Bone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL); however, osteonecrosis of the jaw (ONJ) is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP)-related and denosumab (anti-RANKL antibody)-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC)-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction  in ONJ-altered and healthy periodontal tissue.Twenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio . Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each) to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels.Semi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4  expression in ONJ-adjacent periodontal tissue. ONJ tissue also exhibited decreased relative gene expression for Msx-1 (p < 0.03) and RANKL (p < 0.03) and increased BMP-2/4 expression (p < 0.02) compared to control.These results explain the sclerotic and osteopetrotic changes of periodontal tissue following BP application and substantiate clinical findings of BP-related impaired remodeling specific to periodontal tissue. RANKL suppression substantiated the clinical finding of impaired bone remodelling in BP- and aRANKL-induced ONJ-affected bone structures. Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to maxillofacial tissue. Numerous attempts have targeted explaining the etiology of the restriction of amino-bisphosphonate (BP)-associated osteonecrosis of the jaw (BONJ) to the jaws, but an accepted model of formal pathology has been lacking ,2. ExistPotential factors to consider include the unique biological features of the alveolar bone of the jaw. Impairment of cranial neural crest (CNC)-specific RANKL-associated cell signaling as an underlying mechanism of ONJ is an attractive hypothesis because CNC-derived periodontal progenitor cells are involved in remodeling of both hard and soft jaw tissues -9. ImpaiThis study included oral mucoperiosteal specimens from 40 patients. Of these, 20 were from periodontal soft tissue adjacent to clinically and histologically confirmed BONJ of 20 consecutively treated patients undergoing radical sequestrotomy, taken as part of the tissue samples provided for routine histopathological diagnostics. The study was approved by the ethical committee of the University of Erlangen-Nuremberg. All patients gave their informed consent to participation. Additional criteria for specimen inclusion were intravenous application of either pamidronate or zoledronate for at least 12 months and clinical evidence of an exposed jaw bone for at least 8 weeks. Any former radiotherapy was excluded. Details about patient data, surgical treatment, and the follow-up period were previously documented . ControlTissue samples were processed for immunohistochemistry as previously described. AntibodSections were examined qualitatively under a bright-field microscope  at 100-400× magnification for number and localization of stained osteoblast progenitors and fibroblasts. In healthy periodontal samples, subepithelial tissue was observed, including connective, submucous, and periosteal structures. Mature bone tissue, including osteocytes, was excluded from any analysis. In BONJ samples, soft tissue adjacent to the necrotic zone was identified, and three visual fields per section for each sample were digitized at 200× magnification using a CCD camera  and the program AxioVision . For this purpose, randomized systematic subsampling was performed as previously described . SemiquaFrozen tissues were agitated  in lysis buffer , and whole RNA from tissues was extracted using the RNeasy Kit according to the manufacturer's protocol. Quantitative measurement of mRNA in each probe was performed using a commercial microfluid Lab-on-a-Chip technology . The cDNAs from total RNA were synthesized using the High Capacity cDNA Archive Kit  according to the manufacturer's protocol. Real-time RT qPCR analyses were done using QuantiTect Primer Assay (200) . For normalization, GAPDH was used . The QuantiTect TM SYBR Green PCR kit  was used for PCR amplification. The relative quantification of mRNA was performed with the ABI Prism 7300 Sequence Detection System (Applied Biosystems). In total, 40 ng of cDNA was used for each PCR reaction in a total volume of 25 μl. Each PCR run included a 15-min activation time at 95°C, followed by a three-step cycle: denaturing at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 34 s. Formation during PCR of undesired side products that contribute to fluorescence was assessed by melting curve analysis after PCR. Msx-1, BMP-2, and RANKL mRNA quantities were analyzed in duplicate, normalized against GAPDH as an internal control, and expressed in relation to mRNA isolated from healthy periodontal tissue as a calibrator. Relative gene expression was determined using the ΔΔCt method. RNA isolated from healthy oral periodontal tissue (pool of 20 patients) was used as controls.To analyze the immunohistochemical cytoplasmic staining and the spatial pattern of expression, the labeling index of positively stained cells per visual field was assessed. Comparing the relative gene expression, addressed by the real-time RT-PCR, the median gene expression for Msx-1, BMP-2, and RANKL in the pool of healthy oral mucoperiosteum was set as 1. Gene expression in both groups was stated as relative expression compared to healthy mucoperiosteal expression. Multiple measurements per group of investigation were aggregated prior to analysis. Descriptive analysis of labeling index and relative gene expression data were performed using the median (ME) and the interquartile range (IQR). Graphical representations use diagrams representing ME, IQR, minimum, and maximum. Confirmatory comparisons were made between treatment and control groups using generalized estimating equations with "treatment modality" and "subject id" as independent factors for appropriate analysis of repeated measurements per individual. Multiple p values were adjusted according to Bonferroni by multiplying each p value obtained by the number of confirmatory tests performed (n = 10). Two-sided adjusted p values of p ≤ 0.05 were considered to be significant. All calculations were made using SPSS 18.0 for Windows .All examined BONJ samples had multinucleated cells and a thickened epithelial layer above necrotic tissueareas between vital zones Figures , 2b, 3b.In control jaw periodontal tissue, Msx-1 expression was localized in the nucleus and cytoplasm of osteoblasts, fibroblasts, and progenitors within the connective tissue layer Figure . In the RANKL expression was present throughout the soft tissue in normal jaw samples Figure , includiThe labeling index of Msx-1-expressing Figure  and RANKThe patterns for mRNA expression reflected those for protein expression. Msx-1 mRNA levels were significantly suppressed 6.8-fold in BONJ samples compared to control periodontal tissue Figure . BMP-2/4This study identified a significantly diminished expression of Msx-1, a cellular plasticity and proliferation-mediating transcription factor, in BONJ-affected jaw periodontal tissue at the protein and mRNA levels. Significantly elevated expression of BMP-2/4 in the BONJ-related periodontal and periosteal tissue revealed an increased osseous differentiation stimulation in progenitors of osteoblastic lineage in BP-compromised jaw mucoperiosteal tissue. As with Msx-1 expression, RANKL expression in the jaw bone overlying mucoperiosteal tissue was significantly reduced, suggesting suppressed osteoclast activation by osteoblasts .BP-related Msx-1 loss in the PDL can explain the sclerotic, periapical hypermineralized thin lines around dental roots of BP-altered PDL tissue, which is known for having the highest endogenous Msx-1 expression in the jaw ,12,13,20The significantly increased expression of BMP-2/4 identified here at the cellular and mRNA levels in BONJ-affected jaw periosteum is consistent with the clinical and radiologic observation of the osteopetrotic aspect of ONJ-related jaw bone: BMP-2/4 is an essential osteoinductive factor and induces terminal osseous differentiation through DLX5 signaling in the absence of Msx-1 ; 25]. I. I25]. Iin vivo is described here for the first time and indicates the relevance of BP effects on cellular differentiation in explaining the etiology of BONJ. The significantly reduced expression of RANKL in ONJ-adjacent periodontal tissue at the protein and mRNA levels demonstrates the effect of BP action on soft-tissue remodeling. Suppression of RANKL has been described as the main action of BP, preventing osteoclast activation and bone resorption in malignancies and osteoporosis [The finding of BP-related RANKL suppression in periodontal progenitor cells oporosis -31. Thisoporosis ,6. The cThese findings help to explain some of the molecular underpinnings of the restriction of BONJ to the jaw bone. Jaw restricted osteopetrosis implicated in BONJ can be explained by loss of Msx-1. Msx-1, known to be a key regulator of cellular plasticity and constitutively expressed in CNC-derived jaw hard and soft tissue progenitor cells, could be of relevance in jaw-restricted diseases associated with impaired bone and soft tissue remodeling -34. AddrThere are no competing interests of the authors to be declared.This study was funded by the ELAN-Fonds of the University of Erlangen-Nuremberg, Germany.FW was responsible for the application of grant support , the conduction of study, built the hypothesis, established and conducted the methods and analytic procedures and wrote the manuscript. PH built the hypothesis and did the interpretation of the data. JR established the m-RNA analysis and RT-PCR and wrote the manuscript, section RT-PCR. PS and KS did the immunohistochemistry analysis.FN interpreted the data and wrote the manuscript, section discussion. EN interpreted the data and conducted the study by harvesting samples. KA established immunohistochemistry, analysed the tissue samples, interpreted the data and was responsible for the histopatholgical analysis of ONJ- and control tissue samples. All authors read and approved the final manuscript.


Tyrosine Kinase Inhibitors (TKI) have significantly changed the landscape of current cancer therapy. Understanding of mechanisms of aberrant TK signaling and strategies to inhibit TKs in cancer, further promote the development of novel agents.in vitro and in animal cancer xenograft models using tumor cell lines that were "addicted" to signaling of kinases targeted by ABT-869. When given together with chemotherapy or mTOR inhibitors, ABT-869 showed at least additive therapeutic effects. The phase I trial for ABT-869 was recently completed and it demonstrated respectable efficacy in solid tumors including lung and hepatocellular carcinoma with manageable side effects. Tumor cavitation and reduction of contrast enhancement after ABT-869 treatment supported the antiangiogenic activity. The correlative laboratory studies conducted with the trial also highlight potential biomarkers for future patient selection and treatment outcome.ABT-869, a novel ATP-competitive receptor tyrosine kinase inhibitor is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families. ABT-869 showed potent antiproliferative and apoptotic properties in vitro studies on ABT-869 resistance phenotype identified novel resistance mechanism that may be applicable to other TKIs. The future therapeutic roles of ABT-869 are currently been tested in phase II trials.Parallel to the clinical development,  Receptor tyrosine kinases (RTKs) and protein phosphatases control reversible protein phosphorylation ,2. This The common molecular mechanisms underlying such aberrant activities are point mutation, duplication, and amplification of the RTK, which leads to gain-of-function and consecutive activation of the kinases in general. The fms-like tyrosine kinase 3 (FLT3) is a class III RTK family and shares strong structural similarity with other family members including receptors for platelet-derived growth factors A (PDGFRA) and B (PDGFRB), the colony-stimulating factor 1 receptor (CSF1-R) and steel factor receptor (KIT) . FLT3 muABT-869, a novel ATP-competitive RTK inhibitor, is active against all VEGFRs and PDGFR families, but minimally active against unrelated RTKs and cytosolic tyrosine kinases and serine/threonine kinases . The goa50, but much less activity to other nonrelated tyrosine kinase  . Th. Th19]. Pfizer]) .Another potentially important aspect of the distinctive activity profile of ABT-869 is the molecule's activity against CSF1R . This acin vivo mechanism-based targeting, including acute myeloid leukemia with FLT3 mutation (MV4–11), highly angiogenic fibrosarcoma (HT1080), small cell lung carcinoma , colon adenocarcinoma (DLD-1), epidermoid carcinoma (A431) and breast cancinoma (MX-1). In addition to flank xenografts, ABT-869 has demonstrated dose dependant efficacy in orthotopic tumor growth models with the breast carcinoma cell lines MDA-231  and MDA-435LM  as well as a rat glioma cell line (9L). ABT-869 was also efficacious at inhibiting the growth of prostate cancer cells in a bone environment, thereby demonstrating potential therapeutic utility in a metastases setting [Initial nonclinical studies demonstrated potent antiproliferative and apoptotic effects of ABT-869 on cancer cells whose proliferation is dependent on mutant kinases, such as FLT3 ,20,22. Atation MV–11, highIn addition to single agent activity ABT-869 also exhibited antitumor activity when given in combination with chemotherapy agents, including: carboplatin, cisplatin, docetaxel, gemcitabine, irinotecan, paclitaxel, rapamycin, TMZ and Ara-C ,22,24,25In light of its preclinical activity profile, ABT-869 underwent the industrial standard pre-clinical toxicology, metabolism, and pharmacology studies and the compound was deemed to be suitable to further clinical development (see below).Approximately, 25% of AML patients have acquired FLT3 internal tandem duplications (FLT3-ITDs), varying from 3 to ≥ 400 base pairs in the juxtamembrane domain, and 7% of AML patients harbor activating point mutations in the second kinase domain (FLT3-TK) . FLT3 mu50 less than 10 nM [1 cell cycle arrest and apoptosis in these FLT3-ITD positive cells [1/S cyclins, and progressive increases in cyclin dependent kinase inhibitors (CDKIs) p21waf1/Cip, p27kip1 contributed to the blockage of G1/S progression induced by ABT-869 [For leukemia cell lines with FLT3-ITD such as MV4–11 and MOLM-14, ABT-869 potently inhibits their proliferation at ICan 10 nM ,27. ABT-ve cells ,27. Anal ABT-869 . ABT-869 ABT-869 .ABT-869, as predicted from its kinase inhibition profile, targets the FLT3 signaling pathway. In MV4–11 cells, ABT-869 inhibits phosphorylation of FLT3 receptor (p-FLT3), as well as downstream signaling effectors p-AKT, p-ERK, p-STAT5 and PIM-1 kinase at a concentration of 1 nM ,27. Impo®, Cephalon, Inc., Frazer, PA, USA) [However, considering the complexity of the disease, ABT-869 as a single agent is unlikely to deliver complete or lasting responses in AML. We demonstrated that ABT-869 also produces synergistic antileukemic effect with chemotherapy in a sequence dependent manner . This sePA, USA) . For simPA, USA) . In contPA, USA) . In a MVPA, USA) . Low denPA, USA) . CollectNeoangiogenesis plays an important role in the pathogenesis of AML, so targeting VEGF/VEGFR receptors appears to be an alternative approach for treating AML . Based oExpression of VEGF, the primary pro-angiogenic factor, has higher in HCC than in normal hepatic parenchyma cells and has been shown to positively correlate with vascularization of HCC ,34. HCC In addition, the mammalian target of rapamycin (mTOR), a cytosolic serine/threonine kinase, has emerged as an attractive anticancer target in recent years . mTOR plin vivo. This is supported by immunohistochemistry (IHC) analysis that shows ABT-869 significantly down-regulates VEGF and reduces the formation of Microvessel density (MVD). Bevacizumab, a specific anti-VEGF antibody, was also compared with ABT-869 in a Sk-hep-1 mouse xenograft. The antitumor activity of ABT-869 is significantly higher than bevacizumab in this model [We demonstrated that oral administration of ABT-869 as a single agent at a dose of 10 mg/kg/day effectively inhibits the growth of Huh7 and Sk-hep-1 tumors in mouse xenograft models . ABT-869is model . Furtheris model . The addCombination of ABT-869 (10 mg/kg/day) with Rapamycin (2 mg/kg/day) shows significant tumor volume reduction in both Huh7 and Sk-hep-1 animal models when compared to either of the single drug treatments (p < 0.05). Up-regulation of the cell cycle inhibitor, p27, and inhibition of the MAPK pathway contribute to the synergistic antitumor effect observed in combination therapy .Taken together, these results support the rationale for clinical development of combination therapy of ABT-869 and other chemotherapies such as Rapamycin in HCC.In contrast to their potent efficacy in cellular based assays and xenograft models, in clinical trials, FLT3 inhibitors alone only achieve moderate and transient responses in the majority of AML patients ,42-45. FAs ABT-869 was entering early phase clinical development with continuous daily dosing schedule, we investigated some of the mechanisms that could potentially be used by leukemia cells to overcome the cytotoxic effect under long-term use of ABT-869. Three resistant cell lines  were developed by over three-month co-culture of the human leukemia cell line, MV4–11  with increasing concentrations of ABT-869 . These rMembrane-bound and soluble forms of FLT3 ligand are both biologically active . FLT3 liSurvivin (encoded by BIRC5), the smallest member of the inhibitor of apoptosis protein (IAP) family, has been regarded as one of the classic fetal oncoproteins -61. SurvAfter screening for compounds which could potentially reverse the resistance phenotype in MV4–11R, Indirubin derivative (IDR) E804 was identified. As an inhibitor of the SRC-STAT3 pathway , IDR E80In summary, over expression of FLT3 ligand, methylation silencing of the SOCS family and overexpression of survivin all together integrate leading aberrant STAT signaling activity and contribute to resistance to FLT3 inhibitors. The discovery of this novel mechanism of resistance to FLT3 inhibitors, as described in Figure ®. Preliminary PK at doses of 10 mg showed a modest correlation between oral clearance and body-weight; thus subsequent dose escalations in segment A were based on bodyweight. The most common drug-related adverse events were fatigue, proteinuria, hypertension, myalgia, skin toxicity (hand and foot blisters) and oral hypersensitivity, and these toxicities increased in frequency and intensity with increasing doses. The maximal tolerated dose (MTD) was determined to be 0.3 mg/kg/day. In general, the treatments are well tolerated in this patient population with either refractory disease or no standard therapy.In 2006, Abbott made a strategic decision and partnered with the clinical team at National University Hospital in Singapore and conducted the first in man study for ABT-869. The first in man study was started in patients with solid malignancies refractory to or for which no standard effective therapy exists who were enrolled in escalating dose cohorts and treated with oral ABT-869 once daily continuously. This study was designed as a single-arm, open-label Phase I trial and was conducted in three segments in order to determine the maximum tolerable dose (MTD), tolerability, and pharmacodynamics of a lower dose cohort to better define dose-effect relationships. ABT-869 lacks high aqueous solubility, therefore, the study drug was diluted in 60 mLs of Ensure PlusThe treatment response of this phase I trial is encouraging. Three (10%) out of 29 patients achieved partial response (PR); two had non-small cell lung cancer (NSCLC) treated at 0.3 mg/kg/day and 10 mg/day respectively, and one had colorectal cancer (CRC) treated at 0.1 mg/kg/day. An additional sixteen patients had stable disease lasting longer than 12 weeks, among which were patients with CRC (5), NSCLC (2), ovarian cancer (2), hepatocellular carcinoma (HCC) (2) and neuroendocrine tumour (2).Tumor cavitation in the lungs and reduction of contrast enhancement in tumor on post-treatment CT scans after ABT-869 treatment suggesting central necrosis supported antiangiogenic activity, and has been observed with other VEGF antagonists  from this trial were similar between Asian and Caucasian populations  and met the exposure targets derived from nonclinical efficacy studies ,64. DynaA multi-center phase I study was also initiated in patients with refractory or relapsed AML or myelodysplastic syndrome (MDS) as FLT-3 is an obvious therapeutic target of ABT-869. Based on our pre-clinical study , the triThe promising anti-cancer properties of ABT-869 identified at the early phase trial facilitate further clinical development of this novel agent. In June 2007, Abbott and Genentech Inc. formed collaboration for the global research, development and commercialization for ABT-869. Phase II clinical trials evaluating ABT-869 for advanced or metastatic hepatocellular carcinoma, metastatic breast cancer, metastatic colorectal cancer, metastatic non-small cell lung cancer, and advanced renal cell carcinoma are ongoing. A summary of current ABT-869 clinical trials listed on the National Institutes of Health Website is shown in Table Preliminary clinical data on single agent ABT-869 was presented in the 2009 ASCO annual meeting. Encouraging clinical activity has been observed in non-small cell lung cancer (NSCLC) and advanced hepatocellular carcinoma (HCC) trials as well as in a renal cell carcinoma (RCC) trial after Sunitinib failure -67. Howein vivo angiogenesis effect has already produced a promising clinical response in early phase clinical development.In summary, ABT-869 is a novel inhibitor that simultaneously provides potent and selective inhibition of the VEGFR and PDGFR kinase families and has demonstrated activity in patients with solid tumors who failed standard regimen. Optimal dosing and scheduling are being investigated and the potent Based on the Population PK analysis presented in an abstract , ABT-869The reported side effects such as fatigue, proteinuria, hypertension, myalgia, skin toxicity (hand and foot blisters) are similar to commonly described toxicity in other FDA approved oral tyrosine kinase inhibitors such as Sunitinib. Long term dosing of ABT-869 did not appear to pose problems of cumulative toxicity in patients who received more than a year of dosing. The nonclinical studies on combination therapies have demonstrated synergy and are likely to be more effective than monotherapy. Clinical studies of ABT-869 in combination with chemotherapy or other novel targeted therapies, will further our understanding of how to optimize this exciting new therapy. The recent identification of the critical role of survivin in the regulation of ABT-869 resistance is interesting and is therapeutically relevant. Mechanisms of resistance to ABT-869 remain under active investigation.DHA is an employee of Abbott laboratories. The other authors declared no conflict of interest.JZ wrote the review of pre-clinical studies. BCG contributed the phase I trial data, and DHA contributed non-clinical studies, discussion and revision. CSC conceived of the study, wrote the review of clinical trials and organized the manuscript. All authors read and approved the final manuscript.


These observations with essentially non-immunogenic tumours are comparable with early findings showing that embryonic antigens are also expressed on aminoazo dye induced rat hepatomata, and sarcomata induced with 3-methylcholanthrene. Re-expression of embryonal components, therefore, may be a concomitant of neoplastic transformation.


Drosophila gene scribble (scrib) encodes a PDZ-domain scaffolding protein that regulates cell polarity and acts as a tumour suppressor in flies. Increasing evidence also implicates the loss of human Scrib in cancer. In this report, we investigate how loss of Scrib promotes epithelial tumourigenesis in Drosophila, both alone and in cooperation with oncogenic mutations.Metastatic neoplasias are characterized by excessive cell proliferation and disruptions to apico-basal cell polarity and tissue architecture. Understanding how alterations in cell polarity can impact upon tumour development is, therefore, a central issue in cancer biology. The scrib mutants drive epithelial tumourigenesis. First, we show that over-expression of the apical cell polarity determinants Crumbs (Crb) or aPKC induces similar cell morphology defects and over-proliferation phenotypes as scrib loss-of-function. However, the morphological and proliferative defects in scrib mutants are independent of Crb function, and instead can be rescued by a dominant negative (kinase dead) aPKC transgene. Secondly, we demonstrate that loss of Scrib promotes oncogene-mediated transformation through both aPKC and JNK-dependent pathways. JNK normally promotes apoptosis of scrib mutant cells. However, in cooperation with oncogenic activated Ras or Notch signalling, JNK becomes an essential driver of tumour overgrowth and invasion. aPKC-dependent signalling in scrib mutants cooperates with JNK to significantly enhance oncogene-mediated tumour overgrowth.We find that genetically distinct atypical protein kinase C (aPKC)-dependent and Jun N-terminal kinase (JNK)-dependent alterations in Drosophila. This is likely to have a direct relevance to the way in which human Scrib can similarly restrain an oncogene-mediated transformation and, more generally, on how the outcome of oncogenic signalling can be profoundly perturbed by defects in apico-basal epithelial cell polarity.These results demonstrate distinct aPKC and JNK-dependent pathways through which loss of Scrib promotes tumourigenesis in  Drosophila, Scribble (Scrib), Discs large (Dlg) and Lethal giant larvae (Lgl), cooperatively establish and maintain apico-basal cell polarity and repress inappropriate cell proliferation and neoplasia (invasive overgrowth with a failure to differentiate) in both epithelial and neuronal tissues .Drosophila, homozygous scrib, dlg or lgl mutants develop to the third instar larval stage but fail to pupate and die as overgrown larvae. Some of the mono-layered epithelial imaginal discs, notably the wing discs, become multi-layered, fail to differentiate and over-proliferate throughout the extended larval stage of development. These overgrown masses of tissue exhibit characteristics of human cancers, including a failure to cease proliferation and differentiate, loss of tissue structure and a propensity to fuse and invade the surrounding tissues. Using clonal analysis in the eye imaginal disc we have previously examined scrib mutant clones and shown that, although loss of Scrib is associated with altered cell morphology (indicative of aberrant cell polarity), ectopic expression of Cyclin E (CycE) and excessive cell proliferation, the mutant clones of tissue do not become overgrown because they are removed by Jun N-terminal kinase (JNK)-dependent apoptosis . Genetiiewed in ,9. The Ciewed in .lgl mutants, tumour overgrowth can be rescued through reduced levels of aPKC [scrib, dlg or lgl [scrib mutants as has been suggested for mutants in the syntaxin avalanche (avl) [scrib mutant epithelial neoplasias is required.In contrast to what is known about how Scrib regulates cell polarity, much less is known about how it acts to restrain tissue overgrowth. Studies have suggested that the proliferation and polarity functions of Scrib can be separated . However of aPKC , and aPK of aPKC . However of aPKC  and Crb g or lgl . Thus, dhe (avl) . Deciphescrib mutants cooperate with oncogenes in mediating transformation in Drosophila. A number of studies have shown how RasACT subverts the pro-apoptotic JNK signalling response in scrib mutants into a potent inducer of tumour overgrowth and invasion through the JNK-dependent expression of Matrix metalloproteinase 1 (Mmp1) [ACT [ACT [ACT-mediated transformation [Drosophila Scrib exerts its tumour suppressor function.Similarly, clarification is needed of how 1 (Mmp1) -18. Howep1) [ACT , while oACT [ACT  and, thuormation , it is iscrib mutant clones in the eye disc, we found that although the over-expression of Crb or aPKC mimics many of the scrib mutant defects, the excessive proliferation and alterations in cell morphology in scrib mutants are independent of Crb but can be rescued through the expression of a dominant negative aPKC transgene. Furthermore, we identified distinct aPKC and JNK-dependent modes by which scrib mutants promote oncogene-mediated transformation. Our data support the critical role of JNK signalling in scrib mutants in mediating cooperation with RasACT and show that JNK is also essential for NACT-driven tumourigenesis. However, our studies also show that aPKC signalling can play a pivotal role in promoting oncogene-mediated tumour overgrowth and these findings are likely to be of relevance to the way in which loss of human Scrib can similarly potentiate oncogene-mediated transformation.In this study, we define for the first time the relationship between Scrib and other cell polarity regulators in the control of cell polarity and proliferation in imaginal discs. Analysing mosaic analysis with repressible cell marker) [FRT82B and eyeless-FLP1 to induce clones and UAS-mCD8-GFP to visualize mutant tissue.Fly crosses were carried out at 25°C and grown on standard fly media. All clonal analysis was carried out using MARCM ( marker)  with FRTDrosophila stocks were used: eyFLP1, UAS-mCD8-GFP;;Tub-GAL4 FRT82B Tub-GAL80 [msn06946 [scrib1 [UAS-P35 [UAS-bskDN [crbA22 11[UAS-crbwt2e [UAS-DaPKCN Δ[UAS-DaPKCCAAXWT and UAS-DaPKCCAAXDN [UAS-dRas1V12 [UAS-Nintra [UASp-scribFL19.2 .The following ub-GAL80 ; msn0694msn06946 ; scrib1  [scrib1 ; UAS-P35[UAS-P35 ; UAS-bskAS-bskDN ; crb11A2crb11A22 ; UAS-crb-crbwt2e ; UAS-DaP-DaPKCΔN ; UAS-DaPKCCAAXDN ; UAS-dRadRas1V12 ; UAS-NinS-Nintra ; UASp-scin situ cell death detection kit TMR-Red from Roche). F-actin was detected with phalloidin--tetramethylrhodamine isothiocyanate  at 0.77 μM. Samples were mounted in 80% glycerol.Eye/antennal discs and brain lobes were dissected in phosphate-buffered saline (PBS) from wandering third instar larvae and fixed in 4% formaldehyde in PBS. Samples were blocked in either 2% goat serum in PBT (PBS 0.1% Triton X-100) or 5% milk powder/bovine serum albumin in PBS 0.3% Triton X-100. For the detection of S phase cells, a 1 h BrdU (bromodeoxyuridine) pulse was followed by fixation, immuno-detection of green fluorescent protein (GFP), further fixation, acid treatment and immuno-detection of the BrdU epitope. Primary antibodies were incubated with the samples in block overnight at 4°C. Primary antibodies used were: mouse anti-β-galactosidase (Rockland) at 1 in 400, mouse anti-Elav  at 1 in 20, rat anti-Cyc E (Helen McNeill) at 1 in 400, rabbit anti-GFP (Invitrogen) at 1 in 1000, mouse anti-BrdU (Becton-Dickinson) at 1 in 50, rabbit anti-Paxillin at 1 in 400 . Seconda® or Flouroview® software before being processed in Adobe Photoshop® CS2 and assembled into figures in Adobe Illustrator® CS2.Samples were analysed by confocal microscopy using either Bio-Rad MRC1000 or Olympus FV1000 microscopes. Single optical sections were selected in Confocal Assistantscrib mutant cells, within clones of tissue in the eye disc, have severely altered cell morphology and exhibit ectopic cell proliferation. However, they do not overgrow because cells die via JNK-mediated apoptosis. Levels of apoptosis were increased in scrib mutant mosaic discs and blocking JNK signalling in scrib mutant clones by expressing a dominant-negative form of Drosophila JNK, Basket dominant negative (BskDN), dramatically increasing the scrib mutant clonal tissue size [DN in scrib mutant clones reduced apoptosis in the mutant tissue, although cell death was still observed in some wild type cells abutting the mutant clones -lacZ [scrib mutant cells, including those undergoing apoptosis, and that expressing BskDN in scrib mutant clones effectively prevented the ectopic expression of msn-lacZ in the mutant tissue -lacZ , we alsoscrib mutant cells promoted cell death, we next wished to determine if any of the other scrib mutant defects, including the ectopic cell proliferation and altered cell morphology, were also dependent upon JNK. Proliferation in the eye disc follows a stereotypical pattern that can be visualized by CycE levels and bromedeoxyuridine (BrdU) incorporation. Cells normally arrest cell proliferation within the morphogenetic furrow (MF) and undergo a synchronous S phase just posterior to the MF before commencing differentiation, although some unspecified cells undergo a further round of division more posteriorly  and the Baz complex . To determine if this hierarchal relationship is also operative in the eye disc, we began by examining the effects of Crb loss-of-function and Crb over-expression in the eye disc.crb clones, using the null allele crbA22 11[DN, clones became considerably larger and also exhibit ectopic cell proliferation posterior to the MF  in eye disc clones [ΔN) [ΔN-expressing clones restored clone viability and most of the mutant tissue had aberrant morphology and was extruded basally to form large masses of undifferentiated tissue that ectopically proliferated posterior to the MF  . This rescrib mutant defects could be due to deregulated aPKC activity we utilized a transgene expressing a kinase-dead, CAAX membrane-tethered, allele of aPKC (aPKCCAAXDN) [CAAXDN in otherwise wild-type clones of tissue produced no discernible defects in cell morphology, proliferation or differentiation during larval stages of development (data not shown). Strikingly, however, the expression of aPKCCAAXDN in scrib mutant clones restored normal cell morphology to the mutant tissue posterior to the MF. Elav and phalloidin staining generally revealed a normal regular array of differentiating ommatidial clusters in scrib mutant clones expressing aPKCCAAXDN, although sometimes clonal borders showed separation between the mutant and wild-type tissue resulting in tissue scars (data not shown) and occasional basally retracted mutant photoreceptor nuclei  . The expi Figure . FurtherF Figure , althougN Figure  and the h Figure . The faic Figure . Thus, wscrib mutants expressing aPKCCAAXDN, we co-expressed the apoptosis inhibitor P35 in the mutant clones. This, however, failed to significantly rescue clone size and only served to enhance the mutant phenotype. Non-cell autonomous tissue folding that distorted the shape of the disc was apparent and, in some clones, cells adopted a more rounded morphology , the data suggest that blocking cell death in scrib mutants is not enough to fully rescue the mutant phenotype if JNK remains active.In an attempt to rescue the cell death phenotype of y Figure . As the scrib mutant cells expressing aPKCCAAXDN, by co-expressing BskDN, not only was cell viability dramatically restored but the mutant tissue also exhibited normal morphology  RasACT or Notch (NACT) to repress pupal development and, throughout an extended 'giant larvae' phase of development, form massive and invasive tumours [scrib- + RasACT tumour cells grow out basally from the eye disc, fail to differentiate , it was unable to repress RasACT-induced tumour development. Examination of differentiation by Elav staining confirmed that scrib- + RasACT + aPKCCAAXDN tumour tissue remained undifferentiated in the basal sections of the eye disc -mediated inhibition. Our own work indicates that ectopic aPKC expression can induce JNK-dependent cell death, although whether the kinase dead form of aPKC can block this death is not known. In any event, the relationship between scrib and JNK is not likely to be direct since JNK was not activated in all scrib mutant tissue and was often associated with clonal borders. This is more consistent with JNK being indirectly activated due to either alterations in cell adhesion or signalling. Consistent with this, scrib mutant clones expressing both aPKCCAAXDN and BskDN still showed occasional scaring at clonal edges suggestive of an impaired cell adhesion.If Scrib does function upstream of aPKC in Drosophila and, apart from the junctional/scaffold tumour suppressors of scrib, dlg and lgl, the other group of genes, Rab5, avl, erupted and vps25, regulate endocytic pathways. Interestingly, avl mutant hyperplasia is also rescued by the expression of aPKCCAAXDN and this was suggested to reflect the ability of aPKCCAAXDN to reduce Crb activity, since Crb levels were elevated and mislocalized in both avl and Rab5 mutants [avl or Rab5 neoplasias, clearly an intimate relationship exists between the different neoplastic tumour suppressors and the polarity complex proteins. Understanding the mechanistic links between these different proteins is therefore required.A relatively small number of neoplastic tumour suppressor mutants have been described in  mutants . Whetherscrib mutants with RasACT [scrib- + RasACT tumours are the matrix metalloproteinase protein, Mmp1 [our unpublished observations, [scrib- + RasACT tumour cells from fusing with and invading the brain lobes but did not abrogate tumour overgrowth or restore pupal development [our unpublished observations, [scrib or Ras, since clones of cells expressing an activated allele of the Drosophila JNKK homologue, hemipterous (HepACT), also showed strong up-regulation of Pax . This is consistent with JNK being activated indirectly as a consequence of disturbed cell polarity/morphology and further suggests that JNK alone might be sufficient for cooperation. Indeed, co-expression of Hep with RasACT has been shown to result in invasive neoplasia [scrib mutant clones are rescued by aPKCCAAXDN, however, the mutant cells still undergo JNK-mediated apoptosis and can still cooperate with RasACT in tumourigeneis. As neoplastic cells between the brain lobes have an elongated mesenchymal-like appearance, JNK and Ras may promote an epithelial-to-mesenchymal transition by impacting on cell shape and/or cell fate pathways irrespective of the loss of Scrib or the blockade in aPKC activity mediated by the dominant negative aPKC transgene.Many different cell polarity mutants apart from h as sdt  and crb eoplasia . Our resACT alone in clones may induce some JNK activation, as judged by the expression of msn-lacZ, but is clearly not sufficient to cause neoplastic tumours. In contrast, co-expressing HepACT with RasACT inefficiently results in neoplastic transformation, presumably because the levels of JNK signalling are too high and this restrains overgrowth or leads to cell death [ACT.While JNK is clearly an essential component to neoplastic transformation, the level of JNK activation appears to be critical. Ectopic expression of Rasll death . A loss ACT-driven tumourigeneisis suggests that JNK activation is both necessary and sufficient for RasACT cooperation. However, the fact that blocking aPKC signalling in scrib- + NACT tumours retards tumour overgrowth suggests that loss of Scrib can also contribute an aPKC-dependent increase in either cell proliferation or cell survival that can profoundly influence the rate of tumour overgrowth. Ras-driven tumours also showed a slight retardation in tumour development with the addition of aPKCCAAXDN, although this was much less striking than the effects with N. As JNK signalling remains activated in scrib mutant clones expressing aPKCCAAXDN, it is likely that JNK can restrain NACT-driven tumour overgrowth, and RasACT is more effective than NACT at counteracting such a JNK-mediated restraint. The aPKC-dependent effects on CycE and increased cell proliferation in scrib mutants could help overcome this restraint. Further analysis will be required in order to elucidate the mechanisms involved.The analysis of RasDrosophila. aPKC signalling in scrib mutants promotes loss of cell polarity and proliferation, while JNK can either restrain tumour development through cell death or, in cooperation with RasACT or NACT, promote aggressive neoplastic tumour overgrowth.These results demonstrate distinct aPKC and JNK-dependent pathways through which loss of Scrib promotes tumourigenesis in ACT or Myc in promoting transformation. In the case of RasACT expression with Scrib knockdown, cells grown in three-dimensional (3D) culture failed to form the normal polarized acini structures with a central luminal and, instead, adopted a highly invasive morphology [ACT was linked to the ability of Scrib knockdown to potentiate MAPK signalling [Drosophila, its role can be complex as it also promotes tumour regression through cell death in different contexts [Drosophila observations, that loss of Scrib promotes JNK-mediated cell death, JNK activation in scrib mutant clones was variable and possibly regulated through interactions with neighbouring wild type cells rather than through a cell autonomous up-regulation in JNK signalling [Drosophila inhibitor of apoptosis 1 (Diap1) is upregulated in scrib mutants [Drosophila cells from apoptosis in some contexts. Our own studies have also revealed that a loss of Scrib can promote NACT-driven tumour overgrowth through aPKC-dependent pathways involving either increased cell survival or increased cell proliferation. Clearly, further work is required to determine how closely related the tumour suppressor function of Scrib in flies is to its mammalian counterpart. Nevertheless, despite undoubted differences that will exist between the Drosophila and the mammalian systems, studies in both organisms have the potential to allow important insights into how the outcome of oncogenic stimulus can be profoundly affected by perturbations in cell polarity networks.Growing evidence links increased levels of aPKC with the development of human cancers ,39 and arphology . Cooperagnalling , howevergnalling -42, althcontexts . In factgnalling . Further mutants  and, thuaPKC: atypical protein kinase C; Avl: avalanche; Baz: bazooka; BrdU: bromodeoxyuridine; Bsk: basket; Crb: crumbs; CycE: cyclin E; Diap1: Drosophila inhibitor of apoptosis 1; Dlg: discs large; DN: dominant negative; Hep: hemipterous; JNK: Jun N-terminal kinase; Lgl: lethal giant larvae; MARCM: mosaic analysis with repressible marker; Mmp1: matrix metalloproteinase 1; MF: morphogenetic furrow; Msn: misshapen; N: notch; Pax: paxillin; PBS: phosphate-buffered saline; Scrib: scribble; Sdt: stardust.ΔN. HER assisted in the interpretation of experiments and contributed editorial guidance. AMB conceived of the study, designed and carried out experiments, collected and interpreted the data, and wrote the paper. All authors read and approved of the final manuscript.GRL carried out experiments investigating the role of JNK. KRG assisted with the setting up of experiments and in the preparation of the manuscript. NA carried out experiments with aPKCscrib mutant cells are eliminated by JNK-dependent apoptosis. eyFLP-induced MARCM clones expressing mCD8-GFP (green). TUNEL detection marks apoptotic cells , and β-Gal detects msn-lacZ enhancer trap activity . A white bar indicates the location of the MF. (A) FRT82B. In control mosaic eye discs, TUNEL positive cells are most prominently observed in the eye disc just posterior and anterior of the MF. (B) FRT82B scrib1. In scrib mutant mosaic eye discs, the normal pattern of cell death is largely disrupted and TUNEL positive cells are observed both within (arrow) and surrounding (arrowhead) the GFP positive mutant clones. (C) FRT82B scrib1 UAS-bskDN. scrib mutant clones expressing BskDN contain very few TUNEL positive cells, although dying cells are still observed in wild-type tissue adjacent to the mutant tissue (arrow). (D) msn06946 FRT82B. Control mosaic eye discs show low-level expression of msn-lacZ posterior to the MF. (E) msn06946 FRT82B scrib1. In scrib mutant clones, msn-lacZ is ectopically expressed in some patches of mutant tissue (arrow) and in some wild type cells bordering the mutant clones. (F) msn06946 FRT82B scrib1 UAS-bskDN. Expressing BskDN in scrib mutant clones completely abrogates the activation of msn-lacZ in the mutant clones, although msn-lacZ expressing cells are still sometimes observed in the wild-type tissue adjacent to the mutant tissue (arrow). (G) msn06946 FRT82B scrib1. In scrib mutant clones, TUNEL positive and msn-lacZ positive cells do not generally overlap, although occasional cells (arrow) express both markers.Click here for fileEctopic Crb disrupts cell morphology and induces JNK-dependent apoptosis and proliferation. eyFLP-induced MARCM clones (green). Grey scale is Elav , phalloidin to mark F-actin  and BrdU (F). A white bar marks the location of the MF. (A-B) FRT82B crbA2211. crb mutant clones show no defects in the pattern of differentiation in the eye disc (A) and no alterations to the normal columnar epithelial cell morphology (B).  UAS-crbwt2e; FRT82B. Crb-expressing clones are under-represented relative to the surrounding non-clonal tissue and mildly disrupt the normal pattern of photoreceptor differentiation (C) and the normal columnar cell morphology resulting in cells being excluded from the epithelium (D).  UAS-crbwt2e; FRT82B UAS-bskDN. Co-expression of BskDN with Crb disrupts the normal pattern of differentiation (E), and results in large clones of mutant cells that ectopically proliferate posterior to the MF (F).Click here for fileEctopic expression of activated aPKC disrupts cell morphology and results in ectopic cell proliferation. eyFLP-induced MARCM clones (green). Grey scale is Elav  and BrdU . Phalloidin marks F-actin in red . A white bar indicates the location of the MF. (A-D) FRT82B UAS-DaPKCΔN. Ectopic expression of aPKCΔN in clones results in reduced amounts of clonal tissue that is mostly excluded basally from the epithelium and does not express Elav  and does not noticeably over-proliferate (C-D), although any proliferative defects are likely to be masked by cell death. (E-I) FRT82B UAS-DaPKCΔN UAS-bskDN. The co-expression of BskDN with aPKCΔN rescues the small clone phenotype of aPKCΔN clones alone and most of the mutant tissue has aberrant cell morphology and is extruded basally to form large masses of undifferentiated tissue beneath the dorsal and ventral sides of the eye disc epithelium (E-G). The mutant cells ectopically proliferate posterior to the MF, but not within the MF, in both apical and basal sections .Click here for fileDN represses scrib1 + RasACT tumour overgrowth and invasionJNK. Pairs of larval eye/antennal imaginal discs attached to brain lobes (bl) containing eyFLP-induced MARCM clones (green) at day 5 , day 7  and day 8 (E). Grey scale is Elav . Red is phalloidin to mark F-actin. (A-B) UAS-dRas1V12; FRT82B. RasACT-expressing clones do not massively overgrow and mutant cells are not observed between the brain lobes. Note the F-actin rich cables (arrows) extending from between the eye/antennal disc to the region between the brain lobes. (C-D) UAS-dRas1V12; FRT82B scrib1. scrib1 + RasACT tumours massively overgrow by day 7 and tumour cells appear to migrate between the brain lobes (arrow in C) along F-actin rich cables (arrow in D). (E) UAS-dRas1V12; FRT82B crbA22 11scrib1. Loss of crb does not abrogate scrib1 + RasACT tumour overgrowth.  UAS-dRas1V12; FRT82B scrib1 UAS-bskDN. Expression of BskDN in scrib1 + RasACT tumours prevents tumour overgrowth throughout an extended larval stage of development and blocks invasion of tumour cells between the brain lobes.Click here for filePaxillin is a downstream target of JNK signalling. Larval eye/antennal imaginal discs containing eyFLP-induced MARCM clones (green), with attached brain lobes (bl) in (E). Grey scale is Paxillin. (A) FRT82B scrib1. scrib mutant clones have elevated levels of Paxillin in some mutant cells (arrow). (B) FRT82B UAS-bskDN. Ectopic expression of BskDN in clones does not alter Paxillin levels in the eye disc. (C) FRT82B scrib1 UAS-bskDN. scrib mutant clones expressing BskDN no longer show elevated levels of Paxillin, although non-clonal tissue adjacent to mutant clones sometimes does ectopically express Paxillin (arrow), presumably reflecting JNK activation in the non-mutant tissue. (D) UAS-dRas1V12; FRT82B. Ectopic expression of RasACT in clones does not generally elevate Paxillin levels. (E) UAS-dRas1V12; FRT82B scrib1. Paxillin levels are increased in scrib1 + RasACT tumours, most notably at the invasive front, as shown by tumour cells invading into the brain lobes. (F) UAS-hepACT; FRT82B UAS-P35. Clones of tissue expressing an activated allele of the Drosophila JNKK homologue, hemipterous, Hep (HepACT), kept alive through the co-expression of the caspase inhibitor P35, ectopically express elevated levels of Paxillin.Click here for filescrib1 + RasACT tumour cellsJNK signalling is activated in . Larval eye/antennal imaginal discs containing eyFLP-induced MARCM clones (green) at day 5  and at approximately day 7 (D). A pair of eye discs attached to the brain lobes (bl) is shown in (D). Grey scale is β-Gal to detect msn-lacZ enhancer trap activity. Red is phalloidin to mark F-actin. (A) UAS-dRas1V12; msn06946 FRT82B. RasACT-expressing clones show a mild increase in msn-lacZ reporter activity in eye disc clones, especially in the central region of the disc. (B-D) UAS-dRas1V12; msn06946 FRT82B scrib1. scrib1 + RasACT tumours express elevated levels of the msn-lacZ enhancer trap, most prominently in cells located basally within the eye disc (B compared to C) and in the region between the eye and antennal (ant) discs (arrow in B). Tumour cells appear to migrate between the brain lobes and ectopically express the msn-lacZ enhancer trap (D). (E) UAS-dRas1V12; msn06946 FRT82B scrib1 UAS-bskDN. Expression of BskDN in scrib1 + RasACT tumours prevents msn-lacZ expression within the tumour cells.Click here for file


As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.


Drug resistance is one of the problems severely limiting chemotherapy in cancer patients. Thus, it is very important to develop new drugs that are effective against drug-resistant tumour cells. The novel anti-tumour agent NK109 has been developed from benzo[c]phenanthridine derivatives by Nippon Kayaku . We have confirmed that NK109 shows anti-tumour effects against a number of human tumour cell lines by inhibiting DNA topoisomerase II activity through the stabilization of the cleavable complex. Further, its efficacy against several drug-resistant tumour cell lines was also shown. NK109 showed potent anti-tumour activity against doxorubicin-resistant human tumour cell lines that have a typical multidrug resistance phenotype caused by P-glycoprotein. NK109 was not pumped extracellularly by P-glycoprotein and, consequently, NK109 accumulated in resistant cells. Cisplatin-resistant human tumour cell lines, which demonstrated decreased cisplatin accumulation, were sensitive to NK109. NK109 non-cross-resistance was confirmed using xenografts of tumour cells that were resistant to cisplatin in SCID mice. Furthermore, etoposide-resistant cells, with decreased topoisomerase II activity, were markedly sensitive to NK109 when compared with their parent cells, suggesting the possibility that the cytotoxic mechanism of NK109 differs from that of etoposide. In conclusion, NK109 is a very promising new anti-tumour drug for clinical use, because the efficacy of NK109 is not susceptible to several known molecular alterations that are associated with drug resistance. A clinical study of this compound is now in progress in Japan.


To the Editor: Studies indicate that Plasmodium falciparum infection increases HIV replication in adults . When 15 malaria episodes with malaria-free visits 1 month before and after the episode were compared, mean difference (signed-rank test) in viral load “before” and “after” malaria was not significant.After adjusting for age and baseline viral load, we assessed logOur findings suggest that low-density malaria infection may not dramatically affect plasma HIV-1 levels in infants. This finding is similar to results of studies of perinatally HIV-infected children in which, although viral loads were unavailable, number of malaria episodes did not significantly affect development of AIDS-related symptoms  for malaria . Most ofOur assessment was limited in size and duration. Furthermore, in attempting to provide optimal patient care through conducting monthly surveillance and encouraging mothers to bring children in during febrile episodes, ability to assess the effect of high-density malaria was diminished because parasitemia levels never reached clinically significant levels. Finally, because malaria was diagnosed by microscopy, rather than PCR, some subclinical malaria infections may have been misclassified as malaria-negative.Although we found no evidence of an association between subclinical, low-density malaria and infant HIV-1 viral load, the consequences of high-density or clinical malaria need to be explored. If clinical malaria in infants increases HIV-1 viral load as it does in adults (


H2 cytokines in development of allergen-induced airway disease. In contrast, the role of mast cells for the development of allergic airway disease has been controversial. Recent studies in murine models demonstrate a significant contribution of mast cells during the development of airway hyperresponsiveness and airway inflammation. Furthermore these models have allowed identifying certain mast cell-produced mediators (e.g. histamine and leukotriene B4) to be involved in the recruitment of effector T-cells into the lung. Additionally, mast cell-produced TNF can directly activate TH2 cells and contribute to the development of allergic airway disease. These new findings demonstrate a complex role of mast cells and their mediators, not only as effector cells, but also during sensitization and development of allergic airway disease. Therefore mast cells and certain mast cell-produced mediators might be an interesting target for the prevention and treatment of allergic asthma.Murine models have highlighted the importance of T-cells and T For decades mast cells have been known as important effector cells for adaptive immune responses. Functions of mast cells include both IgE-dependent mucosal immunity to parasites but also dysregulated allergic responses to environmental antigens. In patients with allergic asthma, allergen-specific activation of mast cells through IgE bound to the high-affinity IgE receptor (FcΕRI) induces early airway responses following allergen exposure. Also increased numbers of mast cells are found in close proximity to airway smooth muscle in patients with allergic asthma, suggesting their potential role in the development and maintenance of allergic airway disease .W/KitW-v and more recently the C57BL/6-KitW-sh/KitW-sh are used [W/KitW-v mice are sterile, anaemic, and lack intestinal cells of Cajal, whereas the C57BL/6-KitW-sh/KitW-sh are not sterile or anaemic [Murine models have helped to gain insights into the pathogenesis of airway inflammation and airway responsiveness . These mare used . Both st anaemic .The role of mast cells for the development of allergic airway disease in murine models is critically dependent on the sensitization and allergen exposure protocol. In studies using systemic sensitization with adjuvant hyperresponsiveness (AHR) and airway inflammation are similar in mast cell- or IgE-deficient mice compared to wild-type mice -6. HowevMast cells not only play an important role during the effector phase of allergic airway disease but also could be involved during sensitization to an allergen. Intranasal exposure with allergen alone does not induce a specific T-cell response. In contrast, exposure with allergen in combination with a low dose of bacterial lipopolysaccharides (LPS) induces a specific T-cell response  and consMast cells, following activation, degranulate and produce a plethora of lipid mediators, cytokines, and chemokines . Severalin vitro studies have shown that migration of CD8-positive T-effector cells can be initiated by mast cell activation. Furthermore T-cell migration was regulated by leukotriene B4 (LTB4) produced by mast cells and the expression of the receptor 1 (BLT-1) on the T-cells [Other mechanisms could be the secretion of chemo-attractants and chemokines, which lead to the recruitment of other cell populations, such as effector T-cell into the lung and airways. Recently an interaction with mast cells has been described for allergen specific CD8-positive T-effector cells. These  T-cells . CD8-pos T-cells ,21 and e T-cells . Followi T-cells  and this T-cells . The con T-cells . Mice we T-cells . A poten T-cells .et al. directly evaluated the role of mast cell-produced TNF for the induction of allergic airway disease using a mast cell reconstitution model. Similar to previous studies, mast cell-deficient animals fail to develop allergic airway disease whereas mice reconstituted with mast cells derived from wild-type mice developed allergic airway disease. In contrast, mice reconstituted with mast cells derived from TNF-deficient donors have less airway inflammation and AHR, demonstrating that mast cell-produced TNF is pivotal for the induction of allergic airway disease [H2 cells induced by mast cell-produced TNF, suggesting that mast cells induce activation of allergen specific T-cells in the sites of inflammation. These findings are of clinical interest as recent studies in patients with moderate [Also other mast cell-produced mediators are of interest. Histamine receptor 1 has recently been shown to be important for the development of allergic airway disease and also for T-cell trafficking . Another disease . Furthermoderate  and alsomoderate ,41 descrRecent studies have outlined a critical role of mast cells for the development of acute allergic airway disease, especially in sensitization protocols without an adjuvant. Furthermore there is evidence that mast cells are also involved during the sensitization to inhaled allergens by IgE-independent activation through TLR. Still little is known about the role of the different mediators produced and secreted by mast cells. Several mast cell-produced mediators like LTB4, histamine and TNF have been identified to contribute to the development of allergic airway disease. Further research is needed as increasing knowledge about these pathways might allow a more targeted therapy of allergic asthma.S.R. designed and wrote the manuscript, C.T. designed and wrote the manuscriptThe authors declare that they have no competing interests.


Obesity is associated with inflammation of visceral adipose tissues, which increases the risk for insulin resistance. Animal models suggest that T-lymphocyte infiltration is an important early step, although it is unclear why these cells are attracted. We have recently demonstrated that dietary triglycerides, major components of high fat diets, promote intestinal absorption of a protein antigen . The antigen was partly transported on chylomicrons, which are prominently cleared in adipose tissues. We hypothesized that intestinally absorbed gut antigens may cause T-lymphocyte associated inflammation in adipose tissue.Triglyceride absorption promoted intestinal absorption of OVA into adipose tissue, in a chylomicron-dependent manner. Absorption tended to be higher in mesenteric than subcutaneous adipose tissue, and was lowest in gonadal tissue. OVA immunoreactivity was detected in stromal vascular cells, including endothelial cells. In OVA-sensitized mice, OVA feeding caused marked accumulation of CD3+ and osteopontin+ cells in mesenteric adipose tissue. The accumulating T-lymphocytes were mainly CD4+. As expected, high-fat  diets promoted mesenteric adipose tissue inflammation compared to low-fat diets , as reflected by increased expression of osteopontin and interferon-gamma. Immune responses to dietary OVA further increased diet-induced osteopontin and interferon-gamma expression in mesenteric adipose. Inflammatory gene expression in subcutaneous tissue did not respond significantly to OVA or dietary fat content. Lastly, whereas OVA responses did not significantly affect bodyweight or adiposity, they significantly impaired glucose tolerance.Our results suggest that loss or lack of immunological tolerance to intestinally absorbed T-lymphocyte antigens can contribute to mesenteric adipose tissue inflammation and defective glucose metabolism during high-fat dieting. Obesity is an important component of the metabolic syndrome Studies with mice on high-fat diets have demonstrated that expanding adipose tissues become infiltrated with macrophages, which may be responsible for most of the inflammatory events in these tissues Whereas the discovery of the involvement of lymphocytes, macrophages, and other immune cells, such as mast cells We observed that fat absorption indeed promoted antigen absorption into adipose tissue. Moreover, mice previously sensitized to the antigen showed significant inflammatory responses in mesenteric, but not subcutaneous, adipose tissues, and these responses were further enhanced during high-fat dieting. Over time, these responses resulted in a decrease of glucose tolerance. We propose that intestinal antigen absorption may be a contributor to inflammation of visceral adipose tissue during high-fat feeding.125-I as described elsewhere Medium-chain triglyceride (MCT) oil was from Novartis, the long-chain triglyceride (LCT) consisted of food grade soybean oil. Intralipid (20%) was from Glaxo-Welcome. Pluronic L-81, a surfactant blocking chylomicron secretion from enterocytes at pharmacological dose 125I-OVA in various vehicles (0.2 ml). Tissues were harvested from humanely killed mice, after cardiac perfusion with 10 ml cold phosphate-buffered saline . For feeding experiments, OVA-naïve mice were sensitized by two intraperitoneal injections with 10 µg OVA in 0.2 ml alum , with one week between injection. Control mice were injected with alum only. Mice received OVA-containing diets one week after the second injection for indicated durations. For glucose tolerance tests, mice were fasted (4 h) before being intraperitoneally injected with 2 g glucose in PBS/kg bodyweight. Blood samples were obtained from the tail vein at several time points and were tested for glucose concentration with a TrueTrack glucose meter from Home Diagnostics Inc. Adiposity was measured using a EchoMRI-5000 Whole Body Composition machine . All animals were handled in strict accordance with good animal practice as defined by the relevant national and local animal welfare bodies, and all animal work was approved by the Institutional Animal Care and Use Committee of the University of Kentucky .Male BALB/C mice and C57Bl/6 mice, ordered at 6 weeks of age from The Jackson Laboratory, were held in a room of a specific pathogen-free animal facility with a 12 h light/dark cycle, and were used at 8 weeks of age. For OVA absorption experiments, mice were fasted (4 h), then gavaged with Adipose tissue samples, isolated while strictly avoiding lymph node material, were fixed in 10% formalin, embedded in paraffin, and cut in 5 µm sections. Antigens were retrieved in deparaffinized and rehydrated sections by boiling in citrate buffer  for 30 minutes. CD3 was stained by successive incubation with anti-CD3 and Alexa-568 labeled secondary antibody, whereupon the slides were mounted in 4′,6-diamidino-2-phenylindole- (DAPI) containing mounting medium (Vectashield). The slides were observed with an Olympus BX51 fluorescence microscope equipped with a digital camera. Osteopontin (OPN) was detected by using a chromogenic substrate for the secondary antibody according to the kit manufacturer's instructions.Stromal vascular cells were isolated from adipose tissue by collagenase digestion as described elsewhere AGC-AAG-AAA-CTC-TTC-CAA-GCA-A/GTG-AGA-TTC-GTC-AGA-TTC-ATC-CG (OPN), TTG-GCC-AGC-GCC-ATC-TT/CCT-GTT-GCT-GTA-GCC-GTA-TTC-A ), ATG AAC GCT ACA CAC TGC ATC/CCATCC TTT TGC CAG TTC CTC (Interferon gamma (IFNγ)), and TTG GCC AGC GCC ATC TT/TGC CTC CTC CAG AGA GAA GTG (Forkhead Box P3 (FOXP3)).RNA from adipose tissue samples was extracted with the Trizol-reagent based method. RNA was transcribed into cDNA with the iScript kit from Quanta Biosciences, and the resulting cDNA was amplified using Quanta Bioscience's Perfect Sybr mix using a Biorad iQ5 multicycler. Primer pairs were, in 5′-3′ direction, Comparison of gene expression between naïve or sensitized mice on low- or high-fat OVA diets was performed with two-way ANOVA and Bonferroni's post-hoc tests using XLstat Software (Addinsoft SARL). Plasma glucose levels over time were compared between groups with a linear mixed model using SAS software. OVA absorption data were tested for statistical significance by ANOVA with a Bonferroni post-hoc test, and T-lymphocyte subtype percentages with Student's T-test, using Graphpad Prism v5. All bar graphs represent average values ± S.D.125I- OVA in 0.2 ml LCT, MCT (medium-chain triglycerides), or LCT to which the chylomicron secretion inhibitor Pluronic L-81 (Pl-81) was added. We observed that gonadal adipose tissue, isolated 60 minutes after gavage, contained significantly more 125I when the OVA was gavaged with LCT compared with MCT or LCT plus Pl-81, suggesting that chylomicron formation, essential for dietary fat absorption, promotes absorption of gut antigen into adipose tissue (We have recently demonstrated in experiments with mice that dietary long-chain triglycerides (LCT) significantly enhanced intestinal absorption of concomitantly ingested dietary OVA, and that a significant fraction of the OVA was associated with chylomicrons e tissue .125I-OVA in 0.2 ml of 20% Intralipid, and 125I levels were measured in mesenteric , gonadal , and inguinal (subcutaneous) adipose tissue fifteen minutes after gavage. Pilot experiments had revealed similar levels of radioactivity in the tissues 15 minutes and 1 h after gavage. Gonadal adipose tissue contained the least 125I, and mesenteric adipose tissue the most, although the difference between mesenteric and subcutaneous fat did not reach statistical significance , and has been implicated in adipose tissue inflammation in diet-induced obesity To further investigate the potential contribution of T-lymphocyte responses to intestinally absorbed antigens in adipose tissue inflammation, we fed OVA-naïve or OVA-sensitized mice with low- or high- fat diets containing 1% egg-white powder. Mesenteric and inguinal adipose tissue samples were obtained after two or ten weeks. As expected, osteopontin gene expression increased significantly in mesenteric adipose tissue in mice on high-fat diets. Differences were already apparent after two weeks. However, osteopontin expression in subcutaneous fat was not significantly affected by dietary fat content . These oTo test the physiological implications of apparent inflammatory immune responses to gut antigens in adipose tissue, we sensitized BALB/c mice with OVA plus alum or with alum only (control) and fed them low or high-fat diets enriched with 1% OVA. Both naïve and sensitized mice (n = 6 per group) showed similar weight gain within their dietary treatment over the course of the experiment, and total fat weight gain was similar at 2 and 10 weeks . HoweverMounting evidence indicates that T-lymphocytes are involved in the early stages of visceral adipose tissue inflammation in diet-induced obesity. However, it is still unclear why T-lymphocytes are attracted. In this study, we provide evidence for a potential role of antigenic material absorbed from the gut in T-lymphocyte activation in visceral adipose tissue. First, we show that intestinal absorption of dietary fat also stimulates the intestinal absorption of a gut antigen, ovalbumin (OVA), into adipose tissue. Second, we show that under conditions of lack of immunological tolerance to such an antigen, mesenteric adipose tissue is a site of T-lymphocyte dependent inflammation. Third, we show that prolonged exposure to an antigenic insult from the gut can lead to a loss of glucose tolerance, an important feature of metabolic syndrome. Taking these observations into consideration, we propose that T-lymphocyte activation in visceral adipose tissue during diet-induced obesity could be, at least in part, a response to influx of antigenic material absorbed from the gut.Central adiposity is an important component of metabolic syndrome. The problem with expanded visceral fat is that it is frequently inflamed, and this can affect other tissues and lead to deregulation of lipid and carbohydrate metabolism. A hallmark of adipose tissue inflammation in diet-induced obesity is the infiltration of macrophages We and others have previously demonstrated that there is a link between fat absorption and the absorption of bacterial LPS from the gut The apparent absorption of an antigen and its uptake into tissues does not necessarily mean that it will cause inflammatory immune responses. In fact, naïve mice typically develop oral tolerance to absorbed antigens, which protects against allergic or inflammatory responses Our model could be seen as a rather artificial situation, with an antigen not known to affect obesity and metabolic syndrome and with an artificially induced immunological sensitivity to the antigen. Moreover, the mice were sensitized intraperitoneally, which may have biased the response somehow to the viscera. Nevertheless, we clearly observed that even OVA-naïve mice showed significant inflammation in mesenteric adipose tissue during high-fat feeding, in contrast to subcutaneous adipose tissue, which was refractive. We speculate that this difference is due to increased exposure of mesenteric fat to antigenic material from the gut. We believe that our work is novel in that it shows, for the first time (as far as we know), that antigenic material can be absorbed from the intestine into adipose tissue, and that high-fat diets and such antigens mutually reinforce mesenteric adipose tissue inflammation. Since our intestines contain large amounts of microbial antigens with inflammatory potential, and since their presence is required for high-fat diet-induced obesity and metabolic syndrome In summary, we propose a novel mechanism for T-lymphocyte dependent mesenteric adipose tissue inflammation in obesity: High-fat diets promote intestinal absorption of gut antigens, and the delivery of the antigens to adipose tissues, preferentially visceral depots. This leads to early infiltration of T-lymphocytes into the tissue. A pro-inflammatory milieu within the visceral fat, perhaps through increased free fatty acids, might increase the risk for loss of tolerance to the antigens, favoring chronic inflammation.


We attempted to determine whether calcium channel blockers (CCBs) enhance the anti-tumour activity of cis-diamminedichloroplatinum (cisplatin) against both cisplatin-sensitive human glioblastoma U87 MG cells and cisplatin-resistant U87-MG-CR cells, the latter of which we developed for resistance to cisplatin. Nifedipine, a dihydropyridine class CCB, significantly enhanced the anti-tumour effect of cisplatin on these two cell types in vitro and in vivo. Our findings also indicated that, in the absence of normal extracellular Ca2+ nifedipine was capable of enhancing the cytotoxicity of cisplatin. In addition, this anti-tumour activity was partially inhibited by actinomycin D and cycloheximide, suggesting that it is possibly dependent upon new RNA and protein synthesis. Interestingly, ultrastructural analysis, DNA fragmentation assay and cell cycle analysis demonstrated that synergism between cisplatin and nifedipine results in apoptosis (programmed cell death) at a relatively low concentration of cisplatin, which when tested alone did not induce apoptosis. Furthermore, we demonstrated that nuclei from these cells lack a Ca(2+)-dependent endonuclease that degrade chromatin in the linker region between nucleosomes. In conclusion, our studies suggest that the non-cytotoxic agent nifedipine is able to synergistically enhance the anti-tumour effects of cisplatin on U87-MG and U87-MG-CR cells lacking a Ca(2+)-dependent endonuclease and subsequently to induce apoptosis via interaction of nifedipine with an as yet uncharacterised functional site other than a calcium channel on target cells.


Morphologically, no differences were seen between treated and untreated cells up to the time when the cells were transplanted into syngeneic hosts. However, the treated cells grew in soft agar and once injected subcutaneously or intraperitoneally into newborn rats, developed tumours after a latent period of 9-12 weeks. The tumours obtained with DMN-treated cells were well differentiated adenocarcinomata, whereas carcinosarcomata were observed with the MNNG-treated cells.Epithelial-like cells originating from 10-day and 8-week old BD rats were treated


Bony metastases were occasionally the initial presentations of malignancy. Overlooking of bony changes on radiographs in cancer patients with non-specific clinical symptoms may lead to delayed diagnosis.We presented a 70-year-old male having hypercalcemia and diffuse osteoblastic bone metastases on routine plain films. Finally, prostate cancer was diagnosed with a prostate needle biopsy.Although the modern radionuclide bone scanning is useful in diagnosis, osteoblastic bone changes are occasionally seen in plain films, which are frequently overlooked. To avoid delayed diagnosis, bony structures should be carefully examined in all plain-film radiographs. Bone metastases of malignant neoplasm are common, especially in breast, lung and prostate cancer. Bone metastases of prostate cancer are usually osteoblastic which might be related to osteoblasts . The recA 70-year-old man presented with poor appetite and general malaise of two month's duration. Physical examination revealed mildly distended abdomen and hypoactive bowel sounds. Hemogram showed high white blood cell count 22070/μl) with 91.5% segment form and low hemoglobin level (7.0 g/dl). Biochemical examination showed elevated serum calcium level (2.7 mmole/l). Urinary analysis showed hematuria (numerous red blood cells per high power field in sediment examination). The serum prostate-specific antigen (PSA) was 100 ng/ml. A chest radiograph  bone scan and positron emission tomography with 18F-2-deoxyglucose (FDG-PET) were popular in the detection of malignant bone metastases . In addiAlthough the modern radionuclide bone scanning is useful in diagnosis, osteoblastic bone changes are occasionally seen in plain films, which are frequently overlooked. To avoid delayed diagnosis, bony structures should be carefully examined in all plain-film radiographs.PSA: prostate-specific antigen; Tc-99m MDP: technetium-99m methylene diophosphate; FDG-PET: positron emission tomography with 18F-2-deoxyglucose.A fully informed written consent was obtained form the patient family for the publication of this case and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.WHL, SHL and TTW have made significant contributions by making diagnosis and intellectual input in the case and writing the manuscript.


Immunoglobulin (Fc) receptors were detected on leucocytes from patients with acute myeloid leukaemia (AML) by rosette formation with human cDE/cDE erthyrocytes (HE) sensitized with Rhesus (Rh) antisera (HEA). Of 7 Rh antisera tested, erythrocytes sensitized with anti-d (Gm10) detected the highest numbers of rosette-forming cells (HEA-RFC) in normal and AML leucocyte preparations. Using this assay, HEA-RFC was studied in 22 untreated AML patients and ce assay detected 11-6% lymphocyte and 2-1% granulocyte HEA-RFC in normal peripheral blood. Leucocytes from 16 to 22 AML patients had a similar or lower percentage than normal lymphocyte HEA-RFC, which could be explained by the dilution of peripheral blood leucocytes by poorly or non-rosetting leukaemic blasts. Ten of these 16 patients were diagnosed as having acute myeloblasts leukaemia. Six of the 22 AML patients had high HEA-RFC values of which 5 were diagnosed as having myelomonocytic leukaemia. Cytocentrifuge preparations of HEA-RFC showed that the proportion able to form rosettes was lower in myeloblasts than in monoblasts. Enzyme treatment (pronase), inhibition or simultaneous labelling of surface Ig and Fc receptors showed that the characteristic surface Ig found to AML cells is, at least in part, bound to Fc receptors. The HEA-RFC test described in this paper could be useful in the immuno-diagnosis of myelomonocytic leukaemia.


Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4.Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE  as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue.Because claudin-3 and -4 are the epithelial receptors for in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations  or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy.Using a FITC-conjugated CPE peptide we show specific Based on the high levels of claudin-3 and -4 expression in chemotherapy-resistant ovarian cancer and other highly aggressive human epithelial tumors including breast, prostate and pancreatic cancers, CPE peptide holds promise as a lead peptide for the development of new diagnostic tracers or alternative anticancer agents. Ovarian carcinoma remains the most lethal of gynecologic malignancies . AlthougClostridium perfringens enterotoxin (CPE), a potent cytolytic toxin [in vitro [290-319 peptide) is sufficient for high-affinity binding to target cell receptors and small complex formation, although this fragment is not toxic because it is incapable of initiating large complex formation and subsequent cytolysis [290-319 peptide to target ovarian cancer cells based on their high levels of claudin-3 and -4 may represents a novel, potentially highly effective diagnostic and therapeutic approach to identify and treat chemotherapy-resistant ovarian cancer.Claudin-3 and -4 are the epithelial receptors for ic toxin -16. CPE in vitro . Unfortuytolysis . Becausein vitro in multiple cell binding assays against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy experiments showed tumor internalization of the CPE peptide in a time-dependent manner. Biodistribution experiments in highly relevant clinical models of chemotherapy-resistant freshly explanted human ovarian cancer in SCID mouse xenografts were done demonstrating high uptake of the FITC-conjugated CPE peptide in established tumor tissue as well as tumor spheroids floating in the ascitic fluid, recommending this peptide as a promising lead structure for improved targeting of chemotherapy-resistant ovarian cancer.In this study we have carefully assessed the accuracy and specificity of the CPE peptide Clostridium Perfringens carboxy-terminal fragment peptide (CPE290-319 peptide) was synthesized by InVitrogen Corporation , with the sequence SLD AGQ YVL VMK ANS SYS GNY PYS ILF QKF and a purity level >95%. The CPE peptide and its FITC-conjugated form were purified by high performance liquid chromatography and their sequence and structure were confirmed by mass spectrometry.The in vitro Extreme Drug Resistance (EDR) assay  [Primary ovarian carcinoma cell lines used in this study were established from samples obtained either at the time of primary surgery or collected at the time of tumor recurrence from patients harboring advanced stage ovarian carcinoma. All primary cell lines were evaluated for claudin-3 and claudin-4 expression by real time-PCR and flow cytometry. Five primary high grade serous papillary carcinomas (OSPC) and two clear cell ovarian cancers (CC) were available for this study. The characteristics of the primary specimens used in our experiments are depicted in Table ine, CA) . Other cine, CA) . All samine, CA) .CT) method (PE Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in the nonmalignant ovarian epithelial cells, using GAPDH (Assay-on-Demand Hs99999905_m1) RNA as internal controls.RNA isolation from primary cell lines was performed using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. q-PCR was performed with an ABI Prism 7000 Sequence Analyzer using the manufacturer's recommended protocol  to evaluate expression of claudin-3 and claudin-4 in all the samples. Each reaction was run in triplicate. Briefly, five μg of total RNA from each sample was reverse transcribed using SuperScript III first-strand cDNA synthesis . Five μl of reverse transcribed RNA samples  were amplified by using the TaqMan Universal PCR Master Mix (Applied Biosystems) to produce PCR products specific for claudin-3 and claudin-4. The primers for claudin-3 and claudin-4 were obtained from Applied Biosystems as Assay-on-Demand products. Assays ID were Hs00265816_s1 (claudin-3) and Hs00433616_s1 (claudin-4). The comparative threshold cycle  that contained 4 μg of FITC-labeled C-CPE peptide or primary scFv antibody (positive control) and cells were then incubated for 1 hour at room temperature. For scFv antibody staining, after further washing, cells were resuspended with 100 μL PBS 1% BSA solution that contain 0.7 μg of an anti-myc tag antibody 9E10 (Roche Diagnostics) for 1 hour at 4°C, followed by washing and incubation with 2 μg of fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin G  for 30 minutes at 4°C. Cells stained with FITC-labeled C-CPE peptide or scFv and unstained control cells were then washed with PBS, resuspended in 500 μL PBS, and immediately analyzed by FACScalibur .We used FACS analysis to evaluate binding of the FITC-labeled C-CPE peptide to the surface of primary chemotherapy-resistant ovarian carcinoma cell lines overexpressing claudin-3 and -4 receptors. As positive control, we used a single chain fragment variable (scFv) specific against claudin-3 recently developed and characterized in our laboratory . Negativ290-319 peptide on chemotherapy resistant ovarian tumors was further evaluated by fluorescence microscopy. Briefly, unfixed ovarian cancer cells or control cells in suspension or adherent to plastic were incubated with different concentrations of FITC-conjugated-C-CPE290-319 peptide for 30 to 60 minutes at 37°C and binding to cell surface expressed claudin-3 and claudin-4 was analyzed by fluorescent microscopy using a Nikon fluorescence microscope Eclipse E400. Images were captured digitally and analyzed using Metamorph 3.6a version software.The surface binding localization of FITC-conjugated-C-CPE290-319 peptide was able to internalize after binding to claudin-3 and claudin-4 receptors on the surface of chemotherapy-resistant ovarian cancer cells. Briefly, 30,000 ovarian cancer cells were seeded in 3 cm petri dishes. After 24 hours of cultivation, the medium was replaced by fresh medium and FITC-conjugated CPE 290-319 peptide (10 μg/ml) was added to the cells. The FITC-labeled peptide was incubated with the tumor cells for the indicated times . The medium was then removed and the cells were washed thrice with 1 mL PBS before being analyzed by confocal imaging on an inverted microscope (Leica DM IRBE) with a confocal laser scanning unit (Leica SP2 MP). Some of the experiments were also carried out using the unlabeled CPE 290-319 peptide as competitor for the binding of FITC-conjugated CPE290-319 peptide while samples without FITC-conjugated CPE 290-319 peptide were analyzed to determine autofluorescence of tumor cells. Processed serial sections were constructed into three-dimensional images using VoxelView  on a Silicon Graphics Indy workstation .We used confocal microscopy to evaluate whether the FITC-conjugated-C-CPE 6 into C.B-17/SCID mice in groups of three to five. Six weeks after i.p. tumor injection when carcinomatosis nodules had grown to 0.5 to 0.8 cm, 100 μg FITC-CPE peptide was injected into the tail vein and allowed to circulate for 12, 24 or 48 hours. In some experiments mice were perfused at the time of sacrifice with PBS through the left ventricle to remove blood and unbound peptide. Tumors and control organs were excised after the injection of the fluorescent peptide and examined for fluorescence using a whole-body optical imaging system. Selective excitation of FITC was produced through a D455/70 nm band-pass excitation filter . Emitted fluorescence was collected through a long-pass filter  on a 3.3 MP digital camera .C.B-17/SCID female mice 5-7 weeks old were obtained from Harlan Sprague-Dawley  and housed in a pathogen-free environment at Yale University. They were given commercial basal diet and water ad libitum. The experimental protocol for the use of the animals for these studies was approved by the Institutional Animal Care and Use Committee. Animals were used to generate ovarian tumor xenografts as previously described [Statistical comparisons between groups were done by the unpaired Student's t test using SPSS version 15 . P ≤ 0.05 was considered statistically significant.We used q-RT-PCR assays to get highly sensitive measurements of claudin-3 and claudin-4 CPE receptor expression in normal tissue cells and primary human tumors derived from patients harboring chemotherapy-resistant ovarian carcinomas. Both claudin-3 and/or claudin-4 genes were found highly expressed in all primary ovarian cancer studied including five high grade serous papillary tumors and two high grade clear cell carcinomas when compared to normal ovarian epithelial cells (NOVA) as well as other normal cells or other gynecologic tumors . To accomplish this goal, we evaluated the ability of the FITC-conjugated CPE peptide to label the freshly established primary ovarian tumor cultures in flow cytometry experiments; in parallel, we also evaluated VERO cells , and heontrols, .in vitro. As representatively shown for OSPC-ARK-1, a high grade serous papillary ovarian carcinoma  with FITC-conjugated CPE peptide (100 μg). The peptide was then allowed to circulate in the blood stream for various time periods ranging from 12 to 48 hrs before tumors, ascites and controls organs were excised and processed from experimental and control animals. Mean fluorescence intensity was calculated over the ovarian tumors as well as multiple explanted organs/tissues including lungs, liver, spleen, brain, heart and kidney at 12 hrs, 24 hrs and 48 hrs after subtraction of background. Strong and specific fluorescence was detected in OSPC-ARK-1-derived tumor xenografts after12 hrs (mean MFI ± SE = 1178 ± 406), 24 hrs (mean MFI ± SE = 2592 ± 1303), and 48 hrs (mean MFI ± SE = 1808 ± 677) after i.v. injection with FITC-conjugated CPE peptide , these findings imply that ovarian cancer refractory to standard treatment modalities may be susceptible to CPE-based therapeutic approaches. Consistent with this view, in the last few years CPE structure/function relationship has been extensively investigated, mainly by characterizing the functional properties of enterotoxin fragments and point mutants [290-319 COOH-terminus fragment is sufficient for high affinity binding to target cell receptor and small complex formation, although this fragment is incapable of initiating large complex formation and cytolysis [Claudin-3 and claudin-4 are tight junction proteins overexpressed in ovarian carcinomas , the mos mutants -16. In tytolysis -16.in vitro and in vivo resistance to chemotherapy. Secondly, using these freshly established biologically aggressive ovarian tumor cell lines, we have carefully assessed the accuracy and specificity of the binding of the carboxy-terminal fragment of CPE conjugated to a fluorescent tag  as well as its internalization properties in vitro as well as in in vivo experiments using a highly relevant clinical model of chemotherapy resistant freshly explanted human ovarian cancer in SCID mouse xenografts.In this study, we have taken advantage of the functional properties of enterotoxin fragments and we have first confirmed at both RNA and protein levels a high expression of the CPE receptors in multiple primary ovarian carcinomas characterized by high Our results established, to our knowledge for the first time, that the CPE peptide is capable of specifically targeting chemotherapy resistant ovarian carcinoma cells overexpressing the CPE receptors claudin-3 and claudin-4. However, it did not bind to normal tissue cells or tumors not expressing significant levels of claudin-3 and/or claudin-4. These findings clearly indicate that the CPE peptide possesses specific targeting and/or homing properties for chemotherapy-resistant ovarian tumors. The remarkable tumor-homing efficiency of the peptide may be due to its propensity to be internalized by cells. Indeed, cells that bind CPE peptide due to the overexpression of the claudin-3 and claudin-4 receptors may transport it across the cell membrane and into the cytoplasm. Thus, peptide internalization is likely to contribute to its effectiveness in becoming concentrated in the targeted tumors. In this regard, it is worth noting that the CPE peptide used in our experiments was conjugated to fluorescin, a complex organic molecule with 44% of the molecular mass of paclitaxel . While win vivo experiments, we found even discrete accumulation  or single chemotherapy-resistant ovarian cancer cells floating in the ascites of OSPC-ARK-1 xenografted animals to be specifically targeted by the FITC-conjugated-C-CPE peptide. These experiments suggest that CPE peptide may have a remarkable ability to target very early stage of occult metastasis foci and/or recurrence disease by detecting concealed tumor cells before they become clinically evident in vivo.Occult micro-metastatic chemotherapy-resistant ovarian disease, by definition, is not detected by even the most sophisticated imaging techniques such as standard MRI and positron emission tomography (PET). To determine whether CPE peptide localizes to tumor, SCID mice harboring established peritoneal carcinomatosis of OSPC-ARK-1-derived tumor xenografts were injected i.v. with FITC-conjugated CPE peptide. Importantly, tumor explanted 12, 24 and 48 hrs after peptide injection showed fluorescing tumor cells while untreated tumor cells did not significantly autofluoresce. Moreover, brain, heart, lungs, kidney, spleen and liver from FITC-conjugated CPE peptide injected tumor bearing mice showed little labeling. A similar result was also obtained with FITC-conjugated CPE peptide injected tumor-free mice, which suggested that the inefficient labeling was not attributable to peptide depletion by the tumor. Remarkably, in our in vivo in patients harboring incurable chemotherapy-resistant ovarian disease or other human malignancies overxpressing the CPE receptors such as breast, prostate, lung, endometrial, thyroid and pancreatic cancer [in vitro experiments or in CPE-peptide injected mice), while is able to behave as a shuttle during transit, and accumulates efficiently within the tumor in a few hours. As a result of our experiments, efforts to conjugate the CPE peptide to diagnostic and therapeutic radionuclides are currently in progress in our laboratory.Eradication of occult micrometastases of chemotherapy-resistant disease by coupling radioactive isotopes or anticancer agents to the CPE peptide would be greatly beneficial in protecting patients with microscopic residual disease or small-volume macroscopic cancer resistant to standard chemotherapeutic agents. Consistent with this view, the potential of the CPE peptide as a carrier to specifically deliver therapeutic drugs c cancer ,23-25 isOur results demonstrate for the first time that the CPE peptide binds and internalizes in ovarian tumor cells and therefore possesses excellent targeting or homing properties for the early diagnosis and/or treatment of chemotherapy-resistant ovarian carcinoma. These data combined with the high expression of claudin-3 and -4 receptors recently reported in other biologically aggressive epithelial human tumors, such breast, prostate, lung, endometrial, thyroid and pancreatic cancer ,23-25 prOSPC: ovarian serous-papillary carcinoma; CPE: Clostridium Perfringens Enterotoxin; FBS: fetal bovine serum; mAb: monoclonal antibody; scFV: single chain fragment variable; PBL: peripheral blood lymphocytes; q-rtPCR: quantitative real-time polymerase chain reaction; FITC: Fluorescein isothiocyanate.The authors declare that they have no competing interests.EC, FC, SB, MB, PT carried out the molecular in vitro studies including RT-PCR, flow cytometry and cell binding assays. EC, CR, JH, HF, MM, GM carried out the in vivo molecular studies with the CPE peptide in mice harboring multiple ovarian cancer xenografts. PS, DS MA, TR, MA-K, SP and AS participated in the design of the study and drafted the manuscript. AS conceived the study. All authors read and approved the final manuscript."The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/349/prepub