Haut PDF Engrailed genes in mammary development and tumorigenesis

Engrailed genes in mammary development and tumorigenesis

Engrailed genes in mammary development and tumorigenesis

HOXC6 is oflen overexpressed in human leukemia yet Hoxc6 transcripts were found to decrease during pregnancy when the mammaiy gland is highly proliferative and HOXC6 is expressed at low [r]

158 En savoir plus

Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis

Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis

Panx1 is dynamically expressed in the mammary gland Panx1 has an ubiquitous expression profile and has been reported in the mouse mammary gland based on expression profiling arrays in NCBI ’s gene expression Omnibus database (ID 1416379, 78225667 [ 17 ]). In the murine mammary gland, Panx1 is expressed and upregulated during pregnancy where it remains elevated during lactation. Developmental regulation of Panx1 is associated with higher expression at earlier stages of development in many organs including the neonatal rat brain and murine newborn skin compared to aged counterparts [ 14 , 16 , 24 ]. Importantly, primary human muscle myoblasts induced to differentiate in culture upregulate the expression of Panx1 while ectopic expression of Panx1 in these cells induces dif- ferentiation in vitro [ 15 ]. Collectively, these results suggest a critical role for Panx1 in cell dif- ferentiation [ 15 ]. Unlike other organs, the mammary gland develops only a rudimentary ductal structure in prenatal mice and requires the onset of pregnancy to induce terminal differentia- tion of the gland [ 1 ]. Therefore, expression of Panx1 during pregnancy and lactation builds on the idea that Panx1 is upregulated in organs undergoing development and differentiation. As such, it might be expected that Panx1 is expressed in the embryonic mammary gland. While we cannot rule this out, loss of Panx1 does not significantly impair the ability of the gland to develop a rudimentary ductal structure and undergo normal ductal development during puberty in the virgin mammary gland. As a result, Panx1 may be more important in the preg- nant than the embryonic mammary gland. This is similar to the gap junction, channel protein, Cx26, which has a critical role after the onset of pregnancy while being less important at earlier cytokeratin (green) revealed a significant decrease in the amount of epithelium in the mammary gland of Panx1 -/- mice compared with control mice. (E) Quantification of the average number of adipocytes, as assessed with perilipin (green), revealed similar cell numbers in the mammary gland of Panx1 -/- mice compared with control mice. Hoescht (blue) denotes nuclei. Values are means ± SEM. N = 6. Scale bars = 50 um.
En savoir plus

23 En savoir plus

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

[ 31 ] and examined the clonal growth of Notch1-expressing cells in the presence of three differ- ent hormones: β-estradiol (β-2E), progesterone (Pg) or prolactin (Prl). After 10 d in culture, we observed a net and prominent expansion of GFP pos cells only in response to estrogen ( β-estra- diol) ( Fig. 6E,F ). To confirm that each exogenously added hormone efficiently activated its spe- cific pathway, we assessed by qRT-PCR the expression of the immediate target genes activated in response to each hormonal treatment: progesterone receptor (Pgr) for estradiol signaling, Rank ligand (Tnfs11) for progesterone stimulation, and β-Casein (Csn2) for Prolactin [ 32 – 34 ]. A significantly elevated expression of mRNAs for each of these target genes, in response to stimulation with the corresponding hormone, clearly shows that cells grown as mammary organoids correctly respond to physiologic hormones ( S6C Fig .). Of note, we could not detect any synergistic effect when we concomitantly treated the organoids with both estrogen and progesterone ( Fig. 6E,F ). These findings in ex vivo cultures, while not able to recapitulate the sequential waves of hormones that occur in vivo, corroborate the results we obtained in vivo during pregnancy and are consistent with the identification of Notch1-expressing cells as the estrogen-responsive cells of the mammary gland, stimulated in a paracrine fashion by neigh- boring ERα pos cells [ 17 ]. As further discussed below, the ability of these cells to respond to hor-
En savoir plus

25 En savoir plus

BRCA1 expression during prenatal development of the human mammary gland

BRCA1 expression during prenatal development of the human mammary gland

Robert Dante* ,1 1 Laboratoire de GeÂneÂtique, UMR 5641 CNRS, Domaine Rockefeller, UCBL1, 8 avenue Rockefeller, 69373 Lyon cedex 08, France Germ-line alterations of BRCA1 are associated with elevated risk of breast cancer. Evidence for the involvement of Brca1 in cellular di€erentiation and morphogenesis has been obtained in mouse models during embryogenesis. Although the presence of well-conserved functional domains might suggest a similar function for both human and mouse genes, very few data on BRCA1 expression in human fetal tissues are available. We have, therefore, investigated the expression of BRCA1 in the mammary gland from human female fetuses aged between 15 and 33 weeks. Quanti®cation of BRCA1 transcripts, using a competitive reverse transcriptase PCR method, indicates a progressive decrease in BRCA1 expression with increasing fetal age between the 15th and 30th week of gestation. Subsequently, the amount of BRCA1 transcripts becomes similar to that found in adult mammary gland. Analysis of BRCA1 protein revealed, in fetal samples, a 220 kDa band corresponding to the 220 kDa BRCA1 protein described in human cell lines. These later experiments con®rm that the relative level of the 220 kDa BRCA1 protein is highest in the early stages of mammary gland development. The temporal patterns of BRCA1 expression in human fetuses suggest a role for BRCA1 in the morphogenesis and di€erentiation of the human mammary gland. Keywords: BRCA1; mRNA; protein; prenatal develop- ment; human mammary gland
En savoir plus

6 En savoir plus

The Prion-like protein Shadoo is involved in mouse embryonic and mammary development and differentiation

The Prion-like protein Shadoo is involved in mouse embryonic and mammary development and differentiation

transcriptomic analyses. Transcriptomic analyses were performed using Affymetrix MouseGene 2,OSt microarray on the Institute Cochin Genom’ic platform. Each analyzed embryonic RNA sample derived from the equal mixing of 6 individual total RNA preparations from E7.5 embryos. Different embryonic samples were used for each mixture. Similarly, each analyzed placenta RNA sample derived from the equal mixing of 6 indi- vidual total RNA preparations from E14.5 placentas. Different individual samples were used for each mixture. Each MG RNA sample derived from the equal mixing of 3 individual total RNA preparations from G7.5 MGs. Different individual samples were used for each mixture. Three biological replicates per tissue and genotype were performed. Differentially expressed genes between FVB/NJ and FVB/NJ Sprn-knockout tissues were identified following normalization of the raw data, with fold chance ratios and p values determined as described in the results section. Data were analyzed by Gene set enrichment analysis (GSEA, http://software.broadinstitute.org/ gsea/index.jsp ). Differentially expressed genes were clustered and classified in pathways and networks by using Ingenuity ( http://www.ingenuity.com/ ).
En savoir plus

14 En savoir plus

Identification, for ruminants, of genes and gene networks involved in differentiation and activity of the mammary gland

Identification, for ruminants, of genes and gene networks involved in differentiation and activity of the mammary gland

lacking the DGAT1 enzyme have impaired mammary gland development, characterized by decreased epithelial proliferation and alveolar development (Cases et al., 2004). Nevertheless, genes involved in milk lipid biosynthesis as LPL, the long chain acylCoA synthetase homolog 1 (ACSL1) and lipin1 (LPIN1), as well as in lactose synthesis, such as UGP2 and B4GALT1 (metacluster D and cluster 17) are only expressed in lactation in the goat mammary gland (Table II.5). Likewise, Akt1, a serine/threonine protein kinase which plays a key role in the regulation of glucose transport and lipid metabolism (Anderson et al., 2007; Schwertfeger et al., 2003) displays a significant increase in expression, between P110 and L40 (metacluster D). Butyrophilin (BTN1A1) which is supposed to be an activator of milk secretion (Ogg et al., 2004) followed the same pattern (Table II.5 and Figure II.3A). SREBF1 (Sterol regulatory element binding transcription factor 1) which represents a central node in the milk lipid metabolism network and controls transcription of most of the genes which regulate milk fat synthesis in mice (Rudolph et al., 2003) and cattle (Bionaz & Loor, 2008), was also shown in our study to be a critical regulator of transcription as many genes regulated by this factor showed the same expression profile (ACACA, FASN, LPL, ACSL1 and INSIG1).
En savoir plus

236 En savoir plus

Multimodal In Vivo Imaging of Tumorigenesis and Response to Chemotherapy in a Transgenic Mouse Model of Mammary Cancer

Multimodal In Vivo Imaging of Tumorigenesis and Response to Chemotherapy in a Transgenic Mouse Model of Mammary Cancer

important to assess response to neoadjuvant drug therapy as early as possible in order to avoid unnecessary drug exposure. Functional and metabolic properties of tissues, including tumors, respond more rapidly than gross anatomy to changes in the environment; therefore, functional and molecular imaging modalities likely to monitor tumor response to treatment more precociously than tumor size are an important field in cancer research. Because they are based essentially on the same techni- ques than clinical imaging, small animal imaging devices are able to provide longitudinal follow-up in preclinical studies of new anticancer drugs [ 16 ]. Here, we compared the ability of several imaging methods to document the natural course of mammary tumor development in PyMT mice. We then tested the ability of standard imaging observables derived from these methods to document precociously, precisely, and predictively the response to chemotherapy.
En savoir plus

11 En savoir plus

Myc is required for beta-catenin-mediated mammary stem cell amplification and tumorigenesis.

Myc is required for beta-catenin-mediated mammary stem cell amplification and tumorigenesis.

We recently showed that Myc deletion from the mam- mary basal cell layer affects stem cell activity [22]. Consist- ent with data for other tissues [7,28] microarray analysis of Myc-deficient mammary basal cells showed impaired expression of numerous genes involved in important cell functions, including metabolism, replication, protein syn- thesis and the cell cycle, relating to the capacity of the cell to proliferate (Additional file 5: Table S1). Many of these genes were deregulated in the mammary tumors devel- oped by K5ΔNβcat mice (Additional file 6: Table S2). Interestingly, about half of these genes (40 out of 85) were found to be upregulated in the basal-like human breast tumors [26], (Additional file 6: Table S2). The expression of a set of proliferation-related genes upregulated in K5ΔNβcat tumors was analyzed by qPCR in mammary basal cells from K5ΔNβcat;K5Cre;Myc F/F mice and control basal cells. In the absence of Myc, these genes were down- regulated (Figure 4D), suggesting that Myc deletion im- peded β-catenin-induced tumorigenesis, probably by restricting the acute growth program required for the amplification of basal progenitors and tumor formation.
En savoir plus

12 En savoir plus

Mechanotransduction's Impact on Animal Development, Evolution, and Tumorigenesis

Mechanotransduction's Impact on Animal Development, Evolution, and Tumorigenesis

HYPERPROLIFERATION AND INCREASED STIFFNESS IN TUMOR DEVELOPMENT MECHANICALLY REACTIVATES EMBRYONIC β-CATENIN–DEPENDENT DEVELOPMENTAL PATHWAYS Our understanding of tumor progression has progressed considerably as a result of intensive biochemical and genetic studies of the pathways and master genes involved in the deregulation of tissue homeostasis (Hanahan & Weinberg 2011). The role of the microenvironment in tu- mor progression and invasion is being intensively studied (Bissell & Radisky 2001). Within this context, pioneering approaches have implicated the fibrotic rigidity of the tumor microenviron- ment in enhancing late-stage tumor progression (Butcher et al. 2009, Ghajar & Bissell 2008, Wozniak & Chen 2009). This increase in rigidity may be characterized by an anomalous boost of actomyosin activity in cells adapting to the fibrotic microenvironment. Increased actomyosin activity is believed to activate mechanotransduction pathways as a result of the local increase in tension applied to cell junctions, particularly adherens junctions, possibly leading to subsequent cytoskeleton reorganization (Butcher et al. 2009, Ghajar & Bissell, 2008, Grashoff et al. 2010, Sawada et al. 2006). Following these results, a cascade of studies has focused on the role of the microenvironment in tumor progression from the nanoscale level to the tissue level. The evolving extracellular matrix, the scaffold for tissue organization, provides both biochemical and biome- chanical signals that modulate tissue development and homeostasis and, when altered, critically influence tumor evolution (DuFort et al. 2011, Pickup et al. 2014, Yu et al. 2011). For example, the oncogenic mechanical engagement of vinculin enhances PI3-K activation of phosphatidyl- inositol (Rubashkin et al. 2014). Most recently, a 3D substrate with low stiffness was shown to
En savoir plus

29 En savoir plus

RIP140 increases APC expression and controls intestinal homeostasis and tumorigenesis.

RIP140 increases APC expression and controls intestinal homeostasis and tumorigenesis.

was associated with the accumulation of β -catenin and overexpression of the β - catenin target genes : Cyclin D1 and c-Myc (5). The transcription cofactor RIP140 (receptor interacting protein, 140 kDa), also known as NRIP1 (nuclear receptor-interacting protein 1), was first identified in human cancer cells through its interaction with estrogen receptor α (6). RIP140 was also shown to interact with many other nuclear receptors and transcription factors (for a review see (7)). More recently, we demonstrated that RIP140 also behaves as an Rb-like regulator of the E2F pathway by directly binding to E2Fs and repressing their transactivation potentials (8). RIP140 mainly acts as a transcriptional repressor by means of four inhibitory domains that recruit histone deacetylases or C-terminal binding proteins (9). Several post-translational modifications, such as sumoylation and acetylation, also play important roles in controlling the subcellular location and repressive activity of RIP140 (for a review (10)). RIP140 is a ubiquitously expressed gene whose transcription is finely regulated at the transcriptional levels both by nuclear receptors and E2Fs (11). The physiological importance of RIP140 has been evaluated using mice that lack the Rip140 gene (RIPKO mice). These animals are viable but display a wide range of phenotypic alterations in various tissues and organs such as infertility of female mice (12) or reduced body fat content (13), and, more recently, severe cognitive impairments (14) and mammary gland morphogenesis (15).
En savoir plus

50 En savoir plus

Sexual dimorphism in adrenal gland development and tumorigenesis

Sexual dimorphism in adrenal gland development and tumorigenesis

administration) [24] . Hierarchical clustering revealed 269 genes differentially expressed in males vs females, 71 of which were regulated by changes in the androgenic milieu, suggesting that in addition to male sex hormones, other potential factors including sex chromosomee encoded information could explain those differences. In coherence with these early studies, gene expression data obtained from wild-type and mutant mice developing Cushing syndrome confirmed that samples from cas- trated males clustered close to female samples. This indicates that in mice, androgens should be the main determinant of adrenal sex dimorphism in both physi- ology and disease [15] . The time points of sexual dimorphism in gene expression can be seen until em- bryonic day 14.5 for Cyp17, which is weakly expressed in males and intensively in females. By embryonic day 18.5, Cyp17 is no longer expressed in male adrenals but is still highly expressed in those of females [25] . The role of this early dimorphic expression is not well
En savoir plus

7 En savoir plus

Denervation suppresses gastric tumorigenesis

Denervation suppresses gastric tumorigenesis

availability of metastatic models of gastric cancer, the effect of denervation in metastatic lymph nodes or other organs remains unclear and needs to be further investigated in suitable models. Previous studies suggested that nerves contribute to the normal stem cell niche (1, 2, 38), and a recent report has linked sympathetic nerves to prostate cancer progression (7). However, the stomach differs from other solid organs in that its autonomic innervation is largely parasym-pathetic in nature, and cholinergic nerves have been shown to regulate gastrointestinal proliferation (39). The present study demonstrated that Lgr5 + gastric stem cells express the M 3 receptor, and that Wnt signaling in those cells is directly activated by cholinergic vagus stimulation, resulting in epithelial proliferation and stem cell expansion. Gastrointestinal stem cells are supported by a number of niche cells including Paneth cells, mesenchymal stem cells, myofibroblasts, smooth muscle cells, lymph and vascular endothelial cells, and bone marrow–derived stromal cells (40–42). Here, we identified nerves regulating gastric stem cell expansion during the tumorigenesis.
En savoir plus

26 En savoir plus

Chromatin assembly factors and heterochromatin organization during cell prolifertaion, tumorigenesis and in quiescence

Chromatin assembly factors and heterochromatin organization during cell prolifertaion, tumorigenesis and in quiescence

Histone   modifications   are   established   and   removed   by   a   series   of   enzymes,   including   histone   acetyl   transferases   (HAT),   histone   deacetylases   (HDAC),   histone   methyl  transferases  (HMT)  and  histone  demethylases  (HD).  In  each  group  of  enzymes,   different   members   often   show   specificity   toward   a   certain   histone   residue.   The   most   diverse   group   is   the   histone   deacetylases,   which   contains   18   members   in   mammalian   cells  (reviewed  in  Witt  et  al,  2009).  This  group  is  further  divided  into  four  classes,  based   on  sequence  homology  with  their  yeast  orthologues.  HDACs  of  class  I  (HDAC1,  2,  3  &  8)   are   expressed   in   a   ubiquitous   manner,   while   HDACs   of   classes   II,   III   and   IV   are   tissue   specific   and   might   thus   contribute   to   development   and   cell   identity.   Many   HDACs   not   only  deacetylate  histone  proteins,  but  also  other  nuclear  or  cytoplasmic  proteins  such  as   the  tumor  suppressor  protein  p53  (Luo  et  al,  2000)  or  tubulin  (Zhang  et  al,  2003a).  It  is   noteworthy  that  histone  methylation,  which  can  occur  on  lysine  and  arginine  residues,   was   until   recently   considered   as   a   very   stable   mark   with   low   turnover   (Byvoet   et   al,   1972).  The  discovery  of  Lysine-­‐Specific  Demethylase  1  (LSD1)  (Shi  et  al,  2004),  which   demethylates  H3K4me2  though  an  oxidation  reaction,  therefore  constituted  a  landmark   in  our  understanding  of  chromatin  dynamics.  Since  then,  a  large  family  of  other  histone   demethylases,   which   share   a   so-­‐called   Jumonji   C   catalytic   domain,   has   been   described   (reviewed   in   Agger   et   al,   2008).   The   reversibility   of   all   histone   modifications   underscores   the   dynamic   nature   of   chromatin   organization   and   raises   again   the   question   of   which   marks   could   be   considered   as   epigenetic,   and   how   these   can   be   transmitted  through  cell  division.    
En savoir plus

251 En savoir plus

Comment faire beaucoup avec peu dans le système nerveux - Engrailed recrute la mitochondrie

Comment faire beaucoup avec peu dans le système nerveux - Engrailed recrute la mitochondrie

distinctes nécessaire à l’élaboration d’un réseau synaptique complexe (10 15 connexions chez l’homme) semble excé- der les capacités codantes du génome (30 000 gènes). Aujourd’hui, le problème ne se pose plus en ces termes : les molé- cules impliquées dans le guidage se sont avérées être polyvalentes, donc le concept de gènes exclusivement dédiés à une fonction de guidage semble avoir fait long feu. Des protéines d’abord décrites comme facteurs de guidage axonal (éphrines et sémaphorines) ont en effet été impliquées ensuite dans d’autres fonctions, comme l’angioge- nèse ou la plasticité synaptique. Inver- sement, des protéines dont on ne soup- çonnait pas initialement le rôle dans le guidage interviennent dans ce proces- sus. C’est le cas de morphogènes (Shh [sonic hedgehog], certaines homéopro- téines, Wnt, FGF [fibroblast growth fac- tor], BMP [bone morphogenic protein]) dont la fonction classique est la mise en place précoce des structures primaires de l’embryon. Utilisées plus tardive- ment au cours du développement, ces protéines vont en fait participer aussi à l’élaboration de la circuiterie ner- veuse. Engrailed, une homéoprotéine qui intervient très tôt dans la spécification du territoire mésencéphalique dans le cerveau embryonnaire, contribue plus tard au guidage et à la stabilisation des axones rétiniens par des mécanismes originaux mis à jour récemment.
En savoir plus

4 En savoir plus

The roles of the NOOT-BOP-COCH-LIKE genes in the symbiotic organ identity and in plant development

The roles of the NOOT-BOP-COCH-LIKE genes in the symbiotic organ identity and in plant development

Figure 6 | Indeterminate nodule organization General anatomical features of the indeterminate root nodules. Infection events occur generally in front of a xylem pole but sometimes the infection occurs facing a phloem pole (Hirsch 1992; Heidstra et al., 1997; Guinel, 2009). Here the nodule developed from an infection opposite a phloem pole. The indeterminate nodule show 6 different zones from young to old (right to left). The zone I consists in an uninfected nodule meristem. Discrete cell domains add cells for growth of various nodule tissues. The zone II corresponds to the infection thread penetration zone. The inter-zone II/III is enriched in large amyloplasts. The zone III corresponds to the nitrogen fixation zone. The zone IV corresponds to the senescence zone and the last zone V corresponds to a saprophytic zone. Zones II–V represent stages of differentiation of the bacteroid-containing tissue. This tissue is surrounded with cortical layers (from outer to inner layer): parenchymatous outer cortex (OC) of non-specialized cells, cortical endodermis (CE), which is a monolayer of lignified cells that together with specialized cells located within the inner cortex (IC) contribute to the maintenance of micro-oxic conditions necessary for nitrogenase activity. The CE is continuous with the root endodermis. Vascular system of the nodule consists of inner cortex- located nodule vascular bundles (NVB) that bifurcate from a nodule vascular trace (NVT), which connects the nodule with the root vascular tissues. Each NVB possess its own nodule vascular meristem (NVM). The bifurcation, which usually occurs close to the base of the bacteroid containing tissues, has been omitted in the drawing to show that apical NVB portions are connected with the nodule meristem. NVT and NVB are sheathed in a vascular endodermis (VE, not to the scale) continuous with the root endodermis and of similar ultrastructure. Ep, root epidermis; Rc, root cortex, En, root endodermis; Pe, root pericycle; ph, phloem pole; x, xylem pole. Adapted from Lotocka et al. (2012), Guinel, (2009) and Bond, (1948).
En savoir plus

314 En savoir plus

Complex interplay between {beta}-catenin signalling and Notch effectors in intestinal tumorigenesis.

Complex interplay between {beta}-catenin signalling and Notch effectors in intestinal tumorigenesis.

We then investigated whether the deregulation of Notch signalling occurred early in b -catenin-induced intestinal tumori- genesis, by analysing Hes1 expression in a conditional mouse model with acute APC loss in the adult intestinal epithelium (Apc lox/lox Vil-CreER T2 ). 3 In this system, Apc was deleted in response to tamoxifen injection (Apc-null mice, Apc / ). This system is highly efficient, with almost 100% recombination of the target allele in the small intestine of adult mice and a rapid loss of Apc function. 3 This genetic deletion system did not mimic the stochastic process of tumour initiation occurring in Apc +/ mice, but it did reproduce all the phenotypes previously identified as associated with early intestinal lesions. As previously reported, 5 days after tamoxifen injection, considerable enlargement of the crypt compartment, with intense cell proliferation, apoptosis and severe defects in differentiation, was observed in the intestine of mutant mice. 3 This enlargement was accompanied by Hes1 induction, at both the mRNA and protein levels (figure 3AeC). Accordingly, we observed nuclear staining for Hes1 in all dysplastic cells with nuclear b -catenin, consistent with Apc loss (figure 3C). This increase in Hes1 expression could result from hyperactive b -catenin signalling or it could occur because of an expansion of cells that normally express Hes1. To distinguish between these two hypotheses, we analysed Hes1 expression in a human CRC cell line in which hyperactive b -catenin signalling was blocked. SW480 cells, which produce a truncated form of APC that cannot target b -catenin for degradation, were transfected with two different small interfering RNAs (siRNAs) targeting b -catenin. We found that Hes1 expression was markedly decreased by b -catenin depletion (figure 3D).
En savoir plus

13 En savoir plus

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development

Western blots After perfusion, a portion of the ventricles from TRECUGBP1/MHC or MHC control animals were lysed in HEPES-sucrose buffer (10 mM HEPES pH 7.4, 0.32 M sucrose, 1 mM EDTA, and proteases inhibitors) using Bullet blender (Next Advance) equipment and SDS was added (final concentration: 1% SDS). Samples were sonicated (3 min at 75 V: 30 sec ON and 30 sec OFF) and centrifuged for 10 min at 14,000 r.p.m at 4°C. Supernatants were transferred to new tubes and protein concentration was estimated with Pierce BCA protein assay kit (Thermo Scientific). Samples were diluted in loading buffer (100 mM Tris- HCl pH 6.8, 4% SDS, 0.2% Bromophenol blue, 20% glycerol, 200 mM β-mercaptoethanol), boiled for 5 min, and total proteins (40 µg) were assayed by 10% SDS-PAGE. After transfer, membranes were blocked in 5% non-fat dried milk/0.1% Tween-TBS buffer (TTBS) for 1 hour, washed and incubated overnight at 4°C with primary antibodies diluted in 5% milk/ TTBS: mouse monoclonal anti-CUG-BP1, clone 3B1 (Milipore, #05–621) (1:1,000), rabbit polyclonal anti-sarcomeric alpha actinin (Abcam, #ab72592) (1:2,000). The following day, membranes were incubated with secondary antibodies (1:5,000) for 1 hour at room temperature: peroxidase-conjugated goat anti-mouse IgG light chain specific (Jackson Immunoresearch, #115-035-174), goat anti-rabbit IgG horseradish peroxidase-conjugated (Invitrogen, # 621234). Flag-tag was detected by monoclonal anti-FLAG M2 peroxidase
En savoir plus

32 En savoir plus

Expression profiling of development related genes in rice plants ectopically expressing AtTOR

Expression profiling of development related genes in rice plants ectopically expressing AtTOR

The seven times diluted cDNA was used for qRT-PCR analysis of different developmental genes using SYBR Green ® Premix (Takara Bio, Clonetech, USA). The primers used in qRT-PCR have been listed in Table 1. The qRT PCR reaction conditions included an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 15 sec, with annealing temperature ranging

18 En savoir plus

Alkylation-induced colon tumorigenesis in mice deficient in the Mgmt and Msh6 proteins

Alkylation-induced colon tumorigenesis in mice deficient in the Mgmt and Msh6 proteins

Abstract O 6 -methylguanine DNA methyltransferase (MGMT) suppresses mutations and cell death that result from alkylation damage. MGMT expression is lost by epigenetic silencing in a variety of human cancers including nearly half of sporadic colorectal cancers, suggesting that this loss maybe causal. Using mice with a targeted disruption of the Mgmt gene we tested whether Mgmt protects against azoxymethane (AOM) induced colonic aberrant crypt foci (ACF), against AOM and dextran sulfate sodium (DSS) induced colorectal adenomas, and against spontaneous intestinal adenomas in Apc Min mice. We also examined the genetic interaction of the Mgmt null gene with a DNA mismatch repair null gene, namely Msh6. Both Mgmt and Msh6 independently suppress AOM-induced ACF, and combination of the two mutant alleles had a multiplicative effect. This synergism can be explained entirely by the suppression of alkylation-induced apoptosis when Msh6 is absent. In addition, following AOM+DSS treatment Mgmt protected against adenoma formation to the same degree as it protected against AOM-induced ACF formation. Finally, Mgmt deficiency did not affect spontaneous intestinal adenoma development in Apc Min/+ mice,
En savoir plus

14 En savoir plus

B Cells Promote Pancreatic Tumorigenesis

B Cells Promote Pancreatic Tumorigenesis

Main text Treatment of pancreatic cancer has proven very difficult because of both the non-immunogenic nature of the tumor and the late stages at diagnosis. This is reflected in the abysmal 5-year survival rate of ~7% and the harsh standard-of-care (SOC) for patients. The most common treatment, aside from palliative care or surgery in eligible patients, is gemcitabine, which is a general DNA damaging agent. However, those patients healthy enough to handle strong treatment side effects are given a cocktail of four drugs called FOLFIRINOX in the hope of stalling tumor development (FDA.gov). Other FDA-approved drug combinations may be tried as the first-line treatment, but all have appalling side effects impacting patient quality of life without much measurable benefit. Although no immunotherapies are currently approved for treating pancreatic cancers, there are 16 clinical trials of various immunotherapies for pancreatic cancer (clinicaltrials.gov). These trials fall into two categories: combination therapies and vaccines. Most of the combination therapies involve adding an immunotherapy like checkpoint blockade, vaccine, or cytokines to the SOC, aiming to first make PDAC immunogenic, and then promote anti-tumor immune responses (1).
En savoir plus

10 En savoir plus

Show all 10000 documents...