Methylation alterations are not a major cause of PTTG1 misregulation.

BACKGROUND On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. METHOD We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. CONCLUSION Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of misregulation associated to PTTG1 over-expression.

Expression profiling of rectal tumors defines response to neoadjuvant treatment related genes.

To date, no effective method exists that predicts the response to preoperative chemoradiation (CRT) in locally advanced rectal cancer (LARC). Nevertheless, identification of patients who have a higher likelihood of responding to preoperative CRT could be crucial in decreasing treatment morbidity and avoiding expensive and time-consuming treatments. The aim of this study was to identify signatures or molecular markers related to response to pre-operative CRT in LARC. We analyzed the gene expression profiles of 26 pre-treatment biopsies of LARC (10 responders and 16 non-responders) without metastasis using Human WG CodeLink microarray platform. Two hundred and fifty seven genes were differentially over-expressed in the responder patient subgroup. Ingenuity Pathway Analysis revealed a significant ratio of differentially expressed genes related to cancer, cellular growth and proliferation pathways, and c-Myc network. We demonstrated that high Gng4, c-Myc, Pola1, and Rrm1 mRNA expression levels was a significant prognostic factor for response to treatment in LARC patients (p<0.05). Using this gene set, we were able to establish a new model for predicting the response to CRT in rectal cancer with a sensitivity of 60% and 100% specificity. Our results reflect the value of gene expression profiling to gain insight about the molecular pathways involved in the response to treatment of LARC patients. These findings could be clinically relevant and support the use of mRNA levels when aiming to identify patients who respond to CRT therapy.

Identification et caractérisation de gènes suppresseurs de tumeurs dans le glioblastome

Introduction: Glioblastoma (WHO Grade IV glioma) is the most frequent and most¦malignant primary tumor of the brain. With a mean survival of 15 months despite¦multidisciplinary management combining surgery, chemo- and radiotherapy, the prognosis¦is poor. Different studies measured a down-regulation of Wnt Inhibitory Factor 1 (WIF1)¦expression in a majority of gliobastoma due to genetic and epigenetic regulation. Recently,¦a focus on chromosome 12 identified WIF1 as a potential tumor suppressor gene. In¦previous results, transfected glioblastoma cells with ectopic expression of WIF1 had a¦decreased growth rate and adopted a senescence-like phenotype. In this report, we first¦investigated the effect of WIF1 re-expression in glioblastoma cell lines to see if Wnt¦inhibition by WIF1 can lead to senescence. To look further, we assessed p21 and c-Myc¦expression. p21 has a key role in senescence onset and is directly inhibited by c-Myc,¦itself a target of Wnt-pathway. We thus looked if a variation of expression of these genes is¦triggered by WIF1 activity. Finally, as autophagy is thought to play a role in senescence¦onset, we analyzed the expression of different autophagy genes. We therefore looked for¦an association between autophagy activity and senescent phenotype in WIF1-¦overexpressing cell lines.¦Methods: WIF1-overexpressing clones were selected after transfection of stable¦glioblastoma cell lines. Analysis were made through quantitative Polymerase Chain¦Reaction (qPCR), Fluorescence-activated Cell Sorting (FACS) and histochemistry.¦IGFBP7 and ALDH1A3 have been selected to reflect senescence. ATG5, ATG7 and ULK3¦have been selected to reflect autophagy activity.¦Results: Using FACS analysis, we found a higher percentage of large cells with increased¦granularity amongst WIF1-overexpressing cell lines, which are characteristics of¦senescence. In addition, histochemistry showed a higher percentage of multi-nucleated,¦beta-galactosidase positive cells in the same cell lines. An increased expression of genes¦associated with senescence was found as well. All characteristics were correlated with¦levels of WIF1 expression. We did not find any association between p21 and WIF1¦expression. No correlation between WIF1 and c-Myc expression was noticed either. In one¦of the two cell lines analyzed, the expression of autophagy genes showed some¦correlation with expression of WIF1 and expression of genes associated with senescence.¦Discussion: After investigations and characterizations on multiple levels, we have¦evidence for a senescence phenotype upon WIF1-overexpressing cell lines. This gives a¦role to Wnt pathway in the tumorigenicity of glioblastoma. Further experiments are¦required to investigate how Wnt inhibition leads to senescence. The role of autophagy in¦our senescent cells is here still unclear. Some correlations can be found, letting us think¦that there is indeed some involvement of autophagy. However, it is yet to soon to explain¦this relationship. Further experiments are required again to confirm the preliminary results¦and analyze the variations of autophagy activity within time.

Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector

Induced pluripotent stem (iPS) cells have generated keen interestdue to their potential use in regenerative medicine. They havebeen obtained from various cell types of both mice and humans byexogenous delivery of different combinations of Oct4, Sox2, Klf4,c-Myc, Nanog, and Lin28. The delivery of these transcription factorshas mostly entailed the use of integrating viral vectors (retrovirusesor lentiviruses), carrying the risk of both insertional mutagenesisand oncogenesis due to misexpression of these exogenousfactors. Therefore, obtaining iPS cells that do not carry integratedtransgene sequences is an important prerequisite for their eventualtherapeutic use. Here we report the generation of iPS cell linesfrom mouse embryonic fibroblasts with no evidence of integrationof the reprogramming vector in their genome, achieved by nucleofectionof a polycistronic construct coexpressing Oct4, Sox2, Klf4,and c-Myc

p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation.

In keratinocytes, the cyclin/CDK inhibitor p21(WAF1/Cip1) is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function was associated with down-regulation of Wnt signaling. Here, we show that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independently of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control.

Integrated methodologies to study human transcriptional regulation from functional genomics data

Abstract : The human body is composed of a huge number of cells acting together in a concerted manner. The current understanding is that proteins perform most of the necessary activities in keeping a cell alive. The DNA, on the other hand, stores the information on how to produce the different proteins in the genome. Regulating gene transcription is the first important step that can thus affect the life of a cell, modify its functions and its responses to the environment. Regulation is a complex operation that involves specialized proteins, the transcription factors. Transcription factors (TFs) can bind to DNA and activate the processes leading to the expression of genes into new proteins. Errors in this process may lead to diseases. In particular, some transcription factors have been associated with a lethal pathological state, commonly known as cancer, associated with uncontrolled cellular proliferation, invasiveness of healthy tissues and abnormal responses to stimuli. Understanding cancer-related regulatory programs is a difficult task, often involving several TFs interacting together and influencing each other's activity. This Thesis presents new computational methodologies to study gene regulation. In addition we present applications of our methods to the understanding of cancer-related regulatory programs. The understanding of transcriptional regulation is a major challenge. We address this difficult question combining computational approaches with large collections of heterogeneous experimental data. In detail, we design signal processing tools to recover transcription factors binding sites on the DNA from genome-wide surveys like chromatin immunoprecipitation assays on tiling arrays (ChIP-chip). We then use the localization about the binding of TFs to explain expression levels of regulated genes. In this way we identify a regulatory synergy between two TFs, the oncogene C-MYC and SP1. C-MYC and SP1 bind preferentially at promoters and when SP1 binds next to C-NIYC on the DNA, the nearby gene is strongly expressed. The association between the two TFs at promoters is reflected by the binding sites conservation across mammals, by the permissive underlying chromatin states 'it represents an important control mechanism involved in cellular proliferation, thereby involved in cancer. Secondly, we identify the characteristics of TF estrogen receptor alpha (hERa) target genes and we study the influence of hERa in regulating transcription. hERa, upon hormone estrogen signaling, binds to DNA to regulate transcription of its targets in concert with its co-factors. To overcome the scarce experimental data about the binding sites of other TFs that may interact with hERa, we conduct in silico analysis of the sequences underlying the ChIP sites using the collection of position weight matrices (PWMs) of hERa partners, TFs FOXA1 and SP1. We combine ChIP-chip and ChIP-paired-end-diTags (ChIP-pet) data about hERa binding on DNA with the sequence information to explain gene expression levels in a large collection of cancer tissue samples and also on studies about the response of cells to estrogen. We confirm that hERa binding sites are distributed anywhere on the genome. However, we distinguish between binding sites near promoters and binding sites along the transcripts. The first group shows weak binding of hERa and high occurrence of SP1 motifs, in particular near estrogen responsive genes. The second group shows strong binding of hERa and significant correlation between the number of binding sites along a gene and the strength of gene induction in presence of estrogen. Some binding sites of the second group also show presence of FOXA1, but the role of this TF still needs to be investigated. Different mechanisms have been proposed to explain hERa-mediated induction of gene expression. Our work supports the model of hERa activating gene expression from distal binding sites by interacting with promoter bound TFs, like SP1. hERa has been associated with survival rates of breast cancer patients, though explanatory models are still incomplete: this result is important to better understand how hERa can control gene expression. Thirdly, we address the difficult question of regulatory network inference. We tackle this problem analyzing time-series of biological measurements such as quantification of mRNA levels or protein concentrations. Our approach uses the well-established penalized linear regression models where we impose sparseness on the connectivity of the regulatory network. We extend this method enforcing the coherence of the regulatory dependencies: a TF must coherently behave as an activator, or a repressor on all its targets. This requirement is implemented as constraints on the signs of the regressed coefficients in the penalized linear regression model. Our approach is better at reconstructing meaningful biological networks than previous methods based on penalized regression. The method is tested on the DREAM2 challenge of reconstructing a five-genes/TFs regulatory network obtaining the best performance in the "undirected signed excitatory" category. Thus, these bioinformatics methods, which are reliable, interpretable and fast enough to cover large biological dataset, have enabled us to better understand gene regulation in humans.

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1.

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317-326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317-326 sequence of RasGAP (TAT-RasGAP₃₁₇₋₃₂₆), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP₃₁₇₋₃₂₆ did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP₃₁₇₋₃₂₆ was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.

GLUT8 subcellular localization and absence of translocation to the plasma membrane in PC12 cells and hippocampal neurons.

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

Maf1, a new player in the regulation of human RNA polymerase III transcription.

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the alpha-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.

Human Gene Expression in Uncomplicated Plasmodium falciparum Malaria.

To examine human gene expression during uncomplicated P. falciparum malaria, we obtained three samples (acute illness, treatment, and recovery) from 10 subjects and utilized each subject's recovery sample as their baseline. At the time of acute illness (day 1), subjects had upregulation of innate immune response, cytokine, and inflammation-related genes (IL-1β, IL-6, TNF, and IFN-γ), which was more frequent with parasitemias >100,000 per μL and body temperatures ≥39°C. Apoptosis-related genes (Fas, BAX, and TP53) were upregulated acutely and for several days thereafter (days 1-3). In contrast, the expression of immune-modulatory (transcription factor 7, HLV-DOA, and CD6) and apoptosis inhibitory (c-myc, caspase 8, and Fas Ligand G) genes was downregulated initially and returned to normal with clinical recovery (days 7-10). These results indicate that the innate immune response, cytokine, and apoptosis pathways are upregulated acutely in uncomplicated malaria with concomitant downregulation of immune-modulatory and apoptosis inhibitory genes.

A biologia molecular no prognóstico do carcinoma da tireóide

This overview examines some selected genetic mechanisms of cancer development. Strong evidence has been accumulated suggesting that alteration in either the struture or activity of proto-oncogene contributes to the development and for the maintenance of the malignant phenotype. Many factors are known to interfere with both normal and pathological controls of growth and differentiation of thyroid cells. Among them, some are oncogenes, like those encoding g-proteins (ras, gsp, TSH-R), encoding thyrosino kinases receptors (RET, trk, c-met, c-erb, BRAF) and encoding nuclear proteins (c-myc, e-fós). Others are anti-oncogenes (p53, p15, RB), by loss of the growth suppression ativity of the suppressive gene. Cancer cell invasion and metastasis are the major causes of morbidity and mortality in cancer patients. Many genes are involved in the mechanism of invasion and metastasis of thyroid tumors, like Nis, b-catenina, E-caderina, galectina-3, GLUT, telomerase, VEGT, nm-23. All these oncogenes, antioncogenes and tumor invasion and metastasis-related genes are analysed. Several clinical and prognostic factors have been proposed to identify patients at risk for the development of metastasis and death. The role of molecular genetics in this issue is discussed. However, other studies are needed to validate molecular alterations as an independent prognostic factor in thyroid cancer.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Proliferative signaling initiated in ACTH receptors

This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a) rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) (2 to 10 min), b) transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min), c) induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d) onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3% positive cells), alpha3 (93.3 ± 7.0%), alpha5 (50.4 ± 12.0%) and alpha6 (34.1 ± 4.9%) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0%) and fibronectin (40.0 ± 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4% vs DMSO: 70.7 ± 2.5%) while simultaneously reducing alpha5 (24.2 ± 19%) and alpha6 (14.3 ± 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.