulatory elements in skin cells. This interaction has important biological relevance since CDC42 and SCIN proteins have opposite effects in actin cytoskeleton organization and dynamics, a key mechanism underlying melanoma cell migration and invasion.

A phloem-limited fijivirus induces the formation of neoplastic phloem tissues that house virus multiplication in the host plant.

A number of phloem-limited viruses induce the development of tumours (enations) in the veins of host plants, but the relevance of tumour induction to the life cycle of those viruses is unclear. In this study, we performed molecular and structural analyses of tumours induced by rice black-streaked dwarf virus (RBSDV, genus Fijivirus) infection in maize plants. The transcript level of the maize cdc2 gene, which regulates the cell cycle, was highly elevated in tumour tissues. Two-dimensional electrophoresis identified 25 cellular proteins with altered accumulation in the tumour tissues. These proteins are involved in various metabolic pathways, including photosynthesis, redox, energy pathways and amino acid synthesis. Histological analysis indicated that the tumours predominantly originated from hyperplastic growth of phloem, but those neoplastic tissues have irregular structures and cell arrangements. Immunodetection assays and electron microscopy observations indicated that in the shoots, RBSDV is confined to phloem and tumour regions and that virus multiplication actively occurs in the tumour tissue, as indicated by the high accumulation of non-structural proteins and formation of viroplasms in the tumour cells. Thus, the induction of tumours by RBSDV infection provides a larger environment that is favourable for virus propagation in the host plant.

Learning from the Uncontrollable

Research papers are usually revisionist histories, imagined accounts driven by impeccable logic and unbroken experimental success, which in no way reflect the slowness, messiness, and serendipity of real life in the lab. To give a different hopefully more balanced perspective than what is often told, I want to tell you the story behind some of the discoveries that shaped my early career.“In reality, of course, I had stumbled on the control by complete accident.”

Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms

SummaryAlternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3′-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3′-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons.

Molecular Analysis of Adaptational Strategies under Heat Stress by Whole‐Genome Sequencing of Laboratory‐Evolved Saccharomyces cerevisiae

Experimental evolution is an effective method to connect genotypes to phenotypes. The outcomes obtained from the method potentially facilitate the rational engineering of useful strains for bioproduction.In this study, we carried out experimental evolution of Saccharomyces cerevisiae under heat stress [1, 2]. In order to track the cell populations, intermediate strains obtained during the adaptational experiments have been preserved. Whole‐genome sequencing of 19 intermediate strains revealed that a total of 49 genetic mutations had emerged during the experimental evolution. A phylogenetic tree analysis showed that there were at least 5 events in which these strains had acquired mutations in a CDC25 gene. One of the CDC25 mutants successfully adapted to a 38°C‐environment. These mutations led to the downregulation of cAMP‐dependent protein kinase (PKA) signaling pathway, which controls variety of processes such as cell‐cycle progression and stress tolerance. In general, excessive downregulation of this pathway causes a trade‐off between induced thermotolerance and diminished growth rate. However, these strains have acquired thermotolerance without any growth defects. Our study demonstrates that experimental evolution leads to fine tuning of signaling pathways and engineering of beneficial cellular traits [3].Support or Funding InformationThis work was supported by a Grant‐in‐Aid for JSPS Fellows (No. 26‐1413) from Japan Society for the Promotion of Science, the commission for Development of Artificial Genes Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan, and, JST‐CREST.

Potential of GRID2 receptor gene for preventing TNF-induced neurodegeneration in autism

Autism is one of the most common subtypes of autism spectrum disorder (ASD). Recent studies suggested a relationship between immune-dependent coding genes and ASD, indicating that long term neuroimmunological anomalies affect brain development and synaptic transmission among neural networks. Furthermore, various studies focused on biomarker potential of TNF-α in autism. Ionotropic receptors are also studied as potential marker for autism since altered gene expression levels are observed in autistic patients. GRID2 is a candidate ionotropic receptor which is involved glutamate transfer. In this study, to propose TNF-α dependent cellular processes involved in autism aetiology in relation to GRID2 we performed a bioinformatic network analysis and identified potential pathways and genes that are involved in TNF-α induced changes at GRID2 receptor levels. As a result, we ascertained the GRID2 receptor gene as a candidate gene and further studied the association between GRID2 expression levels and TNF-induced neurodegeneration. Our bioinformatic analyses and experimental results revealed that TNF-α regulates GRID2 gene expression by activating Cdc42 and GOPC genes. Moreover, increased TNF-α levels leads to increase of caspase-3 protein levels triggering neuronal apoptosis leading to neuronal deficiency, which is one of the major symptoms of autism. The study is the first to show the role of TNF-α in regulation of GRID2 gene expression and its signalling pathway. As a result, GRID2 gene can be a suppressor in TNF-induced neurodegeneration which may help to understand the main factors leading to autism.

Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains.

Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction.

Identification and evolution of two insulin receptor genes involved in Tribolium castaneum development and reproduction

The insulin and insulin-like signaling (IIS) pathway exists in a wide range of organisms from mammals to invertebrates and regulates several vital physiological functions. A phylogenetic analysis have indicated that insulin receptors have been duplicated at least twice among vertebrates, whereas only one duplication occurred in insects before the differentiation of Coleoptera, Hymenoptera, and Hemiptera. Thus, we cloned two putative insulin receptor genes, T.cas-ir1 and T.cas-ir2, from T. castaneum and determined that T.cas-ir1 is most strongly expressed during the late adult and early pupal stages, whereas T.cas-ir2 is most strongly expressed during the late larval stage. We found that larval RNAi against T.cas-ir1 and T.cas-ir2 causes 100% and 42.0% insect death, respectively, and that parental RNAi against T.cas-ir1 and T.cas-ir2 leads to 100% and 33.3% reductions in beetle fecundity, respectively. The hatching rate of ds-ir2 insects was 66.2%. Moreover, RNAi against these two genes increased the expression of the pkc, foxo, jnk, cdc42, ikk, and mekk genes but decreased erk gene expression. Despite these similarities, these two genes act via distinct regulatory pathways. These results indicate that these two receptors have functionally diverged with respect to the development and reproduction of T. castaneum, even though they retain some common regulatory signaling pathways.

Influence of Toxoplasma gondii on invitro proliferation and apoptosis of hepatoma carcinoma H7402 cell.

To discuss the influence of tachyzoite of Toxoplasma gondii (T.gondii) RH strain on proliferation and apoptosis of hepatoma carcinoma (HCC) H7402cell. The HCC H7402cell in logarithmic phase and tachyzoite of T.gondii RH strain in different concentrations (1×10(7)/mL, 2×10(7)/mL, 4×10(7)/mL, 8×10(7)/mL and 16×10(7)/mL) were co-cultured. CCK-8 was utilized to determine the inhibition rate of T.gondii tachyzoite on H7402 cell growth. Flow cytometry was used to detect the change of cell cycle. RT-PCR method was used to detect the expression of cyclinB1 and cdc2-two genes related to cell cycle. Western blot method was used to detect the expression of apoptosis-related proteins Caspase-3 and Bcl-2. The tachyzoite of T.gondii RH strain can inhibit the proliferation of HCC H7402 cells. The inhibition rate of tumor cell growth increased with the increase of concentration of T.gondii tachyzoite. With the increase of concentration of T.gondii tachyzoite, the proportion of G0/G1 phase of H7402 cell increased, the proportion of S phase decreased, and PI value decreased accordingly. The expression of cyclinB1 and cdc2 genes decreased with the increase of the concentration of T.gondii tachyzoite. With the increase of the concentration of tachyzoite of T.gondii RH strain, the expression quantity of Caspase-3 in H7402 cell increased, but the expression quantity of Bcl-2 protein decreased. T.gondii can inhibit the invitro proliferation of HCC H7402 cell, and induce its apoptosis. This effect shows a trend of concentration-dependent increase. Moreover, it is related to the down-regulation of cyclinB1 and cdc2 (cell cycle-related genes), the increase of apoptosis-related protein Caspase-3, and the decrease of Bcl-2 expression.

A novel approach to the expression and purification of recombinant Xenopus Cdc25C

The Cdc25 family encodes dual specificity protein phosphatases that play critical roles in cell cycle progression. Activation of the Cdc25C represents a primary driver for meiosis progression in Xenopus oocytes. Given its central role in meiosis the Xenopus Cdc25C has been studied extensively, however purification of the recombinant protein is difficult thus preventing better characterization of its function. Here we describe methods to overcome these difficulties resulting in the production of high purity and yield recombinant Xenopus Cdc25C. We use a synthetic Xenopus Cdc25C gene that was codon optimized for expression in E. coli. We further combine an N-terminal His-tag with a C-terminal Strep-tag II, to isolate extremely pure full-length Cdc25C protein. The recombinant Xenopus Cdc25C is active both in vitro using a phosphatase assay and in vivo when injected into Xenopus oocytes. This new approach should be applicable to the purification of other members of the Cdc25 gene family.

CYP1B1 promotes tumorigenesis via altered expression of CDC20 and DAPK1 genes in renal cell carcinoma.

BackgroundCytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC.MethodsExpression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses.ResultsFirst, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples.ConclusionsCYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.