While BxPC-3 (KRAS wild type) and MIA PaCa-2 (KRAS mutated) cell lines were sensitive to GDC0941 and AZD6244 as single agents, synergistic inhibition of tumor cell growth and induction of apoptosis were observed in both cell lines when the two drugs were combined. Interestingly, phosphorylation of the cap-dependent translational components, 4E-binding protein (p-4E-BP1) and S6 was found to be closely associated with sensitivity to GDC0941 and AZD6244. In BxPC-3 cell xenografts, survival differences were observed between the control and the AZD6244, GDC0941, and combination groups. Our study provides the rationale for concurrent targeting of the PI3K and MEK pathways, regardless of KRAS status, and suggests that phosphorylation of 4E-BP1and S6 can serve as a predictive biomarker for response to treatment.Patients with pancreatic cancer have dismal prognoses, and novel therapies are urgently needed. Mutations of the KRAS oncogene occur frequently in pancreatic cancer and represent an attractive target. Direct targeting of the predominant KRAS pathways have been challenging and research into therapeutic strategies have been now refocused on pathways downstream of KRAS, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK [MEK]). We hypothesized that concurrent inhibition of the PI3K and MEK pathways would result in synergistic antitumor activity, as it would circumvent the compensatory feedback loop between the two pathways. We investigated the combined effect of the PI3K inhibitor, GDC0941, and the MEK inhibitor, AZD6244, on cell viability, apoptosis and cell signaling in a panel of pancreatic cancer cell lines. An  Pancreatic cancer is the fourth leading cause of cancer-related deaths in men and women in the United States. An estimated 43,140 people were diagnosed with and 36,800 died of pancreatic cancer in 2013  × 100. Tumor sizes were calculated by the standard formula of Tumor Size = Length x Width2 x 0.5. Mice that developed tumors reaching 100-150 mm3 in size were randomized into the following four groups with 8 mice in each group: vehicle, GDC0941, AZD6244, or the combination treatment. Both drugs were administered once or twice daily by oral gavage in a volume of 10 ml/kg body weight. Drugs were dissolved in a vehicle of 0.5% (w/v) methycellulose/0.2% Tween 20 for administration. The PI3K inhibitor was delivered daily at a final concentration of 50 mg/kg [2 overdose. All studies were carried out in accordance with the protocol approved by the Washington University Institutional Animal Care Facility (Protocol number: 20100114).Nude mice, 4 or 5-weeks-old, were purchased from the National Cancer Institute. They were acclimated for 1-2 weeks before they were subjected to any experimental procedures. BxPC-3 cells . Cell apoptosis data and tumor growth experiments were represented as the mean ± standard error of the mean (s.e.m.). Kaplan-Meier curves were used for survival analyses. Statistical comparisons were made using the ANOVA (single factor) and t-test (paired two samples for means and unpaired t-test), as indicated. A p value of < 0.05 was considered statistically significant.in vitro, four pancreatic cancer cell lines were selected for study. BxPC-3 is a KRAS wild type pancreatic cancer cell line, while the other three cell lines harbor the KRAS mutation. All four cell lines are not known to carry either PI3K or BRAF mutations [50) after 72 hours exposure were 376.4 nM in BxPC-3 cells and 754.6 nM in MIA PaCa-2 cells . In addition, we compared control vs 1 nM AZD6244 in PANC-1 and MIA PaCa-2 cells, and no significant differences were observed To determine the anti-tumor activity of the PI3K and MEK inhibitors alone and in combination utations . The ant-2 cells . AZD6244ectively . The IC550s in the BxPC-3 and MIA PaCa-2 cell lines. Cell growth in both cell lines was markedly decreased following combination treatment at multiple paired concentrations when compared with either single agent alone. Data were evaluated to get the CI under the corresponding effective dose (ED) in BxPC-3 and MIA PaCa-2 cell lines  according to the Chou-Talalay method (20) using a fixed dose ratio. Both GDC0941and AZD6244 were introduced to cell cultures at 0.25×, 0.5×, 1×, 2× and 4× their respective ICll lines  by Calcull lines .Interestingly, while GDC0941 or AZD6244 alone did not impact PANC-1 and Capan-2 cell growth, administration of these two drugs in combination mildly inhibited cell growth. Cell growth was reduced to 71.4% and 67.0% in the PANC-1 and Capan-2 cell lines, respectively, following administration of the drugs in combination  .To determine the apoptotic effect of the combined therapy, two different concentrations of GDC0941 and AZD6244 were used alone and in combination. While the apoptosis rate of BxPC-3 cells at baseline was 17.0%, it increased significantly to 34.0% and 47.8% following administration of GDC0941 at 380 nM and 1,520 nM concentrations, respectively . AZD6244 alone at 600 nM and 2,400 nM increased the apoptosis rate of BxPC-3 cells to 26.5% and 27.2%, respectively . A combination of GDC0941 and AZD6244 resulted in a much higher rate of apoptosis in BxPC-3 cells compared with the control group or inhibitor alone. The combination of GDC0941 at 380 nM and AZD6244 at 600 nM or the combination of GDC0941 at 1,520 nM and AZD6244 at 2,400 nM increased the BxPC-3 cell apoptosis rate to 63.3% and 82.8% respectively  . The ratTo assess the impact of both drugs on downstream effectors of the PI3K and MEK pathways, we used Western blot analysis to observe total protein expression and phosphorylation status. The total protein levels of ERK, S6 and 4E-BP1 remained unchanged after treatment with GDC0941 and AZD6244 in each cell lines . p-ERK, To understand the phenotypic differences seen in the MIA PaCa-2 (sensitive to GDC0491 and AZD6244) and PANC-1 (resistant to GDC0491 and AZD6244) cell lines, both of which harbor the KRAS mutation, we examined the differences in their downstream effectors following GDC0941 and AZD6244 administration . In bothin vivo, we used GDC0941, AZD6244, and a combination of GDC0941and AZD6244 to treat BxPC-3 xenograft mice for 18 days. Compared to the control (vehicle) group, tumor volumes decreased significantly in the AZD6244 and combination groups  but not in the GDC0941group as compared to control  and MIA PaCa-2 (sensitive) cell lines. These two cell lines differ in their phenotypic response to PI3K and ERK inhibitors, despite harboring similar major genetic alternations. Both cell lines are reported to contain KRAS mutations, p53 mutations, wild type P16 and wild type DPC-4 . While inslation . Our obsnslation ,30.4E-BP1 plays a major role in cap-dependent translation. It binds to the elF4E-mRNA cap complex to inhibit cap-dependent translation, and phosphorylated 4E-BP1 then falls out of the translation complex, so initiation of translation can begin . mTORC1,In summary, we have explored the benefit of concurrent pathway blockade by PI3K and MEK inhibitors alone and in combination for pancreatic cancer. Synergy in decreasing cell growth was observed and the effects were not KRAS dependent. Persistent phosphorylation of S6 and 4E-BP1 appeared to be associated with resistance to the PI3K and MEK inhibitors. Future investigations into the alternative mechanisms of 4E-BP1 phosphorylation and targeting of 4E-BP1 in pancreatic cancer are warranted.


Canada is a nation of trees. Canadian forests cover 50% of Canada's land area and represent 10% of world’s forested area. Canada exports more processed forest products than any other country. It is also a host to a variety of forest insects both native and non native that limit the economic, recreational and wildlife habitat use. Our research focuses on initiating insect cell lines, determining nutritional requirements of cells, and developing low cost media for large scale propagation of insect pathogenic viruses as ecologically sound alternatives to chemical pesticides. Cell lines are used for bioassay and strain selection of viruses and bacterial toxins and for production of foreign gene products using baculovirus- and entomopoxvirus expression vectors. They offer a cleaner, viable alternative to insect larvae for producing viral pesticides.Choristoneura fumiferana), western spruce budworm , forest tent caterpillar , tobacco hornworm (Manduca sexta), white-marked tussock moth (Orgyia leucostigma), red-headed pine sawfly (Neodiprion lecontei), gypsy moth (Lymantria dispar), white pine weevel (Pissodes strobi), the tarnished plant bug (Lygus lineolaris) and the ash and privet borer (Tylonotus bimaculatus). These cell lines represent six tissues of origin, namely neonate larvae, pre-pupae, embryos, ovaries, hemocytes, and midgut and four Insect Orders namely Lepidoptera, Hymenoptera, Coleoptera and Hemiptera.Since 1969, over 150 continuous cell lines have been produced for forest insect pest research at GLFC. This collection represents one of largest single site repository of frozen insect cell lines in the world. Cell lines developed include tissues of the eastern spruce budworm  and the budded virus (BV) in vitro. During the in vivo infection process hemolymph (BV) is collected and cultures are infected producing both phenotypes. BV, released in the media is used to infect other cells; ODV produced in the nuclei to infect other insects.Although chemical pesticides are generally more reliable and effective control agents, they are not environmentally acceptable as their negative impact far out weigh their benefits. We have developed methodologies to duplicate the in vitro agarose lawn assay for testing the toxicity of freeze-dried ethanolic leaf extracts and secondary compounds. Foliage from sugar maple, trembling aspen and mulberry was assayed for possible insecticidal properties against cells from two lepidopteran defoliators, spruce budworm and fall armyworm. Cells were suspended in buffered agarose and spread in a petri dish. Leaf extracts solubilized in 50-70% DMSO were applied directly to the cells. Trypan blue staining was used as an indicator of toxicity. Quantitative comparisons were determined by threshold doses that elicited positive responses. Toxicity and validity of the assay were further confirmed by the presence of membrane disruption and cellular lysis. The activity of trembling aspen was markedly enhanced when the pH was raised from 7 to 10.5, a level similar to that of the larval midgut, but the effect was largely abolished in the presence of gut juice. Analysis of a crude mulberry extract treated with neat gut juice suggested that most of the active material in the lepidopteran leaf diet is either insoluble or precipitated in the larval midgut, while the activity of any solubilized material is suppressed through interaction with gut-juice proteins.Trees produce numerous compounds with toxic and growth regulating properties to protect themselves against insect attack. We have developed a rapid in vivo within the whole insect, CF-203 cells are an invaluable in vitro tool for this function.A spruce budworm midgut cell line, FPMI-CF-203 has been shown to respond to the molting hormone ecdysone, juvenile hormone and other chemicals. Not only was gene expression stimulated in these treated cells, but they allowed for the analysis of house keeping genes or molting gene promoters as well as the study of cell signaling pathways such as protein kinase C and its targets, steroid hormone and molting gene expression. In addition, GFP tag fused target proteins were readily visible in single living CF-203 cells. They were permissive for the production of active foreign gene proteins using recombinant baculoviruses vector systems. We have observed that while RNA interference (RNAi) technology for the study of gene function does not work well Developing insect cell lines suitable for a specific application is a very slow and challenging process. Primary cultures started may or may not develop into cell lines. Those that do might take months and the resulting cells may not be suitable for a desired function. Of the 4-6 M insect species, we currently have 800+ cell lines from 100 species. There are presently no cell lines from exotic or invasive species. Once established, insect cells, like insects, must be fed to be kept alive. Suitable culture medium for the growth of tissues of different insects is currently not available. Large scale production of insect viruses would require the media to be optimal for cell growth as well as for replication of the virus or other control agents. Each cell line/virus combination requires its own unique media composition. Two pressing concerns requiring immediate attention are cross and misidentification contamination of existing cell lines and retiring of key researchers. With few insect cell lines in public collections, private collections are often inaccessible after the principal investigator leaves. Retiring scientist equals lost insect cell cultures.More dedicated researchers developing new lines from different species, routine and aggressive identification, characterization and verification of cells lines employing new molecular techniques and maximizing the potential of insect cells as viable commercial ventures are needed.


Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), a metastasis suppressor gene of human cancer, is the most abundantly expressed member of the ceramide synthase gene family. Expression of LASS2 has been reported in carcinomas of the prostate, liver and breast. However, there has been no report on the expression of LASS2 in human bladder cancer cell lines. In order to investigate the expression and potential role of this new tumor metastasis supressor gene in human bladder cancer, we compared the proliferation, metastasis and invasion among the BIU-87, T24, EJ and EJ-M3 human bladder cancer cell lines. The mRNA expression levels of the LASS2 gene were examined using real-time quantitative PCR (qPCR). The expression levels of LASS1 and LASS3 mRNA were used as references. The protein expression level of the LASS2 gene was detected using western blotting. The most aggressive of these four human cancer cell lines was observed to be EJ-M3. The expression of LASS2 mRNA was significantly correlated with diverse proliferation, metastasis and invasion. The expression levels of LASS1 and LASS3 mRNA were not correlated with these parameters. At the protein level, we observed that the more aggressive the cancer cell line, the lower the LASS2 protein expression level. Therefore, LASS2 expression may be correlated with the development and progression of human bladder cancer and may be a prognostic indicator for this cancer. Bladder cancer is one category of common genitourinary cancers and recurrence with metastasis is the main reason for treatment failure . MetastaTumor metastasis suppressor genes are a relatively new class of genes. These genes, which include NM23 , KISS-1 Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 , is a gene isolated from a human liver cDNA library by the laboratory of Shanghai Medical College, Fudan University , and it is a human homolog of the yeast (Saccharomyces cerevisiae) longevity assurance gene, LAG1. LASS2 has been observed to correlate with the degree of invasion and recurrence in carcinomas of the prostate (The LASS (longevity assurance homolog) family members are highly conserved from yeasts to mammals. prostate ,16, liveprostate  and breaIn our previous study, we demonstrated that LASS2-negative bladder cancer was associated with poor clinical prognosis. The expression of LASS2 mRNA was significantly correlated with clinical stage (P<0.001), depth of tumor invasion (P<0.001) and recurrence (P<0.001) .In order to fully understand the biological importance of LASS2, we examined the mRNA and protein expression of LASS2 in human bladder cancer cell lines  with diverse proliferation and invasion potential, and analyzed the potential role of the tumor metastasis supressor gene LASS2 in these human bladder cancer cell lines.2/95% air at 37°C.The EJ, T24 and BIU-87 human bladder cancer cell lines were preserved by our department . The highly invasive human bladder carcinoma EJ-M3 cell line was established in previous studies ,21. The 4) were plated in seven 24-well culture plates after the cell count. The cells were harvested on days 1, 2, 3, 4, 5, 6 and 7 for cell counting experiments, and the values were normalized to untreated controls. This experiment was repeated three times. We then created growth curves and compared the proliferation ability among the four cell lines.The growth of the BIU-87, T24, EJ and EJ-m3 cells was evaluated by a cell proliferation assay. Briefly, cells . The target gene sequences were obtained from the National Center for Biotechnology Information GenBank databases. The purity of RNA samples was determined by the OD260/OD280 (between 1.7 and 2.0) and the OD260/OD230 (>1.7) values and by analysis of the ribosomal RNA band integrity by conventional denaturing agarose RNA electrophoresis . First-strand cDNA was synthesized in a volume of 20 t method .μg of protein from whole cell extracts, according to the procedure described by Baron et al was separated by SDS-PAGE gels and transferred to a membrane. LASS2 was detected in 100 ron et al and Calo al of at least three separate experiments. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. The correlation between gene expression and potential causative variables, were evaluated with the Chi-square test. P<0.05 was considered to indicate a statistically significant result. Each statistical analysis was performed using the SPSS 11.0 software for Windows .The population-doubling time of BIU-87, EJ, T24 and EJ-M3 cell lines were 31.7±0.1, 27.6±0.2, 28.5h±0.1 and 20.8±0.2h, respectively. The cell growth rate was significantly different between EJ-M3 and the other three cell lines (P<0.05). There was no significant difference between the growth rates of BIU-87 and T24 (P>0.05). The growth curves are shown in The numbers of EJ-M3, EJ, T24 and BIU-87 cells that attached to the lower side of the membrane were 122.8±16.8, 78.6±14.1, 32.9±10.7 and 16.0±8.1, respectively. The numbers of EJ-M3, EJ, T24 and BIU-87 cells that invaded into the 24-well plate were 32.8±9.8, 7.5±4.2, 10.2±5.1 and 8.0±4.9, respectively. Using the method of analysis of variance (ANOVA), we demonstrated that there was no statistic difference of invasiveness between EJ, T24 and BIU-87. However, the invasiveness of EJ-M3 was significantly different from that of EJ, T24 and BIU-87 (P<0.001) and are shown in Prior to the quantitative analysis, optimization procedures were carried out for qPCR reactions on EJ-M3, EJ, T24 and BIU-87 cell lines, using specific primers for human LASS2, LASS2 and LASS3 genes, separately. Since LASS1, LASS2 and LASS3 have been reported to be expressed at the mRNA level in normal human tissues, we used LASS2 expression as a control in qPCR reactions. To optimize our quantitative technique, a cDNA synthesized from the cell sample was used in serial dilution and a PCR efficiency close to 100% was obtained. After optimization of the qPCR, expression of LASS2, LASS2 and LASS3 genes were analyzed using qPCR in the bladder carcinoma cell lines EJ-M3, EJ, T24 and BIU-87. The result is shown in The differences among the four bladder carcinoma cell lines were calculated using the ANOVA method. The differences in LASS2 mRNA expression levels among the four cell lines were statistically significant (P<0.001). LASS1 and LASS3 expression levels among the four cell lines were not statistically significant (P>0.05).The expression of LASS2 protein was observed in the four bladder carcinoma cell lines, EJ-M3, EJ, T24 and BIU-87. The highest expression level of LASS2 was detected in the human bladder carcinoma cell line EJ-M3 by western blot analysis. Moderate LASS2 protein expression was observed in EJ and T24 cell lines, and weak LASS2 expression was observed in the BIU-87 cell line by western blotting. The result is shown in The present study identified the presence of LASS2 mRNA and protein in four bladder carcinoma cell lines. The expression levels of LASS2 among these human bladder cancer cell lines, which have diverse reproductive activity and invasive abilities, are distinct at the mRNA and protein level.It is well known that an imbalance of cellular growth regulation results from certain genetic changes in the oncogenic process, which lead to uncontrolled tumor growth. However, unrestrained proliferation does not, by itself, result in invasion and metastasis. Certain additional genetic changes are required for tumor cells to be able to invade and metastasize . A tumoret al(et al(LASS2 has previously been demonstrated to function as a tumor metastasis suppressor gene. Previous studies have shown that LASS2 gene plays a key role in carcinomas of the prostate ,16, liveet al observed al(et al,16 demon al(et al.This is the first study investigating the expression of LASS2 in human bladder carcinoma cell lines. In this study, we examined the protein and mRNA expression of LASS2 in four bladder carcinoma cell lines using the methods of qPCR and western blotting.We also demonstrated that LASS2 expression correlated with the biological characteristics of these human bladder carcinoma cell lines. The results suggest that LASS2 expression is downregulated in cell lines with a high degree of malignancy and that the more malignant tumor cells expressed lower amounts of LASS2 at the protein and mRNA levels. This study supports the role of LASS2 as a metastasis suppressor gene and its potential utility as a clinical prognostic marker in human bladder carcinoma.The results of qPCR are coincidental to the results of western blotting. LASS2 may be a useful indicator of tumor invasion and progression. However, due to the increasing appreciation of the importance of LASS2 in human bladder carcinoma, as well as other cancers, future studies focusing on how LASS2 is regulated and the mechanisms of its anti-metastatic functions, with the goal of critically examining the possibilities of exploiting LASS2 as a therapeutic target, are required.In conclusion, using qPCR and western blotting, we have shown that LASS2 expression may be correlated with the development and progression of human bladder cancer and may be a prognostic indicator for this cancer.


Data within the cells of Table 2 should appear on separate lines within the cell. Please see the correct Table 2 here:


Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology , entitled "Ao38, a new cell line from eggs of the black witch moth,  Trichoplusia ni cell line virus (TNCLV), an alphanodavirus that was previously reported in a T. ni derived cell line [Ascalapha odorata. In the course of our subsequent characterization, we discovered that the source of the Ao38 cell line was not A. odorata as reported, but was instead T. ni. A careful analysis of archived intermediate stages in the production of the cell line indicated that the line resulted from primary A. odorata cultures that were contaminated with T. ni cells. While the source of the contamination is not known with certainty, further analysis suggests that Ao38 cells are likely a clonal derivative of the High Five (Tn-5B1-4) cell line. While we stand behind the analysis of the characteristics of this cell line, we are providing a correction to the above study, and we sincerely apologize for any problems or difficulties caused by our error in identification of the origin of this line. Below, we provide representative data establishing the identity of this line as a T. ni derived cell line. We also reexamined the cell line for the presence of TNCLV under conditions of defined sensitivity, and provide additional data confirming the absence of detection of the TNCLV alphanodavirus from this line.After publication of this work , we discell line . We repocadherin gene [T. ni) cells, and nearly identical alignment with sequences from T. ni larvae . Thus, the T. ni origin of the Ao38 cells was established using these sensitive marker genes. To examine the origin of the Ao38 cell line within the species T. ni, we examined cadherin gene sequences. A fragment of the T. ni cadherin gene sequence that contains an intron, was previously found to be highly polymorphic in sequence [cadherin sequences from several sources of T. ni. The target cadherin sequence from Ao38 cells was identical to that from High Five cells but differed from that of other T. ni sources. Figure cadherin sequence. In contrast, sequences derived from larvae of a laboratory strain of T. ni (Cornell strain [T. ni cell line (QB-9-4s) [To identify the species of origin of the Ao38 cell line, we amplified sequences from three marker genes that have been used to distinguish between and within lepidopteran species: a) mitochondrial cytochrome c oxidase subunit 1 (COI) , b) interin gene  , and frQB-9-4s) , showed in vitro transcribed and purified an RNA representing that region of the TNCLV genome. The in vitro transcribed TNCLV RNA was subsequently used for spiking experiments with Sf9 cell RNA, to determine the sensitivity of TNCLV RNA detection in our RT-PCR studies (Figure T. ni or other cell lines suspected to harbor this alphanodavirus.The alphanodavirus TNCLV is readily detected by RT-PCR in growing High Five cells. We previously reported that we were unable to detect TNCLV in the Ao38 cell line by RT-PCR. We therefore performed a careful reexamination of the Ao38 cells for the presence of TNCLV RNA, and measured the sensitivity of our methods. For these studies, we amplified and cloned a 566 nt region of the TNCLV genome, then s Figure . Under ts Figure , lane 8.To reflect the significant contributions of scientists involved in the extended analysis of the origin of the Ao38 cell line, and the analysis of TNCLV RNA detection and sensitivity, Shiying Zhang and Yun-Ru Chen were included as authors of this study.GWB conceived the initial project. YH developed methods and designed and performed experiments for cell line generation. GWB and YH designed experiments on cell line characterization, virus infectivity, and recombinant protein production. YH performed those experiments and GWB and YH analyzed the data and wrote the manuscript. Sheng Z, YH, and GWB designed glycan analysis studies. Sheng Z performed the experiments, analyzed glycan data, and wrote a portion of the manuscript. Sheng Z, YH, and GWB edited, read and approved the original manuscript. YC and Shiying Z designed experiments and performed analysis of species-specific marker genes. Shiying Z and GWB designed experiments on alphanodavirus detection and Shiying Z performed the experiments and analyzed the data. GWB wrote the correction and all authors edited, read and approved the correction.


The random nature of transgene integration harbours various pitfalls for development of production cell lines including clonal variation in expression level and growth characteristics. The CEMAX system is an expression system for targeted integration of expression cassettes via DNA double-strand break induced homologous recombination. Stable high producers are available within 4 weeks without the need of extensive clone screening and process development.Stable and high expression rates are ensured with the CEMAX system due to targeted integration of a single copy of the gene of interest at a transcriptionally highly active site in the host cell genome. Producer cell lines for various therapeutic product candidates were established. These cell lines produced antibodies and highly glycosylated antibody fusion proteins and showed high clonal similarity. This made a platform fed-batch process profitable.The CEMAX expression system is based on a genetically modified CHO cell line bearing a tag at a highly active genomic transcription site. The gene of interest (GOI) is integrated via site-directed DNA double-strand break-induced homologous recombination. The target site comprises a screening and selection cassette, which was used for initial development of the high producer host cell. This exchangeable cassette is flanked by elements that facilitate site-directed integration by homologous recombination. These elements include regions of homology for recombination with the CEMAX vector, rudimentary selection markers that are activated during recombination, and cleavage sites for the homing endonuclease I-SceI (Meganuclease).Transfection of the CEMAX vector comprising the GOI along with transient Meganuclease activity triggers a chain of events after cotransfection: DNA gets cleaved by I-SceI and cellular DNA repair machinery is induced by free DNA double-strand breaks. The CEMAX vector comprising the GOI functions as a repair matrix using recombination between homologous elements flanking the DNA lesion. The GOI gets integrated and the rudimentary selection markers become activated upon homologous recombination. CEMAX producer cells were selected at multi-well plate scale followed by analysis for outgrowth of stable producer cells. The platform process for fed-batch cultivation of various CEMAX producer cells was applied after expansion to the scale needed for production of protein material.The idea of a platform process that is suitable for all CEMAX producer cells was based on theoretical consideration about similarity of cells due to site-directed integration and the observation that cell growth and metabolism of CEMAX cell lines were comparable. This was verified by the results of this study. Applying the fed-batch process cell growth of CEMAX host cells and CEMAX producer cells was comparable Figure . The devThe fed-batch process comprises a chemically defined and commercially available basal medium and the chemically defined feed solution CeloFeed (Celonic) supplemented according to the improved feeding regime. The glucose level was maintained between 4.5 g/L and 7.5 g/L. Glutamine was kept at a concentration above 0.8 mM. By applying the platform fed-batch process product concentrations up to 690 mg/L were achieved with CEMAX cell lines producing a glycosylated Fc fusion protein after site-directed integration of the GOI Figure . AchievaThe platform fed-batch process was developed by optimization based on commercial available and proprietary basal and feed media formulations. It was suitable for all producer cell lines derived of a particular CEMAX host cell due to highly similar growth behavior after site-directed integration of the gene of interest. The platform process made product concentrations of up to 0.7 g/L achievable. Animal derived component free chemically defined medium and feed solution ensure a robust and regulatory compliant process. By combining the platform process with site-directed integration timelines in cell line development and process development were shortened for faster entry in first-in-man studies.


Chronic kidney disease (CKD) is regarded as a state of Klotho deficiency and FGF23 excess. In patients with CKD a strong association has been found between increased serum FGF23 and mortality risk, possibly via enhanced atherosclerosis, vascular stiffness, and vascular calcification. The aim of this study was to examine the hypothesis that soluble Klotho and FGF23 exert direct, rapid effects on the vessel wall. We used three in vitro models: mouse aorta rings, human umbilical vein endothelial cells, and human vascular smooth muscle cells (HVSMC). Increasing medium concentrations of soluble Klotho and FGF23 both stimulated aorta contractions and increased ROS production in HVSMC. Klotho partially reverted FGF23 induced vasoconstriction, induced relaxation on phosphate preconstricted aorta and enhanced endothelial NO production in HUVEC. Thus Klotho increased both ROS production in HVSMC and NO production in endothelium. FGF23 induced contraction in phosphate preconstricted vessels and increased ROS production. Phosphate, Klotho and FGF23 together induced no change in vascular tone despite increased ROS production. Moreover, the three compounds combined inhibited relaxation despite increased NO production, probably owing to the concomitant increase in ROS production. In conclusion, although phosphate, soluble Klotho and FGF23 separately stimulate aorta contraction, Klotho mitigates the effects of phosphate and FGF23 on contractility via increased NO production, thereby protecting the vessel to some extent against potentially noxious effects of high phosphate or FGF23 concentrations. This novel observation is in line with the theory that Klotho deficiency is deleterious whereas Klotho sufficiency is protective against the negative effects of phosphate and FGF23 which are additive. Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in patients with chronic kidney disease (CKD) CKD is considered as a state of Klotho deficiency. α-Klotho (Klotho), originally identified as an anti-aging gene, is predominantly expressed in the kidney but is also detectable in other organs such as parathyroids, choroid plexus and human vascular tissue Klotho deficiency leads to FGF23 resistance since Klotho acts as an obligatory co-receptor in various tissues probably including the vessel wall Collectively, ample evidence suggests that Klotho and FGF23 play a role in vascular function. However, the interaction between Klotho and FGF23 has recently been found to be more complex than previously thought, requiring further clarification. In the present study, we aimed to determine direct, rapid effects of Klotho and FGF23 on vascular function and to investigate potentially underlying mechanisms. We first examined the vascular reactivity of mouse aorta to exogenous soluble Klotho and FGF23 and observed direct effects. We next asked whether the effects were related to abnormal NO and ROS generation, and then investigated potentially involved signaling pathways using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC).HVSMC were isolated in our laboratory using an explant technique from aortic tissue of healthy donors. Samples were obtained after aortic valve bypass surgery or other types of surgery on the aorta from patients with various cardiovascular diseases  who gave informed written consent in accordance with French legislation, under PROTOCOL N°2009/19. This protocol was approved by the Ethics Committee, CPP Nord-Ouest II. The investigations were performed according to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects. All of the animal studies were conformed to the principles of European Commission guidelines and all protocols were approved by our Institution's Animal Care and Use Committee .2PO4), Dulbecco's Modified Eagle's Medium (DMEM) and penicillin/streptomycin (P/S) were purchased from Sigma Aldrich . GlutaMAX™ (Glut) and Fluorescent probe, 2′, 7′dichlorodihydrofluorescein diacetate (DCF) were obtained from Invitrogen,  and fetal calf serum (FCS) from Dominique Dutcher Laboratories . FACS Canto II flow cytometer and FACSDiva software were obtained from BD Biosciences . 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate (DAF-FM DA) was obtained from Merck Millipore .For the experiments, we used Recombinant Human soluble form of Klotho and Recombinant intact Human FGF23 . Endothelial Cell Growth kit-BBE was obtained from ATCC . Phosphate .All animal studies were performed using C57/BL-6 wild-type female mice at age 8 weeks. The mice were purchased from Charles Rivers Laboratories . They were housed in polycarbonate cages in temperature- and humidity-controlled rooms with a 12-h light/dark cycle and given free access to water and regular laboratory chow .Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (150 mg/kg) and at the experimental endpoint euthanasia was accomplished by removal of the heart.2 and 95% O2 atmosphere at 37°C. The aorta rings were maintained under a 1.3 g tension  and allowed to equilibrate for 60 min. The aorta rings were constricted with KCl (70 mM) and then washed out to reach the resting level.Wild-type mice were anesthetized and a midline incision was made through the sternum to open up the thoracic cavity, and the descending thoracic aorta was carefully isolated. Each aorta was sectioned into 3.5 mm rings devoid of fat and connective tissue. The rings were placed in Kreb's-Henseleit (KH) solution under 5% COFor the study of potential vasoconstrictor effects of phosphate, Klotho or FGF23, aorta rings were exposed to cumulative treatments with phosphate (1–3 mM), recombinant mouse Klotho (0–2 nM) or recombinant mouse FGF23 (0–400 ng/ml). As the vessels were not preconstricted, only vasoconstriction was observed. To test the implication of oxygen-derived free radicals or ERK, we incubated vessels in the presence of dimethylthiourea (10 mM), a scavenger of hydroxyl radical or the ERK inhibitor U0126 (10 μM) for 40 min.The vascular effect of Klotho in association with FGF23 was studied by exposing aorta rings to both Klotho (0.8–1.6 nM) and FGF23 (10–200 ng/ml). Vascular wall tension was supplied by a force-displacement transducer and changes in isometric force recorded continuously. The contractile response was expressed as percentage of KCl contraction, taking into account resting contraction level.The vascular effect of phosphate in association with Klotho was studied by exposing vessels to phosphate 2.0 mM followed by a range of Klotho concentrations (0.4–2.0 nM). Contraction values were expressed as percentage of contraction obtained with KCl, taking into account resting contraction level. The relaxation obtained with Klotho was calculated taking into account the maximal contraction obtained with phosphate 2.0 mM. A possible involvement of NO was studied by incubating vessels with L-NNA  for 30 min before stimulation with phosphate and Klotho. The role of endothelial cells in arterial relaxation was studied by using aortic rings devoid of endothelium. The endothelium was removed by gently rubbing off the intimal surface of the aortic rings with a wooden stick. The effectiveness of this removal was demonstrated by the lack of response to acetylcholine (up to 1.0 μM).The vascular effect of phosphate in association with FGF23 was studied by stimulating vessels with phosphate 2.0 mM, followed by a range of FGF23 concentrations (10–200 ng/ml). Contraction values were expressed as percentage of contraction obtained with KCl, taking into account resting contraction level.The vasoconstrictor effects of phosphate and Klotho in association with FGF23 were studied by exposing aortic rings to phosphate (2.0 mM), Klotho (1.6 nM) and FGF23 (10 ng/ml) together.−5 M). The contractile response was expressed as percentage of KCl contraction, taking into account resting contraction level.The effects of these 3 drugs were confirmed by concomitant vessel incubation with phosphate (2 mM), Klotho (1.6 nM) and FGF23 (10 ng/ml) followed by stimulation with phenylephrine (3.10−5 M). Relaxation was calculated taking into account the maximal contraction obtained with phenylephrine.The relaxation effects of phosphate (2.0 mM), Klotho (1.6 nM) and FGF23 (10 ng/ml) were studied by incubating vessels with these 3 drugs followed by stimulation with acetylcholine  and anti-p-ERK1/2 (1∶1000). The blots were then incubated with secondary antibody to detect immuno reactive bands. The membranes were developed with the enhanced chemiluminescence detection system and a specific, peroxidase-conjugated anti-IgG antibody. Band intensity was analyzed using the GeneGenius Bio Imaging System and protein levels were normalized against total ERK1/2 for p-ERK1/2.HUVEC, obtained from ATCC, were cultured on 0.1% gelatin-coated flask in a specific culture medium  with endothelial Cell Growth kit-BBE.HVSMC were maintained in DMEM with 1% Glut, P/S and supplemented with 15% FCS.The two cell types were maintained in culture at 37°C  and used from passage 3 to passage 8.After overnight starvation in DMEM containing P/S, 1% Glut, 0.2% Bovine Serum Albumin (BSA) and 0% FCS, cells were incubated for 10 min at 37°C with DMEM 1% P/S, 1% Glut, containing phosphate (2.0 mM) and/or Recombinant Human Klotho (1.6 nM) and/or Recombinant Human FGF23 (10 ng/mL).2O2) levels were monitored using fluorescent probe, DCF. After overnight serum starvation, confluent cells were incubated with the different drugs. H2O2 production was measured by flow cytometry on detached cells resuspended in PBS and analyzed in a FACS Canto II flow cytometer. The analysis was focused on the whole cell population. Mean fluorescence intensity was calculated using FACSDiva software and expressed as percentage of control value.To evaluate ROS production, hydrogen peroxide  for 30 min. Subsequently, cells were stimulated with the different drugs for 10 min. The stimulated cells were then trypsinized and using flow-cytometry (FACSCantoII), a population of 10,000 cells was gated and segregated based on its relative fluorescence intensity. Analyses were done using FACSDiva software.After overnight incubation in starvation medium, HUVECs were stimulated with respective drugs for 10 min. After cell lysis, protein levels were quantified with Bio-Rad Dc Protein Assay kit. Proteins were separated in 10% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blots were then incubated overnight at 4°C with the following primary antibodies: anti eNOS (1∶1000), anti iNOS (1∶1000), and anti phospho-eNOS . The membranes were developed with the enhanced chemiluminescence detection system and a specific, peroxidase-conjugated anti-IgG antibody. Band intensity was analyzed using the GeneGenius Bio Imaging System and protein levels were normalized against eNOS for phospho-eNOS and β-actin for iNOS.U test to evaluate the significance of differences between groups. Differences were considered to be significant when p<0.05.Results were expressed as means ± SEM. Differences between groups were evaluated using analysis of variance (ANOVA) to assess differences between individual mean values. Comparisons between more than two means were performed by using analysis of variance (ANOVA) with or without repeated measurements. If a significant difference was found, Scheffe's test for multiple comparisons was used to identify inter-group differences. For ROS and NO determinations and for western blotting, data were analyzed using a non parametric test  to determine significant differences (p<0.05) in mean values between groups followed by Mann-Whitney 2O2 concentration in HVSMCs by phosphate (2.0 mM), Klotho (1.6 nM) and FGF23 (10 ng/ml) (B). The contractions of aortic rings obtained with phosphate, Klotho or FGF23 were abolished by dimethylthiourea . Moreover, Klotho induced iNOS de novo expression, and this effect was maintained after co-exposure with FGF23  exerted vasoconsctrictive, Klotho independent vascular effects.When Klotho was associated with FGF23, the vasoconstriction induced by FGF23 could be abolished by Klotho, probably via enhanced NO production. Interestingly, Klotho also mitigated the direct effects of combined phosphate and FGF23 on aortic contractility, thereby protecting the vessel wall. Similar complex interactions between Klotho and FGF23 at the site of the VSMC have recently been observed by Lim et al, although they examined longer time periods Finally, when studying the combined effects of phosphate, Klotho and FGF23, we observed no direct change in vascular function despite the vasoconstriction induced by each substance separately.After incubation in presence of phosphate or Klotho or FGF23, the response to phenylephrine was increased by FGF23 and endothelium dependent relaxation was decreased by phosphate. The combined stimulation with phosphate and Klotho and FGF23 did not change the contraction induced by phenylephrine, but this combination decreased the endothelium dependent vasorelaxation induced by acetylcholineMoreover, the combined exposure of these three compounds led to enhanced ROS concentration by HUVECs. As to eNOS activation, it was comparable when HUVEC were exposed to Klotho alone or in association with phosphate and/or FGF23. Thus with triple exposure there is an imbalance between the contraction effect mediated by smooth muscle generated ROS and the relaxation effect mediated by endothelial NO. The predominant phenomenon appears to be endothelial ROS generation opposing the endothelial NO effect. This observation is in accordance with the partial inhibition of endothelium dependent relaxation in response to acetylcholine.The present study has some limitations. We did not test the different compounds in vivo. Moreover, in contrast to the study of Faul et al In conclusion, although separate in vitro exposure to phosphate, soluble Klotho, or FGF23 stimulates aorta contraction directly, Klotho mitigates the vasoconstrictive effects of phosphate and FGF23 and thereby protects the vessel wall against potentially excessive vasoconstrictive effects of high phosphate and FGF23 concentrations. Our findings support the theory that Klotho deficiency in CKD is noxious whereas Klotho sufficiency is protective against the negative effects of high phosphate and FGF23 concentrations, which are additive. Since both Klotho deficiency and FGF23 excess appear to be deleterious one could speculate about the relative merits of potential therapeutic effects of Klotho stimulation versus FGF23 suppression. Since high phosphate, Klotho and FGF23 concentrations combined can induce endothelial cell dysfunction the correction of Klotho deficiency alone may not be sufficient.


Although gemcitabine is highly active in several cancer types, intrinsic and acquired drug resistance remains a major challenge. Overexpression of Bcl-2 has been associated with gemcitabine resistance. The aim of this study is to determine whether gossypol can overcome gemcitabine resistance in cell lines with high level of Bcl-2 expression in combination drug therapy. Our study demonstrated that in 10 cell lines derived from different cancers, high Bcl-2 baseline expression was observed in cell lines that were resistant to gemcitabine (GEM-R). Furthermore, synergistic effect of combination therapy was observed in gemcitabine-resistant (GEM-R) cell lines with high Bcl-2 expression, but not in a gemcitabine-sensitive (GEM-S) cell lines regardless of Bcl-2 expression. Gossypol treatment resulted in the decrease of anti-apoptotic genes such as Bcl-2 and Bcl-xl and an upregulation of the pro-apoptotic gene, Noxa. Furthermore, the addition of gossypol to gemcitabine resulted in lower expressions of anti-apoptotic genes compared to gemcitabine alone. Gene expression profiling in GEM-R and GEM-S cell lines suggest that anti-apoptotic genes such as pAkt and PI3KR2 may play important role in gemcitabine resistance, while pro-apoptotic Bcl-2 related genes  may regulate synergistic interaction in combination therapy. Yu et al demonstrated an inverse correlation between Bcl-2 expression and chemosensitivity to several drugs including adriamycin and 5-Fluorouracil in breast cancer cells. In vitro cultured cell lines derived from primary HCC tumours that express high levels of the anti-apoptotic Bcl-2 and Bcl-xl were also found to be resistant to paclitaxel Gemcitabine  is a pyrimidine nucleoside analogue of deoxycytidine commonly used in breast Gossypium Malvaceae). Initially used as a male fertility-control agent, it was recently discovered to have anti-tumorgenic properties in a variety of cancers Gossypol is a polyphenolic compound isolated from the cotton plant (The aims of this study were to (1) correlate Bcl-2 protein expression with gemcitabine resistance (GEM-R) in gastric, nasopharyngeal and breast cancer cell lines, (2) evaluate the combination of gemcitabine and gossypol in GEM-R cancer cell lines with high level of Bcl-2 expression and (3) determine the mechanisms involved in reversing gemcitabine resistance by gossypol.2 and maintained in RPMI 1640 medium  containing 10% heat inactivated fetal bovine serum  and 1% penicillin/streptomycin . Mycoplasma status of cells was routinely tested using MycoAlert™ Mycoplasma Detection Kit . Mycoplasma status of all the cell lines carried out in this study were negative. Gossypol  and gemcitabine  were dissolved in DMSO at a concentration of 10 mM and stored at −20°C. AT-101 was provided by Ascenta Therapeutics and prepared as 10 mM stock with DMSO. Primary antibodies consisting of anti-caspase-6, anti-Bcl-xl, anti-Bad, anti-Bak, anti-Bax, anti-pAKT, anti-PIK3R2, anti-calpain-1 and anti-Actin were purchased from Cell Signaling Technology . Primary anti-Noxa was purchased from Calbiochem ; and anti-Bcl-2 and anti-Mcl-1 was purchased from Santa Cruz Biotechnology . Secondary antibodies  were purchased from Cell Signaling Technology .Nasopharyngeal cancer cell lines CNE1, CNE2, HONE1 were all derived from undifferentiated Chinese NPC tumors, while HK1 was from a well-differentiated Chinese NPC ueous One Solution Cell Proliferation Assay) . Cell suspension was cultured in 96-well flat-bottomed microtiter plates at a concentration of 2×103 cells/per well and incubated overnight. Drug treatments were carried out as follows: gossypol (0.1 µM –100 µM), gemcitabine (0.001 µM–100 µM) or a combination of both drugs in varies concentrations. Each drug was tested in triplicate. The plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 72 h. Microtiter wells containing tumour cells with no drug treatments were used as controls, and wells containing only complete medium were used as blank controls for nonspecific dye reduction. Cells were incubated for 72 h before the addition of MTS solution (1 mg/mL per well) and absorbance was read at 490 nm using a spectrophotometric microplate reader . The percent cell viability to different drug concentrations was calculated as the inhibition rate of (mean absorbance of treated wells/mean absorbance of control wells)×100%. IC50 was calculated by GraphPad Prism v4.0 .To assess the chemosensitivity of tumor cells, cell viability was measured by MTS  as previously reported 50 was calculated by antilog the value obtained. All data are presented as mean ± standard error (SE) from at least two independent experiments.The dosage increments were log-transformed, allowing equal horizontal spacing of data points on the dose response curve. Spline curve was plotted from fit spline/LOWESS analysis by GraphPad Prism v4.0 and the concentration resulted in 50% cell death was determined from the curve generated. ICCells treated with drug were washed with ice cold PBS and resuspended in lysis buffer  in the presence of a protease inhibitor cocktail . Lysates were sonicated, incubated on ice for 20 min and centrifuged at 14000 rpm for 20 min at 4°C. Protein concentrations were determined using Bradford assay . 20 µg of protein samples were electrophoretically separated on 12% SDS-PAGE and electrotransferred to a PVDF membrane . Membranes were blocked for an hour at room temperature in 5% non-fat dry milk  and subsequently incubated with primary antibodies overnight at 4°C. The respective primary antibodies were used as follows: anti-Bcl-xl  at 1∶1000 dilution, anti-Bad  at 1;1000 dilution, anti-Bak  1∶1000 dilution, anti-Bax  1∶3000 dilution, anti-caspase-6  1∶1000 dilution, anti-P1K3R2  1∶1000 dilution, anti-pAKT  at 1∶1000 dilution, anti-calpain-1  at 1∶1000 dilution, anti-Actin  at 1∶3000 dilution, anti-Bcl-2  1∶3000 dilution, and anti-Mcl-1  at 1∶3000 dilution, and anti-Noxa  1∶1000 dilution. After three washes with Tween 20 in PBS, membranes were incubated for 1 hr at room temperature with corresponding horseradish peroxidase conjugated anti-rabbit and anti-mouse secondary antibodies. The membranes were washed four times with PBS/Tween 20, and the signals were visualized by ECL reagent , followed by exposure to chemiluminescence film . Immunoblot analyses were repeated at least twice for each protein tested.in vitro transcription to synthesis biotin-labeled cRNA using the IIumina® TotalPrep RNA Amplification Kit  as per manufacturer’s instructions. Labeled cRNA were resuspended with hybridization reagents and hybridized to the HumanHT-12 v4 BeadChips for 18 hours in 58°C hybridization oven. The BeadChips were washed, blocked and stained to prepare for scanning. Fluorescence intensity at each probe was scanned using the Illumina BeadArray Reader. Three independent experiments were conducted for each sample.Whole-genome expression profiling of 2 cancer cell lines (CNE2 and SNU1) were carried out using the HumanHT-12 v4 whole-genome gene expression direct hybridization assay . Total RNA from cells treated with drug was extracted using the TRIzol reagent  as per the manufacturer’s instructions. RNA samples were quantitated using a ND-1000 spectrophotometer  and assessed for degradation using the BioAnalyzer 2100  prior to gene expression array analysis. Total RNA (500 ng) was converted to cDNA, followed by an Differentially expressing genes between the drug treatment groups (gemcitabine and combination of gemcitabine and gossypol) in the two cell lines (CNE2 and SNU1 respectively) were determine based on the methodology discussed in Wong et.al 50 of gemcitabine sensitivity whereby CNE2 was found to be resistant to gemcitabine, with IC50 over 100 µM  were treated with various concentrations of gemcitabine (0.001 to 100 µM) to evaluate the chemosensitivity of gemcitabine. There was a wide range in ICr 100 µM  and HK1 r 100 µM  was resir 100 µM  and 3. W50, respectively. As expected, (−)-gossypol was more potent than racemic gossypol but there were generally less than 10fold differences in IC50 between both drugs in gastric cell lines. All subsequent in vitro studies were carried out using the racemic gossypol  were treated with gossypol and AT101 to determine their ICgossypol  as theregossypol . Unlike To determine the possible synergism interaction between gossypol and gemcitabine, combination drug treatment were carried out in all 10 cancer cell lines. With the exception of SNU1, synergism was observed in the cell lines that had high Bcl-2 expression, but not in cell lines that expressed low Bcl-2 . Collects)  and up-regulation of pro-apoptotic proteins (Noxa and Mcl-1S).To investigate the underlying molecular mechanisms of synergistic interaction between gossypol and gemcitabine, we examined the changes of pro-apoptotic and anti-apoptotic proteins in high Bcl-2 expression GEM-R cell lines . As shown in s) . An overWhole genome expression profiling was carried out in SNU1 (GEM-S) and CNE2 (GEM-R) to evaluate alternative mechanisms for their differences in drug response to gemcitabine alone and gemcitabine/gossypol combination . A totalTo further evaluate genes that may be responsible for gemcitabine sensitivity and synergism to combined treatment with gemcitabine and gossypol, we decided to focus only on apoptosis related genes in the KEGG pathway that were differentially modulated after treatment of gemcitabine alone and after combined therapy. We identified 65 genes (31 anti-apoptosis and 34 pro-apoptosis) in the apoptotic pathway that were significantly regulated in CNE2 (GEM-R), and 49 genes (23 anti-apoptosis and 26 pro-apoptosis) that were significantly regulated in SNU1 (GEM-S) , we found that most of the anti-apoptotic genes were significantly up-regulated when cells were treated with gemcitabine . ConversSimilar trend of decreased pro-apoptotic genes expressions was observed in both cell lines treated with combination drug therapy compared to cell lines treated with gemcitabine alone . HoweverTo validate the cluster of anti- and pro-apoptotic genes in CNE2 (GEM-R) and SNU1 (GEM-S) cell lines shown in We observed that there was a minor increase in the protein expression of pro-apoptotic genes, BAD, CASP6 and CAPN1 after gemcitabine treatment in both CNE2 (GEM-R) and SNU1 (GEM-S) cell line . BAD, CAHigh Bcl-2 expression has been correlated with poor clinical prognosis in cancer patients In our study, we found that nasopharyngeal, breast and gastric cancer cell lines resistant to gemcitabine had higher Bcl-2 expression, and treatment with gemcitabine resulted in an up-regulation of anti-apoptotic Bcl-2 or Bcl-xl. These observations suggested that the level of gene expression of anti-apoptotic Bcl-2 and Bcl-xl was important in gemcitabine resistance. In support of this finding, Schniewind et al found a significant inverse correlation between Bcl-xl expression and gemcitabine induced apoptosis Several reports had demonstrated the effectiveness of gossypol in targeting cancer cells with high level of Bcl-2 and its family members, by increasing cellular apoptosis when used as a single agent S was increased in a GEM-R cell line treated with gossypol or combined treatment . In contrast, cell lines that was sensitive to gemcitabine regardless of their Bcl-2 levels, showed antagonism to combination treatment with gossypol. Nonetheless, our findings suggested clinical application for treatment management in patients bearing gemcitabine resistant tumours that expressed Bcl-2, and may benefit from the synergistic interaction of combination therapy with gossypol.Figure S1Isobolograms showing the interactions bwtween gemcitabine and gossypol. These results were generated by CalcuSyn software for (A) CNE2, (B) MCF7, (C) YCC16, (D) SNU1, (E) CNE1, (F) HONE1, (G) HK1, (H) SKBR3, (I) T47D and (J) AGS. The ED points located on the lower left of the diagonal indicating synergism.(TIF)Click here for additional data file.Table S12702 significant genes corresponding to 61 significant pathways differentially expressed between the treated and untreated cell lines.(XLS)Click here for additional data file.Table S2Pro- and anti-apoptosis genes that are significantly regulated in treated and untreated cells lines.(XLS)Click here for additional data file.


The authors retract this publication due to concerns about the cell lines employed in the study.One part of the study involves an assessment of the effects of NDRG2 on the expression of pVHL, HIF-1α and VEGF in 786-O cells. After the publication of the article, a reader raised concerns that the human renal cancer cell lines 786-O and A498 used in the study lack HIF-1α. In addition the cell line 786-O has been reported to be VHL negative. The authors have completed a short tandem repeat analysis on the cell lines and this has revealed cross-contamination, which compromises the relevance of the work to renal cell carcinoma, and thus the conclusions of the study.In the light of this, the authors retract this publication.


Replication-incompetent adenovirus vectors have shown promise as vaccine candidates. We are developing replication-competent adenovirus vectors to increase antigen expression and duration and to facilitate mucosal routes of vaccine delivery. We have developed replication-competent Ad5 (rcAd5) and Ad26 (rcAd26) based vectors, tested their growth in human and simian cell lines, and determined the dynamics of virus shedding after inoculation of rhesus monkeys.Replication-competent Ad5 and Ad26 vectors were produced by adding the E1 region back into the vector. To facilitate rcAd5 growth in rhesus monkey cells, 2 host range mutations were also introduced into the DNA binding protein. The growth of rcAd5 and rcAd26 was tested in human  and simian (CV-1 and Cos7) cell lines. In addition, rcAd5 was administered intranasally to rhesus monkeys, and the kinetics of viral shedding was determined by qPCR on nasal, oral, and rectal swabs, and serum.As expected, replication-incompetent Ad5 and Ad26 grew in the E1-complementing cell lines Per55K and 293, but not in A549 or CV-1 cells. In contrast, rcAd5 and rcAd26 grew in all human and simian cell lines tested, although rcAd26 growth was suboptimal in simian cells. After intranasal inoculation of rhesus monkeys, rcAd5 viral sequences could be detected by qPCR in nasal swabs for 5 weeks post-inoculation.Replication-competent Ad vectors can be produced efficiently by the re-introduction of E1 into standard replication-incompetent vector backbones. However, the extent of replication in simian cells appears to vary based on Ad serotype. Future studies will compare the immunogenicity of replication-competent vs. replication-incompetent Ad vectors.


A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.Q61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAF NRAS and BRAF provide oncogenic driver signals in melanoma of skin origin through the constitutive activation of the mitogen-activated protein kinase (MAPK) pathway -based colorimetric cell proliferation assay  as previously described .Cells were treated with different concentrations of vemurafenib, metformin, the combination or parallel vehicle control for 24 hours, fixed with Cytofix/Cytoperm , washed with Perm/Wash (BD Biosciences), and then resuspended in sterile PBS containing 0.5 μg/mL DAPI (Sigma-Aldrich). Flow cytometry analyses for this and other experiments was performed in an LSR-II (BD Biosciences) and data was analyzed using FlowJo .Cells were plated in 6-well plates and treated with DMSO, vemurafenib, metformin, the combination or 1 μM staurosporine as a positive control. After 72 hours apoptosis analysis was performed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) following the manufacturer's instructions and analyzed by flow cytometry.Staining of phosphorylated AKT (p-AKT Thr308) in melanoma cells exposed to DMSO, vemurafenib, metformin or the combination for 24 hours was performed in permeabilized cells using the phosphoflow technique as previously described  and analWestern blotting was performed as previously described . PrimaryCells were plated in standard high glucose RPMI media (3000 mg/L). After 24 hours, media was aspirated and low glucose media was added to the cells for at least 4 hours before treatment. Low glucose media consisted of RPMI media without glucose, supplemented with L-glutamine (Mediatech), to which 1000 mg/L D-glucose (Sigma-Aldrich), 10% FBS and 1% PSF were added. Cells were treated with different concentrations of DMSO, vemurafenib, metformin or the combination in low glucose media. For incubations longer than 24 hours, drugs were added freshly every day.50] and the shape of the dose-effect curve  to calculate the combination index (CI). A CI equal to 1 indicates an additive effect; a CI less than 1 indicates synergy. With the use of CalcuSyn software, synergy is further refined as synergism (CI = 0.3-0.7), strong synergism (CI = 0.1-0.3), and very strong synergism (CI < 0.1).To determine synergistic, additive, or antagonistic effects of the drug combinations we used the combination index method of Chou and Talalay  using thin vitro exposure to this agent [V600E) and 7 cell lines with an NRAS (NRASQ61L or NRASQ61K) mutation [in vitro derived sub-lines resistant to vemurafenib, harbors mutations both in BRAFV600E and NRASQ61K [V600E mutant cell lines, while NRASQ61 or double wild type cell lines were resistant  or moderate (M257) sensitivity to single agent metformin. In four out of six cell lines with synergistic characteristics, the effect of the combination was greater than the sum of each individual drug effect. In the rest there was either an effect due to the sum of each drug alone or no apoptotic effect at all. Cell lines with antagonistic characteristics showed no effect or an effect less than the sum of single agents. Interestingly, in the cell line SKMEL28 (antagonistic) the combination showed a greater apoptotic effect compared to either single drug treatment.Effects on apoptotic cell death were studied by flow cytometry using co-staining with Annexin V and Propidium Iodide Figure . As expes Figure . Single V600E mutant cell lines is presented in Figure V600E mutant cell lines had significant decrease with either single agent or combination containing vemurafenib. Single agent metformin had minimal effects on pERK or pLKB1, although in some cases , or the sensitivity to single agents or the combination. In fact, the combination of the drugs did not demonstrate differential effects on pAKT compared to either drug alone.Given a lack of a clear correlation between sensitivity to the combination and signaling changes in the MAPK and LKB1-AMPK pathways, we used the quantitative phospho-flow assay to better explore potentially differential effects on AKT phosphorylation that may be missed with Western blot analysis. Vemurafenib, metformin or the combination resulted in decreased phosphorylation of AKT in BRAFStandard cell culture media is supplemented with glucose at concentrations that are much higher than physiological concentrations in tumors. It is possible that the high glucose concentrations in cell culture experiments would offset the effects of AMPK activation by metformin. Therefore, we performed experiments using low glucose media compared to standard high glucose-containing media. Side-by-side studies in the M263 cell line with high or low glucose media demonstrated that there was no change in cell proliferation in MTS assays, cell cycle effects or apoptosis by flow cytometry (data not shown). Western Blot analysis . Interestingly, even though pAMPK levels at 24 hours did not correlate well, pACC levels were always increased when pERK was inhibited. Therefore, effects of vemurafenib blocking oncogenic BRAF studies ,10. An iogenesis .V600E and NRASQ61 mutant melanoma cell lines. A time course experiment with M263 showed that this decrease was observed 8 hours after treatment. Since the metformin-induced increase in pACC was noted earlier, it suggests that the inhibitory effect of metformin on the MAPK pathway may be mediated by a feedback mechanism. Interestingly, pACC was elevated in all cell lines, indicating that AMPK activity must have been increased by metformin, since AMPK is responsible for ACC phosphorylation. ACC phosphorylation was dependent on the sensitivity to the drug, with those sensitive showing the strongest increase in pACC. Of note, research by Guo et al. [The exact mechanism by which metformin activates AMPK, and if AMPK is the primary therapeutic target of metformin, is a matter of debate. One issue that comes up is the relatively high concentrations of the drug (1-20 mM) that are required for AMPK activation in cultured cells. Estimated concentrations in human plasma after a standard therapeutic dose of around 30 mg/kg range from 10-40 μM. An explanation for this could be the low level or lack of expression of the transporter OCT1 in many cultured cell lines . Other mo et al.  with AICV600E and BRAFWT cell lines, including NRASQ61 mutants. However, the differential effects of this combination could not be attributed to known oncogenic mutations nor differential modulation of MAPK, PI3K/AKT or LKB1-AMPK signaling pathways. In particular, the combination did not reverse natural or acquired resistance to vemurafenib in BRAFV600E mutant cell lines, and the effects on BRAFWT cell lines could not be explained by a unified effect on the signaling pathways studied by us. Given the observation of metformin paradoxically stimulating the growth of one cell line in our panel, and the antagonistic effects of the combination of metformin and vemurafenib in several cell lines, the combination of vemurafenib and metformin should not be considered clinically until a more detailed understanding of their differential effects is generated.Our studies provide evidence that the combination of an inhibitor of the MAPK pathway with an AMPK activator has synergistic antitumor effects in some BRAFacetyl CoA carboxylase (ACC); AMP-activated protein kinase (AMPK); cancer Osaka thyroid (COT); combination index (CI); dimethyl sulfoxide (DMSO); 50% inhibitory concentrations (IC50); insulin-like growth factor-1 receptor (IGF1R); liver kinase B1 (LKB1); materials transfer agreement (MTA); tetrazolium compound [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS); organic cation transporter-1 (OCT1); phosphorylated Erk (pErk); platelet-derived growth factor receptor beta (PDGFRβ); receptor tyrosine kinases (RTKs)Antoni Ribas has received honoraria from Roche-Genentech, the maker of vemurafenib.FN, BC-A and AR planned the research. FN performed the majority of the experiments. She received help from EvE, NA and DG on the performance of Western Blot analysis, from DG on the use of metformin, from DM, HS and CN for the care of melanoma cell lines and cell viability studies and from BC-A for the optimization and conduct of cell viability, cell cycle, apoptosis and phosphoflow studies: JAG, JAR, RSL and PSM helped focus the research plan and discussed the proposed experiments, as well as provided interpretation of the emerging results and the studies that should follow. FN and AR wrote the manuscript, with significant contributions and edits from JAR, RSL and PSM. All authors read and approved the final manuscript.Western blot analysis of two cell lines with synergy with the combination. To analyze the phospho-protein signaling events triggered by exposure to vemurafenib and metformin, cell lines were treated with DMSO, vemurafenib (5 μM), and metformin (10 mM), either singly or in combination, for 24 hours. Protein phosphorylation was examined by Western Blot analysis and phospho-specific flow cytometry. Blotting for total proteins and β-actin was used as a loading control. Phosphorylation was analyzed with antibodies against pRAF, pMEK, pERK, pLKB1, pAMPKα, pACC, pS6K, pS6, and their total proteins. a) BRAFV600E mutant cell line M263; b) NRASQ61L mutant cell line M207.Click here for fileTime course analysis of signaling in two BRAFV600E mutant cell lines with different response to the combination. Cells were treated for 2, 4, and 24 hours with 5 μM vemurafenib, 10 mM metformin, or the combination. a) M263, in which the combination showed slight synergistic effects in proliferation assays; b) SKMEL28, in which the combination had slight antagonistic effects in proliferation assays.Click here for filePhospho-specific flow cytometry for p-Akt Thr308. Cells were treated with vemurafenib (5 μM), metformin (10 mM) or the combination for 24 hours, and intracellularly stained with a pAKT Thr308 antibody. Percentages shown are in relation to DMSO controls and were analyzed by flow cytometry.Click here for fileV600E mutant cell lines M263 (top) and SKMEL28 (bottom) in low glucose mediaTime-course Western blot analysis of the BRAF. For these studies, the BRAFV600E mutant cell lines M263, resistant to vemurafenib and sensitive to metformin, and SKMEL28, resistant to vemurafenib and metformin, were treated with agents dissolved in low glucose media (1000 mg/L) instead of normal RPMI (3000 mg/L) and analyzed at 4, 8 or 24 hours.Click here for file


Graft-versus-Host Disease (GvHD) is the most serious complication of allogeneic stem cell transplantation (SCT) and results from an activation of donor lymphocytes by recipient antigen-presenting cells (APCs). For a long time, it has been postulated that the intestinal microflora and endotoxin exert a crucial step in this APC activation, as there is early and severe gastrointestinal damage induced by pretransplant conditioning. With the detailed description of pathogen-associated molecular patterns and pathogen recognition receptors single nucleotide polymorphisms of TLRs and especially NOD2 have been identified as potential risk factors of GvHD and transplant related complications thus further supporting the crucial role of innate immunity in SCT, related complications. Gastrointestinal decontamination and neutralization of endotoxin have been used to interfere with this early axis of activation with some success but more specific approaches of modulation of innate immunity are needed for further improvement of clinical outcome. Graft-versus-Host Disease (GvHD) in its acute and chronic form is the major cause of mortality and morbidity following both, experimental and clinical allogeneic stem cell transplantation (SCT). Donor T-cells activated by major or minor histocompatibility antigens on host antigen presenting cells (APCs) are the essential players in the pathophysiology of GvHD , and T-cMajor target organs of acute GvHD are the skin, the liver, especially bile duct epithelia, the gastrointestinal tract and still controversially discussed, the lung. In chronic GvHD, oral manifestations and again, the pulmonary involvement of bronchiolitis obliterans (BOs) are frequent. A common denominator of these organs is that they present epithelial surfaces with strong immunological interactions between commensal and pathogenic bacteria, epithelial barrier and defence mechanisms and the immune system which are usually in a perfect balance to maintain a status of immunological tolerance . These epithelial defence mechanisms are heavily disturbed by epithelial damage through pretransplant conditioning which is the first step in the SCT procedure and includes high-dose cytotoxic therapy or total body irradiation (TBI). Although conditioning aims to eliminate the recipient's leukemia and achieve myeloablation, in both, experimental and clinical SCT, it has become clear that increasing the dose of TBI from 900 to 1300 cGy in mice and from 12 to 15,5 Gy clinically was associated with increased epithelial damage, more severe GvHD and inferior outcome –6. SimilIn 2 clinical trials, gastrointestinal toxicity was directly assessed by either monitoring diarrhea during the aplastic phase  or direcα axis has been postulated as a major mechanism of acute GvHD. In humans, TLR4 is considered to represent the classical endotoxin receptor. With the characterization of the human genome, single nucleotide polymorphisms (SNPs) for many genes have been described which translate into altered functions of these genes. In the setting of allogeneic SCT, TLR4 SNPs have been assessed by 2 groups which however, reported opposing effects, either protection or enhancement of GvHD in the presence of TLR4 SNPs [In murine GvHD, endotoxin has been identified as a major mediator of inflammation involved in initiation of intestinal and systemic GvHD. Genetic susceptibility as well as direct antagonisms against endotoxin has a major impact on the occurrence and severity of experimental GvHD , 13 and LR4 SNPs , 15.κB and/or activation and cleavage of IL-1β [Beyond TLR4, there is a large set of further TLRs recognizing other microbial patterns including further bacterial and viral ligands. SNPs have been described for most of these TLRs, and recently, presence of the homozygous TLR9 variant in the patient has been associated with improved survival and a reduced relapse rate following allogeneic SCT . In addiof IL-1β . Based oof IL-1β . We and of IL-1β , in largSince our first description of association of NOD2 variants with GvHD and mortality  several −/− mice and bone marrow chimeras were used as recipients in experimental BMT models. These data confirmed an accelerated mortality from GvHD if hematopoietic cells were NOD2 deficient, and O' Penack and his group identified the antigen-presenting cell (APC) as the target of NOD2 deficiency [−/− mice induced a much stronger alloreaction as compared to wild-type mice pointing to a deficiency of immunoregulatory APC molecules. Recently, our group addressed the immunohistopathology of skin and gut GvHD in relation to absence and presence of NOD2 SNPs: In both tissues, skin and gut, recipient NOD2 SNPs had no impact on the extent of apoptosis as the hallmark of GvHD nor on the CD8 and macrophage infiltrate in the biopsies. However, we observed a uniform reduction of CD4 cells in the presence of NOD2 SNPs suggesting that GvHD especially in these patients is characterized by a loss of protective CD4 population including a loss of regulatory T-cells [However, a further and even more important aspect of these observations is the implication of these molecules in pathophysiology. Our observation of an association of bronchiolitis obliterans syndrome with NOD2 SNPs suggest that this interference of SNPs with epithelial defence mechanisms applies to all epithelial tissues , and it ficiency . APCs fr T-cells . NOD2 havan Bekkum's work elegantly elaborated the role of the intestinal microflora and is up to now the basis for the gnotobiotic approach to modulate GvHD. In his early experiments he did not only show protection from GvHD induced by bone marrow transplantation in gnotobiotic mice. He also reported that subcutaneous fetal gut implants revealed attenuated histopathological GvHD if the carrier mice were germ-free prior to allogeneic transplantation indicating a systemic effect of decontamination . SimilarE.coli sera, and an increased antibody titer against a certain E. coli strain in patients was associated with a lower incidence of severe GvHD [Further evidence for a role of the intestinal flora can be derived from attempts to neutralize endotoxin. In 1987, Cohen reported protection of mice by passive immunization with anti-ere GvHD , 41. In ere GvHD .Lactobacillus rhamnosus) in an experimental BMT model. Indeed, feeding of lactobacilli reduced severity of experimental GvHD and improved survival. The systemic effect of probiotic bacteria could be demonstrated by a reduction of splenic donor T-cell proliferation further demonstrating that activation of innate immunity sets the state for subsequent adaptive alloreactions [Based on positive reports in inflammatory bowel disease, our group tested the use of probiotic bacteria  in intestinal antigen-presenting cells. Experimental data suggest a strong impact of this enzyme in GvHD pathophysiology , and ong


Cytokines and growth factors have biologic effects that could stimulate tumor growth, invasion and angiogenesis. The incidence of 24 factors was investigated in 25 cultured human melanoma cell lines and in 62 fixed tissues at different stages of the disease. Over 80% of the human melanoma cell lines expressed TGF-β, IL-8, IL-6, VEGF, PDGF-AA and OPN. Significantly higher TGF-β, IGF-1 and IL-15 were determined in primary lesions compared to distant metastases by immunohistochemistry. Illustrating the complexity of the milieu of the tumor microenvironment, some of these factors may have to be considered in targeted therapy. In vitro studies seem to indicate that cultured melanoma cell lines produce excessive levels of several cytokines and growth factors, which have various biological effects. Some of them function as autocrine factors and/or paracrine factors and are capable of stimulating tumor growth, invasion and angiogenesis [in situ overexpression of these factors was studied in melanoma tissues from various stages of the disease utilizing immunohistochemistry (IHC). We focused our IHC studies on overexpression of cytokines and growth factors in primary and metastatic melanoma in order to delineate potent potential therapeutic targets. Our IHC study excluded stromal expression of these factors to define overexpression of factors only by the melanoma cells in the progression of the disease.Many studies have implicated cytokines and growth factors in tumor growth, survival and migration in various human cancers. Melanoma has multifactorial etiology and its genetic and immunological background has not yet been clearly elucidated. ogenesis ,2. Otherogenesis ,4,5, andogenesis ,7. It isEighteen of 24 factors were detected by ELISA at different incidences and levels in the spent medium from human melanoma cell lines after seven days of culture. Each cell line secreted 5-12 factors. Six factors were not identified in any of the spent medium samples. All cell lines secreted IL-8 and TGF-β, and 95% secreted VEGF-A as can be seen in Melanoma cell lines derived from two patients who had recurrent metastases generated data exemplifying the heterogeneity of melanoma. The results from ELISA assays for platelet derived growth factor (PDGF-AA), osteopontin (OPN), and placenta growth factor (PlGF) can be seen in i.e., extent. The natural history of the melanoma was considered by assessing lesions from primary sites, local recurrences, intransit metastases, lymph node metastases and distant metastases. There was extreme variation in the expression of cytokines and growth factors among different stages of the disease. For statistical analysis of the two extremes, Fisher’s Exact test was applied to the IHC data from the primary lesions and the distant metastases. High levels of expression of cytokines and growth factors were evaluated in terms of intensity of staining as well as the percentage of immunostained tumor cells, in vitro studies utilizing melanoma cultured cell lines revealed that melanoma cells express a variety of cytokines and growth factors, and some of them may stimulate tumor angiogenesis, growth and invasion [Clinical trials in the management of melanoma have shown that some cytokines are beneficial and have anti-neoplastic effect such as interferon α-2b and interleukin-2. The present invasion , can be invasion . These finvasion  for effeinvasion .Ex vivo expansion of tumor specific lymphocytes using IL-15 and IL-21 produced fewer Tregs and more cytolytic cells against melanoma cell lines [Interleukins detected in the culture spent medium were IL-8, IL-6, IL-10, IL-1α and IL-15. All of the melanoma cell lines secreted IL-8. IL-8 is an angiogenic factor expressed at high levels in a variety of vascular cancers . IL-8 bill lines . Howeverll lines .Conversely, the interleukins IL-4, IL-13, IL-17 and IL-18 were not detected in the spent medium from our human melanoma cell lines. These cytokines have immune modulating functions. IL-4 and IL-13 are produced by Th2 cells polarizing a humoral immune response. IL-17 is produced by Th17 effector T helper cells. Its proinflammatory function has dual effects on cancer, promoting antitumor cytotoxic T responses but also promoting angiogenesis and metastasis . IncreasOther cytokines detected in the spent medium were osteopontin (OPN), tumor necrosis factor-α (TNF-α), interferon-alpha (IFN-α) and granulocyte-macrophage colony stimulating factor (GM-CSF). OPN is an extracellular structural protein which is expressed in a wide range of immune cells. It has multiple functions involving cell adhesion, chemotaxis, invasion, anti-apoptosis and regulation of cell signaling in almost all malignancies. The elevated serum level is associated with poor prognosis . TNF-α iTransforming growth factor-beta 1 (TGF-β1) was detected in the spent medium of all 25 of the melanoma cell lines and also was statistically significant in the IHC intensity comparison of primary lesions versus metastases. TGF-β1 is initially a tumor suppressor, but once the tumor is established, its signaling can enhance tumor progression. Some tumors can escape the suppressive effect through mutation of receptor I/II but this mechanism of resistance has not been identified in melanoma . Vascular endothelial growth factor-A (VEGF-A) is secreted by normal and tumor cells, with a significant portion bound to the endothelial surface and extra-cellular matrix . It has Platelet derived growth factors PDGF-AA and PDGF-AB are members of a family of isoforms that stimulate the growth, survival and motility of fibroblasts, smooth muscle cells and other cell types. Beside their mitogenic activity, they activate fibroblasts to produce insulin-like growth factor-1 (IGF-1), which in turn stimulates the growth of some malignant cells that express its receptor . IGF-1 (insulin-like growth factor 1) is a potent growth and survival factor secreted in 33% of the cell lines. It promotes proliferation and cell survival through activation of MEK/ERK and P13-k/Akt signaling cascades in the cell . IGF-1 sPlacenta growth factor (PlGF) was secreted in 59% of the cultured cell lines and seen in the majority of the melanoma tissues by extent of IHC staining in TGF-α contributes to changes in gene expression resulting in uncontrolled growth and avoidance of apoptosis ,45. FGF-b (fibroblast growth factor-basic) stimulates angiogenesis and cell growth of cells from mesodermal and muco-ectodermal origin including endothelial cells and fibroblasts. It regulates proliferation, differentiation and migration . EGF  is an angiogenic factor and very potent mitogen for endothelial cells ,48. It hPDGF-BB was not secreted by any of our cultured cell lines. However, it was evaluated because of the co-expression of PDGF-B chain and its receptor in soft tissue sarcoma . PDGFB dThere were no concordance between the levels of secreted factors of the cultured cell lines and their IHC expression in paraffinized tissues. While cultured cell lines consisted of actively dividing cells in an artificial environment, IHC expressed the results in fixed tissue from a snapshot in time. The cultured cell lines were established from lymph node and distant metastases, whereas IHC expressed factors presented at different stages of the disease. While it was expected that some cytokines/growth factors would increase with the progression of the disease, the opposite results were obtained. The results of IHC revealed that there were no significant differences among local recurrences, intransit, regional lymph node or distant metastases. There were more factors overexpressed in primary lesions  than in metastases . This may indicate that these factors may be required in the early stages of the disease and to a lesser extent in later stages.The results from patients with recurrent metastases at different sites suggest that various clones of tumor cells can be present at various sites, in the same patient and on the same day. This may have an impact on the therapeutic approaches, especially autologous vaccines, and explain the reason for initial failure or initial response then failure to therapy. Repeated biopsies of recurrences and metastases may be required to assess the status of various cytokines/growth factors in order to redirect therapy.GM-CSF is of special interest as it is being administered clinically as adjuvant therapy to induce APC for melanoma vaccines. Dendritic cells and macrophages are effective in capturing dying tumor cells and inducing cellular and humeral immunity . On the Twenty-five human melanoma cell lines were established from metastatic tumors of consenting patients. The majority of the cell lines  were der2, the floating debris was discarded and fresh complete medium was added. The cultures were fed 2–3 times per week, replacing about half of the spent medium. Melanoma cell lines were split when near confluence and subcultured at 4 × 104 viable cells per cm2 surface area in polystyrene culture flasks. Cultures were shown to be free of mycoplasma contamination using the MycoProbeTM mycoplasma detection kit . To insure that each cultured cell line consisted of melanoma cells, each cell line was stained for melanoma-specific antigens MART-1, gp100, TRP75, or melanoma-associated chondroitin sulfate proteoglycan (MCSP) and analyzed by flow cytometry. At least one of these four melanoma antigens was observed in order to include the cell line in the study. All cell lines were early passages, ranging from 2 to 36 passes. Single cell suspensions were prepared from freshly resected specimens by mechanical mincing. No enzymatic dissociation was used. Viable tumor cells were cultured in Iscove’s medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. After overnight incubation at 37 °C with 5% COExpression of melanoma antigens MART-1, gp100 and TRP75 was determined by permeabilizing with Cytofix/Cytoperm (BD Biosciences) and Perm/Wash solution (BD Biosciences), staining 30 minutes at room temperature protected from light and washing with Perm/Wash solution. Surface staining for MCSP included the viability stain 7AAD. Surface antigens were stained for 15 minutes at room temperature protected from light and washed with 0.5% BSA-PBS-0.1% sodium azide solution. Samples were centrifuged at 450× g for 5 minutes, stored in 1% paraformaldehyde-PBS pH7.25 and run through the FACSCalibur flow cytometer (BD Biosciences) within 24 hours of fixation. Isotype controls were performed for each test. Antibodies used to stain for melanoma antigens are listed in After seeding 3–4 million viable cells, cultures were fed 2–3 times per week by measuring the amount of spent medium that was removed and replacing it with fresh medium. Samples of the spent medium were collected after 7 days of culture to allow for approximately 2–3 doublings and concentrations of factors to accumulate. Commercially available ELISA kits  were used to measure the factors secreted by the melanoma cell lines. The lowest standard on the standard curve was considered the ELISA sensitivity. Complete Iscove’s-10% FBS was used in the standard curves and as the blank in the ELISA assays to control for any potential cross reactivity from bovine cytokines/growth factors in the complete medium. Triplicate experiments were performed for each factor. The spent media were evaluated for the presence and levels of 24 factors in 17 to 25 cell lines. This study does not attempt to evaluate the functional level of factors but rather evaluates the complexity of the mixture of factors secreted by melanoma.Five-micron thick sections obtained from 62 fixed melanoma tissues (paraffin blocks) were stained for 17 factors by IHC. The specimens represented 12 primary lesions, 10 local recurrences, 6 intransit metastases, 15 regional lymph node metastases and 19 distant metastases. Each case was individually stained for each antibody utilizing commercially available monoclonal/polyclonal antibodies  and biotA component of the heterogeneity inherent in melanoma is the secretion and expression of various cytokines and growth factors at variable levels. The high incidence of expression of TGF-β, IL-8, IL-6, VEGF, PDGF-AA, OPN, IGF-1 and IL-15 by human melanoma supports consideration of these factors in future planning of therapy. Due to the heterogeneity of melanoma, repeated biopsies of metastases may have to be considered to redirect future therapy as personalized medicine develops. It will be the challenge of personalized medicine to develop appropriate diagnostics for criteria of therapeutic choices.


Consequently, egg cells have become an object of demand for both infertility treatment and stem cell research, and this raises complex legal, ethical, social and economic issues.Since the successful introduction of In this paper we compare how the procurement and use of human egg cells is regulated in two countries: Israel and Austria. Israel is known for its scientific leadership, generous public funding, high utilization and liberal regulation of assisted reproductive technology (ART). Austria lies at the other extreme of the regulatory spectrum in terms of restrictions on reproductive interventions.In both countries, however, there is a constant increase in the use of the technology, and recent legal developments make egg cells more accessible. Also, in both countries the scarcity of egg cells in concert with the rising demand for donations has led to the emergence of cross-border markets and global 'reproductive tourism' practices. In Israel, in particular, a scandal known as the 'eggs affair' was followed by regulation that allowed egg cell donations from outside the country under certain conditions.Cross-border markets are developed by medical entrepreneurs, driven by global economic gaps, made possible by trans-national regulatory lacunae and find expression as consumer demand. The transnational practice of egg cell donations indicates the emergence of a global public health issue, but there is a general lack of medical and epidemiological data on its efficacy and safety. We conclude that there is need for harmonisation of domestic laws and formulation of new instruments for international governance. Consequently, egg cells have become an object of demand for both infertility treatment and stem cell research. However, the procurement of egg cells involves conflicting interests and raises multiple concerns and ethical issues. In this paper we compare the approach to egg cell donations in two countries with populations of comparable size (around 8 million): Israel and Austria. First we provide a general background on the medical practice of egg cell donations for infertility therapy, the commercialization of reproductive medicine and the concerns about the commodification of women's body parts, given the intrinsic scarcity of egg cells and the growing demand for egg cells both for reproduction and for stem cell research. Then we compare ART utilization rates in Israel and Austria, and the different regulatory approaches. Finally we focus on the emergence of cross-border markets in reproductive tourism, in which egg cells become a coveted commodity, as a result of economic gaps and differences in law and policy between countries. We conclude that there is need for harmonisation of domestic laws and formulation of new instruments for international governance.Since the successful introduction of in vitro fertilisation (IVF) baby, Louise Brown, in 1978, it is estimated that 3.75 million babies have been born world wide with the assistance of medical reproductive technologies Health risks of egg cells procurement.Click here for file