Dynamic expression of Ralstonia solanacearum virulence factors and metabolism-controlling genes during plant infection.

BACKGROUND: Ralstonia solanacearum is the causal agent of bacterial wilt, a devastating plant disease responsible for serious economic losses especially on potato, tomato, and other solanaceous plant species in temperate countries. In R. solanacearum, gene expression analysis has been key to unravel many virulence determinants as well as their regulatory networks. However, most of these assays have been performed using either bacteria grown in minimal medium or in planta, after symptom onset, which occurs at late stages of colonization. Thus, little is known about the genetic program that coordinates virulence gene expression and metabolic adaptation along the different stages of plant infection by R. solanacearum. RESULTS: We performed an RNA-sequencing analysis of the transcriptome of bacteria recovered from potato apoplast and from the xylem of asymptomatic or wilted potato plants, which correspond to three different conditions (Apoplast, Early and Late xylem). Our results show dynamic expression of metabolism-controlling genes and virulence factors during parasitic growth inside the plant. Flagellar motility genes were especially up-regulated in the apoplast and twitching motility genes showed a more sustained expression in planta regardless of the condition. Xylem-induced genes included virulence genes, such as the type III secretion system (T3SS) and most of its related effectors and nitrogen utilisation genes. The upstream regulators of the T3SS were exclusively up-regulated in the apoplast, preceding the induction of their downstream targets. Finally, a large subset of genes involved in central metabolism was exclusively down-regulated in the xylem at late infection stages. CONCLUSIONS: This is the first report describing R. solanacearum dynamic transcriptional changes within the plant during infection. Our data define four main genetic programmes that define gene pathogen physiology during plant colonisation. The described expression of virulence genes, which might reflect bacterial states in different infection stages, provides key information on the R. solanacearum potato infection process.

Evaluación de la escala modificada de Cormack-Lehane para visión laringoscópica durante la intubación orotraqueal en una unidad de cuidados críticos. Estudio prospectivo observacional / Evaluation of the laryngoscopy view using the modified Cormack-Lehane scale during tracheal intubation in an intensive care unit. A prospective observational study

Antecedentes y objetivos: La intubación orotraqueal en las unidades de cuidados críticos está asociada a una frecuencia alta de dificultad y complicaciones. Una mala visión glótica durante la laringoscopia directa podría ser la causa. El objetivo de este estudio es evaluar si existe una relación entre la visión laringoscópica valorada por la escala modificada de Cormack-Lehane y la dificultad de intubación y la aparición de complicaciones. Métodos: Se incluyeron todos los pacientes intubados con laringoscopia directa en la Unidad de Cuidados Críticos durante un periodo de 45 meses. En todos los pacientes se valoró la visión glótica medida con la escala modificada de Cormack-Lehane, la dificultad de intubación (intubación al primer intento, dificultad valorada por el anestesiólogo, uso de la guía de Frova) y la aparición de complicaciones (hipotensión, hipoxia, intubación esofágica). Resultados: Se incluyeron 360 pacientes. A medida que aumentó el grado en la escala modificada de Cormack-Lehane, disminuyó la incidencia de intubación orotraqueal al primer intento (1: 97%, 2a: 94%, 2b: 80%, 3: 60%, 4: 0%, p<0,001), aumentó la incidencia de intubación con dificultad moderada o severa (1: 2%, 2a: 4%, 2b: 36%, 3: 77%, 4: 100%, p<0,001), así como la necesidad de uso de Frova (1: 7%, 2a: 8%, 2b: 45%, 3: 60%, 4: 100%, p<0,001). A medida que aumentó el grado en la escala modificada de Cormack-Lehane, aumentó la aparición de hipoxia<90% (1: 20%, 2a: 20%, 2b: 28%, 3: 47%, 4: 100%, p=0,0073) y la hipoxia<80% (1: 11%, 2a: 10%, 2b: 12%, 3: 27%, 4: 100%, p=0,00398). No encontramos relación entre la aparición de hipotensión y el grado en la escala modificada de Cormack-Lehane (p=ns). Conclusiones: Durante la intubación orotraqueal en la Unidad de Cuidados Críticos hemos encontrado una estrecha relación entre una mala visión glótica valorada por la escala modificada de Cormack-Lehane y una mayor dificultad de la técnica. La incidencia de hipoxia se relaciona directamente con un mayor grado en la escala modificada de Cormack-Lehane. No hemos encontrado ninguna relación entre la hipotensión y la escala modificada de Cormack-Lehane

Background and objectvies: Tracheal intubation in the Intensive Care Unit is associated with a high incidence of difficult intubation and complications. This may be due to a poor view of the glottis during direct laryngoscopy. The aim of this study is to determine if there is a relationship between laryngoscopy view using the modified Cormack-Lehane scale with the incidence of difficult intubation and complications. Methods: All patients who were subjected to tracheal intubated with direct laryngoscopy in the Intensive Care Unit over a 45 month period were included in the study. In all patients, an evaluation was made of the laryngoscopy view using the modified Cormack-Lehane scale, as well as the technical difficulty (number of intubations at first attempt, operator-reported difficulty, need for a Frova introducer), and the incidence of complications (hypotension, hypoxia, oesophageal intubation). Results: A total of 360 patients were included. When the grade of the modified Cormack-Lehane scale was increased from 1 to 4, the incidence of first success rate intubation decreased (1: 97%, 2a: 94%, 2b: 80%, 3: 60%, 4: 0%, p<.001), the incidence of moderate and severe difficulty intubation increased (1: 2%, 2a: 4%, 2b: 36%, 3: 77%, 4: 100%, p<.001.), as well as the need for a Frova guide (1: 7%, 2a: 8%, 2b: 45%, 3: 60%, 4: 100%, p<.001). When the grade of the modified Cormack-Lehane scale increased from 1 to 4, the incidence of hypoxia<90% increased (1: 20%, 2a: 20%, 2b: 28%, 3: 47%, 4: 100%, p=.0073), as well as hypoxia<80% (1: 11%, 2a: 10%, 2b: 12%, 3: 27%, 4: 100%, p=.00398). No relationship was observed between the incidence of hypotension and the grade of the modified Cormack-Lehane scale (p=ns). Conclusions: During tracheal intubation in the Intensive Care Unit a close relationship was found between a poor laryngoscopy view using the modified Cormack-Lehane scale and a higher difficulty technique of intubation. A relationship was found between the incidence of hypoxia with a higher grade in the modified Cormack-Lehane scale. No relationship was found between hypotension and the modified Cormack-Lehane scale

Evaluation of the laryngoscopy view using the modified Cormack-Lehane scale during tracheal intubation in an intensive care unit. A prospective observational study. / Evaluación de la escala modificada de Cormack-Lehane para visión laringoscópica durante la intubación orotraqueal en una unidad de cuidados críticos. Estudio prospectivo observacional.

BACKGROUND AND OBJECTVIES: Tracheal intubation in the Intensive Care Unit is associated with a high incidence of difficult intubation and complications. This may be due to a poor view of the glottis during direct laryngoscopy. The aim of this study is to determine if there is a relationship between laryngoscopy view using the modified Cormack-Lehane scale with the incidence of difficult intubation and complications. METHODS: All patients who were subjected to tracheal intubated with direct laryngoscopy in the Intensive Care Unit over a 45 month period were included in the study. In all patients, an evaluation was made of the laryngoscopy view using the modified Cormack-Lehane scale, as well as the technical difficulty (number of intubations at first attempt, operator-reported difficulty, need for a Frova introducer), and the incidence of complications (hypotension, hypoxia, oesophageal intubation). RESULTS: A total of 360 patients were included. When the grade of the modified Cormack-Lehane scale was increased from 1 to 4, the incidence of first success rate intubation decreased (1: 97%, 2a: 94%, 2b: 80%, 3: 60%, 4: 0%, p<.001), the incidence of moderate and severe difficulty intubation increased (1: 2%, 2a: 4%, 2b: 36%, 3: 77%, 4: 100%, p<.001.), as well as the need for a Frova guide (1: 7%, 2a: 8%, 2b: 45%, 3: 60%, 4: 100%, p<.001). When the grade of the modified Cormack-Lehane scale increased from 1 to 4, the incidence of hypoxia<90% increased (1: 20%, 2a: 20%, 2b: 28%, 3: 47%, 4: 100%, p=.0073), as well as hypoxia<80% (1: 11%, 2a: 10%, 2b: 12%, 3: 27%, 4: 100%, p=.00398). No relationship was observed between the incidence of hypotension and the grade of the modified Cormack-Lehane scale (p=ns). CONCLUSIONS: During tracheal intubation in the Intensive Care Unit a close relationship was found between a poor laryngoscopy view using the modified Cormack-Lehane scale and a higher difficulty technique of intubation. A relationship was found between the incidence of hypoxia with a higher grade in the modified Cormack-Lehane scale. No relationship was found between hypotension and the modified Cormack-Lehane scale.

«Doctor, tengo Chikungunya». Sistematización conceptual de las implicaciones epidemiológicas específicas desde la medicina de familia / «Doctor, I have Chikungunya». Conceptual systematisation of specific epidemiological implications in family medicine

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miR-26a enhances miRNA biogenesis by targeting Lin28B and Zcchc11 to suppress tumor growth and metastasis.

Human cancers often exhibit attenuated microRNA (miRNA) biogenesis and global underexpression of miRNAs; thus, targeting the miRNA biogenesis pathway represents a novel strategy for cancer therapy. Here, we report that miR-26a enhances miRNA biogenesis, which acts as a common mechanism partially accounting for miR-26a function in diverse cancers including melanoma, prostate and liver cancer. miR-26a was broadly reduced in multiple cancers, and overexpression of miR-26a significantly suppressed tumor growth and metastasis both in vitro and in vivo, including melanoma, prostate and liver cancers. Notably, miR-26a overexpression was accompanied by global upregulation of miRNAs, especially let-7, and let-7 expression was concordant with miR-26a expression in cancer cell lines, xenograft tumors and normal human tissues, underscoring their biological relevance. We showed that miR-26a directly targeted Lin28B and Zcchc11-two critical repressors of let-7 maturation. Furthermore, we have demonstrated that Zcchc11 promoted tumor growth and metastasis, and it was prominently overexpressed in human cancers. Our findings thus provide a novel mechanism by which a miRNA acts as a modulator of miRNA biogenesis. These results also define a role of the miR-26a and Zcchc11 in tumorigenesis and metastasis and have implications to develop new strategies for cancer therapy.

[Cauda equina syndrome caused by paraganglioma of the filum terminale]. / Paraganglioma del filum terminal como causa de síndrome de cauda equina.

Paragangliomas are neuroendocrine tumors that originate from cells migrating from the neural crest. They have a diverse localizations, and are common in the head, neck, mediastinum or retroperitoneum. Their growth in the filum terminale region is very infrequent. We report the case of a patient who suffered an acute cauda equina syndrome. We give a detailed description of the diagnostic process, radiological characteristics, treatment and the macro and microscopic properties of this tumor.

Paraganglioma del filum terminal como causa de síndrome de cauda equina / Cauda equina syndrome caused by paraganglioma of the filum terminale

Los paragangliomas son tumores neuroendocrinos originados a partir de células que migran de la cresta neural. Su localización es diversa, siendo frecuentes encabeza, cuello, mediastino o retroperitoneo. Su crecimiento en la región del filum terminal es muy poco frecuente. Presentamos el caso de una paciente que debuta con un cuadro agudo de cauda equina. Describimos en detalle el proceso diagnóstico, las características radiológicas, el tratamiento y las propiedades macro y microscópicas de este tumor (AU)

Paragangliomas are neuroendocrine tumors that originate from cells migrating from the neural crest. They have a diverse localizations, and are common in the head, neck, mediastinum or retroperitoneum. Their growth in the filum terminale region is very infrequent. We report the case of a patient who suffered an acute cauda equina syndrome. We give a detailed description of the diagnostic process, radiological characteristics, treatment and the macro and microscopic properties of this tumor (AU)

SIRT1 activation enhances HDAC inhibition-mediated upregulation of GADD45G by repressing the binding of NF-κB/STAT3 complex to its promoter in malignant lymphoid cells.

We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies.

The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival.

IL-6 and downstream JAK-dependent signaling pathways have critical roles in the pathophysiology of multiple myeloma (MM). We investigated the effects of a novel small-molecule JAK inhibitor (AZD1480) on IL-6/JAK signal transduction and its biological consequences on the human myeloma-derived cell lines U266 and Kms.11. At low micromolar concentrations, AZD1480 blocks cell proliferation and induces apoptosis of myeloma cell lines. These biological responses to AZD1480 are associated with concomitant inhibition of phosphorylation of JAK2, STAT3 and MAPK signaling proteins. In addition, there is inhibition of expression of STAT3 target genes, particularly Cyclin D2. Examination of a wider variety of myeloma cells (RPMI 8226, OPM-2, NCI-H929, Kms.18, MM1.S and IM-9), as well as primary myeloma cells, showed that AZD1480 has broad efficacy. In contrast, viability of normal peripheral blood (PB) mononuclear cells and CD138(+) cells derived from healthy controls was not significantly inhibited. Importantly, AZD1480 induces cell death of Kms.11 cells grown in the presence of HS-5 bone marrow (BM)-derived stromal cells and inhibits tumor growth in a Kms.11 xenograft mouse model, accompanied with inhibition of phospho-FGFR3, phospho-JAK2, phospho-STAT3 and Cyclin D2 levels. In sum, AZD1480 blocks proliferation, survival, FGFR3 and JAK/STAT3 signaling in myeloma cells cultured alone or cocultured with BM stromal cells, and in vivo. Thus, AZD1480 represents a potential new therapeutic agent for patients with MM.

Descriptive epidemiology of the outbreak of classical swine fever in Catalonia (Spain), 2001/02.

Spain suffered an outbreak of classical swine fever between June 14, 2001 and May 7, 2002, which affected 49 herds; this paper describes the epidemiological characteristics of the 39 herds that were affected in Catalonia, an area of high pig density in the north east of Spain. The outbreak took place in two waves, which affected first the province of Lleida and then Barcelona. A total of 291,058 animals were slaughtered, 59,595 belonging to infected herds; 22 of the infected herds were detected on the basis of clinical suspicion on the part of the farmer or farm veterinarian, and the other 17 were detected by surveillance methods. The transmission of the virus between herds was attributed to the movement of people in 23 per cent of the cases, to animals in 13 per cent, vehicles in 10 per cent, proximity 18 per cent, the pick-up service of the rendering plant in 8 per cent and slurry in 5 per cent; in the other nine herds (23 per cent) the route of entry of the disease could not be established. The viruses isolated in the two waves of the outbreak were 100 per cent homologous and belonged to subgroup 2.3. The origin of the outbreak remains unknown.

Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors.

Bcr-Abl tyrosine kinase has been validated as a molecular target for the treatment of chronic myelogenous leukemia (CML). More recently, it has been reported that CML patients could develop resistance to the Bcr-Abl tyrosine kinase inhibitor, imatinib (STI571, Gleevec), pointing to the need for development of additional Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies. It was also found that a significant proportion of patients who received the Bcr-Abl inhibitor did not achieve complete cytogenetic response. Mechanisms for incomplete cytogenetic response to Bcr-Abl inhibition are not entirely clear. We report here three new pyrido[2,3-d]pyrimidine Bcr-Abl tyrosine kinase inhibitors, PD164199, PD173952, PD173958, that induced apoptosis of Bcr-Abl-dependent hematopoietic cells. An interleukin-3 (IL-3) autocrine loop was observed previously in primitive CD34(+)/Bcr-Abl(+) leukemic cells in CML patients. Using 32Dp210(Bcr-Abl)and Baf3p210(Bcr-Abl) cells as models, we tested whether IL-3 might protect Bcr-Abltransformed, IL-3-responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibition. Results of trypan blue exclusion, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl] -fluoromethylketone (FITC-VAD-FMK), and Annexin-V/7-amino-actinomycin D (7-AAD) binding assays indicate that IL-3 could protect Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34(+)/Bcr-Abl(+) cells in CML patients could contribute to the incomplete eradication of Bcr-Abl(+) cells by Bcr-Abl inhibition.

Inhibition of JAK kinase activity enhances Fas-mediated apoptosis but reduces cytotoxic activity of topoisomerase II inhibitors in U266 myeloma cells.

Our previous work demonstrated that the Janus kinase (JAK)-Stat3 pathway regulates expression of Bcl-x(L) in the U266 human multiple myeloma cell line and prevents Fas-mediated apoptosis. Inhibition of this pathway by the JAK selective kinase inhibitor AG490 or dominant-negative Stat3 protein results in down-regulation of Bcl-x(L) expression and enhanced sensitivity to Fas-mediated apoptosis. Because Bcl-x(L) has also been implicated in resistance to chemotherapeutic drugs, we investigated whether inhibition of the JAK-Stat3 pathway and subsequent reduction in Bcl-x(L) expression would also enhance cytotoxic drug activity. Contrary to this prediction, pretreatment of U266 myeloma cells with AG490, followed by exposure to topoisomerase II- inhibiting agents, antagonized drug-induced apoptosis. This effect correlated with reduced cyclin D1 expression and cell cycle arrest. The cell cycle arrest following AG490 pretreatment further correlated with reduced mitoxantrone-induced DNA double-strand breaks and reduced cell death, findings consistent with the critical requirement of DNA damage for drug cytotoxicity. These studies demonstrate that inhibition of the JAK-Stat3 pathway can result in paradoxical effects relative to cytotoxic drug response. These paradoxical responses may be explained by the findings that JAK-Stat3 signaling regulates the expression of multiple genes involved in controlling cell proliferation and apoptosis. Thus, understanding the cellular context of inhibiting signal transduction pathways is essential for the design of novel combination therapies for cancer.

Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation.

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis.

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3beta protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3beta protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF --> Src --> Stat3 --> Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils.

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.

Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.