Superoxide reductase from the syphilis spirochete Treponema pallidum

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic nonhaem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P21 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A ° , = 106.9 ) and diffracted beyond 1.60 A ° resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A ° , = 104.9 ) and diffracted beyond 1.55 A ° . A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator ( = 1.542 A ° ) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P21 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

Bisphenol A at the reference level counteracts doxorubicin transcriptional effects on cancer related genes in HT29 cells

Human exposure to Bisphenol A (BPA) results mainly from ingestion of food and beverages. Information regarding BPA effects on colon cancer, one of the major causes of death in developed countries, is still scarce. Likewise, little is known about BPA drug interactions although its potential role in doxorubicin (DOX) chemoresistance has been suggested. This study aims to assess potential interactions between BPA and DOX on HT29 colon cancer cells. HT29 cell response was evaluated after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis of several cancer-associated genes (c-fos, AURKA, p21, bcl-xl and CLU) shows that BPA exposure induces slight up-regulation exclusively of bcl-xl without affecting cell viability. On the other hand, a sub-therapeutic DOX concentration (40nM) results in highly altered c-fos, bcl-xl, and CLU transcript levels, and this is not affected by co-exposure with BPA. Conversely, DOX at a therapeutic concentration (4μM) results in distinct and very severe transcriptional alterations of c-fos, AURKA, p21 and CLU that are counteracted by co-exposure with BPA resulting in transcript levels similar to those of control. Co-exposure with BPA slightly decreases apoptosis in relation to DOX 4μM alone without affecting DOX-induced loss of cell viability. These results suggest that BPA exposure can influence chemotherapy outcomes and therefore emphasize the necessity of a better understanding of BPA interactions with chemotherapeutic agents in the context of risk assessment.

Identification of mating type genes in the bipolar basidiomycetous yeast rhodosporidium toruloides: first insight into the MAT Locus structure of the sporidiobolales

Eukaryotic Cell, Vol.7, Nº6

Anti-neoplastic properties of hydralazine in prostate cancer

Prostate cancer (PCa) is a major cause of cancer-related morbidity and mortality worldwide. Although early disease is often efficiently managed therapeutically, available options for advanced disease are mostly ineffective. Aberrant DNA methylation associated with gene-silencing of cancer-related genes is a common feature of PCa. Therefore, DNA methylation inhibitors might constitute an attractive alternative therapy. Herein, we evaluated the anti-cancer properties of hydralazine, a non-nucleoside DNA methyltransferases (DNMT) inhibitor, in PCa cell lines. In vitro assays showed that hydralazine exposure led to a significant dose and time dependent growth inhibition, increased apoptotic rate and decreased invasiveness. Furthermore, it also induced cell cycle arrest and DNA damage. These phenotypic effects were particularly prominent in DU145 cells. Following hydralazine exposure, decreased levels of DNMT1, DNMT3a and DNMT3b mRNA and DNMT1 protein were depicted. Moreover, a significant decrease in GSTP1, BCL2 and CCND2 promoter methylation levels, with concomitant transcript re-expression, was also observed. Interestingly, hydralazine restored androgen receptor expression, with upregulation of its target p21 in DU145 cell line. Protein array analysis suggested that blockage of EGF receptor signaling pathway is likely to be the main mechanism of hydralazine action in DU145 cells. Our data demonstrate that hydralazine attenuated the malignant phenotype of PCa cells, and might constitute a useful therapeutic tool.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

PIDD orchestrates translesion DNA synthesis in response to UV irradiation.

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location.

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3-T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Although p53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, and p53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclear p53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55 vs. 21 percent; chi-squared test, P < 0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test, P = 0.01; hazard ratio = 2.16; 95 percent confidence interval = 1.12-4.11, P = 0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P = 0.008), degree of histologic differentiation (P = 0.012), p53 protein expression, and gene mutation (P = 0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test, P = 0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent of p53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.

E2F1 mediates DNA damage and apoptosis through HCF-1 and the MLL family of histone methyltransferases.

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti-oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1-to-S phase transition, E2F1 associates through a short DHQY sequence with the cell-cycle regulator HCF-1 together with the mixed-lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF-1-binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF-1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF-1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF-1-binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF-1 association with E2F1 is a regulator of E2F1-induced apoptosis.

Molecular pathways involved in the antineoplastic effects of calcitriol on insulinoma cells.

We have previously reported that in tumorigenic pancreatic beta-cells, calcitriol exerts a potent antitumorigenic effect by inducing apoptosis, cell growth inhibition, and reduction of solid beta-cell tumors. Here we have studied the molecular pathways involved in the antineoplastic activity of calcitriol on mouse insulinoma beta TC(3) cells, mouse insulinoma beta TC expressing or not expressing the oncogene p53, and beta TC-tet cells overexpressing or not the antiapoptotic gene Bcl2. Our results indicate that calcitriol-induced apoptosis was dependent on the function of p53 and was associated with a biphasic increase in protein levels of transcription factor nuclear factor-kappa B. Calcitriol decreased cell viability by about 40% in p53-retaining beta TC and in beta TC(3) cells; in contrast, beta TC p53(-/-) cells were only minimally affected. Calcitriol-induced cell death was regulated by members of the Bcl-2 family of apoptosis regulatory proteins, as shown by calcitriol-induced up-regulation of proapoptotic Bax and Bak and the lack of calcitriol-induced cytotoxicity in Bcl-2-overexpressing insulinoma cells. Moreover, calcitriol-mediated arrest of beta TC(3) cells in the G(1) phase of the cell cycle was associated with the abnormal expression of p21 and G(2)/M-specific cyclin B2 genes and involved the DNA damage-inducible factor GADD45. Finally, in beta TC(3) cells, calcitriol modulated the expression of IGF-I and IGF-II genes. In conclusion, these findings contribute to the understanding of the antitumorigenic effects of calcitriol on tumorigenic pancreatic beta-cells and further support the rationale of its utilization in the treatment of patients with malignant insulinomas.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

Sleep deprivation induces transcriptional modifications of genes related to astrocyte-neuron lactate shuttle in astrocytes

Astrocytes play a key role in the neurometabolic coupling through the glycogen metabolism and the ''Astrocyte-Neuron Lactate Shuttle'' (ANLS). We previously reported that brain glycogen metabolism was affected by sleep deprivation (SD). Therefore, it is of prime interest to determine if a similar sleep loss also affects the ANLS functioning in astrocytes. To address this issue, we sleep deprived transgenic mice expressing the GFP under the control of the GFAP promoter and in which astrocytes can be isolated by FACS. The levels of expression of genes related to ANLS were assessed by qRT-PCR in the GFP-positive cells (GFPþ). The FVB/NTg( GFAP-GFP)Mes14/j mice were weaned at P20-P21 and underwent an instrumental 6 h SD at P23-P27. The SD was realized using the ''CaResS'' device which has been designed to minimize stress during SD. Control group corresponds to undisturbed mice. At the end of SD, mice were sacrificed and their cerebral cortex was rapidly dissected, cut in small pieces and enzymatically digested. After cell dissociation, GFPþ and GFP- cells were sorted by FACS and treated for RNA extraction. A quantitative RTPCR was realized using specific probes against different genes involved in ANLS. Results indicate that genes encoding the LDHb, the GLT1, the alpha2 subunit of the Na/KATPase pump as well as the GLUT1, were significantly increased in the GFPþ cells from SD mice. No significant change was observed in the GFP- cells from the same group. These results indicate that this approach is suitable to determine the transcriptional signature of SD in glial cells from juvenile animals. They also indicate that sleep loss induces transcriptional changes of genes involved in ANLS specifically in astrocytes. This could suggest that an adaptation of the ANLS at the transcriptional levels exists in pathophysiological conditions where neuronal activity is enhanced.

Alterations of specific biomarkers of metabolic pathways in vascular tree from patients with Type 2 diabetes.

The aims of this study were to check whether different biomarkers of inflammatory, apoptotic, immunological or lipid pathways had altered their expression in the occluded popliteal artery (OPA) compared with the internal mammary artery (IMA) and femoral vein (FV) and to examine whether glycemic control influenced the expression of these genes. The study included 20 patients with advanced atherosclerosis and type 2 diabetes mellitus, 15 of whom had peripheral arterial occlusive disease (PAOD), from whom samples of OPA and FV were collected. PAOD patients were classified based on their HbA1c as well (HbA1c ≤ 6.5) or poorly (HbA1c > 6.5) controlled patients. Controls for arteries without atherosclerosis comprised 5 IMA from patients with ischemic cardiomyopathy (ICM). mRNA, protein expression and histological studies were analyzed in IMA, OPA and FV. After analyzing 46 genes, OPA showed higher expression levels than IMA or FV for genes involved in thrombosis (F3), apoptosis (MMP2, MMP9, TIMP1 and TIM3), lipid metabolism (LRP1 and NDUFA), immune response (TLR2) and monocytes adhesion (CD83). Remarkably, MMP-9 expression was lower in OPA from well-controlled patients. In FV from diabetic patients with HbA1c ≤6.5, gene expression levels of BCL2, CDKN1A, COX2, NDUFA and SREBP2 were higher than in FV from those with HbA1c >6.5. The atherosclerotic process in OPA from diabetic patients was associated with high expression levels of inflammatory, lipid metabolism and apoptotic biomarkers. The degree of glycemic control was associated with gene expression markers of apoptosis, lipid metabolism and antioxidants in FV. However, the effect of glycemic control on pro-atherosclerotic gene expression was very low in arteries with established atherosclerosis.