Ras-related Nuclear Protein Research Articles

Interaction of Transportin-SR2 with Ras-related Nuclear Protein (Ran) GTPase

The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are capable of infecting non-dividing cells and, therefore, need to be imported into the nucleus before integration into the host cell chromatin. Transportin-SR2 (TRN-SR2, Transportin-3, TNPO3) is a cellular karyopherin implicated in nuclear import of HIV-1. A model in which TRN-SR2 imports the viral preintegration complex into the nucleus is supported by direct interaction between TRN-SR2 and HIV-1 integrase (IN). Residues in the C-terminal domain of HIV-1 IN that mediate binding to TRN-SR2 were recently delineated. As for most nuclear import cargoes, the driving force behind HIV-1 preintegration complex import is likely a gradient of the GDP- and GTP-bound forms of Ran, a small GTPase. In this study we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. By size exclusion chromatography we demonstrate stable complex formation of TRN-SR2 and RanGTP in solution. Consistent with the behavior of normal nuclear import cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. Although in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2-RanGTP complex was significantly more compact. Further analysis supported a model wherein one monomer of TRN-SR2 is bound to one monomer of RanGTP. Finally, we present a homology model of the TRN-SR2-RanGTP complex that is in excellent agreement with the experimental small angle x-ray scattering data.

OC-027 Defining Cancer Risk in Barrett’S Oesophagus using a 90-Gene Signature

<h3>Introduction</h3> Barrett’s oesophagus (BE) has a highly variable outcome with 0.12–0.5% of patients per year progressing to oesophageal adenocarcinoma (EA). The histopathological grading of dysplasia is used to detect cancer risk in BE; however, considerable variability exists in the reporting of dysplasia. Molecular biomarkers that can detect BE patients with dysplasia would improve risk stratification in BE, enabling clinicians to focus on high risk patients requiring treatment and reduce endoscopic surveillance in the low risk group. The aim of this study was to identify and validate a gene expression signature as a biomarker that can objectively determine dysplastic status and thereby determine the risk of cancer progression. <h3>Methods</h3> Microarray gene expression profiling was done using 59 oesophageal samples with strict consensus diagnosis by expert pathologists (21 BE with no dysplasia, 10 BE with low grade dysplasia, 13 BE with high grade dysplasia and 8 EA). This data was used to identify a gene signature that separated non-dysplastic BE from high grade dysplasia. Gene expression data from publically available datasets were used to validate the signature. An independent set of 135 fresh frozen samples covering a spectrum of dysplastic Barrett’s stages and control tissue (40 BE with no dysplasia, 21 BE with low grade, 33 BE with high grade dysplasia, 32 EA and 9 duodenum) were used for validation using the high throughput 96:96 microfluidic Fluidigm® chip on the BioMark™ PCR system. <h3>Results</h3> A set of 90 genes was identified that separated BE with no dysplasia from BE with high grade dysplasia. This 90-gene signature was able to separate the remaining untrained samples on the microarray dataset (7 non-dysplastic, 10 low grade dysplasia and 8 EA). The signature also separated non dysplastic BE samples from EA samples on 2 external published datasets (p ≤ 0.0012). With the fresh frozen samples, the signature separated BE with no dysplasia from BE with dysplasia and EA with an area under the curve of 0.87 (95% CI, 0.80–0.93). Pathway analysis revealed that the RAN (RAs-related Nuclear protein) regulation pathway (p &lt; 0.0001) was the most significant pathway in this gene set. Furthermore, MYC was found to be the most significant transcription factor regulating at least 30% of these genes (p &lt; 0.0001). <h3>Conclusion</h3> The 90 gene-expression profile can reliably identify BE samples with dysplasia and cancer. This approach has the potential to provide robust risk stratification in BE samples as it overcomes the problems with variability in the reporting of dysplasia. <h3>Disclosure of Interest</h3> None Declared

644 Defining Cancer Risk in Barrett's Esophagus Using a 90-Gene Signature

Introduction Barrett’s oesophagus (BE) has a highly variable outcome with 0.12–0.5% of patients per year progressing to oesophageal adenocarcinoma (EA). The histopathological grading of dysplasia is used to detect cancer risk in BE; however, considerable variability exists in the reporting of dysplasia. Molecular biomarkers that can detect BE patients with dysplasia would improve risk stratification in BE, enabling clinicians to focus on high risk patients requiring treatment and reduce endoscopic surveillance in the low risk group. The aim of this study was to identify and validate a gene expression signature as a biomarker that can objectively determine dysplastic status and thereby determine the risk of cancer progression. Methods Microarray gene expression profiling was done using 59 oesophageal samples with strict consensus diagnosis by expert pathologists (21 BE with no dysplasia, 10 BE with low grade dysplasia, 13 BE with high grade dysplasia and 8 EA). This data was used to identify a gene signature that separated non-dysplastic BE from high grade dysplasia. Gene expression data from publically available datasets were used to validate the signature. An independent set of 135 fresh frozen samples covering a spectrum of dysplastic Barrett’s stages and control tissue (40 BE with no dysplasia, 21 BE with low grade, 33 BE with high grade dysplasia, 32 EA and 9 duodenum) were used for validation using the high throughput 96:96 microfluidic Fluidigm® chip on the BioMark™ PCR system. Results A set of 90 genes was identified that separated BE with no dysplasia from BE with high grade dysplasia. This 90-gene signature was able to separate the remaining untrained samples on the microarray dataset (7 non-dysplastic, 10 low grade dysplasia and 8 EA). The signature also separated non dysplastic BE samples from EA samples on 2 external published datasets (p ≤ 0.0012). With the fresh frozen samples, the signature separated BE with no dysplasia from BE with dysplasia and EA with an area under the curve of 0.87 (95% CI, 0.80–0.93). Pathway analysis revealed that the RAN (RAs-related Nuclear protein) regulation pathway (p Conclusion The 90 gene-expression profile can reliably identify BE samples with dysplasia and cancer. This approach has the potential to provide robust risk stratification in BE samples as it overcomes the problems with variability in the reporting of dysplasia. Disclosure of Interest None Declared

RanGTPase: A Candidate for Myc-Mediated Cancer Progression

Ras-related nuclear protein (Ran) is required for cancer cell survival in vitro and human cancer progression, but the molecular mechanisms are largely unknown. We investigated the effect of the v-myc myelocytomatosis viral oncogene homolog (Myc) on Ran expression by Western blot, chromatin immunoprecipitation, and luciferase reporter assays and the effects of Myc and Ran expression in cancer cells by soft-agar, cell adhesion, and invasion assays. The correlation between Myc and Ran and the association with patient survival were investigated in 14 independent patient cohorts (n = 2430) and analyzed with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. Myc binds to the upstream sequence of Ran and transactivates Ran promoter activity. Overexpression of Myc upregulates Ran expression, whereas knockdown of Myc downregulates Ran expression. Myc or Ran overexpression in breast cancer cells is associated with cancer progression and metastasis. Knockdown of Ran reverses the effect induced by Myc overexpression in breast cancer cells. In clinical data, a positive association between Myc and Ran expression was revealed in 288 breast cancer and 102 lung cancer specimens. Moreover, Ran expression levels differentiate better or poorer survival in Myc overexpressing breast (χ2 = 24.1; relative risk [RR] = 9.1, 95% confidence interval [CI] = 3.3 to 24.7, P < .001) and lung (χ2 = 6.04; RR = 2.8, 95% CI = 1.2 to 6.3; P = .01) cancer cohorts. Our results suggest that Ran is required for and is a potential therapeutic target of Myc-driven cancer progression in both breast and lung cancers.

Avalanche-like behavior in ciliary import

Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.

Reduced RAN Expression and Disrupted Transport between Cytoplasm and Nucleus; A Key Event in Alzheimer’s Disease Pathophysiology

Transcription of DNA is essential for cell maintenance and survival; inappropriate localization of proteins that are involved in transcription would be catastrophic. In Alzheimer’s disease brains, and in vitro studies, we have found qualitative and quantitative deficits in transport into the nucleus of DNA methyltransferase 1 (DNMT1) and RNA polymerase II (RNA pol II), accompanied by their abnormal sequestration in the cytoplasm. RAN (RAs-related Nuclear protein) knockdown, by siRNA and oligomeric Aβ42 treatment in neurons, replicate human data which indicate that transport disruption in AD may be mechanistically linked to reduced expression of RAN, a pivotal molecule in nucleocytoplasmic transport. In vitro studies also indicate a significant role for oligomeric Aβ42 in the observed phenomena. We propose a model in which reduced transcription regulators in the nucleus and their increased presence in the cytoplasm may lead to many of the cellular manifestations of Alzheimer’s disease.

Structural Determinants of Conformational Flexibility and Long-Range Allostery of the CRM1 Export Complex

In eukaryotes the nucleocytoplasmic transport of macromolecules is mainly mediated by soluble nuclear transport receptors of the karyopherin-β superfamily termed importins and exportins. The prototypical and highly versatile exportin CRM1 (chromosome region maintenance 1) is essential for nuclear depletion of numerous structurally and functionally unrelated protein and RNP cargoes. CRM1 has been shown to bind RanGTP (GTP bound RAs-related nuclear protein) and cargo proteins in an allosteric manner and to adopt a toroidal structure in several functional transport complexes. It was thought to maintain this conformation, with N- and C-terminal regions in close proximity, throughout the entire nucleocytoplasmic transport cycle.A recently solved structure of cargo-free CRM1 however revealed a superhelical, open conformation. Using molecular dynamics simulations, two distinct features of this conformation and their influence on the structural stability were investigated. One of those, the C-terminal helix, was identified as a major stabilising factor of the superhelical conformation.We furthermore showed that the overall configuration of CRM1 influences the local configuration of the cargo binding site. Based on these results we suggest a mechanism for the observed cooperative binding.

Downregulation of the small GTPase Ras-related nuclear protein accelerates cellular ageing

BackgroundThe small GTPase Ran, Ras-related nuclear protein, plays important roles in multiple fundamental cellular functions such as nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, by binding to either GTP or GDP as a molecular switch. Although it has been clinically demonstrated that Ran is highly expressed in multiple types of cancer cells and specimens, the physiological significance of Ran expression levels is unknown. MethodsDuring the long-term culture of normal mammalian cells, we found that the endogenous Ran level gradually reduced in a passage-dependent manner. To examine the physiological significance of Ran reduction, we first performed small interfering RNA (siRNA)-mediated abrogation of Ran in human diploid fibroblasts. ResultsRan-depleted cells showed several senescent phenotypes. Furthermore, we found that nuclear accumulation of importin α, which was also observed in cells treated with siRNA against CAS, a specific export factor for importin α, occurred in the Ran-depleted cells before the cells showed senescent phenotypes. Further, the CAS-depleted cells also exhibited cellular senescence. Indeed, importin α showed predominant nuclear localisation in a passage-dependent manner. ConclusionsReduction in Ran levels causes cytoplasmic decrease and nuclear accumulation of importin α leading to cellular senescence in normal cells. General significanceThe amount of intracellular Ran may be critically related to cell fate determination, such as malignant transformation and senescence. The cellular ageing process may proceed through gradual regression of Ran-dependent nucleocytoplasmic transport competency.

Functions of the small GTPase protein Ran in cell cycle regulation

RAN (Ras-Related Nuclear Protein) is a small GTPase that hydrolyzes GTP and plays a role as a “molecular switch” in cells. According to research using yeast and vertebrate cells, the functions of Ran include two features: for one, controlling nuclearcytoplasm transportation during the interphase stage, for another, mediating spindle assembly in the prophase and the rebuilding of nuclear-membranes during ana- and telophases. Although little is known about the function of Ran in higher plants, according to recent research in modal plants, Ran was found to control the cell cycle in several cell types. Particular research has indicated a relationship between Ran and the auxin signaling pathway. Consequently, the functions of the Ran proteins between different species are essentially conserved and show specificity.

Reduced Ran expression in Ran +/− fibroblasts increases cytokine-stimulated nuclear abundance of the AP-1 subunits c-Fos and c-Jun

Ran (Ras-related nuclear protein), a Ras family GTPase, is involved in multiple cellular functions, including the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing-exportation, and nuclear protein importation. Ran +/− embryonic stem (ES) cells were produced in an attempt to generate Ran null mutant mice. Even after an extremely large number of blastocyst injections, no Ran +/− chimeric mice could be generated. Ran +/− ES cell-derived fibroblasts showed reduced Ran protein expression, and manifested augmented nuclear abundance of AP-1 factors (c-Jun and c-Fos) upon cytokine stimulation. Our experiments demonstrated that intracellular Ran protein levels controlled the nuclear presence of certain transcription factors, such as c-Fos and c-Jun.

A Genetic Defect in Exportin-5 Traps Precursor MicroRNAs in the Nucleus of Cancer Cells

The global impairment of mature microRNAs (miRNAs) is emerging as a common feature of human tumors. One interesting scenario is that defects in the nuclear export of precursor miRNAs (pre-miRNAs) might occur in transformed cells. Exportin 5 (XPO5) mediates pre-miRNA nuclear export and herein we demonstrate the presence of XPO5-inactivating mutations in a subset of human tumors with microsatellite instability. The XPO5 genetic defect traps pre-miRNAs in the nucleus, reduces miRNA processing, and diminishes miRNA-target inhibition. The XPO5 mutant form lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and pre-miRNAs accumulate in the nucleus. Most importantly, the restoration of XPO5 functions reverses the impaired export of pre-miRNAs and has tumor-suppressor features.

Is Increasing the Copy Number of the MicroRNA Precursor in a Vector Really Helpful for Further Improving the Level of Mature Microrna in Stable Cells?

We read with great interest the article by Xu et al.1 The authors used lentiviral vectors to overexpress microRNA 122 (miR-122). They constructed three different vectors carrying one, four, or eight copies of the miR-122 precursor. Finally, the authors generated stable cells possessing eight copies of the miR-122 precursor. This is an ingenious method for further improving the expression level of miR-122. However, we think that some problems need to be discussed. First, how do we determine that the stable HepG2 cells possess eight copies of the miR-122 precursor when the cells are infected with lentiviral vectors carrying multiple copies? Why do we not use lentiviral vectors carrying 10 or more copies to further improve the level of miR-122? Are there data from the experiment using a gradually increasing number of miR-122 copies in vectors to construct miR-122–overexpressed stable cells? Recently, in our experimentation, HepG2 cells were transfected with vectors carrying a single copy of the miR-122 precursor. By screening different single clones, we obtained several HepG2 cell clones that had large differences in the expression level of miR-122. We deduced that the miR-122 precursor sequence could be integrated into several sites of the genome simultaneously. Information about the copy number can help us to choose optimal stable cell lines because of the existence of copy loss in the genome. We think that a stable cell line with a high copy number should be chosen when there are similar expression levels in different cell lines. Second, primary microRNA must be cleaved into precursor microRNA (pre-miRNA) by the nuclear RNase III endonuclease Drosha. Then, the pre-miRNA is actively transported from the nucleus to the cytoplasm by ras-related nuclear protein–guanosine triphosphate and the export receptor exportin 5.2, 3 The processes of enzyme digestion and export depend on not only the transcript sequences but also their space structure.4 We suppose that when eight or more copies of the miR-122 hairpin were chained to express in one primary transcript, the processing or export of pre-miRNA was likely influenced by the changes in the conformational construct of the primary transcript or pre-miRNA. Therefore, we want to know how to determine the optimal copy number of a microRNA precursor in a vector. In many studies, microRNA-overexpressed stable cell lines have been obtained by the integration of the microRNA precursor sequence into the cell genome.5, 6 In most cases, a vector carrying a single-copy precursor sequence has been chosen.7, 8 If the aforementioned problems can be solved with certainty, this novel method will be adopted widely in microRNA and RNA interference research, and a better cell model will be used in RNA-related research. Fang Wang*, Fu Yang*, Ling Zhang*, Shuhan Sun*, * Department of Medical Genetics, Second Military Medical University, Shanghai, China

RCC1 Uses a Conformationally Diverse Loop Region to Interact with the Nucleosome: A Model for the RCC1–Nucleosome Complex

The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is critical for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran ( Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the molecular mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. We have used biochemical approaches to test structural models for how the RCC1 β-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the β-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, we find that RCC1 uses a conformationally flexible loop region we have termed the switchback loop in addition to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.

PPAR Signaling Pathway and Cancer-Related Proteins Are Involved in Celiac Disease-Associated Tissue Damage

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of wheat gliadin and related proteins in genetically predisposed individuals. To find a proteomic CD diagnostic signature and to gain a better understanding of pathogenetic mechanisms associated with CD, we analyzed the intestinal mucosa proteome alterations using two dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF ms) of CD patients with varying degrees of histological abnormalities defined by Marsh criteria and controls. Our results clearly evidenced the presence of two groups of patients: Group A, including controls and Marsh 0-I CD patients; and Group B, consisting of CD subjects with grade II-III Oberhuber-Marsh classification. Differentially expressed proteins were involved mainly in lipid, protein and sugar metabolism. Interestingly, in Group B, several downregulated proteins (FABP1, FABP2, APOC3, HMGCS2, ACADM and PEPCK) were implicated directly in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Moreover, Group B patients presented a deregulation of some proteins involved in apoptosis/survival pathways: phosphatidylethanolamine-binding protein 1 (PEBP1), Ras-related nuclear protein (Ran) and peroxiredoxin 4 (PRDX4). PEBP1 downregulation and RAN and PRDX4 upregulation were associated with more severe tissue damage. Likewise, IgMs were found strongly upregulated in Group B. In conclusion, our results indicate that a downregulation of proteins involved in PPAR signaling and the modulation of several cancer-related proteins are associated with the highest CD histological score according to Oberhuber-Marsh classification.

Ran Overexpression Leads to Diminished T Cell Responses and Selectively Modulates Nuclear Levels of c-Jun and c-Fos

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.

Overexpression of OsRAN2 in rice and Arabidopsis renders transgenic plants hypersensitive to salinity and osmotic stress

Nucleo-cytoplasmic partitioning of regulatory proteins is increasingly being recognized as a major control mechanism for the regulation of signalling in plants. Ras-related nuclear protein (Ran) GTPase is required for regulating transport of proteins and RNA across the nuclear envelope and also has roles in mitotic spindle assembly and nuclear envelope (NE) assembly. However, thus far little is known of any Ran functions in the signalling pathways in plants in response to changing environmental stimuli. The OsRAN2 gene, which has high homology (77% at the amino acid level) with its human counterpart, was isolated here. Subcellular localization results showed that OsRan2 is mainly localized in the nucleus, with some in the cytoplasm. Transcription of OsRAN2 was reduced by salt, osmotic, and exogenous abscisic acid (ABA) treatments, as determined by real-time PCR. Overexpression of OsRAN2 in rice resulted in enhanced sensitivity to salinity, osmotic stress, and ABA. Seedlings of transgenic Arabidopsis thaliana plants overexpressing OsRAN2 were overly sensitive to salinity stress and exogenous ABA treatment. Furthermore, three ABA- or stress-responsive genes, AtNCED3, AtPLC1, and AtMYB2, encoding a key enzyme in ABA synthesis, a phospholipase C homologue, and a putative transcriptional factor, respectively, were shown to have differentially induced expression under salinity and ABA treatments in transgenic and wild-type Arabidopsis plants. OsRAN2 overexpression in tobacco epidermal leaf cells disturbed the nuclear import of a maize (Zea mays L.) leaf colour transcription factor (Lc). In addition, gene-silenced rice plants generated via RNA interference (RNAi) displayed pleiotropic developmental abnormalities and were male sterile.

Screening of potential molecular targets for colorectal cancer therapy

Colorectal cancer is a leading cause of cancer death worldwide. To identify molecular targets for colorectal cancer therapy, we tested small interfering RNAs (siRNAs) against 97 genes whose expression was elevated in human colorectal cancer tissues for the ability to promote apoptosis of human colorectal cancer cells (HT-29 cells). The results indicate that the downregulation of PSMA7 (proteasome subunit, alpha-type, 7) and RAN (ras-related nuclear protein) most efficiently induced apoptosis of HT-29 cells. PSMA7 and RAN were highly expressed in colorectal cancer cell lines compared with normal colon tissues. Furthermore, PSMA7 and RAN were overexpressed in not only colon tumor tissues but also the other tumor tissues. Moreover, in vivo delivery of PSMA7 siRNA and RAN siRNA markedly induced apoptosis in HT-29 xenograft tumors in mice. Thus, silencing of PSMA7 and RAN induces cancer cells to undergo apoptosis, and PSMA7 and RAN might be promising new molecular targets for drug and RNA interference-based therapeutics against colorectal cancer.

Involvement of N-acetyltransferase human in the cytotoxic activity of 5-fluorouracil

N-acetyltransferase human (NATH) participates in a posttranslational modification of the proteins and has been reported to play a role in apoptosis. In this study, the involvement of NATH in the cytotoxic action of 5-fluorouracil (5-FU) in human squamous cell carcinoma HEp-2 cells was examined. We found that 5-FU decreased NATH expression in a dose-dependent and time-dependent manner. No change was observed after treatment with bleomycin, nedaplatin, mitomycin C, or methotrexate. Interestingly, knockdown of NATH by small interfering RNA resulted in the downregulation of thymidylate synthase mRNA expression and induced apoptosis. Conversely, NATH overexpression facilitated cell proliferation independent of the presence of 5-FU. The effect of NATH knockdown on the expression of proteins in HEp-2 cells was examined using two-dimensional gel electrophoresis and mass spectrometry. Profilin 1, CutA, ras-related nuclear protein, annexin A5, enolase 1, and elongation factor 1 alpha 1 were found to be upregulated and 14-3-3eta, tublin, nuclear auto antigenic sperm protein, heat shock protein 70, and heat shock protein 90 were downregulated by knockdown of NATH. The results of this study suggest that NATH plays an important role in the cytotoxic activity of 5-FU.

DNA Replication Licensing and Progenitor Numbers Are Increased by Progesterone in Normal Human Breast

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast, we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing, including E2F transcription factors, chromatin licensing and DNA replication factor 1 (Cdt1), and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone, showing that progesterone influences epithelial cell lineage differentiation. Therefore, we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers.

Ran overexpression leads to a compromised transcription factor nuclear translocation and diminished T-cells response to TCR activation (35.8)

Abstract Ran (Ras-related nuclear protein) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 hours after their activation. Ran's function in the immune system was investigated. In transgenic T cells with actin promoter-driven Ran expression, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor (TCR) activation in vitro, compared with wild-type T cells. At the molecular level, transgenic T cells were defective in nuclear factor kappa-B (NFκB), activator protein 1 (AP-1) and nuclear factor of activated T cells (NFAT) nuclear translocation. Our experiments showed, for the first time, that Ran has important regulatory function in T-cell activation, and such function is probably related to its role in nuclear import of critical transcription factors.