Cdc2 Gene Research Articles

Forty-five years of cell-cycle genetics

In the early 1970s, studies in Leland Hartwell’s laboratory at the University of Washington launched the genetic analysis of the eukaryotic cell cycle and set the path that has led to our modern understanding of this centrally important process. This 45th-anniversary Retrospective reviews the steps by which the project took shape, the atmosphere in which this happened, and the possible morals for modern times. It also provides an up-to-date look at the 35 original CDC genes and their human homologues.

MicroRNA-137 Negatively Regulates H₂O₂-Induced Cardiomyocyte Apoptosis Through CDC42.

BackgroundOxidative stress, inducing cardiomyocyte apoptosis or myocardial ischemia, is the major denominator of many cardiac diseases. In this study, we intended to explore the regulatory function of microRNA-137 (miR-137) in oxidative stress-induced cardiomyocyte apoptosis.Material/MethodsCardiomyocytes were extracted from newborn C57BL/6 mice and cultured in vitro. Apoptosis was induced by H2O2, and evaluated by TUNEL assay. The effect of cardiomyocyte apoptosis on gene expression of miR-137 was evaluated by qRT-PCR. Lentivirus was used to stably down-regulate miR-137, and the subsequent effects of miR-137 down-regulation on cardiomyocyte apoptosis, its targeted gene CDC42, and caspase pathway were evaluated by TUNEL assay, dual-luciferase reporter assay, and Western blot assay, respectively. Finally, CDC42 was down-regulated by siRNA and its effect on miR-137-mediated cardiomyocyte apoptosis protection was examined.ResultsH2O2 induced significant apoptosis and up-regulated miR-137 in cardiomyocytes, whereas lentivirus-mediated miR-137 down-regulation protected against apoptosis. CDC42 was the direct target gene of miR-137 and proteins of CDC42, caspase-3, and caspase-9 were all regulated by miR-137 down-regulation in cardiomyocyte apoptosis. SiRNA-mediated CDC42 down-regulation reversed the protection of miR-137 down-regulation against cardiomyocyte apoptosis.ConclusionsOur work demonstrated miR-137 and CDC42 are critical regulators in cardiomyocyte apoptosis. It may help to identify the molecular targets to prevent myocardial injury in human patients.

Dme-miR-314-3p modulation in Cr(VI) exposed Drosophila affects DNA damage repair by targeting mus309

microRNAs (miRNAs) as one of the major epigenetic modulators negatively regulate mRNAs at post transcriptional level. It was therefore hypothesized that modulation of miRNAs by hexavalent Chromium [Cr(VI)], a priority environmental chemical, can affect DNA damage. In a genetically tractable model, Drosophila melanogaster, role of maximally up-regulated miRNA, dme-miR-314-3p, on DNA damage was examined by exposing the third instar larvae to 5.0–20.0μg/ml Cr(VI) for 24 and 48h. mus309, a Drosophila homologue of human Bloom’s syndrome and predicted as one of the potential targets of this miRNA, was confirmed as its target by 5′RLM-RACE assay. A significant down-regulation of mus309 was observed in dme-miR-314-3p overexpression strain (myo-gal4>UAS-miR-314-3p) as compared with that in parental strains (myo-gal4 and UAS-miR-314-3p) and in w1118. A significant increase in DNA damage including double strand breaks generation was observed in exposed myo-gal4>UAS-miR-314 and mus309 mutants as compared with that in parental strain and in unexposed control. A significant down-regulation of cell cycle regulation genes (CycA, CycB and cdc2) was observed in these exposed genotypes. Collectively, the study demonstrates that dme-miR-314-3p can mediate the downregulation of repair deficient gene mus309 leading to increased DNA damage and cell cycle arrest in exposed organism which may affect Cr(VI) mediated carcinogenesis.

MiR-17 is involved in Japanese Flounder (Paralichthys olivaceus) development by targeting the Cdc42 mRNA.

The expression patterns of 197 miRNAs during Japanese flounder metamorphic development were recently analyzed. miR-17 was differentially expressed during the metamorphic period of the Japanese flounder; however, the role of miR-17 in Japanese flounder development has remained elusive to date. Bioinformatics analysis showed that Cdc42 was a putative target of miR-17. Cdc42 is a gene related to cell adhesion, migration, polarity, cytokinesis, growth, actin cytoskeleton, microtubule dynamics and transcription factor activity; thus, Cdc42 may contribute to metamorphic development. In our study, overexpression of miR-17 in FEC cells suppressed Cdc42 expression. The luciferase reporter assay confirmed that Cdc42 was the target of miR-17. The Cdc42 cDNA from the Japanese flounder was cloned and characterized for the first time. The expression of miR-17 was found to be negatively correlated with Cdc42 mRNA expression during temporal development and in the tissues of adult Japanese flounders. These results indicated that the decrease in miR-17 contributed to the up-regulation of Cdc42 during Japanese flounder metamorphosis. Cdc42 gene expression was down-regulated by thyroid hormone during Japanese flounder metamorphosis, whereas miR-17 was significantly up-regulated by thyroid hormone during these stages. These results indicated that miR-17 was a negative regulator of Cdc42.

Russell's viper venom affects regulation of small GTPases and causes nuclear damage

Russell's viper with its five sub-species is found throughout the Indian subcontinent. Its venom is primarily hemotoxic. However, its envenomation causes damage to several physiological systems. The present work was aimed to study the dose and time dependent cytotoxic effects of Russell's viper venom (RVV) on human A549 cells grown in vitro. Time dependent changes have been observed in cellular morphology following exposure to RVV. Presence of stress granules, rounding-off of the cells, and formation of punctate structure and loss of cell–cell contact characterized the cellular effects. Fluorescence microscopic studies revealed that apoptotic cell population increased on exposure to RVV. Further to understand the mechanism of these effects, status of small GTPase (smGTPases) expression were studied by Western blot and RT-PCR; as smGTPases play pivotal roles in deciding the cellular morphology, polarity, cell movement and overall signaling cascade. It was shown for the first time that expression patterns of Rac, Rho and CDC42 genes are altered on exposure to RVV. Similarly, significant difference in the expression pattern of HSP70 and p53 at the mRNA levels were noted. Our results confirmed that RVV induces apoptosis in A549 cells; this was further confirmed by AO/EtBr staining as well as caspase-3 assay. All experiments were compared using RVV unexposed cells. We propose for the first time that RVV induces morphological changes in human A549 cells through modulation of smGTPase expression and affects the cellular-nuclear architecture which in turn interferes in proliferation and migration of these cells along with apoptosis.

Distinct cell cycle regulation during saprophytic and pathogenic growth in fungal pathogens.

In a number of dimorphic and hemibiotrophic pathogens, cell cycle regulation has been shown to be important for morphological changes related to infectious growth or infection-related morphogenesis. However, the role of mitotic CDK kinase Cdc2, the key regulator of cell cycle, in pathogenic growth is not clear, because most fungal pathogens have a single CDC2 gene that is essential for cell cycle progression and viability. Interestingly, the wheat scab fungus Fusarium graminearum has two CDC2 genes. Although CDC2A and CDC2B have redundant functions in vegetative growth and asexual production, only CDC2A is required for invasive growth and plant infection. In this study, we showed that Cdc2A and Cdc2B interacted with each other and may form homo- and heterodimers in vegetative hyphae. We also identified sequence and structural differences between Cdc2A and Cdc2B that may be related to their functional divergence. These results, together with earlier studies with cyclins, important for differentiation and infection in Candida albicans and Ustilago maydis, indicated that dimorphic and hemibiotrophic fungal pathogens may have stage-specific cyclin-CDK combinations or CDK targets during saprophytic and pathogenic growth.

Abstract 4559: Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor

Abstract Although several nuclear regulatory proteins may be targeted by estrogen, our efforts have focused on the redox nuclear respiratory factor-1 (NRF-1), because our recent study showed that NRF-1 gene expression significantly increases with the progression of breast tumor grades. NRF-1 overexpression supported in vitro tumor formation. To determine if NRF-1 is required for 17 beta estradiol (E2)-induced neoplastic phenotype, we have generated NRF-1 mutants by site directed mutagenesis. We have generated stable clones that express NRF-1 mutant. The in vitro tumor formation was detected by anchorage independent growth and 3D spheroid assays. NRF-1 overexpression enhanced in vitro tumor spheroid formation, cell migration and cell invasion. Our flow cytometry analysis showed that overexpression of wild-type NRF-1 increased the percent of transformed MCF-10A cells in S phase compared to vector alone. Dominant negative NRF-1 protein showed reduction in the tumor formation. In addition, shRNA targeting NRF-1 resulted in the inhibition of anchorage independent growth of MCF-10A cells in both vehicle control and E2-treated cells. Since NRF-1 is a substrate of the kinase AKT, we determined whether NRF-1 phosphorylation was increased in MCF-10A cells after treatment with a carcinogenic regimen of E2. We observed more than a 2-fold increase in phospho-NRF-1 in E2 treated (100pg/ml for 30 min) MCF-10A cells and phosphorylation of NRF-1 was inhibited by co-treatment with either biological (CAT or MnSOD) or chemical (20μM ebselen) ROS modifiers. These results suggest that E2-induced phosphorylation of NRF-1 is influenced by ROS signaling messengers. We found that E2 treated MCF-10A cells showed increased NRF-1 binding to the promoters of Cdc2, PRC1, PCNA, Cyclin B1, and CDC25C genes. NRF-1 binding induced by E2 treatment was inhibited by the overexpression of CAT and MnSOD. NRF-1 phosphorylation site specific to kinase AKT, Thr-109, was mutated to Asp (D) or Ala (A), and the NRF-1 acetylation site specific to acetyl-transferase PCAF, Lys- 89, was mutated to Gln (glutamine)-Q or Ala (A). We examine the effect of one of the NRF-1 mutants on E2-induced in vitro tumor formation. We observed that the expression of NRF-1 (T109A) phosphodeficient mutant (NRF-1PMT) significantly inhibited E2-induced cell transformation, whereas empty vector did not influence E2-induced colony formation. Together these findings support the idea that NRF-1 may play an important role in E2-induced malignant transformation of breast epithelial cells. Thus, our data is consistent with the hypothesis that in addition to the estrogen receptor activity, NRF-1 activation contributes to the susceptibility to develop malignant phenotype in response to exposure to estrogen. This work was in part supported by a VA MERIT Review (VA BX001463) grant to DR Citation Format: Lazaro Mesa, Jayanta Das, Alok Deoraj, Victor Okoh, Deodutta Roy. Phosphodeficient NRF1 mutant suppresses the susceptibility of the breast epithelial cells to develop tumors when exposed to estrogen - a major breast cancer risk factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4559. doi:10.1158/1538-7445.AM2015-4559

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum.

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

Bioinformatics analysis of transcription profiling of sepsis

Sepsis is a fatal whole-body inflammatory response that complicates a serious infection. To elucidate the molecular mechanism of sepsis, transcription profile data of GSE12624 which included a total of 70 samples (34 sepsis samples and 36 non-sepsis samples) was downloaded. The t test based on Bayes method in limma package was used to identify differentially expressed genes (DEGs) between sepsis and non-sepsis samples (criterion: P value <0.05). Gene Ontology (GO) enrichment analysis was conducted to investigate the biological processes involved DEGs. Protein-protein interaction (PPI) network and sub-network analysis were conducted to investigate the interactions between DEGs. A total of 894 DEGs, including 479 downregulated DEGs and 415 upregulated DEGs, were identified in sepsis samples comparing with non-sepsis samples. GO enrichment analysis showed that DEGs mainly involved in cellular metabolic process, primary metabolic process, and response to organic cyclic compound. In the PPI network, four genes of CDC2, GTF2F2, PCNA, and SMAD4 with degrees more than 10 were identified. Subsequently, four sub-networks, in which genes of PTBP1, PSMA3, PSMA6, PSMB9, PSMB10, and GADD45 had relative high degrees were identified from the PPI network. After the discussion referring to previous studies, we suggested that CDC2, GTF2F2, PCNA, SMAD4 PSMA3, PTBP1, and GADD45 might be used as new therapeutic targets for sepsis. However, experiments should be further performed to prove the practical utility of these candidates.

Cellular and molecular markers in monitoring the fate of lymphoid cell culture from Penaeus monodon Fabricius (1798).

Lymphoid cell culture from penaeid shrimps has gained much acceptance as an in vitro platform to facilitate research on the development of prophylaxis, and therapeutic strategies against viruses and for cell line development. However, lymphoid cells can be used as platform for in vitro research, only if they are in metabolically and mitotically active state in vitro with unaltered cell surface receptors. Through this study, we addressed the response of lymphoid cells to a new microenvironment at cellular and molecular levels; including the study of mitotic events, DNA synthesis, expression profile of cell cycle genes, cytoskeleton organization, metabolic activity and viral susceptibility. The S-phase entry and synthesis of new DNA was recorded by immunoflourescent technique. Cdc2, CycA, CycB, EF-1α and BUB3 genes involved in cell cycle were studied in both the cells and tissue, of which EF-1α showed an elevated expression in cells in vitro (∼ 19.7%). Cytoskeleton network of the cell was examined by studying the organization of actin filaments. As the markers for metabolic status, mitochondrial dehydrogenase, protein synthesis and glucose assimilation by the cells were also assessed. Viral susceptibility of the cell was determined using WSSV to confirm the preservation of cellular receptors. This study envisages to strengthen the shrimp cell line research and to bring forth lymphoid cell culture system as a 'model' in vitro system for shrimp and crustaceans altogether.

MiR-30c Mediates Upregulation of Cdc42 and Pak1 in Diabetic Cardiomyopathy.

Cardiac hypertrophy and myocardial fibrosis significantly contribute to the pathogenesis of diabetic cardiomyopathy (DCM). Altered expression of several genes and their regulation by microRNAs has been reported in hypertrophied failing hearts. This study aims to examine the role of Cdc42, Pak1, and miR-30c in the pathogenesis of cardiac hypertrophy in DCM. DCM was induced in Wistar rats by low-dose streptozotocin-high-fat diet for 12 weeks. Cardiac expression of Cdc42, Pak1 and miR-30c, and hypertrophy markers (ANP and β-MHC) was studied in DCM vs control rats and in high-glucose (HG)-treated H9c2 cardiomyocytes. Diabetic rats showed cardiomyocyte hypertrophy, increased heart-to-body weight ratio, and an increased expression of ANP and β-MHC. Cardiac expression of Cdc42 and Pak1 genes was increased in diabetic hearts and in HG-treated cardiomyocytes. miR-30c was identified to target Cdc42 and Pak1 genes, and cardiac miR-30c expression was found to be decreased in DCM rats, patients with DCM, and in HG-treated cardiomyocytes. miR-30c overexpression decreased Cdc42 and Pak1 genes and attenuated HG-induced cardiomyocyte hypertrophy, whereas miR-30c inhibition increased Cdc42 and Pak1 gene expression and myocyte hypertrophy in HG-treated cardiomyocytes. Downregulation of miR-30c mediates prohypertrophic effects of hyperglycemia in DCM by upregulation of Cdc42 and Pak1 genes.

Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16.

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-β1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.

Dissection of Lung Tumorigenesis Induced by Human Vascular Endothelial Growth Factor (hVEGF)‐A165 Overexpression in Clara Cells of Transgenic Mice

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF‐A165 (hVEGF‐A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF‐A165 in lung‐specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf‐A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF‐A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT‐PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up‐regulated genes (hvegf‐A165, cyclin b1, cdc2, egfr, mmp9, nrp‐1, and kdr) in VEGF tumors compared with wild‐type lung tissues. In conclusion, hVEGF‐A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF‐targeted therapies in lung adenocarcinoma.

ArchaeProfile: A Database of Archaea and their Origins of Replication

Archaea are single cell microorganisms having several unique characteristics which differentiate them from bacteria. One of the key features which make archaea distinct from bacteria is the replication process, which is very different and resembles that of the eukaryotes. In-vivo mapping of the ori site in Archae is a time consuming and tedious job due to complexity involved in the culture of archaeal colony, which puts challenges as well as opportunity to scientist to devise in-silico method to map the Ori site in archaeal genomes. Z-curve approach is a widely used insilico method to predict the Ori site in archaea, but it is not equally successful for all archaeal genomes. Several other parameters like copy number and location of the cdc6 gene, AT rich region with the presence of origin recognition boxes (ORB) provide a better estimate of the Ori site in archaea. The motivation behind development of Archae Profile database is to predict the location and the number of putative Ori sites in archaeal genomes based on purinepyrimidine ( R-Y) and amino-keto(M-K) disparity curve along with the consensus ORB sequences, cdc6 gene copy number, their location and upstream AT richness. Quick update cycle and easy browser interface makes Archae Profile distinct from other databases. Another important feature is the integration of tools for plotting disparity plot of a given genome sequence and finding specific repeats with copy number and location in a sequence. ArchaeProfile will be updated timely and the emphasis will be to integrate other tools for genome analysis as well as new search features in the database. Presently Archaea Profile has Ori related data of 122 archeal genome which is likely to increase with time. Availability: ArchaeProfile can be accessed freely from http://www.bioinfommv.in/archaeprofile. Data available on the database could be used for further analysis and tutorial purpose.

Yeast as a model system to screen purine derivatives against human CDK1 and CDK2 kinases

Cyclin-dependent kinases (Cdk) play crucial roles in cell cycle progression. Aberrant activation of Cdk1 has been observed in a number of primary tumors and Cdk2 is deregulated in various malignancies. The therapeutic value of targeting Cdk1 and Cdk2 has been explored in a number of experimental systems. In the present study, taking advantage of the fact that deletion of the yeast CDC28 gene is functionally complemented by human CDK1 or CDK2, we set up an in vivo screen system to evaluate the inhibitory potency of purine derivatives against these two human Cdks. We constructed three isogenic strains highly sensitive to small molecules and harboring genes CDK1, CDK2 or CDC28, under the control of the CDC28 promoter. In a proof of principle assay, we determined the inhibitory effect of 82 purine derivatives on the growth rate of these strains. Thirty-three of them were revealed to be able to inhibit the Cdk1- or Cdk2-harboring strains but not the Cdc28-harboring strain, suggesting a specific inhibitory effect on human Cdks. Our data demonstrate that the yeast-based assay is an efficient system to identify potential specific inhibitors that should be preferentially selected for further investigation in cultured human cell lines.

A role of Candida albicans CDC4 in the negative regulation of biofilm formation.

The CDC4 gene is nonessential in Candida albicans and plays a role in suppressing filamentous growth, in contrast to its homologues, which are involved in the G1-S transition of the cell cycle. While characterizing the function of C. albicans CDC4 (CaCDC4), we found that the loss of CaCDC4 resulted in a reduction in cell flocculation, indicating a possible role for CaCDC4 in biofilm formation. To elucidate the role of CaCDC4 in biofilm formation, Cacdc4 null mutant strains were constructed by using the mini-Ura-blaster method. To create a CaCDC4 rescued strain, the plasmid p6HF-ACT1p-CaCDC4 capable of constitutively expressing CaCDC4 was introduced into the Cacdc4 homozygous null mutant. To determine the biofilm formation ability, an in vitro XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-5-carboxanilide) reduction assay was used. Compared with the parental auxotrophic strain BWP17, the Cacdc4 homozygous null mutant was able to enhance biofilm formation significantly. This enhancement of biofilm formation in the Cacdc4 homozygous null mutant could be reversed by constitutively expressing CaCDC4. We conclude that CaCDC4 has a role in suppressing biofilm formation in C. albicans.

Cancer specificity of promoters of the genes controlling cell proliferation.

Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.

The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.

A novel role for lncRNAs in cell cycle control during stress adaptation.

Eukaryotic cells have developed sophisticated systems to constantly monitor changes in the extracellular environment and to orchestrate a proper cellular response. To maximize survival, cells delay cell-cycle progression in response to environmental changes. In response to extracellular insults, stress-activated protein kinases (SAPKs) modulate cell-cycle progression and gene expression. In yeast, osmostress induces activation of the p38-related SAPK Hog1, which plays a key role in reprogramming gene expression upon osmostress. Genomic analysis has revealed the existence of a large number of long non-coding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Upon osmostress, hundreds of lncRNAs are induced by the SAPK p38/Hog1. One gene that expresses Hog1-dependent lncRNA in an antisense orientation is the CDC28 gene, which encodes CDK1 kinase that controls the cell cycle in yeast. Cdc28 lncRNA mediates the induction of CDC28 expression and this increase in the level of Cdc28 results in more efficient re-entry of the cells into the cell cycle after stress. Thus, the control of lncRNA expression as a new mechanism for the regulation of cell-cycle progression opens new avenues to understand how stress adaptation can be accomplished in response to changing environments.

MiR-630 inhibits proliferation by targeting CDC7 kinase, but maintains the apoptotic balance by targeting multiple modulators in human lung cancer A549 cells.

MicroRNAome analyses have shown microRNA-630 (miR-630) to be involved in the regulation of apoptosis. However, its apoptotic role is still debated and its participation in DNA replication is unknown. Here, we demonstrate that miR-630 inhibits cell proliferation by targeting cell-cycle kinase 7 (CDC7) kinase, but maintains the apoptotic balance by targeting multiple activators of apoptosis under genotoxic stress. We identified a novel regulatory mechanism of CDC7 gene expression, in which miR-630 downregulated CDC7 expression by recognizing and binding to four binding sites in CDC7 3'-UTR. We found that miR-630 was highly expressed in A549 and NIH3T3 cells where CDC7 was downregulated, but lower in H1299, MCF7, MDA-MB-231, HeLa and 2BS cells where CDC7 was upregulated. Furthermore, the induction of miR-630 occurred commonly in a variety of human cancer and immortalized cells in response to genotoxic agents. Importantly, downregulation of CDC7 by miR-630 was associated with cisplatin (CIS)-induced inhibitory proliferation in A549 cells. Mechanistically, miR-630 exerted its inhibitory proliferation by blocking CDC7-mediated initiation of DNA synthesis and by inducing G1 arrest, but maintains apoptotic balance under CIS exposure. On the one hand, miR-630 promoted apoptosis by downregulation of CDC7; on the other hand, it reduced apoptosis by downregulating several apoptotic modulators such as PARP3, DDIT4, EP300 and EP300 downstream effector p53, thereby maintaining the apoptotic balance. Our data indicate that miR-630 has a bimodal role in the regulation of apoptosis in response to DNA damage. Our data also support the notion that a certain mRNA can be targeted by several miRNAs, and in particular an miRNA may target a set of mRNAs. These data afford a comprehensive view of microRNA-dependent control of gene expression in the regulation of apoptosis under genotoxic stress.