Climate Change and the Impact of Greenhouse Gasses: CO2 and NO, Friends and Foes of Plant Oxidative Stress.

Here, we review information on how plants face redox imbalance caused by climate change, and focus on the role of nitric oxide (NO) in this response. Life on Earth is possible thanks to greenhouse effect. Without it, temperature on Earth's surface would be around -19°C, instead of the current average of 14°C. Greenhouse effect is produced by greenhouse gasses (GHG) like water vapor, carbon dioxide (CO2), methane (CH4), nitrous oxides (NxO) and ozone (O3). GHG have natural and anthropogenic origin. However, increasing GHG provokes extreme climate changes such as floods, droughts and heat, which induce reactive oxygen species (ROS) and oxidative stress in plants. The main sources of ROS in stress conditions are: augmented photorespiration, NADPH oxidase (NOX) activity, ß-oxidation of fatty acids and disorders in the electron transport chains of mitochondria and chloroplasts. Plants have developed an antioxidant machinery that includes the activity of ROS detoxifying enzymes [e.g., superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PRX)], as well as antioxidant molecules such as ascorbic acid (ASC) and glutathione (GSH) that are present in almost all subcellular compartments. CO2 and NO help to maintain the redox equilibrium. Higher CO2 concentrations increase the photosynthesis through the CO2-unsaturated Rubisco activity. But Rubisco photorespiration and NOX activities could also augment ROS production. NO regulate the ROS concentration preserving balance among ROS, GSH, GSNO, and ASC. When ROS are in huge concentration, NO induces transcription and activity of SOD, APX, and CAT. However, when ROS are necessary (e.g., for pathogen resistance), NO may inhibit APX, CAT, and NOX activity by the S-nitrosylation of cysteine residues, favoring cell death. NO also regulates GSH concentration in several ways. NO may react with GSH to form GSNO, the NO cell reservoir and main source of S-nitrosylation. GSNO could be decomposed by the GSNO reductase (GSNOR) to GSSG which, in turn, is reduced to GSH by glutathione reductase (GR). GSNOR may be also inhibited by S-nitrosylation and GR activated by NO. In conclusion, NO plays a central role in the tolerance of plants to climate change.

S-nitrosylation influences the structure and DNA binding activity of AtMYB30 transcription factor from Arabidopsis thaliana.

MYB proteins are a family of transcription factors that play an important role in plant development and regulatory defense processes. Arabidopsis thaliana MYB30 (AtMYB30), a member of this protein family, is involved in cell death processes during the hypersensitive response (HR) of plants. HR is characterized by a vast production of reactive oxygen species (ROS) and nitric oxide (NO). NO may thus influence the binding of AtMYB30 to DNA. In this work we evaluated the effect of NO on AtMYB30 DNA binding activity, and also in the protein structural properties. A fully active minimal DNA-binding domain (DBD) of AtMYB30 (residues 11-116) containing two cysteine residues (C49 and C53) was overexpressed and purified. Site-directed mutagenesis was used to obtain AtMYB30 DBD mutants C49A and C53A. The DNA binding activity of AtMYB30 DBD, and Cys single mutants is clearly inhibited upon incubation with a NO donor, and S-nitrosylation was confirmed by the biotin switch assay. Finally, in order to understand the mechanism of NO effect on AtMYB30 DNA binding activity we performed circular dichroism analysis, to correlate the observed protein function inhibition and a potential structural impairment on AtMYB30 DBD. Indeed, NO modification of C49 and C53 residues promotes a subtle modification on the secondary structure of this transcription factor. We thus demonstrated, using various techniques, the in vitro effect of NO on AtMYB30 DBD, and thus the potential consequences of NO activity on plant metabolism influenced by this transcription factor.

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.

Inhibition of AtMYB2 DNA-binding by nitric oxide involves cysteine S-nitrosylation.

Nitric oxide (NO) can influence the transcriptional activity of a wide set of Arabidopsis genes. The aim of the present work was to investigate if NO modifies DNA-binding activity of AtMYB2 (a typical R2R3-MYB from Arabidopsis thaliana), by a posttranslational modification of its conserved Cys53 residue. We cloned a fully active minimal DNA-binding domain of AtMYB2 spanning residues 19-125, hereafter called M2D. In EMSA assays, M2D binds the core binding site 5'-[A]AACC[A]-3'. The NO donors SNP and GSNO inhibit M2D DNA-binding. As expected for a Cys S-nitrosylation, the NO-mediated inhibitory effect was reversed by DTT, and S-nitrosylation of Cys53 in M2D was detected by biotin switch assays. These results demonstrate that the DNA-binding of M2D is inhibited by S-nitrosylation of Cys53 as a consequence of NO action, thus establishing for the first time a relationship between the redox state and DNA-binding in a plant MYB transcription factor.

Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting.

PURPOSE: The epidermal growth factor receptor (EGFR) is abnormally activated in cancer and two classes of anti-EGFR agents, monoclonal antibodies and low-molecular-weight tyrosine kinase inhibitors, have shown antitumor activity in patients. Because these two classes of antireceptor agents target the EGFR at different sites, we decided to explore whether the combined administration of gefitinib, a tyrosine kinase inhibitor, and cetuximab, a monoclonal antibody, had superior antitumor activity than either agent given alone. EXPERIMENTAL DESIGN: We studied the effects of the combination of gefitinib and cetuximab in a panel of human cancer cell lines and in an EGFR-dependent human tumor xenograft model (A431). The effects of these two agents on EGFR signaling, proliferation, apoptosis, and vascularization were evaluated. In addition, we analyzed, with cDNA arrays, changes in gene expression profiles induced by both agents. RESULTS: The combined treatment with gefitinib and cetuximab resulted in a synergistic effect on cell proliferation and in superior inhibition of EGFR-dependent signaling and induction of apoptosis. In a series of in vivo experiments, single-agent gefitinib or cetuximab resulted in transient complete tumor remission only at the highest doses. In contrast, suboptimal doses of gefitinib and cetuximab given together resulted in a complete and permanent regression of large tumors. In the combination-treated tumors, there was a superior inhibition of EGFR, mitogen-activated protein kinase, and Akt phosphorylation, as well as greater inhibition of cell proliferation and vascularization and enhanced apoptosis. Using cDNA arrays, we found 59 genes that were coregulated and 45 genes differentially regulated, including genes related to cell proliferation and differentiation, transcription, DNA synthesis and repair, angiogenesis, signaling molecules, cytoskeleton organization, and tumor invasion and metastasis. CONCLUSIONS: Our findings suggest both shared and complementary mechanisms of action with gefitinib and cetuximab and support combined EGFR targeting as a clinically exploitable strategy.

Cyclin E gene (CCNE) amplification and hCDC4 mutations in endometrial carcinoma.

Cyclin E overexpression occurs in a subset of endometrial carcinomas (ECs), but the molecular mechanisms underlying this alteration remain to be established. The present study has analysed amplification of the cyclin E gene (CCNE) and mutation in hCDC4, the gene coding for the F-box protein, which tags phosphorylated cyclin E for proteosomal degradation, to ascertain whether these alterations might be responsible for cyclin E overexpression in ECs. Cyclin E and p53 expression was studied by immunohistochemistry in eight atypical endometrial hyperplasias (AEHs), 51 endometrioid endometrial carcinomas (EECs), and 22 non-endometrioid endometrial carcinomas (NEECs). CCNE amplification was analysed by fluorescence in situ hybridization (FISH). Mutations in exons 2-11 of the hCDC4 gene were screened by PCR-SSCP-sequencing. Finally, the polymorphic marker D4S1610 was used to assess loss of heterozygosity (LOH) in the hCDC4 gene. Cyclin E overexpression was found in 26/81 (32%) cases and was associated with the histological type of the lesion, since it was not found in any AEHs but was present in 27% of EECs and 54.5% of NEECs (p=0.035). Cyclin E overexpression was associated with histological grade (p=0.011) and p53 immunostaining in EECs (p=0.033). CCNE amplification was found in 6 of 37 (16%) ECs examined. There was a significant association between CCNE amplification and the histological type of the lesion, since five (83%) of the six cases with amplification were NEECs (p=0.008). One EEC harboured an hCDC4 mutation: a CGA to CAA (Arg/Gln) change at codon 479. In addition, D4S1610 LOH was found in 7 of 23 (30%) informative cases analysed, but no correlation with cyclin E overexpression was found. However, the tumour with hCDC4 mutation also showed LOH. This is the first study demonstrating that cyclin E overexpression is associated with gene amplification in ECs, these alterations being more frequent in NEECs. Although hCDC4 exhibits a low mutation frequency in ECs overexpressing cyclin E, it seems to function as a tumour suppressor gene that is involved in endometrial carcinogenesis.

Differential gene expression profile in endometrioid and nonendometrioid endometrial carcinoma: STK15 is frequently overexpressed and amplified in nonendometrioid carcinomas.

Endometrial carcinoma (EC) comprises at least two types of cancer: endometrioid carcinomas (EECs) are estrogen-related tumors, which are frequently euploid and have a good prognosis. Nonendometrioid carcinomas (NEECs; serous and clear cell forms) are not estrogen related, are frequently aneuploid, and are clinically aggressive. We used cDNA microarrays containing 6386 different genes to analyze gene expression profiles in 24 EECs and 11 NEECs to identify differentially expressed genes that could help us to understand differences in the biology and clinical outcome between histotypes. After supervised analysis of the microarray data, there was at least a 2-fold difference in expression between EEC and NEEC in 66 genes. The 31 genes up-regulated in EECs included genes known to be hormonally regulated during the menstrual cycle and to be important in endometrial homeostasis, such as MGB2, LTF, END1, and MMP11, supporting the notion that EEC is a hormone-related neoplasm. Conversely, of the 35 genes overexpressed in NEECs, three genes, STK15, BUB1, and CCNB2, are involved in the regulation of the mitotic spindle checkpoint. Because STK15 amplification/overexpression is associated with aneuploidy and an aggressive phenotype in other human tumors, we used fluorescence in situ hybridization to investigate whether STK15 amplification occurred in ECs. We found that STK15 was amplified in 55.5% of NEECs but not in any EECs (P

Abnormalities of E- and P-cadherin and catenin (beta-, gamma-catenin, and p120ctn) expression in endometrial cancer and endometrial atypical hyperplasia.

Abnormal expression of cadherins and catenins plays a critical role in the initiation and progression of multiple human tumours. This study aimed to evaluate the immunoreactivity of E- and P-cadherin, beta- and gamma-catenin, and p120ctn in premalignant and malignant endometrial lesions and to correlate their membranous expression with clinicopathological features. In addition, we examined whether or not LOH and promoter hypermethylation of the CDH1 gene were associated with E-cadherin expression and clinicopathological variables. Finally, we studied the frequency of beta-catenin mutations in premalignant endometrial lesions. Immunohistochemical staining was performed in 21 atypical endometrial hyperplasias (AEHs), 95 endometrioid carcinomas (EECs), and 33 non-endometrioid carcinomas (NEECs). Reduced E-cadherin expression was observed in 57.8% of the cases, being more frequent in NEECs (87.1%, p = 0.001) and carcinomas of more advanced stage (85.7% of stage III-IV carcinomas, p = 0.01). LOH of CDH1 gene was found in 57.1% of NEECs but only in 22.5% of EECs (p = 0.011) and showed a trend towards association with reduced E-cadherin expression (p = 0.089). CDH1 promoter hypermethylation was found in 21.2% of endometrial carcinomas but was not associated with clinicopathological or immunohistochemical variables. Reduced expression of beta- and gamma-catenin and p120ctn was found in 76.1%, 94.3%, and 63.6% of the cases, respectively, being more frequent in lesions with reduced E-cadherin expression. In addition, beta-catenin, but not gamma-catenin or p120ctn expression, was associated with the histology of the lesion, since it was reduced in 35% of AEHs, 80.3% of EECs, and 96.9% of NEECs (p = 0.000). Mutations in exon 3 of the beta-catenin gene, associated with beta-catenin nuclear expression, were detected in 3 (14.0%) AEH, a frequency similar to that previously reported in this series of ECs. Finally, upregulation of P-cadherin was observed in 28.6% of cases. This alteration was associated with the histology of the lesion, since it was found in 9.5% of AEHs, 27.7% of EECs, and 46.2% of NEECs (p = 0.021).

Abnormalities of the APC/beta-catenin pathway in endometrial cancer.

The activation of the APC/beta-catenin signalling pathway due to beta-catenin mutations has been implicated in the development of a subset of endometrial carcinomas (ECs). However, up to 25% of ECs have beta-catenin nuclear accumulation without evidence of beta-catenin mutations, suggesting alterations of other molecules that can modulate the Wnt pathway, such as APC, gamma-catenin, AXIN1 and AXIN2. We investigated the expression pattern of beta- and gamma-catenin in a group of 128 endometrial carcinomas, including 95 endometrioid endometrial carcinomas (EECs) and 33 non-endometrioid endometrial carcinomas (NEECs). In addition, we evaluated the presence of loss of heterozygosity and promoter hypermethylation of the APC gene and mutations in the APC, beta- and gamma-catenin, AXIN1, AXIN2, and RAS genes, and phospho-Akt expression. No APC mutations were detected but LOH at the APC locus was found in 24.3% of informative cases. APC promoter 1A hypermethylation was observed in 46.6% of ECs, and was associated with the endometrioid phenotype (P=0.034) and microsatellite instability (P=0.008). Neither LOH nor promoter hypermethylation of APC was associated with nuclear catenin expression. Nuclear beta-catenin expression was found in 31.2% of EECs and 3% of NEECs (P=0.002), and was significantly associated with beta-catenin gene exon 3 mutations (P<0.0001). beta-catenin gene exon 3 mutations were associated with the endometrioid phenotype, and were detected in 14 (14.9%) EECs, but in none of the NEECs (P=0.02). gamma-catenin nuclear expression was found in 10 ECs; it was not associated with the histological type but was associated with more advanced stages (P=0.042). No mutations in gamma-catenin, AXIN1 and 2 genes were detected in this series. Neither RAS mutations nor phospho-Akt expression, which were found in 16 and 27.6% of the cases, respectively, were associated with beta-catenin nuclear expression. Our results demonstrated a high prevalence of alterations in molecules of the APC/beta-catenin pathway, but only mutations in beta-catenin gene are associated with aberrant nuclear localization of beta-catenin.

Proteolytic activities in kidney and liver during refeeding of protein depleted mice

Los contenidos renal y hepático de proteinas disminuyen significativamente en ratones sometidos a una dieta aproteica durante cinco días. La realimentación con una dieta completa induce una rápida recuperación de la masa proteica perdida por ambos tejidos. Esta recuperación es consecuencia de una marcada inhibición de la proteólisis intracelular. El objetivo de este trabajo fue estudiar la contribución de los sistemas proteolíticos lisosomal o ácido y neutro al proceso de recuperación aludido. Para ello se evaluaron las actividades proteolíticas a pH 5.0 y pH 7.4 presentes en los tejidos homogeneizados. La actividad ácida disminuyó en ambos tejidos como consecuencia de la desnutrición proteica y se recuperó luego de 12 horas de realimentación. Sin embargo, las actividades neutras de ambos organos disminuyeron debido a la desnutrición y permanecieron en niveles bajos luego de 12 horas de realimentación. Se estudió, además, el efecto de las dietas sobre la estabilidad osmótica de los lisosomas hepáticos y renales. Esta aumentó durante la realimentación, indicando que se produce una disminución en la actividad autofágica de dichos tejidos. Estos hallazgos indican que tanto la baja actividad del sistema lisosomal vacuolar como la baja actividad del sistema proteolítico neutro serían responsables de la inhibición de la proteólisis in vivo exhibida por los riñones e higados durante la recuperación de su masa proteica