Resistance to cancer chemotherapy is a major problem. Herb-drug combinations can offer a new potential to overcome such resistance. To quantitatively assess chemosensitizing effects of herb-drug combinations (HDC), a new approach that takes into account both the chemosensitizing effect (CE) and “safety” considerations of the HDC is proposed and used in describing the cytotoxic activity of Tripterygium wilfordii-docetaxel (TW-Dtx) combination on prostate cancer cell lines.50 of herb (H), drug (D) alone and in combination  in resistant cells were obtained. CE and chemosensitizing utility index (CUI) were calculated as: CE = IC50D IC50CD; CUI=CE (IC50H/ConH) whereas ConH is the H concentration in the combination.The effect of two extracts of TW with Dtx on Dtx resistant PC3 and DU145 cell lines were compared. Cell viability (cytotoxicity) was determined using sulforhodamine B assay after incubation of the cell line. The IC50CD (nM) from TW extract A-Dtx treatment in resistant PC3 cells were 3.8 , 5.8, and 5.8, respectively versus 17.1, 22.0, and 1.5, respectively from TW extract B-Dtx treatment when low ConH relative to IC50H was used. The corresponding values from extract A versus B-Dtx treatment in resistant DU145 cells were >1.3, >4.5 and 75.3 versus >27.6, >55.2 and 3.63, respectively. However, CE values can dramatically increase with higher ConH. The values for CE (fold change), CUI (fold change), and IC50CD are preferred and may prove to be a useful practical tool for assessing chemosensitizing effect of other HDCs.Based on the above CE, CUI or ICCD values, TW extract B-Dtx appeared to be consistently superior to extract A-Dtx combination. However, assessment based on CE value alone may be misleading since it can change dramatically with ConH used. CUI together with IC


Saccharomyces cerevisiae. HTZ1, a regulator of chromatin structure and gene expression, represents a class of genes implicated in mutational robustness. It had previously been shown to increase robustness of yeast cell morphology to fluctuations in the external or internal microenvironment. We measured morphological variation within and among 79 MA lines with and without HTZ1. Analysis of within-line variation confirms that HTZ1 increases microenvironmental robustness. Analysis of between-line variation shows the morphological effects of eliminating HTZ1 to be highly dependent on the line, which implies that HTZ1 interacts with mutations that have accumulated in the lines. However, lines without HTZ1 are, as a group, not more phenotypically diverse than lines with HTZ1 present. The presence of HTZ1, therefore, does not confer greater robustness to mutations than its absence. Our results provide experimental evidence that revelation of cryptic genetic variation cannot be assumed to be caused by loss of robustness, and therefore force reevaluation of prior claims based on that assumption.Biological systems produce phenotypes that appear to be robust to perturbation by mutations and environmental variation. Prior studies identified genes that, when impaired, reveal previously cryptic genetic variation. This result is typically interpreted as evidence that the disrupted gene normally increases robustness to mutations, as such robustness would allow cryptic variants to accumulate. However, revelation of cryptic genetic variation is not necessarily evidence that a mutationally robust state has been made less robust. Demonstrating a difference in robustness requires comparing the ability of each state (with the gene perturbed or intact) to suppress the effects of new mutations. Previous studies used strains in which the existing genetic variation had been filtered by selection. Here, we use mutation accumulation (MA) lines that have experienced minimal selection, to test the ability of histone H2A.Z (HTZ1) to increase robustness to mutations in the yeast  Natural populations typically harbor much genetic variation. Some of this variation is cryptic — it does not affect observed traits except if the organism is exposed to a major environmental or genetic perturbation. One often-proposed explanation for the revelation of cryptic genetic variation is that the perturbation has made the organisms less robust to mutations, thereby revealing effects of previously neutral mutations that natural selection had allowed to accumulate. Such effects would be dependent on genetic background, as particular cryptic variants will be present in some individuals and not others. We show that a perturbation of chromatin architecture caused by mutation of the budding-yeast gene HTZ1, encoding histone H2A.Z, does alter phenotypes in a genetic background-dependent manner but does not reduce mutational robustness. Using a large set of yeast lines that accumulated mutations with minimal natural selection, we compared variation in cell morphology with and without HTZ1. The effect of eliminating HTZ1 was highly line dependent, suggesting that HTZ1 interacts extensively with genetic variation in the lines. However, HTZ1+ lines span a range of phenotypes similar to that of the corresponding HTZ1− lines. Our results therefore call into question prior studies linking revelation of cryptic genetic variation with reduced mutational robustness. Biological systems produce phenotypes that appear to be robust to genetic and non-genetic sources of variation Drosophila melanogaster only after heat stress and only in some individuals Caenorhabditis elegansSaccharomyces cerevisiaeEscherichia coliD. melanogaster whose impairment can reveal cryptic variation is poised to expand as well: a genetic screen using deficiency chromosomes recently showed that there are at least 10 regions of the D. melanogaster genome containing a gene that reveals cryptic variation in wing morphology when hemizygous A particularly important gap in our understanding is that no specific gene product has been shown to confer robustness against naturally occurring mutations, over and above some baseline level of robustness that would exist in the absence of the gene product The decades of studies revealing cryptic variation have been taken as evidence that the wild-type state is more robust to mutations than the perturbed state, and that the perturbed gene products normally contribute to this robustness. However, this logic is flawed D. melanogasterE. coliTo ascertain the relative amount of mutational robustness conferred by alternative alleles, it is necessary to measure, in the context of each allele, the phenotypic effects of a large sample of spontaneous mutations. As has been noted S. cerevisiae. Yeast, due to their rapid growth rate, ease of handling, and routine genetic techniques, are highly amenable to large-scale experiments such as this one. To avoid the problem of using organisms maintained under selection when testing the extent of mutational robustness conferred by a candidate gene, we use yeast lines generated in a mutation accumulation (MA) experiment Here, we perform this test using strains of We chose to test if the presence of a particular chromatin regulator, HTZ1, confers greater robustness to new mutations than its absence. HTZ1 encodes a histone variant, H2A.Z, that can take the place of histone H2A in nucleosomes. Nucleosomes containing HTZ1 are usually found in the promoter regions of repressed or stress-responsive genes, and HTZ1 is necessary for these genes' full activation S. cerevisiae and its close relative S. paradoxusS. cerevisiae found that although chromatin regulators did not directly interact with many chemicals, their presence increased resistance to many chemicals The prediction is especially strong for chromatin regulators, which have been found in other studies to affect levels of phenotypic variation due to genetic and systematic environmental differences We knocked out HTZ1 in 79 MA lines. We first converted the existing diploid MA lines We measured phenotypic variation in cell morphology in each strain, using an established assay The main goal of this study was to test whether the chromatin protein HTZ1 increases robustness of morphological phenotypes to new mutations, by collecting morphological data on 79 pairs of HTZ1+ and HTZ1− MA lines. As described in Because the morphological assay consists of partially redundant phenotypes, we used principal component analysis (PCA) to identify orthogonal linear combinations of the phenotypes to use for downstream quantifications of morphological variation. PCA was performed separately for the three cell types , because each type has its own suite of phenotype measurements. Only principal components that explained more variance than the random expectation were used in the analysis see . This reWe estimated variance parameters by fitting a linear model. A standard approach to doing so would be to use maximum-likelihood based methods to fit mixed models in which genotype is a fixed effect and MA line is a random effect. However, we chose instead to estimate variance components using a Bayesian approach based on Markov chain Monte Carlo (MCMC) sampling see . The MCMWe compared estimates of within-line variance for HTZ1+ and HTZ1− lines to determine the effect of HTZ1 on microenvironmental robustness. As shown in We next asked if HTZ1 affects between-line morphological variation and consequently affects robustness to new mutations. The absence of a systematic effect on between-line variance supports the scenario diagrammed in Because principal components represent combinations of morphological trait values, as opposed to an individual trait such as cell circumference or budding angle, relating a principal component to a biologically meaningful phenotype can be difficult. However, inspection of individual cells from line pairs with divergent mean principal component values indicates that this difference accurately reflects underlying differences in cell morphology. For example, consistent with which original phenotypes load heavily onto each principal component, principal component 4 for unbudded cells appears to correspond to how elongated a cell is, whereas principal component 1 for large-budded cells appears to correspond to cell size .g × l, is estimated as HTZ1+ is the HTZ1+ between-line variance, VHTZ1− is the HTZ1− between-line variance and CovHTZ1+,HTZ1− is the genetic covariance between HTZ1+ and HTZ1−. A test of the significance of the genotype-by-line interaction is not possible with the MCMC approach, because: 1) the use of an information criterion that penalizes additional parameters, akin to the Akaike information criterion or Bayesian information criterion, is not well established for model selection in this context; and 2) variances are constrained to be positive and therefore credible intervals will not overlap 0 even for negligible variances. Nevertheless, an indication that the interaction variance is substantial is that it is similar in magnitude to the magnitudes of the between-line variances  instead of PCA, as we had done previously to reduce phenotypic redundancies ade2-101 mutation, yet a subset of lines displayed white sectors or colonies, suggesting an epigenetic switch was at play. We therefore repeated analyses with the restricted set of line pairs that showed stable-red inheritance. The restricted analysis yields the same general conclusions as the analysis with the full set of strains. For each principal component, HTZ1− lines have a greater within-line variance. For only two principal components did the 95% credible interval of the difference between within-line variances overlap 0. In addition, for only four principal components did the credible intervals of the difference in between-line variances not overlap 0. For three of these four, the HTZ1− lines had greater between-line variance and in one the HTZ1+ lines did. For each principal component, the magnitude of the between-line variance and genotype-by-line interaction variance is similar. The model-selection analysis showed that for all principal components, the best-fit model included an interaction term.As noted in One potential, related concern about our experimental approach is that estimates of line means and between-line variances might be sensitive to the number of lines assayed. However, it is unlikely that sampling any more lines would change our results. Estimates of the mean and standard deviation of line means for various principal components were recalculated with a line sample size ranging from 5 to 79 pairs. As shown in M, estimated from the HTZ1+ MA lines to that estimated from the HTZ1− MA lines. If HTZ1+ were to contribute to greater robustness to mutations, then the VM should be higher for the HTZ1− lines than the HTZ1+ lines. The magnitude of VM  is also of interest, as it relates to the mutational target size for the trait of interest and the neutral expectation for segregating variation M for cell morphology, which had not been previously estimated, was estimated for lines with and without HTZ1  way of framing the question of whether HTZ1 increases robustness to mutations is to compare the mutational variance, VHTZ1 see . The VM M/VE estimates are lower than those of previous studies measuring a variety of phenotypes in a variety of organisms, which tended to report VM/VE values between 10−4 and 5×10−2M/VE might increase with more precise measurements of VE, which is necessarily a combination of actual biological variation within lines and variation in measurement. Decreased measurement variance could be achieved, for example, by more precisely staging cells (reducing the variance in cell-cycle stage at which cells are measured).Our VPrevious studies have demonstrated the release of cryptic genetic variation after a genetic or environmental perturbation E. coliTheoretical studies have tended to predict congruence between robustness mechanisms, or in other words that mechanisms contributing to robustness to one source of variation will contribute to robustness to other sources S. cerevisiae and S. paradoxus upon deletion of chromatin regulators (including HTZ1) Our results highlight the importance of using MA lines for tests of mutational robustness. The lines used in this study have accumulated extensive genetic variation affecting cell morphology  and 3. IS. cerevisiae has been associated not only with increased between-strain variation for some traits but also with suppression of variation for other traits. For example, wild-type HSP90 function is necessary for some drug-resistance mutations to have their effects Our results are reminiscent of work on Hsp90 in yeast, even though that work was conducted with strains that had been subject to natural selection The present study adds to growing empirical support for the notion that pleiotropy and epistasis are widespread ade2-101, lys2-801, his3-Δ200, leu2-3.112, ura3-52, Gal+, hoHO-expressing plasmid, creating a diploid line homozygous at each locus except the mating-type locus, that served as the ancestor for all the MA lines ade2 mutation build up a red metabolite during respiration, which acts as a visual marker that cells are not “petite” or respiration-deficient Diploid yeast MA lines were provided by David Hall ade2-101 mutation, which causes colonies to be red, we noted the appearance of white or sectored colonies while propagating some of the lines. The loss of red pigmentation can indicate the presence of a petite mutation, a mutation that restores ADE2 function, or an epigenetic mechanism affecting the adenine pathway. The frequency of white-to-red switches and red-to-white switches, as well as sizes of the sectors or colonies, suggested an epigenetic factor was responsible. We hypothesize that the epigenetic factor is [PSI+], a prion form of the translation-termination factor SUP35, because [PSI+] causes increased read-through of stop codons ade2-101 is a premature stop-codon mutation Although the MA lines have the Cell morphology was measured in many cells from each pair of MA lines (HTZ1+ and HTZ1−) using a microscopy-based phenotyping assay Raw cell measurements from CalMorph were analyzed using the R programming environment. CalMorph splits cells into three types: cells with no bud, a small bud, and a large bud. Each type has a unique set of morphological phenotypes and is therefore analyzed separately throughout this study. To allow for comparisons between CalMorph phenotypes of cells of the same type, raw values were transformed using the Box-Cox method The phenotypes measured by CalMorph are not completely independent To confirm that our choice of PCA for eliminating redundancy did not unexpectedly bias results, we performed alternative analyses using partitioning around medoids (PAM), a variant of k-means clustering, as well. PAM clusters phenotypes by similarity and designates a “medoid,” the phenotype that is most representative of the other phenotypes in the cluster. PAM was used previously to eliminate redundancies in CalMorph-generated data Variance-component estimates from linear models were obtained using Markov chain Monte Carlo sampling, with the R package MCMCglmm To test for an interaction between line and genotype, linear mixed models were fitted using the lmer function from the lme4 package in R. Genotype (HTZ1+ or HTZ1−) was modeled as a fixed effect, whereas MA line and a line-by-genotype interaction term were modeled as random effects. Note that lmer assumes a single within-line variance and a single between-line variance.HTZ1+ and sdHTZ1− are the HTZ1+ and HTZ1− between-line standard deviations, respectively. This formula is identical to that given for Vg×l previously HTZ1+,HTZ1−, a term representing the correlation of line means, is replaced by The genotype-by-line interaction variance was partitioned into components that correspond to line crossing and line spreading by this formula:L is the between-line variance, for one or the other HTZ1 genotype, estimated by MCMC, discussed above. t, the number of generations, was estimated as 2062 M is the mutational variance of the haploid lines in which we actually measured phenotypes, not of the diploid lines from which they derive.Estimates of mutational variance were made using the equation At least 150 cells were measured of each cell type from each line. This number was chosen based on a sub-sampling analysis, where an increasing number of cells was randomly drawn (without replacement) and used to calculate a mean and standard deviation value. Each of these estimates tended to converge when sampling more than 150 cells, suggesting this number is sufficient to adequately estimate the line means and within-line variances for a given morphological trait  unbudded cells, (B) cells with small buds, and (C) cells with large buds. The black circles represent principal components obtained by PCA on real data. Red circles represent principal components from data randomly permuted within phenotypes before PCA was performed. Only principal components that explain more variance than the random expectation are studied further in this paper.(PDF)Click here for additional data file.Figure S2Correcting effect of median on MAD by lowess regression. (A) Line MADs plotted against medians for principal component 1 of the unbudded cell type. The black line is the lowess curve. Red circles are HTZ1− lines and blue circles are HTZ1+ lines. (B) The same data from A, with the residuals to the lowess curve, instead of the MADs, plotted against the median.(PDF)Click here for additional data file.Figure S3Mean principal component values of HTZ1+ and HTZ1− lines for each principal component in each cell class: (A) unbudded cells, (B) cells with small buds, and  cells with large buds. Each line connects an HTZ1+ MA line with its HTZ1− derivative. The means and standard deviations of line means are indicated by the black circles and bars. The mean of the ancestral strain is shown to the left in each plot.(PDF)Click here for additional data file.Figure S4Mean principal component values of HTZ1+ and HTZ1− lines, as in (PDF)Click here for additional data file.Figure S5Average silhouette widths for PAM with different numbers of clusters for each cell class: (A) unbudded cells, (B) cells with small buds, and (C) cells with large buds. MAX indicates the number of clusters that maximizes the mean silhouette width. PC indicates the number of significant principal components calculated by PCA see .(PDF)Click here for additional data file.Figure S6Estimates of mean and standard deviation of line means for principal component 1 for unbudded cells. A random subsample containing the given number of MA line pairs  was used. Red represents HTZ1− values and black represents HTZ1+ values.(PDF)Click here for additional data file.Figure S7Trait mean and median absolute deviation (MAD) as a function of sample size. Estimates of unbudded cell area mean (top) and MAD (bottom) with increasing sample size. Samples of the given size were drawn randomly from MA line 12, with HTZ1 intact.(PDF)Click here for additional data file.Table S1Analysis of between-line variance. P-values are given for Levene's test of differences in between-line variance (column 3), likelihood-ratio test for significance of genotype-by-line interaction term (column 4) and likelihood-ratio test for significance of genotype-by-line interaction term after scaling data to remove spreading (column 7) for each principal component (PC). Estimated percentages of total interaction variance explained by line crossing (column 5) and line spreading (column 6) are also given. * = PC for which HTZ1− lines have a significantly greater between-line variance than HTZ1+ lines (p<0.05 by Levene's test). † = PC for which HTZ1+ lines have a significantly greater between-line variance than HTZ1− lines (p<0.05 by Levene's test).(PDF)Click here for additional data file.Table S2Estimates of within-line variance, along with 95% credible intervals (CrI), for HTZ1+ lines and HTZ1− lines, derived from MCMC. * = medoid for which the CrI of the difference between HTZ1+ and HTZ1− in the within-line variance estimates does not overlap 0, and for which the HTZ1+ within-line variance is lower than that of HTZ1−.(PDF)Click here for additional data file.Table S3Estimates of between-line variance, along with 95% credible intervals (CrI), for HTZ1+ lines and HTZ1− lines, derived from MCMC. * = medoid for which the CrI of the difference between HTZ1+ and HTZ1− in the between-line variance estimates does not overlap 0, and for which the HTZ1+ between-line variance estimate is higher than that of HTZ1−. † = medoid for which the CrI of the difference between HTZ1+ and HTZ1− in the between-line variance estimates does not overlap 0, and for which the HTZ1+ between-line variance estimate is lower than that of HTZ1−.(PDF)Click here for additional data file.Table S4Analysis of between-line variance, using PAM instead of PCA for dimensional reduction. P-values are given for Levene's test of differences in between-line variance (column 3), likelihood-ratio test for significance of genotype-by-line interaction term (column 4) and likelihood-ratio test for significance of genotype-by-line interaction term after scaling data to remove spreading (column 7). Estimated percentages of total interaction variance explained by line crossing (column 5) and line spreading (column 6) are also given. * = medoid traits for which HTZ1− lines have a significantly greater between-line variance than HTZ1+ lines (p<0.05 by Levene's test). † = medoid traits for which HTZ1+ lines have a significantly greater between-line variance than HTZ1− lines (p<0.05 by Levene's test).(PDF)Click here for additional data file.Table S5List of MA lines used in this study. Lines that are classified as having a stable-red inheritance pattern are indicated with an X in the stability column (see text).(PDF)Click here for additional data file.Table S6Traits for each cell type measured by CalMorph and used in this study. Each trait is listed by the name used by CalMorph and is accompanied by a brief biological description of what that trait measures.(PDF)Click here for additional data file.Text S1Details of model-independent measures of within-line and between-line variance and linear mixed modeling.(PDF)Click here for additional data file.


Digit ratio (2D:4D) denotes the relative length of the second and fourth digits. This ratio is considered to be a biomarker of the balance between fetal testosterone (T) and estrogen (E) in a narrow window of early ontogeny. Evidence for this assertion is derived from direct and indirect measures of prenatal hormonal exposure  in relation to 2D:4D. In contrast, the relationships between 2D:4D and levels of sex steroids in adults are less clear, as many correlational studies of 2D:4D and adult sex steroids have concluded that this association is statistically non-significant. Here, we suggest that in order to understand the link between 2D:4D and sex hormones, one must consider both fetal organizing and adult activating effects of T and E. In particular, we hypothesize that 2D:4D correlates with organizing effects on the endocrine system that moderate activating effects in adulthood. We argue that this is particularly evident in “challenging” conditions such as aggressive and sexual encounters, in which individuals show increased levels of T. We discuss this refinement of the 2D:4D paradigm in relation to the links between 2D:4D and sports performance, and aggression. The efD:4D is thigh FE . The stCorrelational studies have also focused on the relationship between 2D:4D and the structure of the androgen receptor gene (AR), with emphasis on the number of CAG repeats in the AR.Sensitivity to T is negatively associated to CAGn, such that in general population samples the highest sensitivity is found for CAGn of about 10 and lowest sensitivity for CAGn of about 30. Therefore, we might expect that 2D:4D is positively correlated with CAGn. There is mixed evidence from studies that have investigated this relationship and a recent meta-analysis including 14 samples and 1904 participants found no association between 2D:4D and CAGn . HoweverExperimental studies of the effects of FT and FE on 2D:4D are based on the assumption that the effects of prenatal hormones on human 2D:4D are essentially similar to those observed in other mammals. Consistent with this assumption, the 2D:4D of mammals, such as chimpanzees and bonobos , mice 2, 21, andZheng and Cohn’s  study haDigit ratio has been reported to show associations with a number of behavioral and morphological traits expressed in adults , 26. ThiTestosterone has energetic and immunosuppressive costs and maintaining high resting levels in the absence of challenges may be maladaptive , 34. Thehaka”) and a control video (a blank screen). Testosterone was assayed before and after each video and an aggression questionnaire completed after each video. The aggressive video was associated with a marginally significant increase in T, but the control video was not. Low 2D:4D (this time left 2D:4D) predicted high aggression scores after the aggressive video, and the association was particularly strong in participants showing the highest increase in T. However, there were no associations between 2D:4D and aggression after the control video (There have been two studies that have considered 2D:4D and T spikes induced by exercise or by an aggressive video. (i) The participants in the former were 79 professional rugby union players. Of these, 54 players served as controls and 25 were challenged using a repeated sprint agility test. In the experimental group T was measured immediately before the test and 5 and 20 min after completion. It was found that players with low right 2D:4D relative to left 2D:4D (low Dr–l) produced the highest amount of T at all three time points. The controls showed no association between 2D:4D and testosterone . (ii) WiIf low 2D:4D predicts high T spikes in response to competition in aggressive sports and to aggressive stimuli then we may expect that low 2D:4D is associated with performance in many competitive sports.Digit ratio shows relationships with many traits, but the effect sizes of reported associations are far from uniform. For example, in sports there are considerable relationships with negative correlations of about 0.4–0.6 reported in some sports such as distance running, rowing, rugby, and surfing, but also weak associations in sprinting and strength events [see Ref.  for a meStudies on the associations between 2D:4D and sport are often focused on male participants. However, there is some evidence that low 2D:4D is linked to high levels of performance in females also , yet theWe conclude that 2D:4D is a biomarker for the balance between FT and FE, such that high FT and low FE is linked to low 2D:4D. There is evidence that 2D:4D is fixed in a relatively narrow developmental window at the end of the first trimester of pregnancy and that it does not change substantially with age. Considering 15 years of work on this topic, we feel that there is quite strong evidence for this link. However, more contentiously, we hypothesize that the relative levels of FT and FE have organizing effects on the adult endocrine system, which are particularly evident in “challenging” situations, such as aggressive or sexual encounters. This means that 2D:4D should correlate with T spikes produced under challenge and it may also be linked to response to such spikes. In consequence, low 2D:4D may be a predictor of high performance in sports and high aggression when provoked. We suggest that future studies regarding the links between 2D:4D and such traits as adult T levels, sports performance, and aggression should include aggressive and/or sexual stimuli in their protocols. In this way our hypothesized link between 2D:4D and T spikes may be tested.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation . While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. Conflict of interest:None declared. Allogeneic hematopoietic stem cell transplantation  after high-dose marrow-ablative chemoradiotherapy is an effective treatment method in various hematologic, neoplastic, and congenital disorders. The major complication after allo-HSCT is the development of graft-versus-host disease (GVHD). GVHD is a life-threatening complication even when the major histocompatibility complex is matched ,2. ImmunMultipotent mesenchymal stromal cells (MSCs) are multipotent progenitor cells that can differentiate along multiple mesenchymal lineages including bone, cartilage, or fat and expand extensively in vitro ,5. The iThe aim of this study was to evaluate the prophylactic and therapeutic potential of MSCs against GVHD using an established rat model of acute GVHD. AnimalsFemale Wistar rats of 10-12 weeks old were used as recipients and male Sprague Dawley (SD) rats as donors. All procedures were performed according to the institutional guide for animal experimentation and the study protocol was approved by the institutional ethics committee. Bone Marrow Preparation and Bone Marrow-Derived Rat MSC Generation Bone Marrow Preparation and Bone Marrow-Derived Rat MSC Generation Briefly, SD rats were sacrificed by decapitation and bone marrow (BM) was flushed with L-DMEM  using a 23-gauge needle from femurs and tibias. The BM cells were then pelleted by centrifugation at 1000 rpm for 15 min. The BM cells were gently resuspended using an 18-gauge needle and filtered through a sterile nylon mesh. The viability was consistently >95% as determined by trypan blue exclusion. For the MSC generation, BM cells were plated in 25-cm2 polystyrene flasks in L-DMEM supplemented with 10% fetal bovine serum at 37 °C with 5% CO2 conditions (Gibco). Cells were allowed to adhere for 72 h followed by the removal of nonadherent cells and media were changed every 3 to 4 days. Adherent cells were detached using trypsin-EDTA solution-B  at 37 °C for 10 min and MSCs were expanded 3-4 times to achieve the desired cell numbers for use in in vitro and in vivo experiments. Preparation of Viable Splenocytes8/500 µL. SD rats weighing between 200 and 250 g were sacrificed by decapitation to be used as donors for splenocytes. After sterile splenectomy, spleens were collected and kept on ice with L-DMEM (Gibco) supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL). The spleens were disrupted in the medium by pressing spleen fragments between 2 glass slides. Spleen cell suspensions were filtered through a 100-mesh nylon filter and washed 3 times with L-DMEM medium. Splenocyte viability was consistently >95% as determined by trypan blue exclusion. Viable nucleated cells were counted and adjusted to 4.0x10Allo-HSCT ProcedureWe generated a rat GVHD model in the context of allo-HSCT with infusion of 4x108 donor splenocytes. Allo-HSCT was performed with isolation of 1x108 mononuclear cells from male SD rat donors, dilution in 0.5 mL of phosphate-buffered saline, and infusion to female Wistar rat recipients via the tail veins. The recipient animals were conditioned with a myeloablative regimen consisting of 950 cGy of total body irradiation prior to allo-HSCT. The GVHD prophylactic regimen was arranged as 3 mg/kg/day cyclosporine-A (CsA) and 0.25 mg/kg methotrexate (MTX) intraperitoneally at days +1, +3, and +6. Experimental Design A total of 49 Wistar rats were enrolled in this study and they were randomly assigned to 4 study groups (SGs) and 3 control groups (CGs) (n=7 each). The GVHD model was generated in all SGs but only in one CG. 1. CG-I: Allo-HSCT and GVHD (enforced through donor splenocyte infusion). 2. CG-II: No allo-HSCT, myeloablative regimen only . 3. CG-III: No allo-HSCT, no myeloablative regimen (no GVHD). 4. SG-I: Allo-HSCT, only standard GVHD prophylactic regimen (CsA+MTX) on day -1 of allo-HSCT. 6 cells/kg) on day +1 after allo-HSCT. 5. SG-II: Allo-HSCT, given only MSCs  on day +1 of allo-HSCT. 6 cells/kg) after observation of GVHD findings. 7. SG-IV: Allo-HSCT, given MSCs , hyperkeratosis of the footpads, skin rash, diarrhea, anorexia, and weight loss . ImmunopStatistical AnalysisTo evaluate the effects of several variables on overall survival, Kaplan-Meier survival analysis (log-rank statistics) was performed. Differences among the treatment groups were assessed by nonparametric Mann-Whitney U tests. Analyses were managed with SPSS 10.0  and the significance level was 5% (p<0.05). Wistar recipients from male SD donors were transplanted with allogeneic hematopoietic stem cells  or MSCs as described above. The survival of the Wistar rats as shown in the Kaplan-Meier survival curve following allo-HSCT  was signStatistical evaluation was performed on day 28. Immunophenotypical  examinations and plasma IL-2 levels were performed for all groups following allo-HSCT. The expression of CD25 increased significantly (p<0.001) in all SGs and CG-I compared to CG-III. Intergroup comparisons of all SGs and CG-I in terms of CD25 expression were not statistically significant . The ratAllo-HSCT is an increasingly used treatment modality for hematological malignancies . HoweverAdministration of MSCs is an alternative option in the treatment of steroid-refractory GVHD. Immunomodulatory properties of MSCs have been exploited to reduce the incidence of GVHD after allo-HSCT ,23,24,25MSCs may be used for hematopoiesis enhancement, GVHD prophylaxis, and treatment of established severe acute GVHD in allo-HSCT patients. Previous studies have supported the use of MSCs in steroid-refractory GVHD ,23,24,27The combination of cyclosporine and short course of methotrexate is currently considered the standard prophylaxis of GVHD . There iTo conclude, clinical use of MSCs in both prophylaxis against and treatment of established GVHD seems to be effective. However, MSC infusion combined with the standard GVHD prophylactic regimen causes stronger immunosuppression, with the potential of resultant early mortality. Clinical use of MSCs in prophylaxis and treatment of GVHD after allo-BMT requires further clinical trials.The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences. The technique of SCNT, whereby a clonal embryo is generated after the fusion of a somatic cell with an enucleated oocyte, has been successfully used to generate cloned and transgenic animals in numerous species, and is the preferred tool to achieve modification of the genetic make-up of cattle. However, the generation of transgenic animals other than mice, especially those that require targeted insertion, still remains a lengthy and inefficient process In the present study, QHTMS was used to investigate DNA methylation patterns in a transgene containing sequences from various origins in primary bovine fibroblasts rederived by SCNT. We find that CpG methylation levels increase markedly in transgene sequences in all rederived cell lines and transgene expression is altered compared with the original donor cell lines. From these results, it can be implied that epigenetic processes that occur during embryonic development can influence functionality of transgene sequences which can exclude the reliable extrapolation of functional attributes from transgenic donor cell lines to SCNT derived rejuvenated cells or transgenic animals.As previously published Two of these cell lines, A and B, each originating from an independent insertion event and hence containing unique insertion sites, were selected as acceptor cell lines based on the integration of only one or two transgene copies. Subsequently, both were used as donor cells for SCNT in an attempt to rederive cell lines with rejuvenated proliferative capacity that were genetically and functionally equivalent to the original cell lines A and B. A total of six cell lines were derived from two SCNT fetuses (A1 and A2) generated using cell line A and three cell lines from two SCNT fetuses B1 and B2 produced using cell line B . The celTo evaluate the similarity of original and rederived cell lines, their transgene expression characteristics were initially compared. Under puromycin selection, original cell lines A and B proliferated and remained healthy for the entire selection period, indicative of a resistant phenotype. In contrast, all cells from rederived cell lines cultured under puromycin selection were dead and had lifted off the surface of the culture dish 72 hours after the addition of puromycin.The original cell lines had distinct GFP expression profiles with a geometric mean fluorescence of 5.82 for cell line A and 3.30 for cell line B. The GFP expression profiles of the six rederived cell lines  from fetuses A1 and A2 generated with donor cells from cell line A, were markedly different to the profile of the original cell line and varied considerably between the different cell lines . GFP expThe varied expression profile for the EGFP marker gene in the rederived cell lines, and the complete silencing of the puromycin selection marker in all of the rederived cell lines indicated that differences in the epigenetic modification of the transgene sequences may be present between original and rederived cell lines. DNA methylation profiles of the transgene sequences were therefore determined in all cell lines. Attempts were made to analyze the entire transgene construct, however, as is often the case in amplification of bisulfite converted DNA, in particular sequences of the highly GC-rich (73%) pac gene encoding the puromycin selection marker (not shown), this was not possible. The analysis therefore focused on four regions, representing important functional sections of the transgene construct (completely unmethylated prior to transfection). These regions included the loxP site and 5′ region of the first thymidine kinase (TK) promoter (region I), the second TK promoter and the 5′ region of the EGFP gene sequence (region II), the 3′region of SV40 polyA sequence and the lox2272 site (region III) and an endogenous sequence from the bovine β-casein promoter (BCP region) .Region I contained four CpG sites, two within the loxP site and two within the distal part of the TK promoter copy that drives the expression of the puromycin resistance marker gene. QHTMS analysis generated four CpG containing cleavage fragments, each containing a single CpG. One of the four cleavage fragments could not be analyzed due to a mass/charge ratio outside of the analyzable range and no information was therefore available for the 5′ most CpG of the loxP site. Average methylation across the three CpG sites analyzed revealed an almost complete absence of methylation across Region I in the original donor cell lines A and B . The average methylation of these CpGs was consistently higher in all rederived cell lines compared to their original cell lines . Across The DNA sequence of region II contained a total of 34 methylation target sites, comprising of two CpGs within the intergenic linker sequence upstream of the TK promoter, 19 CpGs in the TK promoter sequence and the first 13 CpGs in the EGFP sequence. Of the 26 CpG containing cleavage fragments generated from Region II analysis, 22 fragments could be analyzed which provided information on 30 of the 34 CpGs. Some of the cleavage fragments contained multiple CpGs. In these cases the methylation proportion represents an average of the methylation level of all CpG sites present on a particular cleavage fragment. Overall, DNA methylation levels were low in original cell lines A and B with an average for the proportional methylation across all 30 analyzed CpGs of 0.09 and 0.17, respectively. This was in stark contrast with the methylation levels determined for the same DNA sequence in the rederived cell lines . Here, tRegion III contained a total of six CpG sites, each in its own cleavage fragment. However, only three of the fragments had suitable mass/charge ratios which limited the measurement of the methylation levels to three CpG sites. Two of these informative CpGs were within a 27bp spacer region between the SV40 polyA sequence and the lox2272 site and the third one within the lox2272 sequence itself. Consistent with our results for regions I and II, the three CpGs in all rederived cell lines showed greatly increased methylation levels compared to the original cell lines A and B . While tWhile most parts of the transgene construct were comprised of sequences derived from organisms other than bovine, including bacteria and viruses, the β-casein promoter is of bovine origin and represents a sequence that is homologous to an endogenous gene locus. The assessment of methylation levels in this study was therefore limited to an average value for the two haploid copies of the endogenous gene locus and the additional copy(s) from the transgene. Compared to the different non-homologous sequence elements of the transgene, the β-casein promoter has a relatively low GC content (37%) and thus contains only a small number of CpGs. Analysis here was concentrated on the proximal promoter region. Unfortunately, the methylation profile of only one out of three CpG sites could be analyzed because the mass/charge ratio of the other two fragments that were generated was outside the analyzable range. Measurement of the level of methylation at this CpG site in the original cell lines A and B revealed a relatively low level of methylation for the β-casein sequence with 0.28 for A and 0.30 for B, respectively. Consistent with the observed hypermethylation in all rederived cell lines for regions I, II and III sequences, all rederived cell lines had elevated methylation levels of the CpG site within the bovine β-casein promoter. The average methylation was determined as 0.78 for derivatives of cell line A and 0.82 for B derived cell lines . HoweverTo determine whether the process of rederiving cell lines from a fetus following SCNT results in the general genome-wide hypermethylation of endogenous sequences, methylation levels of two endogenous sequences were measured.7 that has been previously characterised to show differential methylation levels in response to a specific environmental stimulus in rats The GR-specific assay includes a region equivalent to the rat promoter exon 1In both original donor cell lines A and B, this region was almost unmethylated . These methylation levels remained unchanged in all rederived cell lines compared with the original cell lines A and B , Table 1Sat 1 sequence is an abundant repeat element in the bovine genome and normally found in heterochromatin regions with a reasonably high degree of DNA methylation The Sat 1 sequence analyzed contained a total of 23 CpGs. Of the 21 CpG-containing cleavage fragments that were generated, 17 fragments could be analyzed and provided information on the methylation status for 19 of the CpGs present in the Sat 1 sequence .The Sat 1 repeat sequence was found, on average, to be moderately methylated in both original donor cell lines (A = 0.56 and B = 0.57). The proportional methylation for all of the rederived cell lines (average of 0.66 for A-rederived cells and 0.69 for B-rederived cells) were not significantly different (A-derived cell lines P = 0.09 and B-derived cell lines P = 0.42) to the original cell lines . AnalysiThe introduction of complex modifications into livestock genomes has proven to be challenging because tools such as embryonic stem cell technologies are not available for efficiently performing targeted gene insertions in species other than mice The present study has investigated transgene expression in transgenic cell lines and their rejuvenated counterparts rederived following SCNT. However, despite being isolated from SCNT embryos generated from cells selected for their resistance to puromycin, the rederived transgenic cells were no longer resistant to this antibiotic. Further examination of transgene expression also identified that GFP expression levels varied considerably when compared to levels expressed by primary transgenic cells used for SCNT.Silencing of transgenes is not a new observation The observed hypermethylation was not uniform across the entire length of the transgene, with some CpG sites showing notably greater increases in proportional methylation than others. Previous studies It is believed that the hypermethylation observed across the transgene was specific to the transgene itself and not simply due to global reprogramming errors. Evidence supporting this hypothesis is the analysis of one endogenous single copy gene (GR) and one endogenous repeat sequence (Sat 1). Both GR and Sat 1 sequences displayed levels of methylation equivalent to levels previously measured in control samples The consequences of transgene hypermethylation and corresponding altered gene expression present a new hurdle in producing transgenic cattle. Rederived cells, originally selected for the presence of an antibiotic selection marker gene conferring resistance to the antibiotic puromycin became completely susceptible to the antibiotic, indicating reduced expression of the selection marker gene (driven by the pTK promoter) to levels below the threshold required for puromycin resistance. Notably, absence of resistance was observed even in cell lines A2-4 and A2-5 that showed only a relatively modest increase in the methylation of two CpG sites  located within the 5′ section of the pTK promoter that drives expression of the puromycin resistance gene. This may indicate that hypermethylation of some or all of the 16 CpGs in the more 3′ part of the promoter are more critically important for the activity of this promoter. In contrast, although expression of the EGFP reporter gene was under control of an identical pTK promoter, expression of EGFP varied considerably compared to the original transgenic cell lines. Some rederived cell lines showed markedly lower EGFP levels while some displayed more expression, in spite of the fact that all rederived cells lines were rather uniformly hypermethylated in the pTK promoter  and 5′ region of the EGFP gene . These data indicate that hypermethylation of transgene sequences as a consequence of the SCNT process required for rederivation of cells and the generation of transgenic cows may not necessarily result in the absence of transgene expression and interfere for example with the intended production of a therapeutic protein – but does add an additional layer of complexity and uncertainty.Unpredictable and often suppressed transgene expression, caused by hypermethylation and loss of antibiotic resistance, are not the only extra hurdles that must be overcome in transgenic animal production using a cassette exchange based approach. Methylation of loxP was associated with reduced Cre-lox recombination and thus, may adversely affect the cassette exchange reaction itself All animal studies were undertaken in compliance with New Zealand laws and were approved by the Environmental Risk Management Authority, New Zealand  and the Ruakura Animal Ethics Committee (AE Applications 10724 and 11689).A 7.2 kb SalI-NheI fragment and a 3.5 kb HindIII-SalI fragment comprising 5′ and 3′ regulatory regions of the CSN2 locus were excised from a 17.9 kb DNA fragment containing the entire bovine CSN2 gene 6, passage 6) were electroporated under 400 volts and 250 µF conditions with 10 µg of the gel-purified transgene fragment excised from the plasmid vector with Sal I. Transfected cells were diluted 1∶50 and passaged into 96-well plates. Puromycin selection (2 µg/ml) was applied 24 h post-transfection and maintained until cryopreservation.Cell line BFFL5 was obtained from a day 70 female fetus by disaggregation of lung tissue as previously described While mock transfected cells did not result in any cell growth, cells transfected with the transgene fragment gave rise to 28 individual cell clones. Once the cell clones reached confluency they were trypsinized and passaged into a 24-well plate. Upon confluency, half the cells were cryopreserved and the remainder of the cells used for DNA extraction and PCR analyses.Presence of the transgene was confirmed in the isolated cell clones and all rederived cell populations by PCR and transgene copy number by quantitative real-time PCR as previously described SCNT embryos derived from cell lines A and B were produced using a zona-free reconstruction method essentially as described in For cell line B, two SCNT embryos, designated B1 and B2, were recovered from one recipient. Both fetuses were processed as described above and dissociated cells and small tissue clumps were cultured together. The resulting rederived cell lines were designated as B1-1 and B2-1, respectively. In addition, cells from the amniotic fluid of fetus B1 were cultured to establish another cell line referred to as B1-2.Total genomic DNA was extracted from all cell lines using Qiagen DNeasy extraction kit following the manufacture's protocol . DNA concentration and purity was measured using the Nanodrop spectrophotometer .http://www.epidesigner.com/). Primers were designed for amplification of bisulphite-converted DNA sequences of CpG containing transgene regions as described QHTMS methodology g DNA was bisulfite treated using the EZ-96 DNA Methylation gold kit  to produce methylation-dependent sequence variations of C to T and regions of interest were amplified using T7 tagged PCR primers. PCR was performed using Platinum Taq DNA Polymerase (Invitrogen), with 1 µl bisulfite-converted DNA per reaction in a total volume of 10 µl. The PCR cycling conditions, recommended by Sequenom for amplification of bisulphite-treated DNA, were as follow: 94°C, 15 min followed by 45 cycles of 94°C, 20 sec; 56°C, 30 sec; 72°C, 1 min with a final extension at 72°C for 3 min. PCR products were analyzed by agarose gel electrophoresis to confirm successful amplification. In vitro amplification and transcription was performed on the reverse strand by T7 DNA and RNA polymerases and a simultaneous U specific cleavage by RNAse A. Approximately 20 nl of each sample was spotted onto Sequenom MassArray chips and subjected to mass spectrometry. The efficiency of bisulfite conversion was determined by assessing the quality of the raw data. This protocol enables precise and accurate high-resolution, high-throughput DNA methylation analysis, quantitative to 5% methylation for informative CpG dinucleotides DNA samples were analyzed using previously described methods Occasionally, incomplete bisulfite conversion was observed which can be readily identified as it produces additional, greater-than-expected peaks with mass/charge ratios of multiples of 16. Any data derived from incompletely converted DNA samples were excluded from the analyses.The sequence-specific cleavage can produce fragments containing more than one methylated CpG site which the QHTMS approach is unable to resolve. In these cases, the measured methylation level does not represent the value for a single site but rather the average of the methylation level across all CpGs within that fragment. Similarly, processing of the samples can generate fragments with identical mass/charge ratios which are therefore indistinguishable and the methylation values will be averaged across the CpG sites contained on these fragments. Relevant information about fragments which were affected by averaging has been incorporated into The relative methylation of CpG sites were determined as previously described Pair-wise comparison of the average methylation values for original cell line versus rederived cell lines based on an unequal variance t-test was used to determine significance levels for the observed differences.Figure S1DNA methylation levels for three endogenous genomic DNA sequences, Satellite II, Satellite alpha and SNRPN in original and rederived cells. Methylation levels are shown as heatmap data with each square representing the average methylation level  for a specific cleavage fragment. The numbers below the heatmap identify individual CpGs that are present on each of the analyzed cleavage fragments. The numbering itself refers to the order of the CpG sites as they appear in the DNA sequence. Alphabetic characters highlight fragments of the same mass/charge ratio which cannot be resolved by the QHTMS approach and only allows for conveying an average methylation value for these fragments. White squares depict CpGs where the proportional methylation could not be determined.(TIF)Click here for additional data file.Table S1PCR primer pairs used for the DNA methylation analysis of the endogenous Satellite II, Satellite alpha and SNRPN sequences.(DOC)Click here for additional data file.


Canine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines.473, mTOR complex 1 (mTORC1) at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines.Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Serin vivo and in vitro.Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both  Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells (ECs). Canine HSAs easily metastasize to other organs, and the mean survival time is less than 6 months even with surgical and chemotherapeutic interventions . Human aPTEN, a PI3K antagonist, has been reported in canine HSAs , the cells were incubated with Protein Block Serum-Free  for 30 min at room temperature (RT). The cells were incubated overnight at 4 °C with primary antibodies for VEGF-A  and bFGF . The specific protein signals were visualized using the 3,3′-diaminobenzidinetetrahydrochloride . The cells were counter-stained with Mayer’s hematoxylin.Canine HSA cell lines were cultured to subconfluence under standard conditions in Medium 199 containing 10% FBS and were used for protein expression for VEGF-A and bFGF. After washing with phosphate-buffered saline without CaExpression of mRNA for growth factors and their receptors was examined in the established cell lines. Total RNA was extracted from subconfluent cells grown in Medium 199 containing 10% FBS using TRIzol reagent (Gibco). Reverse transcriptase-polymerase chain reaction was performed as previously described  using th3 cells per well in 200 μL Medium 199 containing 10% FBS in 96-well plates for 24 h. The cells were washed with HBSS, and the medium was replaced with Medium 199 containing 1% FBS. After 24 h of serum starvation, the cells were mixed with 0, 1, 10, 50, or 100 ng/mL of growth factor in Medium 199 containing 1% FBS or were changed to Medium 199 containing 10% FBS. Growth factors included recombinant human VEGF, bFGF, IGF-I, HGF, EGF, or PDGF-BB (R&D Systems), and all of these have been reported to induce cell growth in canine HSA cell lines except VEGF and PDGF-BB [Cell proliferation assays were performed as previously described . Briefly473, rabbit monoclonal antibody clone D9E, 1:1000, and Thr308, rabbit polyclonal antibody, 1:500; Cell Signaling Technology, MA, USA), Akt , p-p44/42 Erk1/2 , p44/42 Erk1/2 , p-mTOR , mTOR , p-p70S6K , p70S6K , p-4E-BP1 , 4E-BP1 , and PTEN . β-actin  was used as a loading control. The specific protein signals were visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL Plus Western Blotting Detection System . CnAOECs were used to examine the protein expression for normal canine ECs.Canine HSA cell lines were cultured to 70–80% confluence under standard conditions in Medium 199 containing 10% FBS. Cells were then washed with HBSS and the medium was replaced with Medium 199 containing 1% FBS. After serum starvation for 24 h, the medium was replaced with Medium 199 containing 1% FBS or Medium 199 containing 10% FBS for 30 min. For PTEN expression, subconfluent cells grown in Medium 199 containing 10% FBS were used. After washing with PBS (−), the cells were lysed with RIPA Lysis Buffer (Santa Cruz Biotechnology Inc.) with Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich). The concentrations of whole cell lysates were determined by modified Lowry method using the DC protein assay kit . Equal amounts of protein (10 μg) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions on 10% polyacrylamide gels. After separation by SDS-PAGE, the proteins were transferred onto a PVFD-membrane . Membranes were blocked with 2% ECL Blocking Agent  in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 1 h at RT. The membranes were then incubated overnight at 4 °C with primary antibodies for phosphorylated Akt (p-Akt Ser6 cells were suspended in 0.2 ml of PBS (−) and injected subcutaneously into the right and left dorsal area of the trunk of 3-week-old male KSN/Slc mice. Five mice were used for each cell line. The mice were observed for tumor development twice a week, and the size of the resulting tumor was measured. After 9 weeks, or when the tumors grew to 10 mm in diameter, the mice were humanely sacrificed, and the tumors were immediately removed. If a detectable tumor was not formed in the mice within 30 days, the mice were sacrificed at this time. The removed tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) or used for immunohistochemical staining. Immunohistochemical staining was performed for CD31 , von Willebrand factor , Ki-67 antigen , p-Akt , and p-4E-BP1  on all tumors formed from the cell injections. The experiments were performed according to the guidelines for the care and use of laboratory animals and approved by the Committee for Animal Research and Welfare of Gifu University (No. 08015).The established cell lines were harvested during logarithmic growth and prepared for injection in mice. Before injection, cells were trypsinized, counted, and washed twice with sterile PBS (−). A total of 1 × 10t test was used to determine statistical significance of the differences between the control and experimental data for the cell proliferation assay. Differences were considered statistically significant at p value of <0.05.Student’s After 60 passages, 3 cell lines were established from the 3 xenograft tumors . After cloning, 7 sub-lines with differential morphologies were established from these 3 initial cell lines Figure A. Three Each sub-line showed variable anchorage-dependent growth as shown in Figure The expression levels of mRNA for growth factors and their receptors were different among the cell lines as measured by RT-PCR Figure . mRNAs f6 cells for 24 h in the cell supernatantas measured by ELISA, but bFGF was not found in the supernatant of any cell line. Immunocytochemical investigations for VEGF-A and bFGF revealed weak to moderate expression of these proteins observed in the cytoplasm of the cell lines  had a VEGF-A concentration of 201 pg/102 had a VAfter 24 h of serum starvation, canine HSA cell lines showed differential response to growth factors, including recombinant human VEGF, bFGF, IGF-I, HGF, EGF, and PDGF-BB, recombinant canine VEGF and HGF, and to FBS as assessed by the WST-1 assay. All the cell lines could proliferate even in serum-starved condition. In KDM/JuB4, which expressed mRNA for all receptors except PDGFR-β, cell proliferation was stimulated by all growth factors except IGF-I and PDGF-BB in a dose-dependent manner, and by FBS. In KDM/JuA1, KDM/Re12, and KDM/Re21, cell proliferation was stimulated only by FBS and not by any growth factors even though these cell lines expressed mRNA for their receptors. Cell proliferation of KDM/JuB2, KDM/Ud2 and KDM/Ud6 was not stimulated by any of the growth factors or by FBS. Similar results were obtained from triplicate experiments. In CnAOECs, cell proliferation was stimulated by all growth factors except PDGF-BB and by FBS (data not shown). Figure 202/Tyr204 levels were low in serum-starved condition and increased in the presence of serum in the KDM/JuA1, KDM/JuB2, KDM/JuB4, and KDM/Re12 cell lines and a similar increase in p-p44/42 Erk1/2 Thr202/Tyr204 was observed in CnAOECs (Figure 473 in any cell line except KDM/Re12 were high in serum-starved condition, and FBS stimulation had no effect on its levels. Similarly, phosphorylation levels of mTORC1 at Ser2448 and 4E-BP1 at all residues were high in unstimulated cells and unchanged by serum stimulation in any of the cell lines. In CnAOECs, phosphorylation levels of these proteins were low in serum-starved condition, and FBS stimulation increased phosphorylation of Akt at Ser473, mTORC1 at Ser2448, and 4E-BP1 at Ser65 but not at Thr37/46 or Thr70. These data suggest that the phosphorylation of Akt at Ser473, mTORC1 at Ser2448, and 4E-BP1 at Ser65 was constitutively activated in the absence of FBS in six cell lines. The levels of p-Akt at Thr308 and p-p70S6K at Thr389 were increased by serum stimulation in KDM/Re12 cells in a manner similar to those of normal canine ECs. Conversely, FBS stimulation decreased phosphorylation of these residues in KDM/Ud2 and KDM/Ud6 cells. In addition, phosphorylation of these two sites was not affected by serum in the KDM/JuB4 cells and was not detected in KDM/JuA1 cells. The present findings suggest that the phosphorylation of p70S6K at position Thr389 may be related to that of Akt at Thr308.Because cell proliferation was stimulated by FBS in 4 cell lines, we further investigated the effect of FBS on the MAPK/Erk and Akt/mTOR pathways, which are major signal transduction pathways associated with cell proliferation [s Figure A. PhosphDeletion or mutation of PTEN is reported in some types of tumors, including vascular tumors ,10,29, wAfter subcutaneous injections of cells from the various cell lines into KSN/Slc mice, tumor masses were formed in all the nude mice that had been injected with KDM/JuA1 or KDM/Re21 cells, and in 2 and 1 nude mice that had been injected with KDM/JuB2 and KDM/JuB4 cells, respectively Table A–D. The 473was observed in both the nuclei and cytoplasm in all tumors (Figure 308 was observed in both the nuclei and cytoplasm (Figure 37/46was highly phosphorylated in both the nuclei and cytoplasm in all tumors (Figure All tumors that formed were examined further for expression of the Akt/4E-BP1 pathway. Moderate to intense degrees of phosphorylation of Akt at Sers Figure H–K. On tm Figure L–N, and m Figure O. 4E-BP1We established 7 canine HSA cell lines from nude mice-xenograft canine HSAs . AlthougThe established canine HSA cell lines expressed differing levels of mRNA for a variety of growth factors and their receptors. Although receptors were expressed in most of the cell lines, cell proliferation was stimulated only by the associated growth factors in the case of KDM/JuB4, in which proliferation was also stimulated by serum. Stimulated proliferation of 3 cell lines was observed in the presence of serum alone. A previous study with a canine HSA cell line showed that proliferation was stimulated by serum and the same growth factors that we used except for human VEGF and PDGF-BB . The preIn the present study, VEGF was detected in culture supernatant only in one cell line, even though mRNA and protein for VEGF was detected in all cell lines, and bFGF was not detected in the supernatant of any cell lines, including two cell lines that expressed mRNA and protein for bFGF. VEGF is known to regulate normal angiogenesis ,38 and i473 was not affected by FBS stimulation in all cell lines except KDM/Re12. In addition, the phosphorylation of mTORC1 at Ser2448 and 4E-BP1 at all residues was unchanged in all cell lines. In normal canine ECs, the phosphorylation of Akt at Ser473, mTORC1 at Ser2448, and 4E-BP1 at Ser65 was increased in the presence of FBS, but not phosphorylation of 4E-BP1 at Thr37/46 or Thr70. 4E-BP1 is known to be sequentially phosphorylated on three residues: phosphorylation of Thr37/46 is followed by Thr70 and then Ser65[37/46 is relatively unaffected by serum [70 and Ser65 are stimulated by serum [65 of 4E-BP1 is an essential site for the control of translation initiation by release of 4E-BP1 from eIF4E [65 was the only site that was regulated in a serum-dependent manner in normal canine ECs, rather than Thr37/46 and Thr70. This indicates that Ser65 of 4E-BP1, Ser473 of Akt, and Ser2448 of mTORC1 were constitutively activated in the present cell lines. mTORC1 and mTORC2 are located both upstream and downstream of Akt, and Ser473 of Akt is directly phosphorylated by mTORC2 [2448 is phosphorylated by Akt [473 and 4E-BP1 is reported in lymphomas [473 in KDM/Re12. Another possibility is that phosphorylation of 4E-BP1 may not be caused by Akt nor mTORC1 because 4E-BP1 is known to be phosphorylated by p44/42 Erk1/2 [The phosphorylation of Akt at Serhen Ser65. The phoby serum , whereasby serum . Howeverby serum . Furtherom eIF4E . Our resy mTORC2 , whereasd by Akt . The preymphomas  and acutymphomas . Since t202/Tyr204, similar to that of normal canine ECs. The MAPK/Erk pathway regulates cell proliferation differently from the PI3K/Akt pathway [Although 4E-BP1 was constitutively activated independent of FBS, cell proliferation was stimulated by serum in 4 cell lines. This stimulation seemed to be related to increased phosphorylation of p44/42 Erk1/2 Thr pathway  and is n pathway . In cont pathway ,13. In K pathway . Therefo473, the phosphorylation of Akt at Thr308 was affected by FBS stimulation and seemed to be correlated with the phosphorylation of p70S6K. Akt is usually phosphorylated at Thr308 by 3- phosphoinositide-dependent kinase, whereas Ser473 is phosphorylated by mTORC2 [As opposed to phosphorylation of Akt at Sery mTORC2 . Althougy mTORC2 , recent y mTORC2 . The mTOy mTORC2 ,41. Take473. It is known that constitutive activation of Akt is not always associated with the deletion or mutation of PTEN [Deletion or mutation of PTEN is known to cause constitutive activation of the PI3K/Akt pathway in some types of tumors, including vascular tumors ,10,29. D of PTEN , and oth of PTEN . However473 and 4E-BP1 at Thr37/46 similar to that of the original cell lines. These in vivo models would be useful tools for evaluating the anti-tumor effect of inhibitors targeting the mTORC2/4E-BP1 pathway. Drugs targeting both mTORC2 and mTORC1 have been studied in acute myeloid leukemia and have shown marked anti-tumor effects [After cell injections into nude mice, HSA tumors developed from 4 cell lines. In these mice with developed tumors, no metastatic lesion was observed, similar to that of original canine HSA xenograft models . Similar effects . Because effects , mTORC1/473, mTORC1 at Ser2448, and 4E-BP1 at Ser65 was not altered by FBS stimulation in 6 cell lines, suggesting that the mTORC2/Akt/4EBP1 pathway was constitutively activated in the present cell lines. After cell injection into nude mice, canine HSA tumors were formed in 4 cell lines. These tumors showed similar expression levels for phosphorylated Akt and 4E-BP1 as the original cell lines. Therefore, the present cell lines are useful models to investigate the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA in both in vitro and in vivo systems.We have established 7 canine HSA cell lines from 3 xenograft canine HSAs. These cell lines showed diverse morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, and PDGF-B and their receptors. Cell proliferation was stimulated by these growth factors and FBS in one cell line, was stimulated by FBS alone in 3 cell lines, and was not stimulated by either growth factors or FBS in the remaining 3 cell lines. Phosphorylation of p44/42 Erk1/2 was increased in the presence of FBS in 4 cell lines and seemed to be related to serum-dependent proliferation. In contrast, phosphorylation of Akt at SerThe authors declare that they have no competing interests.AK was involved in study design, establishment of cell lines, and histopathology. HS and SAA conceived the aims of study and participated in its design and coordination. AH and TY participated in study coordination. All authors have read and approved the final manuscript.


VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation.Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and + KDRand + FLT4cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes.Expression of KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.Our data show that VEGF-Rs  Vascular endothelial growth factors (VEGFs) and their corresponding receptors (VEGF-Rs) are important regulators of angiogenesis and lymphangiogensis. VEGF-A binds VEGF-R1 (FLT1) and VEGF-R2 (KDR). Both tyrosine kinase receptors are expressed on blood vessel endothelial cells. VEGF-C and VEGF-D bind to VEGF-R3 (FLT4) and the fully processed, mature forms also to KDR. FLT4 is primarily expressed on cells of the lymphatic endothelium . The intProx1 have been described as activators of FLT4 in lymphatic endothelial cells -thymidine incorporation were executed as follows: 1.25 × 104 cells (in 100 μl) were seeded in triplicate in 96-well flat-bottom microtiter cell culture plates. Inhibitors were added as 2x concentrated solution in a 100 μl volume. For the last 3 h of the incubation period, 1 μCi [3H]-thymidine  was added to each well.Assays of [Samples were prepared as described previously . Anti FLFor detection of CD31 , FLT4 , KDR  and podoplanin (Reliatech) on the cell surface, cells were washed and incubated with the mouse mAb or with the isotope-matched control mouse immunoglobulin (BD Biosciences) for 30 min at 4°C. Subsequently, cells were treated with FITC conjugated anti-mouse secondary Ab  and propidium iodide. Labeled cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest Pro software.FLT4 and KDR. Cells were seeded at a cell density of 5 × 105 cells/ml, 5-Aza-dC was added at a final concentration of 5 μM. Control cells were treated with 0.05% DMSO. After 2 d, half of the medium was replenished with medium with/without 5-Aza-dC (5 μM). After 3 d, cells were harvested to prepare RNA and protein.5-Aza-dC  dissolved in DMSO was used to verify the effect of methylation on expression of KDR (VEGF-R2) and FLT4 (VEGF-R3) are not only expressed on blood endothelial and lymphendothelial cells, but also on solid tumors and leukemias. Leukemia-derived VEGFs may induce the growth of leukemic cells in an autocrine or paracrine fashion [FLT1 and KDR are controlled by promoter methylation [VEGF-R expression and promoter methylation hitherto.The VEGF-Rs  fashion ,10,18,19 fashion . Recent hylation ,15. HoweVEGF-R regulation, we tested some ninety leukemia and lymphoma cell lines for KDR and FLT4 mRNA expression. Both genes were regularly expressed in leukemia but not in lymphoma cell lines: 10/62 (16%) cell lines from various leukemic entities expressed KDR, 27/65 (42%) expressed FLT4  expressed FLT4 , a technique less costly and laborious than bisulfite sequencing. The majority of KDR negative cell lines were methylated, KDR positive HUVECs were unmethylated  was lower than for KDR M-PCR (88%). Of note was also that TF-1 cells did not express FLT4 although the promoter was unmethylated , demethylation did not induce mRNA expression that would translate into protein levels detectable by Western blot analysis (data not shown). These results suggest that other mechanisms than DNA methylation are also involved in the regulation of KDR and FLT4.To test whether on Table . FLT4 exes Table . KDR anded Table . Althouges Table . FurtherBesides DNA methylation, also histone modifications are epigenetic mechanisms that affect the expression of individual genes. Just to mention two examples, acetylated histone H3 (at lysine 9 and 14) is a marker for gene activation , tri-metKDR and FLT4. Thus, it has been shown that transcription factor binding sites  are essential for the base-line activity of the KDR promoter [FLT4.However, besides epigenetic mechanisms, also the presence or absence of trans-acting factors may govern the expression of promoter . Here, wFLT4 on lymphatic endothelial cells [FLT4 expression in leukemic cells, we stimulated the FLT4 negative cell line EM-2 and the FLT4 positive cell line OCI-AML1 with synthetic MALP-2. MALP-2 binds to toll-like receptors-2 and -6 [TNFα [During inflammation, new lymphatic vessels are formed. NF-κB is a key mediator of inflammatory processes and has recently been identified as inducer of al cells . To test2 and -6 . MALP-2 2 and -6  which le-6 [TNFα .TNFα  and IP-10  in both cell lines. However, the expression of FLT4 was not affected, neither in the FLT4 positive cell line OCI-AML1 nor in the FLT4 negative (methylated) cell line EM-2. MALP-2 did also not increase the FLT4 stimulating effect of 5-Aza-dC on EM-2 cells (data not shown). Thus, our results do not support the view that NF-κB is a transactivator of FLT4. We observed a 10-fold increase in KDR in cell lines OCI-AML1 and EM-2 (the latter pretreated with 5-Aza-dC). However, as the level reached after stimulation was still extremely low, it appears unlikey that NF-κB is an important regulator for KDR either.Accordingly, MALP-2  induced phosphorylation and degradation of the NF-κB inhibitor IκB and stimulated phosphorylation of p38 in cell lines EM-2 and OCI-AML1 Figure . MALP-2 KDR positive leukemia cell lines exhibited demethylated KDR promoters (Figure KDR and FLT4 gene expression.HDMECs, HUVECs and s Figure , Table 2s Figure . Resultss Figure . HDMECs s Figure . HDMECs,s Figure . Both tys Figure . Also FLs Figure  and 7. TKDR and FLT4 are epigenetically regulated genes. Both genes can be silenced by methylation. However, if the promoters are unmethylated, other factors are responsible for the extent of KDR and FLT4 expression. Furthermore, we show that the KDR negative/FLT4 positive cell line OCI-AML1 is a model system for FLT4 signal transduction studies.Our data obtained from primary endothelial cells and from leukemia/lymphoma cell lines show that The authors declare that they have no competing interests.HQ designed the study, performed data analyses and wrote the manuscript. SE carried out bisulfite conversion and helped to design primers for M-PCR and U-PCR. JR performed Western blot analyses. HAW supplied antibodies, HDMECs and HUVECs and gave good advice. MZ performed PCR and FACS analyses. HGD provided cell lines and good advice. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/19/prepub


Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. Recently, Shafiq and co-workers confirmed the specific effect of this compound on CDK4, which is known to play a key role in cell cycle control and is an important target for anticancer drugs [50 values 0.6–4 μM), although testing was actually restricted to 36/60 of these cell lines [Fascaplysin  could not be obtained for fascaplysin, a therapeutic effect was observed.Fascaplysin was tested in a murine sarcoma S180 experimental animal model . TreatmeIn conclusion, cell line screening of the anticancer activity of fascaplysin is not complete and the mechanism inducing cell death in response to this drug, which may comprise different molecular mechanisms, is not clear. Interaction with other chemotherapeutic drugs to detect possible synergism was not described so far. Thus, in the present study, we screened SCLC cell lines, investigated cell cycle and cytotoxic effects of fascaplysin and used different drug combinations to screen putative synergistic action with established chemotherapeutics. In particular, fascaplysin was combined with camptothecins (CPTs) since we have reported enhancement of the cytotoxicity of the CPT analog topotecan against SCLC cell lines employing CDK4 inhibitors such as roscovitine and olomoucine . 50 values measured ranged from 134 to 1740 nM fascaplysin. GLC14, 16 and 19 comprise a series of SCLC cell lines obtained from a single patient prior to chemotherapy and after first-line and second-line therapy, respectively. Whereas SCLC26A is an untreated chemosensitive SCLC cell line with IC50 values (μM ± SD) for cisplatin of 1.2 ± 0.6 and for etoposide of 0.5 ± 0.2, respectively, NCI-H417 and DMS153 are chemoresistant with IC50 values of 12.0 ± 2.8/3.3 ± 0.9 for cisplatin and 15.1 ± 2.2/12.9 ± 0.5 for etoposide, respectively. Therefore, sensitivity for fascaplysine differs by a factor of 1.2–1.6 between SCLC26A and DMS153/NCI-H417, but cisplatin and etoposide sensitivities differ 2.75–10 fold and 25.8–30 fold, respectively. These IC50 values observed did not exceed the corresponding value for the HEK293 cell line, representing normal kidney epithelial tissue. Fascaplysin IC50 values measured in SCLC cell lines are similar to the two chemoresistant NSCLC cell lines H1299 and A549 and the chemosensitive H23 cell line, respectively. Two pulmonary carcinoid cell lines showed sensitivities in the range of the SCLC cell lines and mean IC50 values for a panel of seven ovarian  and five colon cancer  cell lines indicated high chemosensitivity for fascaplysin.The cytotoxic activity of fascaplysin was assessed using a panel of SCLC cell lines using MTT assays . IC50 vaFor the assessment of effects of fascaplysin on cell cycle distribution in SCLC cells, NCI-H417 and DMS153 cells were treated with 0.25–2 μM of the compound and propidium iodide-stained cells analyzed in flow cytometry . The twoFascaplysin-induced apoptotic cell death was investigated in NCI-H417 cells using annexinV-propidium iodide double staining and flow cytometry A. TreatmN-acetyl-l-cysteine (NAC) is suitable for inactivating ROS and, accordingly, inclusion of 2.5–5 mM NAC in NCI-H417 fascaplysin cytotoxicity tests resulted in a twofold reduction of cell death (data not shown).Fascaplysin-induced generation of reactive oxygen species (ROS) in NCI-H417 and DMS153 cells was tested using fluorescence detection with the DCF dye B. A stabFascaplysin was tested for a possible interaction with camptothecin (CPT) and 10-hydroxycamptothecin (HOCPT) in MTT assays using chemoresistant NCI-H417 SCLC cells . Fascapl50 in presence of fascaplysin plus BYK204165 inhibitor/IC50 value in presence of fascaplysin), all other cell lines  used showed strong antagonism for this combination with 2.9 ± 1.13 (range 1.83–4.46) and 1.9 ± 0.51 (range 1.48–2.6) increased chemoresistance compared to fascaplysin alone. Cytotoxicity assays using a combination of fascaplysin with the BYK 204165 PARP1 inhibitor were performed using a panel of SCLC cell lines. Whereas in chemosensitive NCI-H526 cells 50 and 25 μM BYK204165, respectively, yielded a minor chemosensitizing effect  to MTT tests partially reverse the cytotoxic effect of this compound. NAC can interact directly with reactive oxygen species (ROS) because it is a scavenger of oxygen free radicals [Fascaplysin induces apoptotic cell death in NCI-H417 cells dose-dependently and the generation of ROS seems to support this cytotoxicity since addition of radicals ,29. Drugradicals . In limi+resynthesis and ATP depletion and/or translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus [In order to further check possible DNA damage and repair we applied the isoquinolindione BYK204165 selective PARP1 inhibitor to fascaplysin cytotoxicity tests against different SCLC cell lines . The bas nucleus .2, 95% humidity) and checked for mycoplasma contamination .Stock solutions of all compounds were prepared in DMSO. All other chemicals were purchased from Sigma-Aldrich , except indicated otherwise. The BYK 204165 PARP1 inhibitor was obtained from Tocris Bioscience . Cell lines were obtained from ATCC , except DMS153 cells from ECACC , the GLC14/16/19 series from Dr. Nina Pedersen from the Department of Radiation Biology, The Finsen Centre, National University Hospital, Copenhagen, Denmark and H1299 and H23 from the University of Graz. The SCLC26A line was established from a pleural effusion of an untreated patient with SCLC at our institution. Cells were grown in RPMI-1640 bicarbonate medium  supplemented with 10% fetal bovine serum , 4 mM glutamine and antibiotics  under tissue culture conditions  and the test compound added in another 100 μL. Drugs and solute controls were serially diluted in twofold steps in triplicate. The microtiter plates were incubated under tissue culture conditions for four days and cell viability was measured using a modified MTT -2,5-diphenyl-tetrazolium bromide) assay . Optical density was measured using a microplate reader at 450 nm with an empty well as reference. Values obtained from control wells containing cells and media alone were set to 100% proliferation. For tests of synergy, compounds were diluted individually and in combination, using the same (CPT) or double (HOCPT) initial concentrations. The synergistic effect of drug combinations was assessed using CompuSyn , a software program based on the calculations for synergism developed by Chou et al. [1 × 10u et al. . Combina6 cells per well were incubated with the respective compound in six-well plates for three days. Harvested cells were washed with PBS and fixed with 70% ethanol at −20 °C for 30 min, washed again, transferred into staining solution (20 μg/mL propidium iodide (PI), 5 μg/mL ribonuclease A, 0.05% Nonidet P40 in PBS) and incubated at room temperature overnight. Washed cells were analyzed by acquisition of 1 × 104 cells by flow cytometry  at excitation and emission wavelengths of 488 and 675 nm, respectively. The proportion of subG1 (apoptotic) cells was obtained from the logarithmic PI histograms, and percentages of cells in cell cycle phases G1/0 (resting), S (DNA synthesis) and G2M (mitotic) were calculated from linear PI histograms using MultiCycle AV software . Experiments were done in duplicate.1 × 106 washed cells were preincubated with 5 μg/mL 2′,7′-dichlorodihydrofluorescein diacetate (DCF), a chemically reduced, acetylated form of fluorescein, in phosphate buffered saline (PBS) at 37 °C for 15 min. This indicator is readily converted to a green-fluorescent form following removal of the acetate groups by intracellular esterases and oxidation by ROS. Cells which were washed again were then treated with fascaplysin under tissue culture conditions at 37 °C for 4 h. Identical treatment of the cells with Luperox® TBH70X, Di-tert-butyl peroxide solution, gave the positive controls. Samples were subsequently analyzed by flow cytometry.1 × 10t-test at significance levels of p < 0.05 (indicated in the figures by an asterisk).Statistical differences were calculated using student’s paired Fascaplysin exhibits cytotoxicity against chemoresistant SCLC cell lines via several contributing pathways comprising, among others, generation of ROS and cellular mechanisms affecting topoisomerase I-activity and activation of PARP, possibly linked to its interaction with DNA in absence of a functional CDK4-RB1 axis in SCLC cell lines. Synergistic interaction with camptothecins is of special interest for second-line treatment of SCLC relying on the camptothecin analog topotecan.