Electromagnetic behavior of dielectric objects on metallic periodically nanostructured substrates.

In this research, we investigate the electromagnetic behavior of a metallic thin-film with a periodic array of subwavelength apertures when dielectric objects are located on it. The influence of size, geometry and optical properties of the objects on the transmission spectra is numerically analyzed. We study the sensitivity of this system to changes in the refractive index of the illuminated volume induced by the presence of objects with sizes from hundreds of nanometers (submicron-sized objects) to a few microns (micron-sized objects). Parameters such as the object volume within the penetration depth of the surface plasmon in the buffer medium or the contact surface between the object and the nanostructured substrate strongly affect the sensitivity. The proposed system models the presence of objects and their detection through the spectral shifts undergone by the transmission spectra. Also, we demonstrate that these can be used for obtaining information about the refractive index of a micron-sized object immersed in a buffer and located on the nanostructured sensitive surface. We believe that results found in this study can help biomedical researchers and experimentalists in the process of detecting and monitoring biological organisms of large sizes (notably, cells).

ODZ1 allows glioblastoma to sustain invasiveness through a Myc-dependent transcriptional upregulation of RhoA.

Long-term survival remains low for most patients with glioblastoma (GBM), which reveals the need for markers of disease outcome and novel therapeutic targets. We describe that ODZ1 (also known as TENM1), a type II transmembrane protein involved in fetal brain development, plays a crucial role in the invasion of GBM cells. Differentiation of glioblastoma stem-like cells drives the nuclear translocation of an intracellular fragment of ODZ1 through proteolytic cleavage by signal peptide peptidase-like 2a. The intracellular fragment of ODZ1 promotes cytoskeletal remodelling of GBM cells and invasion of the surrounding environment both in vitro and in vivo. Absence of ODZ1 by gene deletion or downregulation of ODZ1 by small interfering RNAs drastically reduces the invasive capacity of GBM cells. This activity is mediated by an ODZ1-triggered transcriptional pathway, through the E-box binding Myc protein, that promotes the expression and activation of Ras homolog family member A (RhoA) and subsequent activation of Rho-associated, coiled-coil containing protein kinase (ROCK). Overexpression of ODZ1 in GBM cells reduced survival of xenografted mice. Consistently, analysis of 122 GBM tumour samples revealed that the number of ODZ1-positive cells inversely correlated with overall and progression-free survival. Our findings establish a novel marker of invading GBM cells and consequently a potential marker of disease progression and a therapeutic target in GBM.

Blockade of the NFκB pathway drives differentiating glioblastoma-initiating cells into senescence both in vitro and in vivo.

Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy because of its aggressive behavior. Cancer-initiating or progenitor cells have been described to be the only cell population with tumorigenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells or the early precursors may be beneficial. We have established different cultures of glioblastoma-initiating cells (GICs) derived from surgical specimens and found that, after induction of differentiation, the NFκB transcriptional pathway was activated, as determined by analyzing key proteins such as p65 and IκB and the upregulation of a number of target genes. We also showed that blockade of nuclear factor (NF)κB signaling in differentiating GICs by different genetic strategies or treatment with small-molecule inhibitors, promoted replication arrest and senescence. This effect was partly mediated by reduced levels of the NFκB target gene cyclin D1, because its downregulation by RNA interference reproduced a similar phenotype. Furthermore, these results were confirmed in a xenograft model. Intravenous treatment of immunodeficient mice bearing human GIC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultrastructural alterations of the brain parenchyma were detected. These findings reveal that activation of NFκB may keep differentiating GICs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed to promote premature senescence of differentiating GICs by blocking key factors within the NFκB pathway.

PAR bZIP-bik is a novel transcriptional pathway that mediates oxidative stress-induced apoptosis in fibroblasts.

PAR bZIP (cells knockout for PAR bZIP transcription factors) proteins, thyrotroph embryonic factor (TEF), albumin D-site-binding protein (DBP), and hepatic leukemia factor (HLF), are a family of transcription factors that have been shown to contribute to the expression of genes involved in detoxification and drug metabolism. Recently, we showed that PAR bZIP proteins were able to regulate the BH3-only gene bcl-gS in tumor cells. Here, we have extended the role of these transcription factors in the control of apoptosis executors by analyzing the expression of BH3-only genes in PAR bZIP triple knockout mouse fibroblasts. We found that bik was the only BH3-only gene downregulated in knockout cells. Consistently, transfection of TEF or DBP induces the expression of endogenous bik, regardless of the presence of active p53. Moreover, both promoter-reporter and chromatin immunoprecipitation assays indicate that PAR bZIP proteins activate the bik promoter directly. Treatment with different stress stimuli reveals a higher survival of knockout fibroblasts compared with that of wild-type cells, especially after incubation with H(2)O(2), which suggest that PAR bZIP proteins participate in oxidative stress-induced apoptosis. Furthermore, the apoptotic cell death promoted by treatment with H(2)O(2) can be impaired by reducing the expression of Bik in wild-type fibroblasts or enhanced by the overexpression of Bik in knockout cells. These findings reveal a novel transcriptional pathway relevant in transducing the apoptotic response to oxidative stress.

Apoptosis regulators as targets for cancer therapy.

Apoptosis serves to remove excess or damaged cells and its dysregulation may lead to a number of pathological disorders including cancer. Studies during the last 20 years have unravelled much of the molecular mechanisms that control apoptosis. Whether a cell dies in response to diverse apoptotic stimuli, including DNA-damaging agents, is determined largely by interactions between proteins of the Bcl-2 family. A death signal is transmitted through the BH3-only proteins to Bax and Bak which in turn permeabilise the outer mitochondrial membrane allowing the release of apoptogenic factors, which triggers activation of cell-deathpromoting caspases. These proteolytic enzymes are tightly controlled by members of the inhibitor of apoptosis (IAP) family. Activation of the caspase cascade via cell death receptors also represents a key apoptotic pathway in both normal and tumour cells. Basic knowledge of these apoptosis regulators provides the basis for novel therapeutic strategies aimed at promoting tumour cell death or enhancing susceptibility to apoptotic inducers. This review focuses on these strategies.

Apoptosis regulators as targets for cancer therapy

Apoptosis serves to remove excess or damaged cells and its dysregulation may lead to a number of pathological disorders including cancer. Studies during the last 20 years have unravelled much of the molecular mechanisms that control apoptosis. Whether a cell dies in response to diverse apoptotic stimuli, including DNA-damaging agents, is determined largely by interactions between proteins of the Bcl-2 family. A death signal is transmitted through the BH3-only proteins to Bax and Bak which in turn permeabilise the outer mitochondrial membrane allowing the release of apoptogenic factors, which triggers activation of cell-deathpromoting caspases. These proteolytic enzymes are tightly controlled by members of the inhibitor of apoptosis (IAP) family. Activation of the caspase cascade via cell death receptors also represents a key apoptotic pathway in both normal and tumour cells. Basic knowledge of these apoptosis regulators provides the basis for novel therapeutic strategies aimed at promoting tumour cell death or enhancing susceptibility to apoptotic inducers. This review focuses on these strategies (AU)

A novel mutation in the E-cadherin gene in the first family with hereditary diffuse gastric cancer reported in Spain.

AIMS: Mutations of the E-cadherin gene (CDH1) result in dominantly inherited hereditary diffuse gastric cancer (HDGC). We report a study in the first family diagnosed with HDGC in Spain, examining the presence of mutations in the CDH1 gene. METHODS: The presence of mutations was studied by direct sequencing of all CDH1 exons. Immunohistochemical analysis with specific antibodies was used to detect the expression of E-cadherin in normal and tumour tissue. RESULTS: A novel 1610delC mutation in exon 11 has been found in a Spanish family diagnosed with HDGC. This mutation generates a premature stop codon at position 1667 giving rise to a truncated protein that lacks the transmembrane and beta-catenin-binding domains. The presence of a 1610delC germline mutation was confirmed in three family members diagnosed with diffuse gastric cancer, and also in six asymptomatic members. Of note, the diffuse gastric cancer coexisted with a gastric lymphoma in the proband. Furthermore, immunohistochemical analyses of tumour tissue showed the complete absence of E-cadherin in the proband, revealing a second genetic hit at the CDH1 locus. CONCLUSIONS: We have identified a HDGC family in Spain that carries a novel germline truncating mutation in the CDH1 gene.

Case-control study and meta-analysis of low density lipoprotein receptor-related protein gene exon 3 polymorphism in Alzheimer's disease.

The low density lipoprotein receptor-related protein (LRP) may influence both the clearance and the production of beta-amyloid peptide and thus plays a role in Alzheimer's disease (AD) pathogenesis. Previous studies, although inconsistent, have suggested that the LRP exon 3 CC genotype contributes to the risk of AD. A case-control study utilizing a clinically well-defined group of 305 sporadic AD patients and 304 control subjects was performed to test this association in an ethnically homogeneous population from Spain. In the current study, the LRP CC genotype was not over-represented in AD patients compared to non-demented controls. A meta-analysis of previous studies revealed a weak correlation of LRP CC genotype with AD (odds ratio of 1.35, P=0.01).

The --491 TT apolipoprotein E promoter polymorphism is associated with reduced risk for sporadic Alzheimer's disease.

Homozygosity for the A allele of the -491 A/T apolipoprotein E (APOE) promoter polymorphism has recently been reported to be associated with sporadic Alzheimer's disease (AD). Two hundred and fifty one patients with AD and an equal number of controls derived from the same region in a Spanish population, were genotyped for -491 A/T and epsilon2/epsilon3/epsilon4 APOE polymorphisms. We did not detect an elevated -491 AA genotype frequency when comparing AD cases to controls. In contrast, persons homozygous for the T allele were at a significantly reduced risk of AD (odds ratio of 0.10, P=0.006). Multiple logistic regression analysis indicated that the -491 TT polymorphism added information on the risk of AD which was independent of that of the APOE epsilon4 allele.

Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic Hrk gene in hematopoietic progenitor cells.

The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3' untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin-3 (IL-3)-dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL-3 or treated with a calcium ionophore or a phosphatidylinositol 3-kinase-specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3-kinase-dependent, but Akt-independent pathway. In all cases, loss of the DREAM-DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL-3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.

Antiapoptotic protein Bcl-x(L) is up-regulated during megakaryocytic differentiation of CD34(+) progenitors but is absent from senescent megakaryocytes.

OBJECTIVE: The expression of Bcl-x(L) has been shown to be regulated during the maturation process of different hematopoietic cell lineages (i.e., erythroid cells, neutrophils, monocytes/macrophages). In the present study, we examined the expression of Bcl-x(L) in megakaryocytes derived from CD34(+) progenitors and in the megakaryoblastic cell line UT7. MATERIALS AND METHODS: Expression of Bcl-x(L) was analyzed in CD41(+) cells cultured in the presence of thrombopoietin and in UT7 cells treated with phorbol diester by Western blot, flow cytometry, and immunocytochemistry analysis. Apoptosis was determined at different culture times by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and propidium iodide uptake. RESULTS: Bcl-x(L) but not Bcl-2 was up-regulated in the megakaryocytic population (CD41(+)) during the first 15 days of culture, which was consistent with the pattern of Bcl-x(L) expression in UT7 cells differentiated to megakaryocytes by incubation with phorbol diester. However, by day 20 of culture, the levels of Bcl-x(L) in CD41(+) cells were greatly reduced, and this expression pattern was accompanied by an increase in the number of apoptotic cells. At this culture time, we detected the presence of cytoplasmic fragments resembling proplatelets with prominent Bcl-x immunostaining, most likely due to the Bcl-x(L) isoform, in close proximity to Bcl-x(-) senescent megakaryocytes. The presence of Bcl-x(L) but not of Bcl-2 in platelets was confirmed by Western blot analysis. CONCLUSION: Although little is known regarding the functional significance of survival proteins within the megakaryocytic compartment, the changes in the Bcl-x(L) expression pattern observed in UT7 and CD41(+) cells may play a role in the survival of developing megakaryocytes and the lifespan of mature platelets.

Specific and rapid induction of the proapoptotic protein Hrk after growth factor withdrawal in hematopoietic progenitor cells.

Hrk is a newly described proapoptotic member of the Bcl-2 family that is mainly expressed in hematopoietic tissues and cultured neurons. In this study we have examined the expression and activity of Hrk in hematopoietic progenitors. To address these issues, we used 3 growth factor-dependent murine hematopoietic cell lines, HCD-57, FDCP-Mix, and FL5.12. The expression of Hrk was undetectable in cells cultured with growth factors, but it was rapidly up-regulated on growth factor withdrawal. In contrast, the expression of Bcl-x(L) decreased and that of proapoptotic Bax, Bad, and Bak was unchanged or down-regulated after removal of growth factors. This pattern of expression correlated with the induction of apoptosis. Hrk was also up-regulated in human cell lines and in bone marrow-derived CD34(+) cells cultured in the absence of growth factors. In addition, the levels of Hrk were up-regulated after treatment with the chemotherapeutic drug etoposide. Expression of prosurvival Bcl-x(L) or Bcl-2 proteins blocked the induction of Hrk. Hrk was induced in FDCP-Mix cells treated with ionomicin in the presence of IL-3, suggesting that cytosolic calcium may regulate the expression of this proapoptotic protein. Furthermore, ectopic expression of Hrk induced cell death of hematopoietic progenitors in the presence of IL-3. Thus, Hrk is specifically and rapidly induced in hematopoietic progenitors after growth factor deprivation or treatment with chemotherapeutic drugs, and this may be sufficient to induce apoptosis in these cells. (Blood. 2000;95:2742-2747)

Telomerase activity in pulmonary neuroendocrine tumors: correlation with histologic subtype (MS-0060).

The authors measured telomerase activity using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay method in 13 neuroendocrine pulmonary neoplasms and in non-neoplastic frozen lung samples from the same patients. These cases belonged to the complete neuroendocrine neoplastic spectrum: four typical carcinoids, three atypical carcinoids, four large cell neuroendocrine lung carcinomas, and two small cell lung carcinomas. The authors performed the same assay for 52 non-neoplastic lung tissues from the surgical files in their department (negative controls). They verified the presence (or absence) of neoplastic tissue in every case by looking at one frozen section done in the same tissue used for telomerase assay. The telomerase activity level in non-neoplastic tissues (mean, 182 A450nm U) was similar to that obtained in the typical carcinoids (mean, 104.5 A450nm U). All neuroendocrine tumors but the typical carcinoids showed high levels of telomerase activity (mean, 1,750.8 A450nm U). According to the telomerase hypothesis, typical carcinoid cells are mortal pre-M2 stage cells, but atypical carcinoid, large cell neuroendocrine lung carcinoma, and small cell lung carcinoma cells are immortal post-M2 stage cells. This finding may be of important prognostic significance in these kinds of tumors. Measurement of enzyme activity with a good morphologic control could be necessary in telomerase activity assay.

Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL.

Bcr-Abl-expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl-expressing cell lines and CD34(+) cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.

Bcr-Abl and inhibition of apoptosis in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) cells are highly resistant to apoptosis induced by chemotherapeutic drugs. The observation that production of Bcr-Abl is the initiating event in CML has focussed attention on the survival signals triggered by this oncogene. A number of signal transducers and transcription factors have been associated with the antiapoptotic phenotype of CML cells, some of which lead to the expression and/or activation of members of the Bcl-2 family of apoptosis modulators, such as Bcl-xL and Bad. In this article, recent advances in understanding the antiapoptotic pathways triggered by Bcr-Abl in CML cells, are discussed.

Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines.

Erythropoietin (Epo) initiates its cellular response by binding to the Epo receptor, which triggers the activation of signal transducer and activator of transcription (Stat) 5 protein. Cell culture studies of erythroid progenitors have suggested that Epo functions as a survival factor by repressing apoptosis at least in part through Bcl-x(L), an anti-apoptotic protein of the Bcl-2 family. In this report, we examine whether Stat5 can induce transactivation of the bcl-x gene in response to Epo. Two Epo-responsive progenitor cell lines, HCD-57 and Bcl-2-transfected Ba/F3-Epo receptor (Ba/F3-EpoR-Bcl-2), were used in this study. After Epo stimulation, we observed a correlation between expression of bcl-x(L) and activation of Stat5 as assessed by the expression of oncostatin M, a direct target of Stat5, and the phosphorylation and nuclear translocation of Stat5. Moreover, a Stat binding element in the bcl-x promoter was found to be active in response to Epo, a finding that was further confirmed because mutagenesis of this sequence motif abrogated its promoter activity and overexpression of a dominant negative Stat5 protein blocked transactivation. When DNA-protein binding analyses were performed, we found that Stat5, not Stat1 or Stat3, was the protein bound to the bcl-x promoter in response to Epo. These data suggest that Epo-dependent activation of Stat5 is a transcriptional pathway that can be used by Epo-responsive progenitor cells to induce the expression of bcl-x(L) and consequently to inhibit apoptosis.

[The prevalence of the Cys282Tyr mutation in the hemochromatosis gene in Cantabria in patients diagnosed with hereditary hemochromatosis]. / Prevalencia de la mutación Cys282Tyr del gen de la hemocromatosis en Cantabria y en los pacientes diagnosticados de hemocromatosis hereditaria.

BACKGROUND: The aim of our study was to evaluate the prevalence of Cys282Tyr mutation in patients with genetic haemochromatosis (GH) in Cantabria. PATIENTS AND METHODS: The HFE Cys282Tyr mutation was determined in a cohort of 60 patients with GH and 213 controls. RESULTS: The frequency of the Cys282Tyr mutation in control individuals was 4.4%. Sixty-seven percent of patients with GH were homozygous for the Cys282Tyr mutation. Twenty-seven percent of patients were normal at Cys282Tyr loci. CONCLUSIONS: The prevalence of the Cys282Tyr mutation in patients with GH in Cantabria, Spain, seems to be lower than in North America and in North Europe patients.

Apoptosis and polycythemia vera.

Polycythemia vera is an acquired clonal myeloproliferative disorder characterized by increased numbers of erythroid cells, often with a concomitant rise in neutrophils and/or megakaryocytes. Normally, erythropoietin is essential for the survival and proliferation of erythroid progenitors; however in polycythemia vera the erythroid progenitor cells can survive and develop in the absence of erythropoietin. Members of the Bcl-2 family of apoptosis regulators have been shown to mediate the erythropoietin-dependent survival of erythroid cells. In this article, recent advances in understanding the mechanisms used by erythroid progenitors from patients with polycythemia vera to control apoptosis, are discussed.

Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells.

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.