E2F1 regulates cellular growth by mTOR signaling

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.

Transplanted beta cell response to increased metabolic demand. Changes in beta cell replication and mass

We determined the capacity of transplanted beta cells to modify their replication and mass when stimulated by changes in metabolic demand. Five groups of Lewis rats were studied: group 1 (Tx-Px) had a 95% pancreatectomy 14 d after transplantation of 500 islets; group 2 (Px-Tx) had a 95% pancreatectomy 14 d before transplantation of 500 islets; group 3 (Tx) was transplanted with 500 islets; group 4 (Px) had a 95% pancreatectomy; and group 5 (normal) was neither transplanted nor pancreatectomized. Blood glucose was normal in Tx-Px and Tx groups at all times. Px-Tx and Px groups developed severe hyperglycemia after pancreatectomy that was corrected in Px-Tx group in 83% of rats 28 d after transplantation. Replication of transplanted beta cells increased in Tx-Px (1.15 +/- 0.12%) and Px-Tx (0.85 +/- 0.12%) groups, but not in Tx group (0.64 +/- 0.07%) compared with normal pancreatic beta cells (0.38 +/- 0.05%) (P < 0.001). Mean beta cell size increased in Tx-Px (311 +/- 14 microns2) and Px-Tx (328 +/- 13 microns2) groups compared with Tx (252 +/- 12 microns2) and normal (239 +/- 9 microns2) groups (P < 0.001). Transplanted beta cell mass increased in Tx-Px (1.87 +/- 0.51 mg) and Px-Tx (1.55 +/- 0.21 mg) groups compared with Tx group (0.78 +/- 0.17 mg) (P < 0.05). In summary, changes in transplanted beta cells prevented the development of hyperglycemia in Tx-Px rats. Transplanted beta cells responded to increased metabolic demand increasing their beta cell mass.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

The p38/MK2/Hsp25 pathway is required for BMP-2-induced cell migration.

Background: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. Methodology/Principal Findings: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2¿s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38¿ or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. Conclusions: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration.

Desenvolupament de problemes virtuals i interactius per a l’aprenentatge de la Microbiologia

La finalitat del projecte ha estat desenvolupar un espai virtual teòric-pràctic per a l’aprenentatge de la Microbiologia. Aquest espai virtual, basat en l'aprenentatge a través de problemes, s’ha anomenat “Microbiologia Interactiva” i proposa a l’alumne les següents àrees temàtiques: Introducció a les tècniques de la Microbiologia; Estructura i funció de la cèl.lula microbiana; Creixement i control microbià; Microbiologia molecular; Fisiologia i metabolisme microbians; Virologia; Ecologia Microbiana; Diversitat microbiana. Per a cada temàtica s’han definit unes competències a assolir a través de la resolució de problemes teòrics o pràctics. En aquest darrer cas, se li proposa a l’alumne que entri en el laboratori virtual per a la resolució dels casos pràctics plantejats. A més, per a la resolució dels problemes, l’alumne disposa d’un seguit de recursos per a cada temàtica. Finalment, també s’inclouen activitats de relació i d’ampliació per tal d’estimular la discussió, l’esperit crìtic, el treball en grup i la recerca bibliogràfica. A més, per tal de facilitar el seu ús, el web disposa també d'un tutorial. El web “Microbiologia Interactiva” es va introduir de forma pilat en l’ensenyament de l'assignatura de Microbiologia de la Llicenciatura de Biologia i de la de Microbiologia I de la llicenciatura de Biotecnologia de la Universitat Autònoma de Barcelona (UAB) durant el curs 2007-08. Al llarg d'aquest curs es va valorar la seva utilitat i acceptació per part dels alumnes mitjançant enquestes. Els bons resultats obtinguts van aconsellar que aquesta eina fora ja utilitzada en totes les assignatures generals de Microbiologia de les llicenciatures de Biologia, Biotecnologia, Bioquímica, Química, Enginyeria Química, Ciències Ambientals i Ciència i Tecnologia dels Aliments de la UAB. Actualment el web s’està també utilitzant amb molt bons resultats a les assignatures de Microbiologia dels nous graus que ofereix la Facultat de Biociències de la UAB. Així doncs, en aquest projecte s’han assolit amb escreix els objectius previstos. Es pot consultar el web desenvolupat a l’adreça http://microbiologia.uab.cat//Microbiologia_Interactiva_Web/.

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression ofSSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.

miR-143 Interferes with ERK5 Signaling, and Abrogates Prostate Cancer Progression in Mice

Abstract Background: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions: miR-143 is as a new target for prostate cancer treatment.

Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs

Background: Nucleoside analogs used in the chemotherapy of solid tumors, such as the capecitabine catabolite50-deoxy-5-fluorouridine (50-DFUR) trigger a transcriptomic response that involves the aquaglyceroporin aquaporin 3 along with other p53-dependent genes. Here, we examined whether up-regulation of aquaporin 3 (AQP3) mRNA incancer cells treated with 50-DFUR represents a collateral transcriptomic effect of the drug, or conversely, AQP3participates in the activity of genotoxic agents. Methods: The role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches. Results: 50-DFUR and gemcitabine, but not cisplatin, stimulated AQP3 expression and cell volume, which was partially and significantly blocked by knockdown of AQP3. Moreover, AQP3 siRNA significantly blocked other effects of nucleoside analogs, including G1/S cell cycle arrest, p21 and FAS up-regulation, and cell growth inhibition. Short incubations with 5-fluorouracil (5-FU) also induced AQP3 expression and increased cell volume, and the inhibition of AQP3 expression significantly blocked growth inhibition triggered by this drug. To further establish whether AQP3 induction is related to cell cycle arrest and apoptosis, cells were exposed to long incubations with escalating doses of 5-FU. AQP3 was highly up-regulated at doses associated with cell cycle arrest, whereas at doses promoting apoptosis induction of AQP3 mRNA expression was reduced. Conclusions: Based on the results, we propose that the aquaglyceroporin AQP3 is required for cytotoxic activity of 5’-DFUR and gemcitabine in the breast cancer cell line MCF7 and the colon adenocarcinoma cell line HT29, and is implicated in cell volume increase and cell cycle arrest.

Aquaporin 3 (AQP3) participates in the cytotoxic response to nucleoside-derived drugs

Background: Nucleoside analogs used in the chemotherapy of solid tumors, such as the capecitabine catabolite50-deoxy-5-fluorouridine (50-DFUR) trigger a transcriptomic response that involves the aquaglyceroporin aquaporin 3 along with other p53-dependent genes. Here, we examined whether up-regulation of aquaporin 3 (AQP3) mRNA incancer cells treated with 50-DFUR represents a collateral transcriptomic effect of the drug, or conversely, AQP3participates in the activity of genotoxic agents. Methods: The role of AQP3 in cell volume increase, cytotoxicity and cell cycle arrest was analyzed using loss-of-function approaches. Results: 50-DFUR and gemcitabine, but not cisplatin, stimulated AQP3 expression and cell volume, which was partially and significantly blocked by knockdown of AQP3. Moreover, AQP3 siRNA significantly blocked other effects of nucleoside analogs, including G1/S cell cycle arrest, p21 and FAS up-regulation, and cell growth inhibition. Short incubations with 5-fluorouracil (5-FU) also induced AQP3 expression and increased cell volume, and the inhibition of AQP3 expression significantly blocked growth inhibition triggered by this drug. To further establish whether AQP3 induction is related to cell cycle arrest and apoptosis, cells were exposed to long incubations with escalating doses of 5-FU. AQP3 was highly up-regulated at doses associated with cell cycle arrest, whereas at doses promoting apoptosis induction of AQP3 mRNA expression was reduced. Conclusions: Based on the results, we propose that the aquaglyceroporin AQP3 is required for cytotoxic activity of 5’-DFUR and gemcitabine in the breast cancer cell line MCF7 and the colon adenocarcinoma cell line HT29, and is implicated in cell volume increase and cell cycle arrest.

Deposition and characterization of lines printed through laser-induced forward transfer

The possibility of printing two-dimensional micropatterns of biomolecule solutions is of great interest in many fields of research in biomedicine, from cell-growth and development studies to the investigation of the mechanisms of communication between cells. Although laser-induced forward transfer (LIFT) has been extensively used to print micrometric droplets of biological solutions, the fabrication of complex patterns depends on the feasibility of the technique to print micron-sized lines of aqueous solutions. In this study we investigate such a possibility through the analysis of the influence of droplet spacing of a water and glycerol solution on the morphology of the features printed by LIFT. We prove that it is indeed possible to print long and uniform continuous lines by controlling the overlap between adjacent droplets. We show how, depending on droplet spacing, several printed morphologies are generated, and we offer, in addition, a simple explanation of the observed behavior based on the jetting dynamics characteristic of the LIFT of liquids.

TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells

Background and purpose: The TP53 induced glycolysis and apoptosis regulator (TIGAR) functions to lower fructose-2,6-bisphosphate (Fru-2,6-P2) levels in cells, consequently decreasing glycolysis and leading to the scavenging of reactive oxygen species (ROS), which correlate with a higher resistance to cell death. The decrease in intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. Given these good prospects of TIGAR for metabolic regulation and p53-response modulation, we analyzed the effects of TIGAR knockdown in U87MG and T98G glioblastoma-derived cell lines. Methods/results: After TIGAR-knockdown in glioblastoma cell lines, different metabolic parameters were assayed, showing an increase in Fru-2,6-P2, lactate and ROS levels, with a concomitant decrease in reduced glutathione (GSH) levels. In addition, cell growth was inhibited without evidence of apoptotic or autophagic cell death. In contrast, a clear senescent phenotype was observed. We also found that TIGAR protein levels were increased shortly after irradiation. In addition, avoiding radiotherapy-triggered TIGAR induction by gene silencing resulted in the loss of capacity of glioblastoma cells to form colonies in culture and the delay of DNA repair mechanisms, based in c-H2AX foci, leading cells to undergo morphological changes compatible with a senescent phenotype. Thus, the results obtained raised the possibility to consider TIGAR as a therapeutic target to increase radiotherapy effects. Conclusion: TIGAR abrogation provides a novel adjunctive therapeutic strategy against glial tumors by increasing radiation-induced cell impairment, thus allowing the use of lower radiotherapeutic doses.