Abstract:

We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)2-vitamin D3 [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21Waf1/Cip1 and p27Kip1 expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D -induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs -dependent mechanism in hormone modulation of myogenesis.

Author affiliation: Irazoqui, Ana Paula. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina

Author affiliation: Heim, Nadia B.. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina

Author affiliation: Boland, Ricardo Leopoldo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina

Author affiliation: Buitrago, Claudia Graciela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina

Abstract:

We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways. CDKs and MAPKs phosphorylate similar sites yet generally have distinct functions and substrates. Repetto et al. uncover a case where these kinases collaborate to regulate a substrate in a signal transduction pathway by phosphorylating a shared set of sites.

Author affiliation: Repetto, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Author affiliation: Winters, Matthew J.. University of Massachussets; Estados Unidos

Author affiliation: Bush, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Author affiliation: Reiter, Wolfgang. Max F. Perutz Laboratories; Austria

Author affiliation: Hollenstein, David Maria. Max F. Perutz Laboratories; Austria

Author affiliation: Ammerer, Gustav. Max F. Perutz Laboratories; Austria

Author affiliation: Pryciak, Peter M.. University of Massachussets; Estados Unidos

Author affiliation: Colman Lerner, Alejandro Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Abstract:

Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin–CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin–proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.

Author affiliation: Di Renzo, María Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina

Author affiliation: Laverrière, Marc. Instituto Pasteur; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús. Instituto de Investigaciones Biotecnológicas (sede Chascomús); Argentina

Author affiliation: Schenkman, Sergio. Universidade Federal de São Paulo. Departamento de Microbiologia, Imunologia e Parasitologia; Brasil

Author affiliation: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús. Instituto de Investigaciones Biotecnológicas (sede Chascomús); Argentina

Author affiliation: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina

Author affiliation: Potenza, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina

Abstract:

Human PTEFb is a protein kinase composed by CDK9 and Cyclin T that controls the elongation phase of RNA Pol II. This complex also affects the activation and differentiation program of lymphoid cells. In this study we found that several head and neck tumor cell lines overexpress PTE Fb. We also established that Cyclin T1 is able to induce transformation in vitro, as we determined by foci and colony formation assays. Nu/nu mice s.c. injected with stable transfected Cyclin T1 cells (NIH 3T3 Cyclin T1) developed tumors faster than animals injected with control cells (NIH 3T3 β-gal). In vitro, NIH 3T3 Cyclin T1 cells show increased proliferation and CDK4-Rb phosphorylation. Even more, silencing E2F1 expression (shRNA E2F1) in NIH 3T3 cells resulted in a dramatic inhibition of Cyclin T1-induced foci. All these data demonstrate for the first time the Cyclin T1 oncogenic function and suggest a role for this protein in controlling cell cycle probably via Rb/E2F1 pathway.

Author affiliation: Moiola, Cristian Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: de Luca, Paola. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Gardner, Kevin. National Institutes of Health; Estados Unidos

Author affiliation: Vazquez, Elba Susana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: de Siervi, Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha.

Author affiliation: Dekanty, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Giulianelli, Sebastian Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Coso, Omar Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

Author affiliation: Rudland, Phillip S.. University of Liverpool; Reino Unido

Author affiliation: Jimenez de Asua, Luis Adan Felipe. University of Liverpool; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Abstract:

E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

Author affiliation: Carcagno, Abel Luis. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Ogara, Maria Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Sonzogni, Silvina Veronica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Marazita, Mariela Claudia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Sirkin, Pablo Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Ceruti, Julieta María. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Canepa, Eduardo Tomas. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Abstract:

Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.

Author affiliation: Rojas, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. University of Texas; Estados Unidos

Author affiliation: Benavides, Fernando. University of Texas; Estados Unidos

Author affiliation: Blando, Jorge. University of Texas; Estados Unidos

Author affiliation: Pérez, Carlos. University of Texas; Estados Unidos

Author affiliation: Cardenas, Kim. University of Texas; Estados Unidos

Author affiliation: Richie, Ellen. University of Texas; Estados Unidos

Author affiliation: Knudsen, Erik S.. Thomas Jefferson University; Estados Unidos

Author affiliation: Johnson, David G.. University of Texas; Estados Unidos

Author affiliation: Senderowicz, Adrian M.. Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research; Estados Unidos

Author affiliation: Rodriguez Puebla, Marcelo L.. University of North Carolina; Estados Unidos

Author affiliation: Conti, Claudio. University of Texas; Estados Unidos

Abstract:

En líneas celulares de origen leucémico se han encontrado deleciones en el brazo corto del cromosoma 9, donde se localizan los genes del interferón de tipo I, lo que en un principio hizo suponer que el interferón se comportaba como un gen supresor de tumor. Se propuso ver si la producción de interferón estaba alterada en las leucemias linfoblásticas agudas (LLA), y si esas alteraciones génicas estarían relacionadas con la capacidad de producción de interferón a por las células malignas al ser inducidas con virus Sendai. Se determinó la capacidad de las células leucémicas de 21 pacientes de LLA, de la línea celular establecida K 562 y de dadores normales de producir interferón a "in vitro" al ser inducidos con virus Sendai con un priming de interferón α. La producción de interferón a por los blastos leucémicos y células normales fue muy variable oscilando entre 633 a 8850 pg/ml/5x10 6 células, no siendo estadísticamente significativo, tampoco se hallaron diferencias entre los subgrupos de LLA. En 4 de los 7 casos estudiados por Southern blot se pudo estudiar la producción de interferón α, presentando uno de ellos deleción de genes de interferón α. De las 21 leucemias en dos casos produjeron una cantidad de interferón α mínima. Esto sugiere que la malignidad en las LLA con baja producción de interferón α podría deberse a la pérdida de algún gen supresor de tumor. Se investigó la frecuencia y asociación de alteraciones del p 16, el interferón α y el interferón β en un total de 39 casos de LLA. De ellos, diez pacientes (25,6 %) presentaron anormalidades de al menos uno de los tres genes estudiados. Se encontró en un porcentaje del 13% deleciones en los genes del interferón α y β, así como también en los recientemente identificados genes supresores de tumor p15 y p16 (23%) los cuales se encuentran próximos a los genes el interferón. Estos datos indican que en las LLA, representando un cuarto de las LLA totales estudiadas el gen p16 está delecionado con mayor frecuencia que los genes de los interferones de tipo 1. Además de p16, el 37,5% está asociado con deleciones del interferón, sugiriendo una asociación entre ambas. Estos datos sugieren que la ausencia de p16, inhibidor de las ciclinas, podría modificar el ciclo celular normal y favorecer la transformación blástica. Su asociación con la deleción de los genes de los interferones de tipo I podría constituir eventos de la leucemogénesis. El porcentaje de deleciones, a su vez dependió del fenotipo de la célula leucémica, siendo mayor en los casos de LLA T que en aquellas precursoras B. Para un mejor tratamiento de la enfermedad es necesario partir de un buen diagnóstico, para lo cual se buscó detectar clonalidad, de tipo B o T por medio de la identificación de fragmentos reordenados, ya sea de la cadena pesada de las inmunoglobulinas (clonalidad B) o de la cadena γ del receptor de los linfocitos T (clonalidad T). A su vez por medio de un mapeo de restricción se llegó a una correcta identificación del subtipo de reordenamiento Vγl en las LLA T. Esto cobra importancia, sobre todo para el seguimiento posterior de la enfermedad mínima residual, sin embargo no es suficiente para la determinación del linaje celular.

Type I Interferon genes are located in the short arm of chromosome 9, where deletions have been found in cell lines originated from leukemia patients. This arised the idea of interferon acting as a tumor suppressor gene in acute lymphoblastic leukemia (ALL). Interferon production was measured in ALL and compared to normal healthy donors. No significative differences were found between groups. By Southern blot analysis α and β interferon gene deletions were found in 13% of ALL patients. The putative tumor-suppressor gene p16 was mapped to human chromosome 9p2l, close to the interferon α cluster. The frequency and association of gene alterations of pl6, interferon α and interferon β were investigated in a total of 39 Acute Lymphoblastic Leukemia (ALL) patients. Of them, ten patients (25.6%) presented abnormalities of at least one of the three genes studied. We also found p15 and p16 gene deletions in a higher frequency (23%). These fidings supports the idea that the role of the interferon as tumor suppressor gene is more related to its location than its function. To have a more accurate diagnosis of acute leukemias, we investigated B and T cell clonality by identifying PCR segments of rearranged immunoglobulin heavy chain genes and γ T cell receptor (TCR) genes, respectively. In order to identify the rearranged Vγl TCR gene to further detect minimal residual disease a restriction analysis was performed. Although we can detect clonality by PCR amplification of IgH and/or γTCR gene rearrangement, in ALL, it can not be use for cell lineage identification.

Author affiliation: Sternik, Gabriel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

Trypanosoma cruzi CRK3 gene encodes a Cdc2p related protein kinase (CRK). To establish if it has a role in the regulation of the parasite cell cycle we studied CRK3 expression and activity throughout three life cycle stages. CRK3 from epimastigote soluble extracts interacted with p13suc1-beads. Endogenous CRK3 phosphorylated histone H1 and this activity was inhibited by specific CDK inhibitors: Olomoucine, Flavopiridol and Roscovitine. Flavopiridol partially inhibited the growth of T. cruzi epimastigotes at 50 nM, the lowest concentration used, but even with the highest (5 μM), cell growth was not completely arrested. CRK3 from Flavopiridol-inhibited epimastigote extracts exhibited a dose dependent inhibition of histone H1 phosphorylation. T. cruzi p13suc1-binding CRK displayed the same inhibition profile. This suggests that CRK3 is the enzyme responsible for the majority of the kinase activity associated with p13suc1. CRK3 activity of hydroxyurea (HU) synchronized epimastigotes peaked in G2/M boundary while the kinase activity associated to p13suc1-beads increased at the same time point but remained high until late G2/M. In addition, CRK3 expression was constant during the cell cycle. This is a common pattern of CDK activity regulation. Taken together, these results support the idea that CRK3 is involved in control of the cell cycle in T. cruzi.

Author affiliation: Santori, Maria I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Laría, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Gomez, Eliana B.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Espinosa, Ingrid. Universidad de Chile; Chile

Author affiliation: Galanti, Norbel. Universidad de Chile; Chile

Author affiliation: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Abstract:

In eukaryotes, an oscillating network of protein kinase activities drives the order and timing of the cell cycle progression. Complexes formed by cyclins associated to cyclin-dependent kinases (CDKs) are the central components of this network. Cyclins act as the activating subunits and their abundance is regulated by different mechanisms in order to promote or prevent kinase activity. Protein synthesis, proteasomal degradation and/or differential subcellular compartmentalization modulate cyclin expression levels along the cell cycle. We describe in this work the characterization of Trypanosoma cruzi Cyclin 2 (TcCYC2), which contributes to a better understanding of the cell cycle regulation in this protozoan parasite. We found TcCYC2 exhibited cyclin function in a yeast complementation assay and over-expression of hemagglutinin tagged TcCYC2-HA rendered shorter duplication times and smaller cell sizes in the epimastigote form of the parasite. Analysis of synchronized cultures showed that over-expression of TcCYC2-HA altered the timing epimastigotes pass through G2/M boundary or cytokinesis. Taken together, our results showed that TcCYC2 is a functional cyclin whose over-expression modifies the dynamics of the cell cycle as well as the morphology of epimastigote forms of T. cruzi, suggesting it plays an important role in the cell cycle regulation machinery.

Author affiliation: Potenza, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Schenkman, Sergio. Universidade Federal de São Paulo; Brasil

Author affiliation: Laverriere, Marc. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Author affiliation: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina