Conversely, inhibition of phosphorylation by HGF in protein kinase C (PKC), protein kinase R (PKR), and also CDK1 was identified. Phosphoantibody-based immunohistochemical analysis of SCLC tumour tissue and microarray established the role of c-MET in SCLC biology. This supports a role of c-MET activation in tumour invasive front in the tumour progression and invasion involving FAK and AKT downstream. The c-MET serves as an attractive therapeutic target in SCLC, as shown through small interfering RNA (siRNA) and selective prototype c-MET inhibitor SU11274, inhibiting the phosphorylation of c-MET itself and its downstream molecules such as AKT, S6 kinase, and ERK1/2. Investigation of mechanisms of invasion and, ultimately, metastasis in SCLC would be very useful with these signal transduction molecules.The c-MET receptor can be overexpressed, amplified, or mutated in solid tumours including small cell lung cancer (SCLC). In c-MET-overexpressing SCLC cell line NCI-H69, hepatocyte growth factor (HGF) dramatically induced c-MET phosphorylation at phosphoepitopes pY1230/1234/1235 , pY1003 (juxtamembrane), and also of paxillin at pY31 (CRKL-binding site). We utilised a global proteomics phosphoantibody array approach to identify further c-MET/HGF signal transduction intermediates in SCLC. Strong HGF induction of specific phosphorylation sites in phosphoproteins involved in c-MET/HGF signal transduction was detected, namely adducin- The c-MET receptor tyrosine kinase (c-MET RTK) is the receptor for hepatocyte growth factor/scatter factor HGF/SF; .Small cell lung cancer is a very difficult disease with very poor prognosis and distant metastasis is often present at diagnosis  and phosphoantibody immunostaining, we also gained further insight into the role of c-MET/HGF signalling in SCLC biology and tumour invasion. Finally, novel targeted therapeutics against c-MET in SCLC was validated by small interfering RNA (siRNA) and the c-MET inhibitor SU11274.L-glutamate, sodium pyruvate, and HEPES buffer as described previously (−1 for 7.5 min).Small cell lung cancer cell line NCI-H69 was purchased from the American Type Culture Collection  and maintained in RPMI-1640  supplemented with 10% (v/v) fetal calf serum (FCS), −1, 7.5 min) stimulation.SU11274 was provided by Pfizer Inc.  and was used as described previously (http://www.kinexus.ca/KPSS.htm). Kinetworks Phospho-Site Screen 1.3 and KPSS-2.0 track 31 and 37 known phosphorylation sites, respectively in phosphoproteins with antibodies that recognise specific phosphorylated epitopes of the target proteins  that relies on sodium dodecyl sulphate (SDS)-polyacrylamide minigel electrophoresis and multilane immunoblotters to permit the specific and quantitative detection of numerous protein kinases (PK) or other signal transduction proteins simultaneously -1.3 and 2.0 . A wide range of phosphorylation site-specific antibodies were used in a qualitative and quantitative fashion as a specific assay for regulation of diverse cell signalling pathways  as loading control. Lung tumour tissue samples were collected with informed consent and in accordance with Institutional Review Board (IRB) approval protocols at the University of Chicago. Tumour tissue microarray was built using the ATA-27 Arrayer (1-mm punch size) from Beecher Instruments Inc. . The tumour microarray consists of nine SCLC tumour samples, and, as controls, two lung adnocarcinoma specimens. Corresponding normal lung or adjacent normal tissues were included in the microarray as negative controls as well. Each specimen was included in duplicates in the array. Tumour tissue immunohistochemistry (IHC) staining was performed using standard techniques as described previously  1/2 [S221/S225], ERK1 [T185/Y187], ERK2 [T185/Y187], ERK1/2 [T202/Y204]; stress and inflammatory response to cytokines and growth factors: MAPKK (MEK) 3/6 [S189/S207], p38α-MAPK [T180/Y182]; and also JNK [T183/Y185]; cytoskeletal functions: FAK [Y576/S722/S910], adducin-α [S724], and adducin-γ [S662]. Increased adducin expression has also been implicated in cell proliferation , adducin-γ [S662] (125%), CREB [S133] (143%), ERK1 [T185/Y187] (449%), ERK1/2 [T202/Y204] (558%), ERK2 [T185/Y187] (289%), MAPKK (MEK) 1/2 [S221/S225] (183%), MAPKK (MEK) 3/6 [S189/S207] (118%), RB [S612] (146%), RB1 [S780] (197%), JNK [T183/Y185] (163%), STAT3 [S727] 139%), FAK [Y576] (132%), FAK [S722] (123%), FAK [S910] (165%), p38α-MAPK [T180/Y182] (136%), and AKT1 [S473] (166%) and [T308] (207%).Compared with the untreated control of the SCLC NCI-H69 cells, HGF stimulation at 40 ng mlphosites : adducin−1; 7.5 min) caused a reduction of phosphorylation in the following phosphoproteins at the specified phosphosites (α [S657] (39% reduction), PKCα/β [T368/641] (36% reduction), PKCδ [T505] (30% reduction), PKR [T451] (46% reduction), and also CDK1 [T14/Y15] (38% reduction).Treatment of the H69 cells by HGF (40 ng mlphosites : PKCα [Sα [S657] (39% reduction), PKCα/β [T368/641] (36% reduction), and PKCδ [T505] (30% reduction). In SCLC NCI-H69 cells, HGF also induced phosphorylation on adducin-α [S724] (146%), and adducin-γ [S662] (125%), which have not been reported earlier.Substantial evidence has been culminated to support the key role of c-MET/HGF signalling in mediating cell motility and cytoskeletal functions in SCLC . There hin vivo c-MET/HGF signalling in SCLC tumour tissues, we performed IHC analysis in SCLC tumours, as established on a tissue microarray. Various phosphospecific antibodies were used in the IHC analysis to provide both qualitative and quantitative information of the signalling pathways in the tumours (To understand better the role of the  tumours . We founThere were 56% (5/9) SCLC samples that had p-Tyr expression, all with strong (3+) IHC staining. It is interesting to note that p-ERK1/2 staining was uniformly strong (3+) in its staining pattern in 89% (8/9) positive samples. The Ki-67 staining was positive in 89% (8/9) SCLC samples. Positive staining in p-FAK [pY861] and p-AKT [pS473] were seen in 67 and 56% of samples, respectively .Tumour tissue microarray analysis allows simultaneous analysis of a number of different phosphoproteins both within the same tumour tissue core and also among different tumour tissues. Analysis of both the IHC staining patterns of p-MET and Ki-67 indicates that the strong staining intensities do not coincide within the same tumours, suggesting that activated p-MET does not necessarily activate the cell proliferation pathway. On the other hand, p-MET  staining coincided with p-FAK and p-AKT expression, suggesting the role of c-MET activation in cell migration, invasion, and survival. None of the three normal lung or paired normal tissues in the TMA showed any expression of either c-MET or p-MET.We also studied the role of c-MET/HGF signalling pathway in SCLC tumour tissues using phosphospecific antibodies IHC analysis with focus on its topographic pattern of expression. The downstream signalling molecules p-AKT [S473] and p-FAK [Y861] were studied in addition to HGF, c-MET, p-MET (both Y1003 and Y1230/1234/1235), and p-Tyr. In one of the four SCLC tumour tissues (25%) screened, preferential c-MET overexpression and activation of p-MET (both the phosphoepitopes pY1003 and pY1230/1234/1235) along the tumour expanding invasive front were identified . SimilarNext, we investigated the potential role of targeting c-MET to inhibit SCLC. We utilised c-MET-specific siRNA to knock down the c-MET signalling in the SCLC NCI-H69 cells using standardised techniques as described in the Materials and methods . c-MET rα p-MAP kinase [T180/Y182], p-AKT1 [S473], p-RB [S672], p-adducin-γ [S662], and p-CREB [S133]). SU11274 was also effective in abrogating the inhibitory effect of HGF on the specific phosphorylation of p-PKCα [S657], p-PKCα/β [T368], and p-CDK1 [T14/Y15] , and PYK2. Here, we adopted a global phosphoantibody array-based approach to delineate further the c-MET/HGF signal transduction pathway and its downstream signalling intermediates in the SCLC phosphoproteome. Using SCLC NCI-H69 cells with and without HGF stimulation as the model, the screening arrays KPSS-1.3 and KPSS-2.0 together allowed detection of strong HGF-induction of specific phosphorylation sites in phosphoproteins downstream of c-MET itself, that are involved in diverse cellular regulation, including transcriptional control, cell cycle G−1 is associated with a trend towards worse survival  and antisense BCL-2 (Oblisense) has been far from successful . The c-M


The compounds have beencharacterized by elemental analysis and 1H-NMR, Uv-Vis, FT-IR and MOssbauer spectroscopic techniques.Attempted recrystallization of (2) in DMSO/methanol 2:1 solution yielded after several days unexpectedlythe dimeric compound bis(tri-phenyltin)sulphide {[(C6H5)3Sn]2S} (7) which has been characterized by x-rayanalysis. The structure of the parent complex (2) as well as the mechanism of the decomposition of cysteineare being further investigated. The in vitro anticancer activity of complexes (I)- (6), against humanleukemia (HL60), human liver (Bel7402), human stomach (BGC823) and human cervix epithelial humancarcinoma (Hela), nasopharyngeal carcinoma (KB) and lung cancer (PG) tumor cells, were evaluated.The synthesis and characterization of the organotin compounds [(n-C


Drosophila. We found that constitutive coactivation of EGFR-Ras and PI3K pathways in Drosophila glia and glial precursors gives rise to neoplastic, invasive glial cells that create transplantable tumor-like growths, mimicking human glioma. Our model represents a robust organotypic and cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in signature genes and pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR and PI3K initiate malignant neoplastic transformation via a combinatorial genetic network composed primarily of other pathways commonly mutated or activated in human glioma, including the Tor, Myc, G1 Cyclins-Cdks, and Rb-E2F pathways. This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration. In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia. Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development. These and other genes within this network may represent important therapeutic targets in human glioma.Gliomas, the most common malignant tumors of the nervous system, frequently harbor mutations that activate the epidermal growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. To investigate the genetic basis of this disease, we developed a glioma model in  Drosophila melanogaster. We found that constitutive coactivation of the EGFR-Ras and PI3K pathways in Drosophila glia gives rise to highly proliferative and invasive neoplastic cells that create transplantable tumor-like growths, mimicking human glioma. This represents a robust cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in genetic pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR-Ras and PI3K induce fly glial neoplasia through activation of a combinatorial genetic network composed, in part, of other genetic pathways also commonly mutated in human glioma. This network acts synergistically to coordinately stimulate cellular proliferation, protein translation, and inappropriate migration. Rate-limiting genes within this network may represent important therapeutic targets in human glioma.Malignant gliomas, tumors composed of glial cells and their precursors, are the most common and deadly human brain tumors. These tumors infiltrate the brain and proliferate rapidly, properties that render them largely incurable even with current therapies. Mutations in genes within the EGFR-Ras and PI3K signaling pathways are common in malignant gliomas, although how these genes specifically control glial pathogenesis is unclear. To investigate the genetic basis of this disease, we developed a glioma model in the fruit fly,  Malignant gliomas, neoplasms of glial cells and their precursors, are the most common tumors of the central nervous system (CNS). These tumors typically proliferate rapidly, diffusely infiltrate the brain, and resist standard chemotherapies, properties that render them largely incurable. One key to developing more effective therapies against these tumors is to understand the genetic and molecular logic underlying gliomagenesis. The most frequent genetic lesions in gliomas include mutation or amplification of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase. Glioma-associated EGFR mutant forms show constitutive kinase activity that chronically stimulates Ras signaling to drive cellular proliferation and migration Understanding the interplay of these mutations and the neurodevelopmental origins of these tumors could lead to new insights into the mechanisms of gliomagenesis. The mammalian brain contains multiple glial cell types that maintain proliferative capacities, including differentiated astrocytes, glial progenitors, and multipotent neural stem cells. EGFR-Ras and PTEN-PI3K signaling regulates many developmental processes in these cell types, particularly proliferation and self-renewal, which are also properties of glioma cells Drosophila glioma model to facilitate genetic analysis of glial pathogenesis. Drosophila offers many tools for precise manipulation of cell-type-specific gene expression and dissection of multigene interactions. Most human genes, including 70% of known disease genes, have functional Drosophila orthologs Drosophila has emerged as a model system for human neurological diseases because the CNS shows remarkable evolutionary conservation in cellular composition and neurodevelopmental mechanisms Drosophila have multiple glial cell types that require the EGFR pathway for their normal development, and these cells appear homologous to mammalian glia in terms of function, development, and gene expression Drosophila an attractive system for modeling gliomas.With these issues in mind, we developed a Drosophila has a single functional ortholog each for EGFR(dEGFR), Raf (dRaf), PIK3CA(dp110), PTEN(dPTEN), and Akt(dAkt), and two functional orthologs for Ras (www.flybase.org). A diagram of the specific mutant forms of dEGFR used in our assays can be found in repo-Gal4 driver, which gives sustained UAS-transgene expression in almost all glia, from embryogenesis through adulthood. For glial-specific RNAi, we employed UAS-dsRNA constructs repo-Gal4 positive glia Since concurrent activation of EGFR-Ras and PI3K signaling in glial precursors induces glioma in the mouse repo-Gal4-driven co-overexpression of activated dEGFR (dEGFRλ) and dp110 (dp110CAAX) induced progressive accumulation of ∼50-fold excess glia  or its effector dRaf (dRafgof) with dp110CAAX, dAkt, or a dPTENdsRNA, which partially knocked-down dPTEN   , indicatλ or Rafgof, induced 5–10 fold excess glia in the larval brain and later pupal lethality , and these cells typically expressed only dCyclinE but not dCyclinB, and thereby likely underwent ectopic DNA replication without mitosis . In controls, Rbf1 or Dap overexpression in wild-type glia inhibited proliferation -lacZ neuroblast reporter showed that excess dEGFRλ;dp110CAAX glia did not express LacZ, and thus are not directly derived from larval neuroblasts (data not shown). Moreover, constitutive EGFR-Ras and PI3K signaling does not elicit overgrowth in all neural cell types, as assessed with defined cell-type specific Gal4-drivers  and neuroblasts/neuronal precursors  did not induce overgrowth  reduced brain size, perhaps because signaling through these pathways stimulates precocious cell cycle exit of neuronal precursors, as in the developing eye V12 or dEGFRλ and dp110CAAX in embryonic glia  also failed to promote glial overgrowth , some astrocyte-like cortex glia , and peripheral perineurial glia  also failed to become neoplastic in response to EGFR-Ras and PI3K  initiated expression of GFP and UAS-containing transgenes only in V12, dEGFRλ, or dEGFRElp alone typically contained 2-fold more cells than wild-type . Of the brains with clones, 75% had 1–3 clones, and 83% of these clones consisted of 1–3 cells of the same glial subtype . Glial cjections , perhapsV12, dEGFRElp, or dEGFRλ was overexpressed within −/−dPTEN glia, using repo-Gal4. We observed overgrown and invasive V12;dPTEN−/−dRas85D and Elp;dPTEN−/−dEGFR clones .To address the function of EGFR-Ras and PI3K in glioma, we analyzed the genetic basis of glial pathogenesis in our λ;dp110CAAXrepo>dEGFR brains, mutant glia showed high levels of nuclear, activated di-phospho-Erk relative to wild-type glia in control brains (pointed (pnt) locus λ;dp110CAAX glia . −/−dSin1 and −/−dRictor mutants display reduced Akt-dependent phosphorylation and inactivation of dFoxO Drosophila ortholog of FoxO transcription factors. This suggests that TORC2 loss might antagonize glial neoplasia through dFoxO upregulation. However, excess dFoxOSA, which is resistant to dAkt phosphorylation λ;dp110CAAXrepo>dEGFR glial overproliferation , S6-kinase (dS6K), and eIF4E (deIF4E); these all significantly reduced accumulation of dEGFRλ;dp110CAAX mutant glial cells, but only caused mild glial hypoplasia in controls , or deIF4E alone did not produce glial overproliferation, even though these constructs can mimic TORC1 activation , illustrating that additional dTor-dependent outputs synergize with dEGFR signaling to drive neoplasia.TORC1, a complex including Tor and the Raptor regulatory protein, drives cellular growth by stimulating protein synthesis through its effectors S6 kinase and the eIF-4E translation initiation factor controls . Co-overcontrols . Glial-sergrowth . HoweverDrosophila ortholog of the Myc bHLH transcription factors dTor coordinates increased translation, mediated by dS6K and deIF4E, with expression of cell cycle regulators and ribosomal components, through dMyc, the single λ;dp110CAAX glia showed high levels of nuclear dMyc compared to wild-type glia . Thus, dCyclinD-dCdk4 is essential for EGFR-PI3K glial neoplasia, although dCdk4 is not required for development .At least four pathway circuits are necessary for glial neoplasia initiated by EGFR and PI3K signaling, including dRas and dMyc circuits, which induce dCyclinE and dCyclinD to drive cell cycle entry, a Pnt circuit, which induces Stg to promote cell cycle progression, and a Tor-eIF4E-S6K pathway, which provides protein translation necessary for proliferation and growth . When acc-myc is commonly amplified in gliomas c-myc amplification may be selected for this reason. D-cyclins, established Myc target genes, and Cdk4 are also commonly amplified and/or overexpressed in gliomas λ;dp110CAAXrepo>dEGFR neoplasia, although dCdk4 itself is not required for normal glial proliferation. Together with our analysis of TORC2, this illustrates that oncogenic EGFR-PI3K co-opts effectors that do not control normal glial development. Similarly, −/−cdk4 mutant mice show normal proliferation in many tissues, but are resistant to ErbB-2-driven breast cancers Coactivation of EGFR and PI3K signaling upregulates dMyc, which is necessary for glial neoplasia. This is consistent with findings that, in flies and mammals, EGFR-Ras, PI3K, and Tor signaling upregulate Myc protein levels Drosophila, neither a neural stem cell fate nor Myc activity confer competence to undergo EGFR-PI3K neoplastic transformation. Rather, our results suggest that neoplastic cells arise from committed glial progenitors: −/−dEGFR-dRas85D;dPTEN clones derived from progenitor cells produce large tumors, and anaplastic cells in λ;dp110CAAXrepo>dEGFR brains are concentrated in regions enriched for glial progenitors. Notably, regulated developmental signaling through the EGFR pathway promotes proliferation of normal Repo-expressing glial progenitors Drosophila glial progenitors and glioma-like cells may illuminate the cellular origins of human gliomas, which are thought to arise from progenitor-like glial cells. Moreover, our results argue that cell-type specific factors govern glial neoplasia. One such factor may be Dap, the single p21/p27 ortholog, which is normally expressed in only 5% of all glia , Elp, UAS-dEGFRwild-typeUAS-dEGFR (N. Baker), UAS-dPTEN, 40A dPTEN2L117FRT, SAUAS-dFoxO (S. Oldham), UAS-dap (I. Hariharan), wild-typeUAS-dp110, 4UAS-dCycD, UAS-dCdk4, UAS-dMyc, dMyc (B. Edgar), UAS-Rbf1 (N. Dyson), appl-Gal4 (K. Finley), pros-Gal4 (B. Ohlstein), wor-Gal4 (C. Doe), gcm-Gal4 (V. Hartenstein), 2L7, dTorl(2)k17004dTor (R. Bodmer), Δ2dRictor (S. Cohen), and CB03726stg (A. Spradling)Stocks were obtained from the Bloomington Stock Center unless otherwise noted. Other than dsRNAUAS-dMyc, dsRNAUAS-TSC1 lines were validated in prior publications UAS-dsRNA lines were crossed to actin-Gal4, ey-Gal4, and GMR-Gal4 to assess phenotypes. Lines that showed phenotypes inconsistent with known phenotypes for their target genes were excluded from analysis. Gene knock-down in repo-Gal4 glia was verified with immunohistochemical stains for the following constructs: dsRNAUAS-dMyc, dsRNAUAS-dAkt, dsRNA, UAS-eIF4EdsRNA, UAS-Rbf1dsRNAUAS-dS6K, and dsRNAUAS-pnt , rat anti-dCyclinE , anti-BrdU , rat anti-Miranda , mouse anti-diphospho-Erk , mouse anti-Rbf1 , mouse anti-Dap , rabbit anti-PntP1 , rabbit anti-eIF4E , rabbit anti-dMyc , and anti-phospho-MPM2 . Anti-HRP-Cy5 and anti-HRP-Cy3 (Jackson Labs) were used at 1∶250–1∶500. Secondary antibodies were conjugated to Cy3 (Jackson Labs) or Alexa-488 or Alexa-647 (Molecular Probes). Actin was visualized with Rhodamine-labeled phalloidin (Invitrogen).Brains were imaged as whole mounts on a Zeiss LSM 510 confocal system. Images were analyzed in Zeiss LSM Image Browser and processed in Photoshop CS3. For experiments in which protein levels were compared between genotypes, all sample preparation, histochemistry, imaging, and image processing was performed in parallel in the same manner.Figure S1Drosophila EGFR mutant forms. Proteins are shown as horizontal bars along which functional domains are indicated, and the locations of alterations in mutant forms are noted. Wild-type human EGFR and Drosophila EGFR (dEGFR) show extensive conservation, with 55% homology in the kinase domain and 41% homology in the ligand-binding portion of the extracellular domain, which includes extensive cysteine repeats. The signal peptide is labeled in yellow. Following the signal peptide, the entire cytoplasmic domain is replaced with the lambda dimerization domain in dEGFRλ, which causes constitutive activation. Both human EGFR and dEGFR show extensive cysteine repeats in the extracellular domain, indicated in blue. The tyrosine kinase domain is labeled in magenta. The Elp mutant form of dEGFR contains an A887T substitution in the N-lobe of the kinase domain, which causes constitutive activation.Diagram of (0.23 MB TIF)Click here for additional data file.Figure S2Drosophila glia causes neoplasia. 2 µm optical sections of representative larval brain hemispheres from wandering 3rd instar larvae (A–H) and an early 2nd instar larval brain (I), all displayed at the same scale. 20 µm scale bars. Frontal sections; midway through brains. Anterior up; midline to left. Glial cell nuclei are labeled with Repo (red). CD8GFP (green), driven by the repo-Gal4 driver, labels glial cell bodies and membranes. An anti-HRP counterstain (blue) reveals neuropil  at high intensity and some cell bodies of neurons and neuronal precursors at low intensity, and this varies slightly according to exact plane of section and mutant phenotype. repo>dEGFRλ;dp110CAAX (A), repo>dRas85DV12;dPTENdsRNA (B), and repo>dRafgof;dp110CAAX (C) brains show increased numbers of glia relative to wild-type (D), repo>dRas85DV12 alone (E), or repo>dPTENdsRNA alone (F). Compared to wild-type (D), repo>dEGFRwild-type (H) brains show reduced neurons , which renders glia more densely packed and the entire brain smaller than normal. However, repo>dEGFRwild-type brains (H) do not show a substantial increase the number of glia compared to wild-type (D). Brains of repo>dEGFRElp 2nd instar larvae (I) show substantial neuron loss, brain malformation, and excess glia for that stage of development. Genotypes: (A) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/+ (B) UAS-CD8GFP/+; repo-Gal4/UAS-dRas85DV12 UAS-dPTENdsRNA (C) UAS-dp110CAAX; repo-Gal4 UAS-CD8GFP/UAS-dRafgof (D) repo-Gal4 UAS-CD8GFP/+ (E) repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12 (F) UAS-CD8GFP/+; repo-Gal4/UAS-dPTENdsRNA (G) UAS-dEGFRλ/+; repo-Gal4 UAS-CD8GFP/+ (H) UAS-CD8GFP/+; repo-Gal4/UAS-dEGFRwild-type (I) UAS-CD8GFP/+; repo-Gal4/UAS-dEGFRElp.Coactivation of Ras-Raf and PI3K in (10.31 MB TIF)Click here for additional data file.Figure S3λ;dp110CAAX glia.  3rd instar larval brains. Frontal sections, showing medial regions enriched for proliferating glia. Anterior up; midline to left. 4.5 µm optical projections, matched in scale. 20 µm scale bars. Expression of the active form of dMMP1 (red) in wild-type (A) and repo>dEGFRλ;dp110CAAX brains (B), shown alone (left panels) and overlaid with an HRP (blue) neuronal label and a CD8GFP (green) glial label (right panels). In wild-type brains, glia (‘G’) rarely express active dMMP1, although some glia on the surface of the brain show low levels of dMMP1 staining (arrow). In repo>dEGFRλ;dp110CAAX brains (B), some neoplastic glia (‘G’) express active dMMP1 , which is largely membrane-localized in individual cells (arrow). Genotypes: (A) UAS-CD8GFP/+; repo-Gal4/+ (B) UAS-dEGFRλ UAS-dp110CAAX/+; UAS-CD8GFP/+; repo-Gal4/+.dMMP1 expression in wild-type and dEGFR(3.93 MB TIF)Click here for additional data file.Figure S4repo-Gal4. HRP counter-staining (blue) reveals neuropil at high intensity and some cell bodies of neurons and neuronal precursors at low intensity. Rbf1 knock-down in repo>Rbf1dsRNA (B) does not significantly alter glial cell numbers (red nuclei) compared to wild-type (A). Ectopic expression of dCyclinE-dCdk2 (C) or dCyclinD-dCdk4 in repo-Gal4-glia promotes an approximate doubling of glial cells numbers (red nuclei), even when combined with Rbf1dsRNA (D). Continuous expression of Dap (E) or Rbf1 (F) in otherwise wild-type repo-Gal4-glia substantially reduces glial cell numbers, showing that repo-Gal4 glia normally undergo proliferation controlled by dCyclinE-dCdk2 and Rbf1-E2F1. Genotypes: (A) UAS-CD8GFP/+; repo-Gal4/+ (B) UAS-CD8GFP/+; repo-Gal4/UAS-Rbf1dsRNA (C) UAS-CD8GFP/+; repo-Gal4/UAS-dCyclinE UAS-dCdk2 (D) UAS-CD8GFP/UAS-dCyclinD UAS-dCdk4; repo-Gal4/UAS-Rbf1dsRNA (E) UAS-dap/+; UAS-CD8GFP/+; repo-Gal4/+ (F) UAS-CD8GFP/+; repo-Gal4/UAS-Rbf1.Glial-specific Rbf1 knock-down or overexpression of G1 Cyclin-Cdks, Dap, and Rbf1 affects glial proliferation. (A–F) 2 µm optical sections of larval brain hemispheres from wandering 3rd instar larvae displayed at the same scale. 20 µm scale bars. Frontal sections; midway through brains. Anterior up; midline to left. Glial cell nuclei are labeled with Repo (red). Glial cell bodies and membranes are labeled with CD8GFP (green) driven by (8.27 MB TIF)Click here for additional data file.Figure S5λ;dp110CAAX glia. 3rd instar larval brains. Frontal sections, showing superficial dorsal regions enriched for Dap- and Rbf1-expressing cells. Anterior up; midline to left. 7 µm optical projections, all matched in scale. 20 µm scale bars.  Dap expression (red) in wild-type (A) and repo>dEGFRλ;dp110CAAX brains (B), shown alone (left panels) and overlaid with Repo (blue) and repo>CD8GFP (green) glial markers (right panels). In both genotypes, Dap is primarily expressed in neuroblasts (‘NB’) and ganglion mother cell neuronal precursors (‘NP’), rarely in glia (‘G’), and almost never in neurons (‘N’). In repo>dEGFRλ;dp110CAAX brains, Dap is rarely expressed in glia as seen by the lack of substantial overlap between Dap expression and glial markers . White arrows denote rare Dap-positive glia in both wild-type and repo>dEGFRλ;dp110CAAX brains; these glia show lower levels of Dap protein than neighboring neuroblasts and neuronal precursors. Dap-positive glia were counted in 3 repo>dEGFRλ;dp110CAAX brains and an average of 5% of all glia expressed Dap . (C–D) Rbf1 expression (red) in wild-type (E) and repo>dEGFRλ;dp110CAAX brains (F), shown alone (left) and overlaid (right) with the CD8GFP (green) glial marker and HRP (blue) neuronal marker. In both genotypes, Rbf1 is highly expressed in glia (‘G’) and neuroblasts (‘NB’), which were identified by their characteristic positions and large cell bodies. Neurons (‘N’) show lower expression. Genotypes:  UAS-CD8GFP/+; repo-Gal4/+  UAS-dEGFRλ UAS-dp110CAAX/+; UAS-CD8GFP/+; repo-Gal4/+.Dap and Rbf1 expression in wild-type and dEGFR(7.96 MB TIF)Click here for additional data file.Figure S6λ; dp110CAAX glia do not express neuronal and neuroblast markers. Late 3rd instar larval brains. Medial anterior regions shown. Anterior up; midline to left. 2 µm optical sections. CD8GFP (green), driven by the repo-Gal4 driver, labels glial cell bodies and membranes. An HRP counter-stain (blue) reveals neuropil at high intensity and cell bodies of neurons and some neuronal precursors at low intensity.  Repo expression (red) in glial cell nuclei in wild-type (A) and repo>dEGFRλ;dp110CAAX brains (B). In wild-type brains, neuronal fibers and neurons (blue) are enveloped by glial processes (green) to give the brain a honeycombed appearance. In repo>dEGFRλ;dp110CAAX brains, neurons (‘N’) lack GFP or Repo expression, in contrast to glia (‘G’). (C–D) Elav expression (red) in wild-type (C) and repo>dEGFRλ;dp110CAAX brains (D), shown alone (left panels) and overlaid (right panels) with the CD8GFP (green) glial marker and HRP (blue) neuronal marker. In both genotypes, Elav is highly expressed in neurons (‘N’) but absent from glia (green cell bodies indicated by white arrows). (E–F) Miranda expression (red) in wild-type (E) and repo>dEGFRλ;dp110CAAX (F) brains, shown alone (left panels) and overlaid (right panels) with CD8GFP (green) glial marker and HRP (blue) neuronal marker. In both genotypes, Miranda is highly expressed in neuroblasts (‘NB’), which are identified by their characteristic positions and large cell bodies, but is absent from glia . Genotypes:  UAS-CD8GFP/+; repo-Gal4/+  UAS-dEGFRλ UAS-dp110CAAX/+; UAS-CD8GFP/+; repo-Gal4/+.dEGFR(9.24 MB TIF)Click here for additional data file.Figure S7λ and dp110CAAX do not induce neoplasia from neurons, neuroblasts, or certain glia. Late 3rd instar larval brain hemispheres. Medial anterior regions shown. Anterior up; midline to left. 3.5 µm optical projections. Repo expression (red) in glial cell nuclei in all genotypes. An HRP counter-stain (blue) reveals neuropil at high intensity and cell bodies of neurons and some neuronal precursors at low intensity, and this varies slightly according to variance in exact section plane. (A–D) Wild-type (A), dEGFRλ; dp110CAAX overexpressed using the 1407-Gal4 neuroblast driver (B), dEGFRλ; dp110CAAX overexpressed using the pros-Gal4 neural driver (C), and dEGFRλ;dp110CAAX overexpressed using the gcm-Gal4 embryonic glial driver (D). In all cases, dEGFRλ;dp110CAAX overexpression did not induce neoplastic overgrowth of glial or neuronal cell types, as determined by brain size and cell-type specific stains.  Cytoplasmic GFP (green), driven by the Nrv2-Gal4 driver, labels cell bodies and cytoplasmic processes, as seen in wild-type (E). dEGFRλ;dp110CAAX overexpressed using the Nrv2-Gal4 glial driver (B). Nrv2-Gal4 is expressed by post-mitotic cortex glia, which proliferate somewhat in response to dEGFRλ;dp110CAAX to cause slight brain enlargement, but do not become neoplastic like repo>dEGFRλ;dp110CAAX glia. Genotypes: (A) +/CyO (B) UAS-dEGFRλ UAS-dp110CAAX/+; 1407-Gal4/UAS-Gal4 (C) UAS-dEGFRλ UAS-dp110CAAX/+; UAS-Gal4/+; pros-Gal4/+ (D) UAS-dEGFRλ UAS-dp110CAAX/+; gcm-Gal4/+ (E) Nrv2-Gal4 UAS-GFP/+; Nrv2-Gal4 UAS-GFP/+ (F) UAS-dEGFRλ UAS-dp110CAAX/+; Nrv2-Gal4 UAS-GFP/UAS-Gal4; Nrv2-Gal4 UAS-GFP/+.dEGFR(7.96 MB TIF)Click here for additional data file.Figure S8repo-Gal4-expressing cells. CD8GFP (green) marks cell bodies and membranes of glial clones derived by FLP/FRT mitotic recombination (see text). Repo (red) marks all glial cell nuclei, in both clones and surrounding normal tissue. 8.5 µm confocal optical sections through brains of similarly aged adults, all matched to scale. 20 µm scale bars. Each panel shows half brains, including a whole optic lobe and adjacent central brain. dEGFRElp (C) and dPTEN−/− (J) clones are composed of 2–5-fold more cells than wild-type controls (H). In contrast, dEGFRλ;dPTEN−/− (D) and dEGFRElp;PTEN−/− double mutant clones form large tumors visible in adult brains. As with hs-FLP clones, dEGFRλ;dPTEN−/− and dEGFRElp;dPTEN−/− clones are less cellular and more invasive than dRas85DV12;PTEN−/− clones  ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/+ (C) ey-flp/+; FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/UAS-dEGFRElp (D) ey-flp/UAS- dEGFRλ; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS- dEGFRλ (E) ey-flp/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dEGFRElp.Coactivation of EGFR and PI3K in glial progenitors creates invasive neoplastic glia.(A–E) FLP/FRT clones in adult brains derived from a population of ey-FLP and ones see . Genotyp(7.84 MB TIF)Click here for additional data file.Figure S9repo-Gal4. An HRP counter-stain (blue) reveals neuropil at high intensity and neuronal cell bodies at low intensity. HRP stains varied between the two samples according to effects of mutant glia and slight variance in section plane. Stg co-overexpression with dEGFRλ;dp110CAAX (B) enhances neoplasia compared to dEGFRλ;dp110CAAX (A), leading to a dramatic increase in aberrant glia and yielding a phenotype similar to that of repo>Rbf1dsRNA;dEGFRλ;dp110CAAX  UAS-dEGFRλ UAS-dp110CAAX/+; UAS-stg/+; repo-Gal4 UAS-CD8GFP/+.Stg overexpression exacerbates EGFR-PI3K glial neoplasia. 2 µm optical sections of larval brain hemispheres from late 3rd instar larvae, displayed at the same scale. 20 µm scale bars. Frontal sections, midway through brains. Anterior up; midline to left. Glial cell nuclei are labeled with Repo (red); glial cell bodies and membranes are labeled with CD8GFP (green) driven by CAAX see . Genotyp(6.27 MB TIF)Click here for additional data file.Figure S10repo-Gal4, labels glial cell bodies and membranes. An HRP-counter stain (blue) reveals neuropil  at high intensity and some cell bodies of neurons and neuronal precursors at low intensity, and this varies slightly according to exact plane of section, brain orientation, and mutant phenotype. repo>dAktdsRNA brains (B) are smaller and have fewer glia than wild-type controls (A). All other genotypes have relatively normal sized brains. By gross examination, repo>TorTED (C), repo>dRaptordsRNA (E), repo>deIF4EdsRNA (F) and repo>dS6KdsRNA (G) display an estimated 10–20% reduction in glial cell numbers. repo>dTSC1dsRNA (D), repo>Rheb (H) and repo>deIF4E (I) brains contain normal numbers of glia. Genotypes: (A) repo-Gal4 UAS-CD8GFP/+ (B) UAS-CD8GFP/+; repo-Gal4/UAS-dAktdsRNA (C) UAS-TorTED/UAS-CD8GFP; repo-Gal4/+ (D) UAS-CD8GFP/+; repo-Gal4/UAS-dTSC1dsRNA (E) UAS-dRaptordsRNA/UAS-CD8GFP; repo-Gal4/+ (F) UAS-CD8GFP/+; repo-Gal4/UAS-deIF4EdsRNA (G) UAS-dS6KdsRNA; UAS-CD8GFP/+; repo-Gal4/+ (H) UAS-CD8GFP/+; repo-Gal4/UAS-Rheb (I) UAS-CD8GFP//UAS-deIF4E; repo-Gal4/+.Knock-down and/or overexpression of dTor and dTor effectors. 2 µm optical sections of larval brain hemispheres from wandering 3rd instar larvae, approximately 130hr AED, all displayed at the same scale. 20 µm scale bars. Anterior up; midline to the left. Frontal sections; midway through brains. Repo (red) marks glial cell nuclei; CD8GFP (green), driven by (8.83 MB TIF)Click here for additional data file.Figure S11repo-Gal4. In  an HRP counter-stain (blue) reveals neurons. Red marks histochemical stains for each indicated protein in repo>dEGFRλ;dp110CAAX  and repo>dEGFRλ;dp110CAAX with each indicated dsRNA construct . Each dsRNA construct was expressed with repo-Gal4 to yield glial-specific knock-down, which left protein expression in surrounding neuronal tissue intact. For the nuclear protein PntP1, knock-down was verified by the lack of glial-specific staining in the presence of the pntdsRNA (B), although PntP1 is present within neighboring neurons (‘N’). For dAkt, deIF4E, and dS6K, gene knock-down was confirmed using antibodies for total protein. Glial-specific reduction in gene expression by dAktdsRNA and deIF4EdsRNA is highlighted by white outlines in (D) and (F). dAkt protein is higher in neuronal tissue (‘N’) in both repo>dEGFRλ;dp110CAAX (C) and repo>dAktdsRNA;dEGFRλ;dp110CAAX (D). deIF4E is high in dEGFRλ;dp110CAAX glia (E) and low in neurons . repo>deIF4EdsRNA;dEGFRλ;dp110CAAX brains (F) have reduced glial eIF4E. Reduced glial dS6K protein, which is diffusely cytoplasmic, is observed in repo>dS6KdsRNA;dEGFRλ;dp110CAAX brains (H) compared to repo>dEGFRλ;dp110CAAX (G). Reduced glial dMyc expression in repo>dMycdsRNA;dEGFRλ;dp110CAAX brains (J) is noted by white outlines, and the absence of purple nuclei in overlay , relative to repo>dEGFRλ;dp110CAAX . dMyc is highly expressed in neuroblasts (‘NB’) in both samples , which do not express the dMycdsRNA. Glial Rbf1 protein is absent in repo>dRbf1dsRNA;dEGFRλ;dp110CAAX brains (L) in contrast to neighboring neurons (‘N’) in ,repo>dEGFRλ;dp110CAAX brains (K). Genotypes:  UAS-dEGFR, λ UAS-dp110,CAAX/+; UAS-CD8GFP/+; repo-Gal4/+ (B) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-pntdsRNA (D) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-dAktdsRNA (F) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-deIF4EdsRNA (H) UAS-dEGFRλ UAS-dp110CAAX/UAS-dS6KdsRNA; repo-Gal4 UAS-CD8GFP/+ (J) UAS-dEGFRλ UAS-dp110CAAX/+; UAS-dMycdsRNA/+; repo-Gal4 UAS-CD8GFP/+ (L) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-Rbf1dsRNA.Validation of dsRNA constructs. 2 µm optical sections of larval brain hemispheres from wandering 3rd instar larvae. Frontal sections. Anterior up, midline to the left. Each individual staining pattern is shown alone (left panels) and with overlaid glial or neuronal markers (right panels). Repo (blue) in (A–J) marks glial cell nuclei. Glial cell bodies and membranes are labeled with CD8GFP (green) driven by (9.04 MB TIF)Click here for additional data file.Figure S12repo>dRbf1dsRNA;dEGFRλ;dp110CAAX brains. (A–C) phospho-MPM2 expression (red) in wild-type brains (A), repo>dEGFRλ;dp110CAAX brains (B), and repo>dRbf1dsRNA;dEGFRλ;dp110CAAX brains (C). 20 µm scale bars. Anterior up, midline to the left. 6 µm optical projections showing representative superficial dorsal regions enriched for mitotic glia. Phospho-MPM2 shown alone (left panels), overlaid with the Repo , and CD8GFP  glial markers. Phospho-MPM2 nuclear foci are present in S-phase cells (arrows note examples). S-phase glia are clearly visible in the middle panel as purple cells with MPM2 foci, which appear enriched in repo>dRbf1dsRNA;dEGFRλ;dp110CAAX brains (C) relative to repo>dEGFRλ;dp110CAAX brains (B). In all genotypes, high levels of phospho-MPM2 is also expressed in mitotic cells (asterisks note examples). Mitotic glia showed low levels of Repo (middle panels), but are clearly GFP-positive (right panels), suggesting that Repo protein expression is reduced upon mitosis in glia. repo>dRbf1dsRNA;dEGFRλ;dp110CAAX brains (C) also show increased density of phospho-MPM2-positive mitotic glia compared to repo>dEGFRλ;dp110CAAX brains (B). In wild-type (A), the majority of phospho-MPM2-postive cells are not glial and are neuroblasts or neuronal precursors, as revealed by the lack of overlap between phospho-MPM2 staining (middle panel) and the Repo and CD8GFP markers. Genotypes: (A) repo-Gal4 UAS-CD8GFP/+ (B) UAS-dEGFRλ UAS-dp110CAAX/+; UAS-CD8GFP/+; repo-Gal4/+ (C) UAS-dEGFRλ UAS-dp110CAAX/+; repo-Gal4 UAS-CD8GFP/UAS-Rbf1dsRNA.Phospho-MPM2 reveals increased S-phase and M-phase glia in (8.99 MB TIF)Click here for additional data file.Table S1Drosophila glia.Overexpression of EGFR-Ras and PI3K-Akt pathways in (0.08 MB PDF)Click here for additional data file.Table S2Coactivation of EGFR and PI3K does not elicit neoplasia in all neural cell types.(0.08 MB PDF)Click here for additional data file.Table S3Genetic analysis of Akt, Tor, Myc, and CyclinD-Cdk4 pathways in neoplastic and normal glia.(0.10 MB PDF)Click here for additional data file.Video S1λ; dp110CAAX tumor cells derived from transplanted larval glia, pictured in λ;dp110CAAX tumor, visible as hollow actin-positive (red) tubules running through tissues. Genotypes: Hosts were w1118 virgin females. Transplanted glia were UAS-dEGFRλ UAS-dp110CAAX/+; UAS-CD8GFP/+; repo-Gal4/+.An animated 7.5 µm thick confocal z-stack of dEGFR(1.54 MB MOV)Click here for additional data file.Video S2λ; dp110CAAX tumor cells derived from transplanted larval glia, pictured in λ;dp110CAAX glial cells invading an ovary, distinguished by its characteristic actin staining (bright red). An asterisk indicates trachea embedded in this dEGFRλ;dp110CAAX tumor, visible as a hollow actin-positive (red) tubule running through the tissue. Genotypes: Hosts were w1118 virgin females. Transplanted glia were UAS-dEGFRλ UAS-dp110CAAX/+; UAS-CD8GFP/+; repo-Gal4/+.An animated 27 µm thick confocal z-stack of dEGFR(1.16 MB MOV)Click here for additional data file.Video S3dPTEN−/−;dRas85DV12 cells pictured in dPTEN−/−;dRas85DV12 mutant cells are labeled with membrane-bound CD8GFP (green) and penetrate through the entire depth of the brain, some of which is visible as red background. These dPTEN−/−;dRas85DV12 cells appear to originate in the optic lobe (right) and extend variable tendrils into the central brain . Genotype: hs-flp1/+; FRT40A tubGal80/FRT40A PTEN2L117; repo-Gal4 UAS-CD8GFP/UAS-dRas85DV12.An animated 88 µm thick confocal z-stack of a hs-FLP/FRT clone of (1.06 MB MOV)Click here for additional data file.Video S4hs-flp1/+;FRTG13 tubGal80/FRTG13 UAS-CD8GFP; repo-Gal4/+.An animated 16 µm thick confocal z-stack of a hs-FLP/FRT clone of wild-type cells pictured in (0.24 MB MOV)Click here for additional data file.


This open-label, non-randomized, parallel-group trial investigated the pharmacokinetics of raltitrexed  after a single intravenous dose of 3.0 mg m(-2), comparing eight cancer patients with mild to moderate renal impairment (creatinine clearance 25-65 ml min(-1)) with eight cancer patients with normal renal function (creatinine clearance >65 ml min(-1)). The primary end points were area under the plasma raltitrexed concentration-time curve from the start of the infusion to the last determined concentration (AUC(0-tldc)) and AUC to infinity (AUC(0-infinity)); secondary end points were peak concentrations of raltitrexed (Cmax) and elimination half-life (t(1/2gamma)). The groups were compared statistically using analysis of covariance. The AUCs were greater for patients with renal impairment than for patients with normal renal function (2452.2 compared with 1247.3 ng h ml(-1) for AUC(0-tldc) ; 2961.5 compared with 1457.0 ng h ml(-1) for AUC(0-infinity) . These differences were statistically significant (P = 0.002 and P = 0.008 for AUC(0-tldc) and AUC(0-infinity) respectively. Terminal half-life was longer for the renally impaired patients . There was no significant statistical difference between the groups for Cmax (652.9 compared with 564.7 ng ml(-1) for patients with impaired and normal renal function respectively: ratio 1.16; 0.91-1.46; P = 0.204). There was a clear relationship between raltitrexed clearance and creatinine clearance. Adverse events, severe (WHO grade 3 or 4) toxicity and hospitalization due to adverse events were more frequent in the group with renal impairment. Therefore, a reduction in raltitrexed dose and increased interval between doses is recommended for patients with mild to moderate renal impairment.


The relationship between two methods of assessing tumour oxygenation in vivo, namely oxygen electrode measurement and magnetic resonance spectroscopy (MRS) of the fluorinated 2-nitroimidazole SR-4554, was investigated. Using three tumour models (two sites), no linear correlation was observed between 19F retention index and pO2 parameters (r < or = 0.3). Substantial retention of SR-4554 (19F retention index > 0.5) was, however, associated with low tumour pO2 (% pO2 < or = 5 mmHg = 60%). Depending on the pO2 parameters used, SR-4554 administration was shown to produce either a significant or a non-significant increase in tumour oxygenation. We conclude that measurement of SR-4554-related compound(s) by 19F-MRS has the potential to detect clinically relevant levels of tumour hypoxia.


Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.In this study we demonstrate that AtHscB from Arabidopsis can rescue the Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol. Besides harbouring a bacterial SUF-like (mobilization of sulfur) system encompassing the SufA, SufB, SufC, SufD, SufS and SufE proteins Iron-sulfur proteins were first identified in plants and several excellent reviews have summarized both early and recent updates in the field of plant [Fe-S] biogenesis et al pioneered this field by identifying the Arabidopsis Sta1 as an Atm1p-like ABC transporter of yeast supporting the maturation of [Fe-S] protein in mitochondria iron sulfur cluster) system Compared to chloroplasts, [Fe-S] biogenesis in plant mitochondria has attracted much less attention. Kushir In the plant cytosol, [Fe-S] biogenesis is much less well understood. However, recent work by Balk and colleagues In bacteria and yeast, the HscA/Ssq1 chaperones and the HscB/Jac1 co-chaperones are important elements of the ISC-like system. HscA/Ssq1 are ATPases, stimulated by the J-type co-chaperone HscB/Jac1 and have been shown to interact with the scaffold protein IscU/Isu, which is regulated by HscB/Jac1 by binding to IscU/Isu to assist [Fe-S] delivery to the chaperone Here we demonstrate that Arabidopsis contains a functional AtHscA1/AtHscB/AtIscU1 protein cluster involved in [Fe-S] protein biogenesis. In contrast to yeast, the AtHscA1/AtHscB/AtIscU1 protein cluster is localized to both mitochondria and the cytosol of Arabidopsis suggesting a dual action between these two spatially separate compartments.E. coli HscB and yeast Jac1 showing 30% and 24% identity, respectively (A full-length cDNA (759 nt) encoding the At5g06410 open reading frame was cloned and its predicted amino acid sequence compared to ectively . The At5jac1  marked plasmid pRS316, with pGADT7-AtHscB or pGBKT7-AtHscB and positive transformants were screened on synthetic dropout media SD/-Leu (minus L-leucine) or on SD/-Trp (minus L-trypothan), respectively. Once scored the wild-type Jac1 cDNA was removed by streaking Δjac1/pGADT7-AtHscB and Δjac1/pGBKT7-AtHscB colonies onto YPD media containing 1 mg/ml 5-FOA (5-Fluoroorotic Acid). As URA3 encodes orotine-5′-monophosphate dicarboxylase, which convert 5-FOA to toxic fluorodeoxyuridine, colonies will only grow where pRS316 has been removed and functional complementation has occurred. On YPD media, Δjac1/pRS316-Jac1, Δjac1/pGADT7- AtHscB and Δjac1/pGBKT7-AtHscB showed clear growth  and transiently expressed this transgene in tobacco cells  whose products are predicted to be mitochondrial pLysS by auto-induction and affinity purified the protein to >95% purity  directly upstream of the AtHscB start codon was PCR-cloned into the β-glucoronidase (GUS) binary vector pBADG and transformed into wild type Arabidopsis. GUS staining of T2 lines showed that AtHscB is universally expressed at low levels but with relatively high levels of expression in anthers and trichomes .AtHscB-specific primers LP585159 and RP585159. Two PCR fragments were obtained and sequenced revealing the presence of two T-DNAs inserted 0 and 5 nt downstream of the AtHscB stop codon (AtHscB transcript and undetectable AtHscB protein (CUT1 sense suppressed plants AtHscB followed by phenotypic characterization. More than 95% (20 out of 21) of transformed resistant plants showed a wild type phenotype (AtHscB-deficiency is responsible for the observed mutant phenotypes. Due to the high level of AtHscB expression in trichomes (AtHscB gene expression patterns homozygous mutants not only have fewer trichomes than wild-type (An Arabidopsis T-DNA insertion line (SALK_085159) was identified and analyzed by PCR with the T-DNA specific primer LBb1 and the op codon . Althougop codon  several op codon  showing  protein . Phenoty protein , a similhenotype  confirmirichomes , we moniild-type  but thesild-type .To test whether AtHscB indeed has an effect on iron sulfur proteins in Arabidopsis, we assayed both aconitase and succinate dehydrogenase (SDH) activities in wild type plants and in homozygous N585159 mutant plants. jac1 mutant  AtHscB can rescue the yeast Δ1 mutant , (ii) At1 mutant  and . Based on our findings it is reasonable to suggest that AtHscB, AtHscA1 and AtIscU1 may function in both mitochondria, and through retention of AtIscU1 by AtHscB  were grown at 21°C with 16 h of light (100 µmol photons m–2 s–1) per day and 60% humidity unless otherwise stated. The AtHscB T-DNA insertion mutant Salk_085159 (N585159) Wild-type Arabidopsis, transgenic Arabidopsis and the Pac1 and SacI of pBA002a AtHscB ATG was amplified with primers P06410-AscI-L/P06410-PacI-R, cloned into AscI and PacI of pBADG and transformed into wild-type Arabidopsis for GUS expression analysis. AtHscB and AtIscU1 were PCR amplified using primers AtHscB-GBK-L/AtHscB-GBK-R and AtIscU1-GBK-L/AtIscU1-GBK-R, and cloned into NdeI and BamHI of pGBKT7 or pGADT7 (Clontech). AtHscB, AtIscU1 and AtHscA1 were amplified using primers AtHscB-XhoI-L/AtHscB-KpnI-R, AtIscU1-XhoI-L/AtIscU1-KpnI-R and AtHscA1-KpnI-L/AtHscA1-KpnI-L R, digested with XhoI/KpnI or with KpnI and inserted into pWEN18, pWEN-N-YFP (YFP amino acids 1–154) and pWEN-C-YFP (YFP amino acids 155–238). AtHscB, AtIscU1, and AtHscA1 were PCR cloned into pET28a using primers AtHscB-ET-L/AtHscB-ET-R AtIscU1-ET-L/AtIscU1-ET-R and AtHscA1-ET-L/AtHscA1-ET-R. AtHscB was PCR amplified with primers AtHscB-XhoI-L/AtHscB-SpeI-R, digested with XhoI/SpeI and cloned into pBA002 for N585159 complementation experiments. Primers are shown in A GFP-GUS DNA fragment was PCR amplified using primers GFPGUS-L/GFPGUS-R  and pWEN-C-YFP (pWEN-CY) containing AtHscB and AtIscU1 and control vector controls were transformed into HF7c and tested for His auxotrophy restoration following the Matchmaker two-hybrid system III manual (Clontech).pGBKT7 and pGADT7 containing jac1 complementation, Δjac1-pRS316-Jac1 was transformed with pGBKT7-AtHscB or pGADT7-AtHscB and screened on SD/-Trp (minus L-trypothan) or SD/-Leu (minus L-leucine). Colonies were streaked onto 5-FOA media  to screen for complementation. Isolated colonies were verified by RT-PCR using primers Jac1-L/R and AtHscB-L/R.For Δssq1 complementation, Δssq1 was transformed with pGBKT7-AtHscA1 and positive transformants screened on SD/-Trp. Overnight cultures were spotted onto YPD or H2O2 plates  and incubated at 34°C for 4 days.For ΔAtHscB, AtIscU1 and AtHscA1 were transformed into E. coli Rosetta(DE3)pLysS (Novagen) and expression performed for 20 h in auto-induction ZYM-5052 media 2, 100 mM NaCl, 1% Triton X-100 and 10 µM/ml Benzonase® Nuclease (Novagen) and the proteins purified using TALON affinity resin (BD Biosciences). Purity was assessed by SDS–PAGE analysis.pET28a containing 32P] ATP (1 Ci = 37 GBq) (specific activity 10 mCi/mmol). All reactions were terminated using 1 µl of 1 M formic acid. Samples were applied to TLC plates (Macherey and Nagel), developed in 0.5 M LiCl and 0.5 M formic acid buffer, and visualized by autoradiography. The hydrolyzed ATP was quantified using phosphorimager.ATPase assays were performed as described previously et al2, 0.1% Triton X-100), 200 µM cis-aconitate, 1.3 mM NADP+, 400 µU IDH (isocitrate dehydrogenase), and a reference cuvette lacking cis-aconitate and IDH was prepared. To each cuvette, protein extraction (≈40 µg protein) was added and absorption increase at 340 nm in a double-beam spectrophotometer was measured (ε340nm = 6220 M−1 cm−1). SDH assay: Two cuvettes containing 950 µl SDH buffer , 0.25% succinate, 70 µM dichlorophenol-indophenol, and 60 µM decylubiquinone were prepared. 0.25% malonate was added to the reference cuvette and the assay was initated by adding cell suspension (≈40 µg protein) to the second cuvette. The absorption decrease was measured at 600 nm in a double-beam spectrometer .Aconitase and succinate dehydrogenase (SDH) assays were performed following Stehling Table S1Primers used in this study(0.04 MB DOC)Click here for additional data file.Figure S1A. Amino acid sequence alignment of AtHscB with HscB of E.coli and Jac1 of yeast. Accession number of HscB is NP_417022 and Jac1 NP_011497. Filled arrow indicates the possible cleavage site of the signal peptide. * underlines the important motif conserved for all the three proteins. B. Stable expression of AtHscB-YFP in Arabidopsis. C. Stable expression of AtHscA1-YFP in Arabidopsis. D. Western blot showing the specificity of the AtHscB antibody. Lane a) represents wild-type E. coli cell extract, lane b) represents cell extract from E. coli expressing AtHscB and lane c) represents total cell extract from wild-type Arabidopsis. The slight size increase in lane b) is due to the presence of a 6xHis affinity tag on the protein. E. Immunogold labelling using pre-immune serum. F. Western blot to check the AtHscB expression in AtHscB-CY /AtIscU-NY  double transformed plants. WT. wild type plants. OX-1 and OX-2 are two double transformed plants. The upper panel used anti-AtHscB. The lower panel is a loading control stained with coomassie.(9.04 MB TIF)Click here for additional data file.Figure S2Amino acid sequence alignment of HscA-like proteins. Accession number: HscA of Ecoli, NP_417021; Ssq1 of yeast, NP_013473; At4g37910 (AtHscA1), NP_195504; At5g09590 (AtHscA2), NP_196521.(4.85 MB TIF)Click here for additional data file.Figure S3A. Conditional sterile phenotype of N585159. Mut (humid): mutant grown under humid condition; Mut: mutant grown under normal condition; Wild-type plant as positive control. Inserted icon: Siliques of WT (wild-type), Mut(h) (mutant plants grown under humid condition) and Mut . B. SEM of N585159 C. SEM of N585159 complemented with AtHscB.(5.79 MB TIF)Click here for additional data file.


It has been generally accepted that transforming growth factor beta1 (TGF-beta1) has both negative and positive effects on tumour growth and progression. This study analysed the prognostic value of TGF-beta1 mRNA in advanced gastric carcinoma. A reverse transcriptase-polymerase chain reaction analysis (RT-PCR) was used for TGF-beta1 in endoscopic biopsy specimens from 42 advanced gastric carcinomas. Thirty specimens expressed TGF-beta1 mRNA while 12 specimens did not. The follow-up duration ranged from 4 to 37 months (mean 22.8 months). TGF-beta1-positive group demonstrated a shorter overall survival compared with the TGF-beta1 -negative group (P=0.0014). A significant correlation was also found in the 32 patients who underwent curative resection (P=0.0048). Significant correlations were found between TGF-beta1 mRNA expression and both stage (P=0.0015) and nodal involvement (P=0.0060). Multivariate analysis demonstrated that only TGF-beta1 mRNA expression (P=0.0306) was an independent prognostic factor. All of ten patients who underwent non-curative resection expressed TGF-beta1 mRNA. Expression of TGF-beta1 mRNA in gastric biopsy specimens may be an important preoperative prognostic variable for advanced gastric carcinoma.


Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP–responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1–mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1–regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1–regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1–regulated genes observed in the pmk-1 null mutant, the induction of PMK-1–regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.Innate immunity in  Caenorhabditis elegans interacts with pathogenic bacteria. Previously, we have established that a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates immunity in C. elegans, establishing the conservation of key innate immune signaling pathways of mammals in the immune response of C. elegans. Whereas multiple proteins have been identified as potential targets of p38 MAPK in immunity, the identification of physiological substrates of p38 MAPK in mammalian organisms has been challenging. Here, using a forward genetic approach to identify downstream regulators of the C. elegans innate immune response, we have characterized the transcription factor ATF-7, a conserved member of the basic-region leucine zipper (bZIP) transcription factor family orthologous to mammalian ATF2. We find that ATF-7 functions as a transcriptional regulator of PMK-1 MAPK–mediated innate immunity, functioning as a repressor of immune gene expression that undergoes a switch to an activator upon activation by PMK-1. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans and suggests a mechanism by which the protean effects of p38 MAPK on the mammalian innate immune response may be mediated.We have investigated mechanisms of how the soil nematode  Studies of innate immunity in phylogenetically diverse organisms have revealed the conservation of key signaling pathways mediating pathogen defense Caenorhabditis elegans. Previously, we identified a requirement for a conserved NSY-1-SEK-1-PMK-1 MAPK pathway, orthologous to mammalian ASK1 MAPKKK-MKK3/6 MAPKK-p38 MAPK, in C. elegans innate immunity C. elegans, unlike the loss of mammalian p38 MAPK, does not affect growth and development of C. elegans on non-pathogenic bacteria. The ASK1-MKK3/6-p38 MAPK pathway has been shown to be required for innate immune signaling downstream of Toll-like Receptor-4 (TLR4) in mice C. elegans is TLR-independent and functions downstream of a Toll-Interleukin-1 Receptor (TIR) domain protein TIR-1 We have focused on the genetic dissection of innate immunity in the nematode C. elegansC. elegans mutants deficient in PMK-1 pathway activity has identified a number of PMK-1-dependent candidate effector genes, including C-type lectins and putative antimicrobial peptides, many of which are induced by pathogen infection C. elegans innate immunity in response to intestinal infection The TIR-1-NSY-1-SEK-1-PMK-1 pathway acts cell autonomously in the intestine to regulate innate immunity in C. elegans. Our data establish a pivotal role for ATF-7 as a transcriptional regulator of the PMK-1-mediated innate immune response in C. elegans.In this paper we report the results of a forward genetic screen for mutants deficient in immune signaling through the PMK-1 p38 MAPK pathway. We report the identification of ATF-7, a putative ortholog of the mammalian ATF2 family of basic-region leucine zipper (bZIP) transcription factors, as a key downstream target of the PMK-1 p38 MAPK pathway in enhanced susceptibility to pathogens (Esp phenotype) C. elegans. We used as our starting strain a wild-type (WT) N2-derived strain carrying the agIs219 transgene, which is comprised of the promoter of a PMK-1-regulated gene, T24B8.5, encoding a ShK-like toxin peptide, fused to green fluorescent protein (GFP) and provides an in vivo sensor of PMK-1 pathway activity Pseudomonas aeruginosa PA14 for isolation of mutants with diminished PMK-1-dependent reporter expression and an Esp phenotype. From an initial round of high-throughput screening of worms derived from 140 000 mutagenized genomes, we isolated 33 mutants representing five complementation groups using a cutoff for Esp screening that required mutants to be dead prior to the death of any unmutagenized worms treated in parallel  domain-containing protein -based mapping  protein . Phylogehenotype .atf-7(qd22) mutant by RNAi of the atf-7 gene in the WT background (atf-7 in the atf-7(qd22) mutant background resulted in reversion of the diminished GFP reporter gene expression (atf-7 in WT worms resulted in an Esp phenotype (atf-7 in the atf-7(qd22) mutant conferred increased pathogen resistance and partial suppression of the Esp phenotype of the atf-7(qd22) mutant /atf-7(qd22 qd130) trans-heterozygote , which is nearly as susceptible to P. aeruginosa as the atf-7(qd22) mutant (atf-7(qd22) allele is recessive with respect to the Esp phenotype  mutant . These d) mutant . Intereshenotype .atf-7(qd22) was a gain-of-function, and possibly neomorphic, mutant allele of atf-7, we anticipated that we would be able to isolate atf-7 loss-of-function alleles from a screen for suppressors of the attenuated GFP expression phenotype of the atf-7(qd22) mutant. We screened 20 000 haploid genomes for mutants with increased GFP reporter expression and isolated an intragenic suppressor of atf-7(qd22), atf-7(qd22 qd130), which carries a nonsense mutation resulting in an early stop codon in the atf-7 gene (atf-7(qd22 qd130) mutant allele suppressed the diminished GFP fluorescence phenotype of the atf-7(qd22) mutant  mutant , but onled to WT .atf-7(qd22 qd130) mutant was caused by the nonsense mutation in atf-7, we also analyzed a second putative null allele of atf-7, atf-7(qd137) (atf-7(qd137) mutant exhibited the same Esp phenotype as that observed for the atf-7(qd22 qd130) mutant, as well as the trans-heterozygote, atf-7(qd22 qd130)/atf-7(qd137) (atf-7 locus with GFP fused to the 3′ end between the atf-7 stop codon and the 3′-untranslated region (UTR). We observed that this transgene partially rescued the Esp phenotype of the atf-7(qd22 qd130) mutant (atf-7(qd22) and atf-7(qd22 qd130) mutants may reflect the detrimental effects of overexpression of atf-7.To confirm that the observed Esp phenotype of the 7(qd137) , which w7(qd137) . We also) mutant . The paratf-7(qd22 qd130) mutant was consistent with the results of the RNAi experiments (atf-7(qd22) gain-of-function allele confers a strong Esp phenotype, loss of atf-7 activity also compromises pathogen resistance relative to WT. We observed that the longevity of atf-7(qd22 qd130) and atf-7(qd137) mutants on E. coli OP50 was comparable to that observed for WT (The Esp phenotype of the eriments  and suggd for WT .atf-7(qd22) appeared to be a gain-of-function allele of atf-7 that exhibited the same phenotypes as observed for mutants carrying loss-of-function mutations in PMK-1 pathway components, we hypothesized that ATF-7 might be negatively regulated by the PMK-1 pathway and function as a repressor of the innate immune response. To test this hypothesis, we carried out epistasis analysis using the atf-7(qd22 qd130) and pmk-1(km25) null alleles. We observed that the atf-7(qd22 qd130) loss-of-function allele suppressed the diminished agIs219 GFP reporter gene expression phenotype of the pmk-1(km25) mutant (atf-7(qd22 qd130); pmk-1(km25) double mutant had a reduced pathogen susceptibility compared to pmk-1(km25), comparable to that of the atf-7(qd22 qd130) single mutant (pmk-1(km25) mutant by atf-7(qd22 qd130) was rescued by a transgene carrying wild-type atf-7 fused with GFP (atf-7(qd22 qd130) also suppressed the pathogen susceptibility (agIs219 GFP reporter expression (sek-1(km4) mutant.Because ) mutant . Also, te mutant . The parwith GFP . Furthertibility  and dimipression  phenotypatf-7(qd22 qd130); pmk-1(km25) and atf-7(qd22 qd130); sek-1(km4) double mutants are comparable to the Esp phenotype of atf-7(qd22 qd130) single mutant. The lack of effect of the pmk-1(km25) or sek-1(km4) mutations on the Esp phenotype in the atf-7(qd22 qd130) mutant background is particularly noteworthy in view of the strong Esp phenotype conferred by inactivation of the PMK-1 pathway in the WT background.The Esp phenotypes of the atf-7(qd22 qd130) and pmk-1(km25), we anticipated that a screen aimed at isolating suppressors of the diminished agIs219 GFP reporter expression and pathogen susceptibility phenotypes of the pmk-1(km25) mutant would yield additional loss-of-function alleles of atf-7. Indeed, we isolated two more putative null alleles of atf-7, the aforementioned atf-7(qd137) allele and atf-7(qd141)  , from a atf-7(qd22) mutant, consistent with the observed effects on agIs219 GFP reporter expression  mutant (atf-7(qd22) mutation on agIs219 expression reflect a change in the regulation of PMK-1-regulated genes.We used quantitative real-time PCR (qRT-PCR) to measure changes in expression of three representative genes previously identified as being regulated by the PMK-1 pathway pression , exhibit) mutant . These dE. coli OP50, was comparable in the atf-7 loss-of-function mutants and WT  loss-of-function allele suppressed the markedly diminished basal expression of PMK-1-regulated genes in the pmk-1(km25) mutant  mutant . These dP. aeruginosa PA14 also require ATF-7 for pathogen-induced expression (E. coli OP50 in the atf-7(qd22 qd130) and atf-7(qd137) mutants is comparable to WT, no induction of expression is observed in the presence of P. aeruginosa (P. aeruginosa infection. This requirement may contribute to the observed Esp phenotype of the atf-7(qd22 qd130) mutant, as optimal regulation of the C. elegans innate immune response may be dependent on PMK-1 regulation of ATF-7.Upon exposure to pathogen infection, a number of genes are up-regulated in a PMK-1-dependent manner pression . Althougruginosa . These dThe genetic and gene expression data above are consistent with a role for PMK-1 in the modulation of the transcriptional regulator ATF-7. Because the PMK-1 pathway acts in the intestine in a cell autonomous manner to regulate the innate immune response C. elegans atf-7, pmk-1, and sek-1 cDNAs in Cos7 cells and immunoblotting against T7-ATF-7 to detect changes in gel mobility indicative of phosphorylation. Expression of PMK-1 with SEK-1, which results in activated PMK-1, produced a shift in the T7-ATF-7 protein band indicative of a change in the phosphorylation state -T7-ATF-7 interaction that was dependent on the activated form of PMK-1, as determined by the requirement for co-transfection of k-1 cDNA .qd22 mutation into the T7-ATF-7 expressed in Cos7 cells and found that in contrast to the WT ATF-7, the mutant ATF-7 showed no change in gel mobility in the presence of activated PMK-1 (atf-7(qd22) allele may encode a form of the protein that can bind PMK-1 (atf-7(qd22) allele, with respect to the Esp phenotype and effects on PMK-1-regulated gene expression, coupled with the apparent insensitivity of the corresponding mutant ATF-7 protein to PMK-1 activity, further suggests that the phosphorylation of ATF-7 by PMK-1 may function to relieve the transcriptional repressor activity of ATF-7.We introduced the ed PMK-1 , lane 5,nd PMK-1 , lane 3,P. aeruginosa PA14, we examined the role of ATF-7 in pathogen resistance to two other microbial pathogens that cause lethal infections in C. elegans, Serratia marcescens and Enterococcus faecalis. On S. marcescens Db10, the pmk-1(km25) mutant had a weak Esp phenotype (P. aeruginosa PA14 (atf-7(qd22 qd130) mutant also had a similarly weak phenotype, as did the atf-7(qd22 qd130); pmk-1(km25) double mutant ; pmk-1(km25) double mutant are consistent with PMK-1 and ATF-7 functioning as positive regulators of pathogen resistance in the same pathway, or with ATF-7 under negative regulation by PMK-1 with ATF-7 functioning as a transcriptional repressor.In order to determine whether our observations were specific to infection by henotype  comparedosa PA14 . The atfe mutant . These dE. faecalis MMH594, the atf-7(qd22 qd130) mutant does not have an appreciable Esp phenotype  mutation only partially suppresses the Esp phenotype of the pmk-1(km25) mutant . Gene expression studies of genes induced in C. elegans infection with Gram-positive bacteria, and the identification of such genes that are regulated by the PMK-1 pathway may further illuminate the differences in the role of ATF-7 observed between E. faecalis and P. aeruginosa.Interestingly, on the Gram-positive pathogen henotype , suggest) mutant , suggestatf-7(qd22 qd130) mutant did not exhibit enhanced sensitivity to arsenite stress, and in addition, did not suppress the arsenite sensitivity of the pmk-1(km25) mutant or the sek-1(km4) mutant (skn-1 mutants did not exhibit enhanced susceptibility to P. aeruginosa (The PMK-1 pathway regulates the response to arsenite and oxidative stress through regulation of the transcription factor SKN-1 ) mutant . In addiruginosa . WhereasC. elegans ortholog of the mammalian ATF2/ATF7/CREB5 family of bZIP transcription factors, as a transcriptional regulator of PMK-1-mediated innate immunity in C. elegans. We isolated four mutant alleles of C. elegans atf-7 from three different forward genetic screens. First, the gain-of-function qd22 allele was isolated from a large-scale screen for mutants with diminished PMK-1-dependent GFP reporter gene expression and an Esp phenotype. The isolation of qd22 served as a starting point for the characterization of ATF-7, and the analysis of this unusual gain-of-function allele provided insights into the mechanism of ATF-7 regulation by PMK-1. The increased levels of PMK-1 activation in the atf-7(qd22) mutant relative to WT (atf-7(qd22) acts as a gain-of-function allele, we determined that atf-7(qd22) is recessive with regard to pathogen susceptibility (qd22 mutant ATF-7 protein cannot be phosphorylated by PMK-1 and thus functions as a constitutive repressor (atf-7(qd22)/atf-7(+) trans-heterozygote.We have described the identification and characterization of ATF-7, a ve to WT  may refltibility . We suggepressor , the resatf-7(qd22) was a gain-of-function allele, we isolated the atf-7(qd22 qd130) allele as an intragenic suppressor of atf-7(qd22). A third genetic screen aimed at isolating suppressors of pmk-1(km25) yielded two additional putative null alleles of atf-7, qd137 and qd141. The genetic analysis of loss-of-function alleles of atf-7 allowed us to begin to address the physiological role of ATF-7 in innate immunity. Genetic interaction analysis of atf-7 mutant alleles suggests that PMK-1 negatively regulates ATF-7, which in turn functions as a negative regulator of C. elegans innate immunity to P. aeruginosa and E. faecalis. At the same time, the Esp phenotype of atf-7 loss-of-function mutants and the analysis of P. aeruginosa-induced gene expression were suggestive of a requirement for ATF-7 in the activation of the inducible innate immune response, as ATF-7 was shown to be required for the increased expression of PMK-1-regulated genes in response to P. aeruginosa infection. Interestingly, the lack of an Esp phenotype of atf-7(qd22 qd130) on E. faecalis may be suggestive of the absence of a PMK-1-regulated inducible response to E. faecalis that is regulated by ATF-7.Based on evidence that P. aeruginosa-induced gene expression nsy-1(ky397)tir-1(qd4)pmk-1(km25)skn-1(zu67), skn-1(zu135)sek-1(km4)Previously isolated and characterized mutants used: LG II: agIs219 atf-7(qd22) III] was isolated as described below and backcrossed three times to its parental strain, AU78. ZD318 [agIs219 atf-7(qd22 qd130) III] was isolated as described below and outcrossed four times to wild-type strain N2. ZD39  was made by crossing the agIs219 transgene from strain AU78 into pmk-1(km25). ZD395  was made by crossing the agIs219 transgene from strain AU78 into sek-1(km4). ZD332  and ZD402  were isolated as described below. ZD332 was backcrossed three times to ZD39. ZD350 [agIs219 atf-7(qd137) III] was made by removing pmk-1(km25) from agIs219 atf-7(qd137); pmk-1(km25), which had been previously backcrossed twice to ZD39, by outcrossing to N2. ZD326  was made by crossing pmk-1(km25) into agIs219 atf-7(qd22 qd130) and was outcrossed to pmk-1(km25) an additional three times. ZD340  was made by crossing sek-1(km4) into agIs219 atf-7(qd22 qd130) and was outcrossed to sek-1(km4) an additional three times.Mutants described in this study: ZD442 [P. aeruginosa PA14 S. marcescens Db10 E. faecalis MMH594 P. aeruginosa PA14 and S. marcescens Db10 were used to inoculate 3 ml cultures of Luria-Bertani (LB) broth, which were then incubated overnight at 37°C. Five microliters of the S. marcescens Db10 culture was used to seed standard 35-mm slow-kill assay plates, whereas five microliters of the P. aeruginosa PA14 culture was used to seed 35-mm slow-kill assay plates containing 0.05 mg/ml 5-fluorodeoxyuridine (FUDR), used to prevent eggs from hatching. Seeded plates were incubated at 37°C overnight and then incubated at room temperature overnight. A single colony of E. faecalis MMH594 was used to inoculate a 3 ml culture of brain heart infusion (BHI) broth containing 80 µg/ml of kanamycin, which was then incubated at 37°C for 5 hours. Seven microliters of culture was used to seed 35-mm BHI agar plates containing 80 µg/ml of kanamycin, which were incubated at 25°C overnight. In all pathogenesis assays, the size of the bacterial lawn was small, meaning that the culture was seeded in the middle of the plate and was not spread to the edge. For each assay, approximately 20–40 L4-staged worms were picked over to prepared plates, with 3–5 plates per strain. The sample sizes for each assay are provided in S. marcescens plates were transferred to new plates on days 1, 2, and 3 of the assay. All S. marcescens plates in a single assay were seeded on the same day. Worms on P. aeruginosa PA14 plates containing FUDR and E. faecalis MMH594 plates did not require transferring. Statistical analyses of survival curves were performed in Prism 5 (GraphPad) using the log-rank test function, which computes the Mantel-Haenszel method.Pathogenesis assays with 2 generation L1 stage larvae were plated onto NGM plates seeded with E. coli OP50 and incubated for 55 hours at 20°C and subsequently sorted with a Union Biometrica COPAS Biosorter. Worms with diminished fluorescence compared to wild-type worms were directly plated onto a plate seeded with P. aeruginosa PA14, and incubated at 25°C. The plates were screened at 24 hours for dead worms. Following the rationale of our previously reported screen for Esp mutants E. coli OP50, allowing the fertilized eggs inside each carcass to hatch so that the mutant strains could be recovered. In three separate screens a total of 140 000 haploid genomes were mutagenized. We note that the yield of the screen is strongly dependent on the time at which the Esp screening takes place, and that the 24 h time point represented a particularly stringent time such that siblings were rarely isolated among the mutant isolates. The early time of screening also accounts for the relatively low yield of mutants from the number of genomes mutagenized and the high specificity of isolated mutations for the PMK-1 pathway.Mutagenesis using ethyl methane sulfonate (EMS) was carried out following standard methods C. elegans isolate CB4856 was performed as reported tir-1(qd4), nsy-1(ag3) and nsy-1(ky397), sek-1(km4), and pmk-1(km25)). After assignment of the isolated alleles into complementation groups, the open reading frame of the affected gene was sequenced to identify the causative mutation in each allele. Isolates from the screen and the identified mutations are shown in qd22, was isolated. Fine mapping of qd22 was carried out using CB4856 SNP-based mapping. The location of qd22 on the left arm of LG III was in the vicinity of the agIs219 integrated transgenic array, and thus a strain carrying qd22 without agIs219 was generated and SNP mapping was carried out using the Esp phenotype of qd22. In order to facilitate interpretation of the pathogen killing assays with recombinants, a strain carrying the qd22 mutation (without the agIs219 transgene) and the CB4856-derived allele of npr-1, 215F, was utilized for crossing with CB4856 because of the enhanced susceptibility conferred by the 215F allele of npr-1 relative to the Bristol N2 background qd22 was initially isolated.Single-nucleotide polymorphism (SNP)-based mapping using the atf-7(qd22) diminished agIs219 GFP fluorescence phenotype was carried out similarly as above. Briefly, C. elegans atf-7(qd22) hermaphrodites carrying agIs219 were mutagenized with EMS and synchronized larvae of the F2 generation were plated onto NGM plates seeded with E. coli OP50 and incubated for 55 hours at 20°C. The F2 worms were screened for GFP expression from the agIs219 transgene using a Zeiss Stereo V12 Discovery microscope with a GFP wide-band fluorescence cube. Any F2 worm with increased fluorescence compared to atf-7(qd22) was singled to a NGM plate seeded with E. coli OP50. Isolates with increased fluorescence were then tested for suppression of the atf-7(qd22) Esp phenotype to P. aeruginosa PA14 using the PA14 pathogenesis assay described above. The atf-7 coding region was then sequenced in isolates that had both increased fluorescence and diminished pathogen susceptibility.A forward genetic screen to identify suppressors of the pmk-1(km25) mutant was carried out using ZD39 as the starting strain and following a procedure as outlined for the isolation of atf-7(qd22) suppressor mutants.A genetic screen for suppressors of the C. elegans genomic fosmids (Geneservice) were isolated using Qiagen Miniprep Kits following the standard protocol. Fosmid 24cA04 was injected into ZD442 at a concentration of 20 ng/µl, along with 25 ng/µl of Pmyo-2::RFP as a co-injection marker and 55 ng/µl of pBlueScript (Stratagene) as carrier DNA. The fluorescently-tagged atf-7 construct was generated by yeast-mediated ligation of genomic fragments generated by PCR using fosmid 25cA04 as template DNA and Phusion high-fidelity DNA polymerase (New England Biolabs). A 22794 bp genomic region, from 7474 to 30267 with respect to fosmid 25cA04, was covered in the fluorescently-tagged atf-7 construct. This construct was injected into ZD326 at a concentration of 20 ng/µl, along with 32 ng/µl of Pmyo-2::RFP as a co-injection marker and 10 ng/µl of pBlueScript (Stratagene) as carrier DNA. Two independent lines carrying this construct were crossed into strain ZD318 using standard genetic techniques.atf-7::GFP was performed as previously described atf-7 gene was amplified in fragments ranging in size from ∼1 kb to 4 kb in 8 separate PCR reactions with at least a 50 bp overlap between adjacent fragments. The gene encoding GFP was amplified from the Fire vector pPD95.75 Yeast-mediated ligation of P. aeruginosa exposure experiments, L4 stage worms were washed onto plates seeded with E. coli OP50 or P. aeruginosa PA14 in parallel, dried, and incubated for four hours at 25°C. Samples were collected, frozen in liquid nitrogen, and stored at −80°C before RNA extraction using TRI reagent (Ambion). cDNA was prepared with the Retroscript kit (Ambion) using oligo dT primers. The reverse transcription reaction was also performed without reverse transcriptase for each sample, and subsequent qRT-PCR on these control reactions showed that no contaminating genomic DNA was present. qRT-PCR was performed with a Mastercycler Realplex (Eppendorf) with SYBR Green detection in triplicate reactions of 20 µl. All primers were previously reported, and relative expression between samples was determined using snb-1 as a reference gene Synchronized populations of wild-type and indicated mutant strains were grown to the L4 larval stage. For atf-7 coding region, corresponding to bases 11532 to 12303 with respect to cosmid C07G2, was amplified by PCR and subcloned into the Fire vector L4440. RNAi by bacterial feeding using E. coli HT115 bacteria expressing either the L4440-derived atf-7 RNAi vector or the empty L4440 vector (control RNAi) was carried out as reported 1 generation animals were assayed for susceptibility to P. aeruginosa PA14 or analyzed for GFP expression from the agIs219 transgene.A 771 bp segment of the C. elegans PMK-1 and activated PMK-1 (Promega) was carried out as described previously Immunoblotting against agIs219 reporter, L4-staged worms, grown at 20°C, were picked over to normal maintenance plates and placed at 20°C overnight. After approximately 18 h, worms were mounted on 2% agarose pads and immobilized in 10 mM sodium azide. Slides were viewed using an AxioImager Z1 fluorescence microscope (Zeiss) with an A-Plan 10X/0.25 objective (Zeiss) and pictures were taken using an AxioCam HRm camera. To visualize expression of fluorescently-tagged atf-7, L4-staged worms, grown on NGM agar plates seeded with E. coli OP50 at 20°C, were mounted and imaged as described above with a Plan-Apochromat 20X/0.8 objective (Zeiss). Background intestinal autofluorescence was removed by taking a picture with the DAPI filter and subtracting the resulting picture from the image taken with the GFP filter.To visualize expression of the 2. Cos7 cells (1×106) were plated in 6-cm dishes and transfected with a total of 6 µg DNA containing various expression vectors by using FuGENE6 (Roshe). The ATF-7 expression vector contained the atf-7c isoform. After 48 h, cells were collected and washed once with ice-cold phosphate-buffered saline (PBS) and lysed in 0.6 ml of extraction buffer . Cellular debris was removed by centrifugation at 10 000×g for 5 min. Small aliquots of each cell lysate were boiled with SDS-sample buffer and were used as whole cell extracts. Remaining cell lysates were divided into 200 µl and each cell lysate was incubated with 0.5 µg of various antibodies and 10 ml protein G-Sepharose . The immune complexes were washed five times with wash buffer  and then boiled with SDS-sample buffer. Phosphatase treatment was performed on immunoprecipitated samples with Lambda protein phosphatase (New England Biolabs) at 30°C for 10 minutes. Immunoblotting was performed as described previously Cos7 cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 µg/ml penicillin G and 100 µg/ml streptomycin at 37°C and 5% COE. coli OP50. L4-staged animals were transferred to standard slow-killing plates supplemented with 5 mM sodium arsenite and 0.05 mg/ml of FUDR, seeded with concentrated E. coli OP50. The sample sizes for the arsenite stress assay are provided in t-test.Sensitivity of mutant strains to oxidative stress was determined using sodium arsenite. Briefly, mutant worms were grown on E. coli OP50 at 20°C. Approximately 40 L4-staged worms (Day 0) were picked over to NGM plates containing 0.05 mg/ml of FUDR, seeded with E. coli OP50. Four to five plates for each strain were used in each experiment and plates that had become contaminated or plates in which the worms had borrowed were excluded upon the appearance of contamination/borrowing. Worms that had protruding/exploding vulvas and worms that crawled off the plate were censored. The sample sizes for each assay are provided in Strains used in the lifespan assays were maintained on Figure S1atf-7(qd22) and WT worms on E. coli OP50. Lifespan assay of L4 larval stage wild-type worms and atf-7(qd22) mutant animals on E. coli OP50. Both strains carry the agIs219 transgene. Replicate data can be seen in Lifespan of (0.13 MB PDF)Click here for additional data file.Figure S2atf-7(qd22) Esp phenotype. Pathogenesis assay of L4 larval stage wild-type worms, atf-7(qd22) mutant animals, and three independent transgenic lines  of atf-7(qd22) mutant animals carrying fosmid 25cA04. All strains carry the agIs219 transgene. The difference in susceptibility between atf-7(qd22) mutant animals and each transgenic line carrying fosmid 25cA04 is significant (p<0.0001).Rescue of (0.13 MB PDF)Click here for additional data file.Figure S3atf-7(qd22) is a gain-of-function allele. (A) Fluorescence microscopy images of GFP expression from the agIs219 transgene in wild-type worms and atf-7(qd22) mutant worms each exposed to both control RNAi and RNAi of atf-7. (B) Pathogenesis assay comparing the effects of control RNAi and RNAi of atf-7 on survival of wild-type worms on P. aeruginosa PA14. The difference in susceptibility between wild-type worms treated with control RNAi and atf-7 RNAi is significant (p<0.0001). Replicate data can be seen in atf-7 on survival of atf-7(qd22) mutant animals on P. aeruginosa PA14. The difference in susceptibility between atf-7(qd22) mutant animals treated with control RNAi and atf-7 RNAi is significant (p<0.0001). Replicate data can be seen in (0.30 MB PDF)Click here for additional data file.Figure S4atf-7(qd22) confers a recessive Esp phenotype. Pathogenesis assay of L4 larval stage wild-type worms; atf-7(qd22) and atf-7(qd22 qd130) mutant animals; and atf-7(qd22)/atf-7(+) and atf-7(qd22)/atf-7(qd22 qd130) trans-heterozygotes, on P. aeruginosa PA14. All strains carry the agIs219 transgene. The differences in susceptibility between atf-7(qd22) mutant animals and atf-7(qd22)/atf-7(+) trans-heterozygotes, and atf-7(qd22 qd130) mutant animals and atf-7(qd22)/atf-7(qd22 qd130) trans-heterozygotes are significant (p<0.0001 for each comparison). Replicate data can be seen in (0.13 MB PDF)Click here for additional data file.Figure S5atf-7::GFP rescues the atf-7(qd22 qd130) Esp phenotype. Pathogenesis assay of wild-type worms, atf-7(qd22 qd130) mutant animals, and two independent transgenic lines (qdEx17 and qdEx19) of atf-7(qd22 qd130) mutant animals carrying the atf-7::GFP construct under the control of the endogenous atf-7 genomic promoter and 3′- untranslated region, on P. aeruginosa PA14. All strains carry the agIs219 transgene. The difference in susceptibility between atf-7(qd22 qd130) mutant animals and each transgenic line carrying the atf-7::GFP transgene is significant (p<0.0001 for each comparison).Expression of (0.13 MB PDF)Click here for additional data file.Figure S6atf-7 loss-of-function mutants and WT worms on E. coli OP50. Lifespan assay of L4 larval stage wild-type worms, atf-7(qd22 qd130) and atf-7(qd137) mutant animals on E. coli OP50. All strains carry the agIs219 transgene. Replicate data can be seen in Lifespan of (0.13 MB PDF)Click here for additional data file.Figure S7atf-7::GFP rescues the atf-7(qd22 qd130) suppression of pmk-1(km25). Pathogenesis assay of pmk-1(km25) mutant animals and atf-7(qd22 qd130); pmk-1(km25) double mutant animals, along with three independent transgenic lines  of atf-7(qd22 qd130); pmk-1(km25) double mutant animals carrying the atf-7::GFP construct under the control of the endogenous atf-7 genomic promoter and 3′-untranslated region, on P. aeruginosa PA14. All strains carry the agIs219 transgene. The difference in susceptibility between atf-7(qd22 qd130); pmk-1(km25) double mutant animals and each transgenic line carrying the atf-7::GFP transgene is significant (p<0.0001 for each comparison).Expression of (0.13 MB PDF)Click here for additional data file.Figure S8PMK-1 phosphorylation of ATF-7 is sensitive to phosphatase. Cos7 cells were transfected with T7-ATF-7, HA-PMK-1, and FLAG-SEK-1 as indicated. ATF-7 was immunoprecipitated with anti-T7, treated with phosphatase where indicated, and immunoblotted with anti-T7 (top). Whole cell extracts were immunoblotted with antibodies that recognize HA (middle) and FLAG (bottom).(0.20 MB PDF)Click here for additional data file.Figure S9skn-1 mutants do not exhibit an Esp phenotype. Pathogenesis assay of L4 larval stage wild-type worms, skn-1(zu67) and skn-1(zu135) mutant animals on P. aeruginosa PA14.(0.13 MB PDF)Click here for additional data file.Figure S1050 means, LT50 standard deviations (S.D.), and sample sizes from two independent P. aeruginosa pathogenesis assays with wild-type worms, atf-7(qd22) and atf-7(qd22 qd130) mutant animals.Replicate of pathogenesis assay shown in (0.18 MB PDF)Click here for additional data file.Figure S1150 means, LT50 standard deviations (S.D.), and sample sizes from two independent P. aeruginosa pathogenesis assays with wild-type worms treated with control RNAi and atf-7 RNAi, and atf-7(qd22) mutant animals treated with control RNAi and atf-7 RNAi.Replicate of pathogenesis assay shown in (0.19 MB PDF)Click here for additional data file.Figure S1250 means, LT50 standard deviations (S.D.), and sample sizes from two independent P. aeruginosa pathogenesis assays with wild-type worms; atf-7(qd22) and atf-7(qd22 q130) mutant animals; and atf-7(qd22)/atf-7(+), atf-7(qd22 qd130)/atf-7(+), and atf-7(qd22)/atf-7(qd22 qd130) trans-heterozygotes.Replicate of pathogenesis assay shown in (0.20 MB PDF)Click here for additional data file.Figure S1350 means, LT50 standard deviations (S.D.), and sample sizes from two independent P. aeruginosa pathogenesis assays with wild-type worms; atf-7(qd22 q130) and atf-7(qd137) mutant animals; and atf-7(qd22 qd130)/atf-7(+), atf-7(qd137)/atf-7(+), and atf-7(qd22 qd130)/atf-7(qd137) trans-heterozygotes.Replicate of pathogenesis assay shown in (0.20 MB PDF)Click here for additional data file.Figure S1450 means, LT50 standard deviations (S.D.), and sample sizes from two independent P. aeruginosa pathogenesis assays with wild-type worms; atf-7(qd22 q130) and pmk-1(km25) mutant animals; and atf-7(qd22 qd130); pmk-1(km25) double mutant animals.Replicate of pathogenesis assay shown in (0.19 MB PDF)Click here for additional data file.Figure S1550 means, LT50 standard deviations (S.D.), and sample sizes from two independent P. aeruginosa pathogenesis assays with wild-type worms; atf-7(qd22 q130) and sek-1(km4) mutant animals; and atf-7(qd22 qd130); sek-1(km4) double mutant animals.Replicate of pathogenesis assay shown in (0.19 MB PDF)Click here for additional data file.Figure S1650 means, LT50 standard deviations (S.D.), and sample sizes from two independent lifespan assays with wild-type worms, atf-7(qd22), atf-7(qd22 q130), and atf-7(qd137) mutant animals.Replicate of lifespan assay shown in (0.18 MB PDF)Click here for additional data file.Table S1Sample sizes for each pathogenesis, arsenite stress, and lifespan assay. Chart showing the sample sizes for each pathogenesis, arsenite stress, and lifespan assay presented in this work. Sample size does not include censored worms.(0.16 MB PDF)Click here for additional data file.


All the compounds have a mixed ligand composition with salicylhydroxamic acid and di-2-pyridylketonoximeas chelate agents. The compounds construct metallacrown cores {[12-MCNi(n)N(sj02(pko)2-4][12-MCNi(ll)N(shO3(pko)-4]}2+ following the pattern [-Ni-O-N-]4. The neutral decanuclear [Ni(II)(A)]2[12-MCNi(II)N(shi)2(pko)2-4][12-MCNi(II)N(pko)3(pko)-4] fused metallacrown, consists of two [12-MCM(ox)N(ligand)-4] units the {Ni(ll)(A)[12-MCNi(II)N(shi)2(pko)2-4]} and {Ni(II)(A)[12-MCNi(II)N(shi)3(pko)-4]} with 1+ and 1- charge, respectively. Each metallacrown unit has four ring Ni(II) ions and one additional encapsulated Ni(II) ion in planar arrangement. The anionic unit is bonded with cationic one creating binuclear moieties. The herbicide or antiiflammatory carboxylato ligands are bridging the central octahedral nickel atom with a ring metal ionin a bindetate fashion. The effect on DNA and their antibacterial activity was examined. The changes in themobility can be attributed to the altered structures of the pDNA treated with Ni(II) complexes. Evaluating thedata of the antibacterial activity of the compounds tested, we can conclude that nickel complexes presentstrong antibacterial activity.The synthesis, characterization and the biological study of a series of Ni(ll)


Analogues substituted in the 3′ position with I (DF 129), CH 3(DF 203), or CI (DF 229) possess an extended profile of antitumour activity with remarkable selective activity in cell lines derived from solid tumours associated with poor prognosis, e.g. breast, ovarian, renal and colon. Growth inhibition occurs via unknown, possibly novel mechanism(s) of action. Two cell lines have been derived from sensitive MCF-7wtbreast cancer cells  following long-term exposure to 10 nM or 10 μM CJM 126, MCF-710 μM 126 and MCF-710 μM 126respectively, which demonstrate acquired resistance to this agent (IC 50> 30 μM) and cross-resistance to DF 129, DF 203 and DF 229. Sensitivity to tamoxifen, benzo[a]pyrene (BP), mitomyin C, doxorubicin and actinomycin D is retained. Resistance may, in part, be conferred by the constitutively increased expression of bcl-2 and p53 proteins detected in MCF-710 nM 126and MCF-710 μM 126lysates. Significantly decreased depletion of CJM 126 (30 μM) from nutrient medium of MCF-710 nM 126cells was observed with predominantly cytoplasmic drug localization and negligible DNA strand breaks. N -acetyl transferase (NAT)1 and NAT2 proteins were expressed by all three MCF-7 sub-lines, but significantly higher expression of NAT2 was accompanied by enhanced acetylation efficacy in MCF-710 nM 126cells. In contrast, CJM 126 (30 μM) was rapidly depleted from nutrient medium of MCF-710 μM 126culture and accessed nuclei of these cells exerting damage to DNA. The major biotransformation product of CJM 126 in MCF-710 μM 126cells was 2-(4-aminophenyl)-6-hydroxybenzothiazole (6-OH 126). This metabolite possessed no antitumour activity. Accordingly, in this sub-line, low constitutive expression and activity of cytochrome P450 (CYP) 1A1 was detected. © 2000 Cancer Research Campaign2-(4-aminophenyl)benzothiazole (CJM 126) elicits potent growth inhibition in human-derived breast carcinoma cell lines, including oestrogen receptor-positive (ER+) MCF-7