Publications by authors named "José Luis Bocco"

24 Publications

  • Page 1 of 1

Synthetic Lethal Activity of Benzophenanthridine Alkaloids From Against BRCA1-Deficient Cancer Cells.

Front Pharmacol 2020 3;11:593845. Epub 2020 Dec 3.

Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Córdoba, Argentina.

Several plants from South America show strong antitumoral properties based on anti-proliferative and/or pro-apoptotic activities. In this work we aimed to identify selective cytotoxic compounds that target BRCA1-deficient cancer cells by Synthetic Lethality (SL) induction. Using a high-throughput screening technology developed in our laboratory, we analyzed a collection of extracts from 46 native plant species from Argentina using a wide dose-response scheme. A highly selective SL-induction capacity was found in an alkaloidal extract from (Fam. Rutaceae). Bio-guided fractionation coupled to HPLC led to the identification of active benzophenanthridine alkaloids. The most potent SL activity was found with the compound oxynitidine, which showed a remarkably low relative abundance in the active fractions. Further validation experiments were performed using the commercially available and closely related analog nitidine, which showed SL-induction activity against various BRCA1-deficient cell lines with different genetic backgrounds, even in the nanomolar range. Exploration of the underlying mechanism of action using BRCA1-KO cells revealed AKT and topoisomerases as the potential targets responsible of nitidine-triggered SL-induction. Taken together, our findings expose an unforeseen therapeutic activity of alkaloids from -spp. that position them as novel lead molecules for drug discovery.
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http://dx.doi.org/10.3389/fphar.2020.593845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793782PMC
December 2020

Synthesis and structure-activity relationships of novel abietane diterpenoids with activity against .

Future Med Chem 2019 12;11(24):3109-3124

Research Institute of Natural Resources & Sustainability José Sánchez Labrador S.J. (IRNASUS-CONICET), School of Chemistry, Catholic University of Córdoba, Córdoba, Argentina.

To find alternative compounds against methicillin-resistant (MRSA) and methicillin-susceptible (MSSA), novel derivatives from dehydroabietic acid were synthesized. Compound was the most effective against 15 MRSA and 11 MSSA with minimum inhibitory concentration values ranging from 3.9 to 15.6 μg/ml. Although less active than , compound , followed by and , also exhibited anti-staphylococcal activity. Additional studies showed that compound is devoid of toxic effect on non-target cells. A structure-activity relationship study revealed that an oxime at C-13 together with a hydroxyl at C-12 could play a key role in the activity. These structures, in particular compound , could arise as templates for the development of agents against MRSA and MSSA.
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http://dx.doi.org/10.4155/fmc-2019-0192DOI Listing
December 2019

PKCα Modulates Epithelial-to-Mesenchymal Transition and Invasiveness of Breast Cancer Cells Through ZEB1.

Front Oncol 2019 27;9:1323. Epub 2019 Nov 27.

Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Córdoba, Argentina.

ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.
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http://dx.doi.org/10.3389/fonc.2019.01323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890807PMC
November 2019

Krüppel-Like Factor 6 Is Required for Oxidative and Oncogene-Induced Cellular Senescence.

Front Cell Dev Biol 2019 22;7:297. Epub 2019 Nov 22.

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Krüppel-like factor 6 (KLF6) is a transcription factor involved in the regulation of several cellular processes. Regarding its role in tumorigenesis, KLF6 is considered a tumor suppressor. Numerous reports demonstrate its frequent genomic loss or down-regulation, implying a functional inactivation in a broad range of human cancers. Previous work from our laboratory showed that the down-regulation of KLF6 expression in normal fibroblasts leads to cellular transformation, while its ectopic expression interferes with the oncogenic transformation triggered by activated Ras through a cell cycle arrest. We hypothesize that the growth suppressor activity of KLF6 may involve the induction of cellular senescence thereby helping to prevent the proliferation of cells at risk of neoplastic transformation. Here, we explored the association of KLF6 up-regulation in two different cellular senescence scenarios. We found that KLF6 silencing bypasses both oxidative and oncogene-induced senescence. In this context, KLF6 expression was capable to trigger cellular senescence in both normal and tumoral contexts. As such, the findings presented in this report provide insights into a potential mechanism by which KLF6 may play a suppressing role of uncontrolled or damaged cell proliferation.
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http://dx.doi.org/10.3389/fcell.2019.00297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882731PMC
November 2019

Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction.

Clin Cancer Res 2019 07 19;25(13):4049-4062. Epub 2019 Mar 19.

Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds.

Experimental Design: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database.

Results: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells.

Conclusions: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3516DOI Listing
July 2019

AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress.

Oncogene 2019 05 31;38(22):4310-4324. Epub 2019 Jan 31.

Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Córdoba, Argentina.

Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.
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http://dx.doi.org/10.1038/s41388-019-0724-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756059PMC
May 2019

Downregulation of adaptor protein MyD88 compromises the angiogenic potential of B16 murine melanoma.

PLoS One 2017 29;12(6):e0179897. Epub 2017 Jun 29.

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

The mechanisms that link inflammatory responses to cancer development remain a subject of intense investigation, emphasizing the need to better understand the cellular and molecular pathways that create a tumor promoting microenvironment. The myeloid differentiation primary response protein MyD88 acts as a main adaptor molecule for the signaling cascades initiated from Toll-like receptors (TLRs) and the interleukin 1 receptor (IL-1R). MyD88 has been shown to contribute to tumorigenesis in many inflammation-associated cancer models. In this study, we sought to better define the role of MyD88 in neoplastic cells using a murine melanoma model. Herein, we have demonstrated that MyD88 expression is required to maintain the angiogenic switch that supports B16 melanoma growth. By knocking down MyD88 we reduced TLR-mediated NF-κB activation with no evident effects over cell proliferation and survival. In addition, MyD88 downregulation was associated with a decrease of HIF1α levels and its target gene VEGF, in correlation with an impaired capability to induce capillary sprouting and tube formation of endothelial cells. Melanomas developed from cells lacking MyD88 showed an enhanced secretion of chemoattractant ligands such as CCL2, CXCL10 and CXCL1 and have an improved infiltration of macrophages to the tumor site. Our results imply that cell-autonomous signaling through MyD88 is required to sustain tumor growth and underscore its function as an important positive modulator of tumor angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491060PMC
September 2017

P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach.

Food Chem Toxicol 2017 Nov 29;109(Pt 2):888-897. Epub 2017 Apr 29.

Fine Chemicals and Natural Products Laboratory, School of Chemistry, Catholic University of Córdoba, Avda Armada Argentina 3555, X5016DHK Córdoba, Argentina. Electronic address:

This work examines the antitumor activity of an isomeric mixture (1), composed of the limonoids meliartenin and its interchangeable isomer 12-hydroxyamoorastatin. The results obtained showed that 1 displayed outstanding cytotoxic activity against CCRF-CEM, K562, A549 and HCT116 cells, with a highly selective effect on the latter, with an IC value of 0.2 μM. Based on this finding, HCT116 cells were selected to study the mechanism of action of 1. Cell cycle analysis revealed that 1 induced sustained arrest in the S-phase, which was followed by the triggering of apoptotic cell death and reduced clonogenic capacity. This cytotoxicity was seen to be preceded by the upregulation of the tumor suppressor p53 and its target effector p21. In addition, it was found that p53 expression was required for efficient cell death induction, and thus that the toxicity of 1 relies mainly on p53-dependent mechanisms. Taken together, these findings position 1 as a potent antitumor agent, with potential for the development of novel chemotherapeutic drugs based on the induction of S-phase arrest.
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http://dx.doi.org/10.1016/j.fct.2017.04.039DOI Listing
November 2017

Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis.

Evid Based Complement Alternat Med 2015 27;2015:912484. Epub 2015 Dec 27.

Fine Chemicals and Natural Products Laboratory, School of Chemistry, Catholic University of Córdoba, Avda Armada Argentina 3555, X5016DHK Córdoba, Argentina.

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2',4'-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4'-dihydroxy-3'-methoxyflavanone (5). Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1-5 was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6-9.9 μM) and a lower effect against CML cells (IC50 = 27.5-30.0 μM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.
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http://dx.doi.org/10.1155/2015/912484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706877PMC
January 2016

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

PLoS Genet 2016 Jan 14;12(1):e1005792. Epub 2016 Jan 14.

Cell Cycle and Genomic Stability Laboratory, Fundación Instituto Leloir, IIBBA/ CONICET, Buenos Aires, Argentina.

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.
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http://dx.doi.org/10.1371/journal.pgen.1005792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712966PMC
January 2016

S-Nitrosylation of NF-κB p65 Inhibits TSH-Induced Na(+)/I(-) Symporter Expression.

Endocrinology 2015 Dec;156(12):4741-54

Centro de Investigaciones en Bioquímica Clínica e Inmunología (J.P.N., V.P., M.N., A.M.L., M.d.M.M., J.L.B., C.G.P., A.M.M.-R.) and Centro de Investigaciones en Química Biológica (J.M.R.), Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba 5000, Argentina.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.
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http://dx.doi.org/10.1210/en.2015-1192DOI Listing
December 2015

Krüppel-like factor 6 interferes with cellular transformation induced by the H-ras oncogene.

FASEB J 2014 Dec 11;28(12):5262-76. Epub 2014 Sep 11.

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina

KLF6 is a member of the Krüppel-like factor family of transcription factors, with diverse roles in the regulation of cell physiology, including proliferation, signal transduction, and apoptosis. Mutations or down-regulation of KLF6 have been described in several human cancers. In this work, we found that KLF6-knockdown resulted in the formation of transformed foci and allowed the spontaneous conversion of NIH3T3 cells to a tumorigenic state. We further assessed the role of KLF6 in the context of oncogenic Ras. We showed that KLF6 was up-regulated by H-Ras(G12V) expression in a Jun N-terminal kinase (JNK)-dependent manner, correlated with enhanced klf6 promoter activity. We found that ectopic KLF6 expression induced a G1-phase cell cycle arrest, thereby decreasing the cell proliferation rate. In addition, constitutive KLF6 expression impaired H-Ras(G12V)-mediated loss of density-dependent growth inhibition and anchorage-independent growth. Moreover, growth of H-Ras(G12V)-driven tumors was reduced in mice challenged with cells stably expressing KLF6. KLF6 expression correlated with the up-regulation of p21, whereas neither p53 induction nor apoptotic cell death was detected. Further, p21 knockdown impaired KLF6-induced cell cycle arrest. These findings provide novel evidence highlighting KLF6 function in response to malignant transformation, suggesting the relevance of KLF6 in controlling cell proliferation and hindering tumorigenesis.
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http://dx.doi.org/10.1096/fj.14-251884DOI Listing
December 2014

Biology of Krüppel-like factor 6 transcriptional regulator in cell life and death.

IUBMB Life 2010 Dec;62(12):896-905

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

An essential role for the Krüppel-like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel-like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss-of-heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross-talk with the c-Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c-Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.
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http://dx.doi.org/10.1002/iub.396DOI Listing
December 2010

GH3B6 pituitary tumor cell proliferation is mediated by PKCalpha and PKCepsilon via ERK 1/2-dependent pathway.

Cell Physiol Biochem 2010 24;26(2):135-46. Epub 2010 Aug 24.

Centro de Microscopía Electrónica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Background: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated.

Methods: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy.

Results: Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min.

Conclusion: We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.
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http://dx.doi.org/10.1159/000320519DOI Listing
December 2010

Effects of microcystin-LR on the expression of P-glycoprotein in Jenynsia multidentata.

Chemosphere 2009 Mar 4;74(9):1179-86. Epub 2009 Jan 4.

Universidad Nacional de Córdoba-CONICET, Facultad Ciencias Químicas, Dto. Bioquímica Clínica-CIBICI, Haya de la Torre esq. Medina Allende, Ciudad Universitaria, 5000-Córdoba, Argentina.

The multixenobiotic resistance phenomenon (MXR) related to the P-glycoprotein multidrug transporter protein (P-gp) has been identified and characterized in several aquatic organisms. In the present work, we prove the presence of a P-gp in liver, gills and brain of Jenynsia multidentata by Western Blot and RT-PCR. A 170 kDa protein has been found in liver and gills while in brain a approximately 80 kDa protein has been detected. The partial nucleotide sequence obtained in this autochthonous fish showed high similarity ranging from 83% to 92% with other fishes. In addition, P-gp expression in this fish was evaluated after time and dose-dependent exposures to the cyanotoxin microcystin-LR. Individuals were exposed to MC-LR at concentrations of 2, 5 and 10 microg L(-1) for 24h and for 6, 12 and 24h at 2 microg L(-1) MC-LR. Changes in P-gp expression were observed in liver, gills and brain. However, this response was tissue specific. Only in gills of J. multidentata P-gp expression, measured either by real-time RT-PCR or Western Blot, was significantly higher compared to controls at most tested times and doses. A 3-fold increase with respect to controls was found at 12h by RT-PCR and after 24h by Western Blot. In dose-dependent experiments the maximum P-gp expression was observed at 2 microg L(-1) MC-LR, measured by both RT-PCR and Western Blot. In the liver, P-gp protein levels were significantly increased after 24h of exposure, at every toxin dose tested. Thus, probably longer exposures would show also significant increases in this tissue. Considering these results we can propose that P-gp belongs to the defence system involved in the response to MC-LR in J. multidentata.
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http://dx.doi.org/10.1016/j.chemosphere.2008.11.068DOI Listing
March 2009

Emergence and dissemination of a community-associated methicillin-resistant Panton-Valentine leucocidin-positive Staphylococcus aureus clone sharing the sequence type 5 lineage with the most prevalent nosocomial clone in the same region of Argentina.

J Clin Microbiol 2008 May 5;46(5):1826-31. Epub 2008 Mar 5.

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Ciudad Universitaria, Pabellon Argentina, Cordoba, Argentina.

Epidemiological surveillance for community-associated methicillin-resistant Staphylococcus aureus revealed prevalences of 33% and 13% in pediatric and adult patients, respectively, in Cordoba, Argentina, in 2005. This study describes for the first time the emergence and dissemination of the sequence type 5 (ST5) lineage as the most prevalent clone (89%) (pulsed-field gel electrophoresis type I-ST5-staphylococcal cassette chromosome type IVa-spa type 311) harboring the Panton-Valentine leukocidin and enterotoxin A genes.
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http://dx.doi.org/10.1128/JCM.01949-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395097PMC
May 2008

Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139 strain.

Microb Pathog 2008 Feb 31;44(2):118-28. Epub 2007 Aug 31.

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Centro de Investigaciones en Bioquímica e Inmunología ClBICI-CONICET, Córdoba, Argentina.

Cholera toxin (CT) gene-negative Vibrio cholerae non-O1, non-O139 strains may cause severe diarrhea though their pathogenic mechanism remains unclear. V. cholerae cytolysin (VCC) is a pore-forming exotoxin encoded in the hlyA gene of V. cholerae whose contribution to the pathogenesis is not fully understood. In this work, the virulence properties of a CT gene-negative V. cholerae non-O1, non-O139 strain causing a cholera-like syndrome were analyzed. Inoculation of rabbit ileal loops with the wild type strain induced extensive fluid accumulation, accompanied by severe histopathological damage characterized by villus shortening, lymphangiectasia and focal areas of necrosis. These pathogenic effects were abrogated by mutation of the hlyA gene thus pointing out the main role of VCC in the virulence of the strain. Interestingly, this toxin was capable of triggering apoptosis in human intestinal cell lines due to its anion channel activity. Moreover, the wild type strain also induced increased apoptosis of the intestinal epithelium cells which was not observed upon inoculation of the VCC null mutant strain, indicating that VCC may trigger apoptotic cell death during infection in vivo. Altogether, these results support a main role of VCC in the pathogenesis of the CT gene-negative V. cholerae non-O1, non-O139 strain and identify apoptosis as a previously unrecognized cell death pathway triggered by VCC.
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http://dx.doi.org/10.1016/j.micpath.2007.08.013DOI Listing
February 2008

Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae.

Proc Natl Acad Sci U S A 2007 Feb 31;104(6):1829-34. Epub 2007 Jan 31.

Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5-/- cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.
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http://dx.doi.org/10.1073/pnas.0601437104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1794277PMC
February 2007

Activation of the human pregnancy-specific glycoprotein PSG-5 promoter by KLF4 and Sp1.

Biochem Biophys Res Commun 2006 May 20;343(3):745-53. Epub 2006 Mar 20.

INSERM U.384, Laboratoire de Biochimie, Faculté de Médecine, F-63000 Clermont-Ferrand, France.

Pregnancy-specific glycoproteins (PSGs) are major placental proteins thought to be essential for the maintenance of gestation. Little is known about the regulation of expression of the 11 genes encoding these proteins. It was previously demonstrated that Krüppel-like factor 6 (KLF6) and specific-protein 1 (Sp1) bind to conserved sequence within the PSG-5 gene promoter. Informatics analysis revealed the presence of one potential binding site for Krüppel-like factor 4 (KLF4), in the PSG-5 promoter, suggesting a potential transcriptional regulator role for KLF4. Using gene promoter-reporter transfections and X-ChIP assays, we demonstrated that KLF4 is an activator of the PSG-5 promoter by binding to a KLF consensus like binding which includes the Core Promoter Element region (-147/-140). Furthermore, we used previous data showing the binding of Sp1 transcription factor to a GT-box (-443/-437) and co-transfection assays with KLF4 and Sp1 to demonstrate the strong synergic activity of these two factors on the PSG-5 promoter.
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http://dx.doi.org/10.1016/j.bbrc.2006.03.032DOI Listing
May 2006

Evolution and molecular characterization of methicillin-resistant Staphylococcus aureus epidemic and sporadic clones in Cordoba, Argentina.

J Clin Microbiol 2006 Jan;44(1):192-200

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.

Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the "Cordobes clone," has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCCmec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCCmec type related to SCCmec type IVc.
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http://dx.doi.org/10.1128/JCM.44.1.192-200.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351928PMC
January 2006

A new role for the Kruppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function.

Oncogene 2004 Oct;23(50):8196-205

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI - CONICET). Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Kruppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that all KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6's role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.
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http://dx.doi.org/10.1038/sj.onc.1208020DOI Listing
October 2004

In vivo expression of recombinant pregnancy-specific glycoprotein 1a induces alternative activation of monocytes and enhances Th2-type immune response.

Eur J Immunol 2003 Nov;33(11):3007-16

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

It has been proposed that pregnancy-specific factors could be responsible for shift the balance of cytokine profiles during maternal immune response from Th1-type reactivity into a "less-damaging" Th2-type reactivity. In the present work, we investigated the in vivo function of human pregnancy-specific glycoprotein (PSG)1a, the major variant of PSG polypeptides released into the circulation during pregnancy, on the modulation of the innate and adaptive immune response. For this, BALB/c mice were injected with a vaccinia virus-based vector harboring the human PSG1a cDNA (Vac-PSG1a) 4 days before immunization with ovalbumin (OVA) in complete Freund's adjuvant, and the early specific T cell response against OVA was evaluated 8 days post-immunization. We also studied the activation status of spleen and peritoneal monocytes/macrophages (Mo) populations from Vac-PSG1a-treated mice, and explored whether PSG1a-targeted Mo could affect the Th-type commitment by investigating their impact on the differentiation of naive T cells. Our data show that the treatment with Vac-PSG1a is able to induce a state of alternative activation on Mo. Furthermore, the generation of the immune response in the context of these alternatively activated antigen-presenting cells may shift T cell differentiation to Th2-type immunity which is more compatible with a successful pregnancy.
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http://dx.doi.org/10.1002/eji.200323993DOI Listing
November 2003

Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation.

J Leukoc Biol 2002 Sep;72(3):512-21

Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.

It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.
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September 2002

Identification of a novel methicillin-resistant Staphylococcus aureus epidemic clone in Córdoba, Argentina, involved in nosocomial infections.

J Clin Microbiol 2002 Apr;40(4):1427-35

Departamento de Bioquímica Clinica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140375PMC
http://dx.doi.org/10.1128/jcm.40.4.1427-1435.2002DOI Listing
April 2002