This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81–88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines ( Electroporation is increasingly being used in cancer treatment strategies .Nine different human cell lines were used in this study; seven cancer cell lines, an immortalized normal cell line, and one primary normal cell line Table . (1) H692+, containing 2.5 μM FM 1-43 (Invitrogen). Laser injury was produced using a 2-photon laser scanning confocal microscope  coupled to a Vision S tunable laser (Coherent) at 100 % power (one laser iteration and a 15 pixels diameter circle bleach area placed over the membrane edge), creating 1.66 µm diameter plasma membrane disruptions. Fluorescence intensity over time was quantified using ZEN 2012, Zeiss software.A well-characterized assay  and three cancer cell lines  were tested for viability after electroporation. These cell lines were chosen to compare the normal cell line with a few of the cancer cells lines that had shown a clear difference in membrane repair.2 in sterile water) and diluted to 5.5 × 106 cells/ml HEPES buffer. In 4 mm cuvettes with aluminum electrodes  300 µl cooled cells (8 °C) were electroporated by delivering 8 pulses of 100 µs, 1.2 kV/cm, and 1 Hz using a BTX T820 square wave electroporator. After 20 min incubation at 37 °C and 5 % CO2 cells were diluted in culture medium to 3.1 × 105 cells/ml and seeded in 96-well plates (100 µl/well). One day after treatment viability was measured by MTS assay  and the bladder cancer cell line (SW780) were tested for degree of permeabilization after electroporation. These cell lines were chosen since they showed a significant difference in viability after electroporation.2·6H2O, 0.4 mM KH2PO4, 1.6 mM K2HPO4·3H2O, 1.5 mM CaCl2·2H2O, 10 mM glucose·H2O in MiliQ water) before adding 400 µl Krebs–Ringer buffer containing 1 µM YO-PRO-1 (Invitrogen) and incubated at 37 °C and 0 % CO2 for 30 min. YO-PRO-1 is a fluorescent dye that binds to nucleic acids after entering cells when plasma cell membranes are permeabilized . Cells were treated with electroporation  using a Cliniporator  and a custom-made contact copper electrode with 8 mm between the electrodes  one day prior to experiments. Cells were washed in 1 ml Krebs–Ringer buffer . Difference in fluorescence intensity in the different cell lines was evaluated as repeated measurements, validated and analyzed with an exponential decrease model with Bonferroni correction. “Cell line,” “Time,” and “n” were used as factors and baseline level of fluorescence intensity were used as covariant. Difference in viability after electroporation between different cell lines and difference in permeabilization after electroporation between HDF-n and SW780 cell lines were assessed using one-way analysis of variance (ANOVA) with Bonferroni correction. Difference in viability after electroporation with addition of calcium between HDF-n and HT29 cell lines was assessed using two-way ANOVA with Bonferroni correction.p < 0.05) with one exception being the PC3-M cell line derived from a bone metastasis of a prostate cancer (p = 0.29)  (Teissie and Rols p < 0.05). This difference in viability after electroporation in the normal primary cell lines and the SW780 cancer cell line was not caused by lower permeabilization after electroporation of the normal cells. Actually, when testing permeabilization after electroporation in the two cell lines in the presence of the fluorescent dye YO-PRO-1, we showed that uptake of the dye was significantly higher in the normal cell line than in the cancer cell line. This indicates higher degree of permeabilization of the normal cell line after electroporation, when using the same electroporation parameters for both cell lines (Fig. To test if this difference in membrane repair has an effect on viability when permeabilizing the plasma membrane by electroporation, we electroporated four of the used cell lines (three cancer cell lines and the normal primary cell line, previously used in another study (Frandsen et al. nes Fig. . In othe2+-sensors are involved in membrane repair including calpains, annexins, and S100 proteins. These sensors are activated by the high Ca2+ entry at the site of the injured membrane and initiate the membrane repair process (McNeil et al. The results of this study indicate that there is a reduced ability of membrane repair in cancer cells compared with the normal cells. This might contribute to the difference in survival and effectivity of treatment on normal and cancer cells and tissues when using permeabilization methods as reported earlier (Lejbkowicz et al. When treating cutaneous metastases with electrochemotherapy in the clinic, the surrounding normal tissue is less affected than the tumor tissue Fig.  and it hIn conclusion, membrane repair was less effective in six of the seven tested cancer cell lines and the immortalized cell line compared with the normal primary fibroblasts. This result could be part of the explanation why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective in inducing cell death of malignant than normal cells. However, further investigations are needed to substantiate these results and investigate in more detail about membrane repair mechanisms and membrane composition in the different cell lines.


There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good  ES anin vitro in both ES and iPS cell lines from different individuals. Reprogramming of ES-derived MSC did not modify this intrinsic hematopoietic potential and isogenic iPS-derived MSC-ES reproduces a similar hematopoietic outcome as their parental ES cell line. In addition, we investigated whether the variation in hematopoietic differentiation among different ES and iPS cell lines could be predicted by expression of key genes involved in hematopoiesis. A large variation in the level of gene expression at the pluripotent stage was observed but was not able to be correlated to distinguish PSC lines with greater hematopoietic potential. As expected, the expression level of these key hematopoietic factors varied during hematopoietic differentiation. The ability of ES and iPS-derived MSC-ES cell lines to allow in vivo hematopoietic reconstitution in immunodeficient mice was detected at low levels for short-term engraftment. Our results show that there is an intrinsic variability of each cell line regarding the hematopoietic differentiation potential. It appeared that the donor cell of origin is a determinant factor for variations in iPS hematopoietic differentiation rather than the derivation or induction methods. These data underline the importance of cell donors for hematopoietic differentiation potential from iPS cell lines.Many ES cell lines have been characterized for their hematopoietic potential in different studies but only few iPS cell lines have been characterized in detail ,5,7. Linhttp://www.wicell.org) imported after authorization from Biomedicine Agency (number RE10-035R/C) and CL01, ES cell line derived by our laboratory from a PGD embryo harboring a trisomy 1 and monosomie 21 (www.hescreg.eu). In addition, fourteen iPS cell lines were studied: two iPS derived from MSC obtained from SA01 and H9 cell lines (iPS-MSC-SA01 and iPS-MSC-H9), ten iPS from healthy donors and two iPS from sickle cell diseases. CL01 human ES cell line and all iPS cell lines were derived by the StemCell core facility (ESTeam Paris Sud) with written and informed consent from the donors in accordance with the approval from the ethical committee of Department of Medicine at Kremlin Bicêtre University Paris Sud . Detailed information about iPS lines (PB3 to PB33) is described in OCT4, SOX2, NANOG, LIN28, or OCT4, SOX2 under the control of the elongation factor-1 promoter, using published methods ) and vs CL01  and 318±197 (n = 34) respectively, were not considered statistically significant with p values (p>0.05) as determined by unpaired t-test, error bars represent the SEM, whereas H9 and CL01 gave rise to significantly (p>0.05) less CFC than SA01 and H1, 38±12 (n = 3) and 41±27 (n = 3) respectively . Distribvs CL01 . In addivs CL01 . Our resp<0.05)  . We obsep<0.05) . The prop<0.05) . AlthougIn conclusion, using two different hematopoietic differentiation protocols, we identified the existence of an intrinsic heterogeneity in the hematopoietic potential of ES regarding the number and commitment of hematopoietic progenitors.www.hescreg.eu) using different reprogramming methods. Pluripotency of all iPS cell lines was confirmed by: 1) teratoma assay and histology of representative tissue from three germ layers, 2) expression of pluripotent markers  by flow cytometry and 3) expression of transgenes, analyzed by reverse transcription polymerase chain reaction (RT-PCR) and iPS cell lines maintaining the expression of transgenes were exclude from this study , endothelial progenitor cells from normal peripherical blood and CD34+ from normal cytapheresis or BM. These iPS cell lines were derived by ESTeam Paris Sud , 80±40 (n = 3), 0±0 (n = 3), 144±100 (n = 6), 0±0 (n = 3), 0±0 (n = 3), 24±10 (n = 3), 670±395 (n = 12), 249±163 (n = 6), 235±208 (n = 4), 100±30 (n = 3), 139±40 (n = 3) for PB3, PB4, PB5, PB7, PB8, PB9, PB10, PB11, PB13, PB17, PB30 and PB33 respectively . It is noteworthy that in all experiments of CFC hematopoietic differentiation of ES and iPS there is at least one positive control cell line to ensure that the absence of CFCs is not a technical problem .These results indicate that the hematopoietic potential of each iPS was heterogeneous, similarly to ES cell lines. Our data strongly suggests that the variability is not due to lack of pluripotency, the passage number (experiments were performed at various passage numbers for each iPS with similar results), or growth conditions since all cell lines were maintained under the same growth conditions, nor genomic instability since karyotype was normal after iterative passages and concomitant of induction of hematopoiesis . Our expWe investigated whether the reprogramming process would have functional outcomes in the capacity of iPS cell lines to differentiate into hematopoietic myeloid and erythroid lineages. To address this question, we took advantage of the ability to differentiate ES into MSC (non-hematopoietic lineage) and then re-program them to generate isogenic iPS lines that in turn could be directly compared back to their original ES donor state for their hematopoietic potential. For this purpose, we generated isogenic iPS cell lines derived from MSC differentiated from SA01 and H9 (iPS-MSC-SA01 and iPS-MSC-H9) as previously described , and theOCT4, SOX2, NANOG, LIN28 and were tested for differentiation along the osteogenic, chondrogenic and adipogenic lineages [OCT4, SOX2, NANOG, LIN28 as previously described [RUNX1 or AML1, HOXB4, TAL1, PU.1, GATA1, GATA2, GATA3, MPO and IKAROS) from MSC-derived from ES cells (MSC-SA01 and MSC-H9) were markedly different compared to their parental ES cells  and iPS-derived from MSC-ES   altered the ES origin cell’s hematopoietic potential, we analyzed expression profile genes involved in hematopoiesis specification in the parental ES cells (SA01 and H9) at the pluripotent stage and compared them to both MSC derived from these cells (MSC-SA01 and MSC-H9) and to iPS-derived from MSC-ES . As previously demonstrated, the MSC lost their pluripotent potential . MSC genlineages . To the escribed . The exp-MSC-H9) .p<0.05) than parental SA01 (209±90 vs 86±40). Similarly, for iPS-MSC-H9, CFC number was higher than parental H9 line but the difference was not significant  (p>0.05) (vs 15±5% (n = 5) (p>0.05), unlike for H9 and iPS-MSC-H9 cell lines, we found 11±3% vs 20±3% (n = 7) (p<0.05)  was significantly higher ((p>0.05) . The sub(p>0.05) . EB-deri(p<0.05)  respectiFor these experiments, the hematopoietic differentiation protocol was different from that used for the EB differentiation to generate data for Figs In conclusion, despite some differences in CFC number and proportion of CD34+ cells, the reprogramming process does not modify the qualitative hematopoietic potential. The variability between iPS for intrinsic hematopoietic potential is more attributable to genetic donor differences rather than to the reprogramming process.in vivo, the long-term hematopoietic reconstitution potential of ES-derived cells in a xenograft model using immunodeficient NOG mice. We compared EB-derived cells at day 16 from SA01 and H9 with their respective isogenic iPS cell line (iPS-MSC-SA01 and iPS-MSC-H9). When EB’s were intravenously injected, we did not observe any hematopoietic engraftment in the transplanted mice while cord blood CD34+ cells engrafted efficiently (data not shown). To circumvent any caveat due to homing defect, we injected 3 to 5x105 day 16 EB cells directly into one femur of sublethally irradiated NOG mice by intra-BM transplantation (IBMT). Mice were monitored for human reconstitution by peripheral blood analysis at various time points  after transplant. Mice were bled and analyzed for human CD45 expression by FACS analysis and Q-PCR assay (data not shown).Our goal was to assess versus anti-human CD45 staining using non-transplanted control mice  in blood of all mice analyzed by FACS  as shownHoxA3 and RUNX1c gene expression from isolated EB’s CD34+ cells from SA01, H9, iPS-MSC-SA01 and iPS-MSC-H9 cell lines and compared them to human fetal liver CD34+ cells (provided by Dr E. Oberlin) which have a high capacity to reconstitute immunodeficient mice. We found that EB’s CD34+ populations from both ES and iPS-MSC-ES have a much lower expression for HoxA3 and RUNX1c genes than fetal liver CD34+ cells , HOXB4, TAL1, PU.1, GATA1, GATA2, GATA3, MPO and IKAROS was studied at the pluripotent stage for four ES cell lines , and 10 iPS cell lines from different origin   varied according to cell lines and virtually had very little expression in PB17 cell line. Finally, IKAROS had a moderate expression for all lines with collapsed in PB17  . Relativ in PB17 . In concHPRT. All mRNA expression was increased at the EB stage of development compared to the pluripotent stage with the exception of TAL1, which expression was similar at both developmental time points for all cell lines. We also detected very high levels of expression for GATA1 mRNA for iPS-MSC-SA01 EBs compared to the pluripotent state. As expected, the expression level of these key hematopoietic factors in EB varied during hematopoietic differentiation  in comparison to the pluripotent stage for three ES cell lines , two iPS-MSC-ES cell lines (iPS-MSC-SA01 and iPS-MSC-H9) and three iPS cell lines  and human BM as a positive control for hematopoietic gene expression. Gene expression was normalized relative to the endogenous RNA control human ntiation .in vivo engraftment and reconstitution. It is known that not all PSC have the same capacity to differentiate into desired cell types. A previous study using ES cells showed that erythroid differentiation did not occur or albeit at low levels, depending on the cell line studied [in vitro into hematopoietic lineage than other cell lines with quantitative and qualitative differences, in particular a remarkable difference in the ratio of myeloid versus erythroid CFC. It has been recently reported that iPS cells differ and retained residual DNA methylation patterns typical of parental somatic cells [In this study, we found a significant intrinsic variation among human ES and iPS cell lines in hematopoietic differentiation, regarding CFC potential both quantitatively and qualitatively and in hematopoietic  studied . Like ot studied , we haveic cells . Neverthic cells  in additic cells ,23. A siic cells . Other sic cells ,25. Howeic cells . Our stuic cells . The comin vivo [HoxA3 and RUNX1c between EB’s CD34+ from 2 ES and 2 iPS-MSC-ES cell lines compared to human fetal liver CD34+ cells. Kaufman’s group recently demonstrated deficient expression from the HoxA locus in derived progenitors CD34+43+ cells from ES and iPS cell lines compared to CD34+ cells from human umbilical cord blood [RUNX1 and HoxA family gene expression may contribute to the inability of human ES and iPS-derived hematopoietic cells to reconstitute immune deficient mice. Other authors have shown that definitive hematopoiesis is regulated by HoxA cluster genes expressed mainly in hematopoietic stem cells suggesting that HoxA cluster expression is important for adult hematopoiesis in mice [HoxA9 plays a major role in hematopoiesis and leukemogenesis but HoxA9 alone is not sufficient to confer long-term engraftment potential in vivo for human ES-hematopoietic derivatives [Kiba’s group showed that HoxA3 has a key role in the commitment to hematopoiesis versus endothelial differentiation in mice, restrained hematopoietic differentiation of the earliest endothelial progenitors and RUNX1 have a essential role to extinguish an endothelial program [Runx1c expression in the AGM region in mouse embryos and they also showed that in mouse ES cells Runx1c expression to be specific for emerging definitive hematopoiesis, unlike Runx1a and Runx1b which are expressed throughout hematopoiesis [HoxA3 and RUNX1c compared to CD34+ cells that have a high capacity for reconstituting immune deficient mice.To date, PSC-derived cells have demonstrated only limited potential for long-term multilineage hematopoietic engraftment in vivo –30. Generd blood . Our res in mice . Ramos-Mivatives . Kiba’s opoiesis . Our resRUNX1 [in vivo was also attributed to the inability to down-regulate key microRNAs involved in hematopoiesis [The inability to reconstitute long-term hematopoiesis could be explained by the fact that they might contain only embryonic primitive hematopoietic progenitors from corresponding yolk sac ,19,36, wRUNX1 . Failureopoiesis  or a lacopoiesis . Recent opoiesis ,41. Receopoiesis .in vitro. In the four ES and 10 iPS cell lines we examined, none of the analyzed genes demonstrated a specific correlation for determining between good versus poor hematopoietic potential. These findings support the notion that the level of expression for genes involved in early and late stages of hematopoietic differentiation is not a feature of prediction for blood developmental potential of ES and iPS cell lines to differentiate in vitro. Our results are in agreement with the study of Ramos-Mejia [versus poor hematopoietic iPS lines. These results suggests there is an unpredictable variation in hematopoietic differentiation for ES and iPS cell lines that can not be predicted based on phenotype or gene expression of the undifferentiated pluripotent cells lines.We compared the ES and iPS cell lines at the pluripotent stage for the expression of intrinsic hematopoietic factors that could be used as a predictor for determining their hematopoiesis potential. Expression of either early or late hematopoietic transcription factors at the pluripotent stage in ES and iPS could not predict the blood developmental potential os-Mejia  showing in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the ES and iPS cell lines used, in particular there were remarkable differences in the ratio of myeloid versus erythroid CFC. Our results suggest that the genetic background of the original cells has a major influence on the iPS cell lines’ hematopoietic differentiation. This means that the cells’ origin, rather than the re-programming method, passage number or method of differentiation is probably the major determinant of variation in hematopoietic differentiation.In summary, our results demonstrate the capacity of ES and iPS to differentiate into hematopoietic cells S1 FigAll ES and iPS cell lines used in this work were routinely screened for high levels of stem cell pluripotency gene expression. Representative FACS analysis of TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 expression for (A) H1, PB7, PB11 and PB13 cell lines . (B) SA01, iPS-MSC-SA01, H9 and iPS-MSC-H9 cell lines .(TIF)Click here for additional data file.S2 FigRUNX1, HOXB4, TAL1, PU.1, GATA1, GATA2, GATA3, MPO and IKAROS was determined by Q-RT-PCR at the pluripotent stage in the parental ES cells (SA01 and H9) and then compared them to both MSC derived from these cells (MSC-SA01 and MSC-H9) and to iPS-derived from MSC-ES .Relative gene expression level of (TIF)Click here for additional data file.S3 Figversus human CD45 (right) with Ig-isotype controls (left). The mouse was a control for the transplanted experimental group and bled at the 4 weeks experimental time points. Note the human CD45 antibody is extremely specific and no human cells or non-specific background was detected compared to mouse CD45 and isotype controls. (B) Representative FACS analysis for mouse blood at 4 weeks post-transplant with EB’s from H9 cell line double stained for mouse-CD45 and human-CD45 antibody. Note the specificity of the human-CD45 to detect a small but distinct cell population as shown in the bottom right dot plot.EB cells were injected directly into the femur of non-lethally irradiated NOG mice. (A) Representative FACS analysis for non-transplanted control mouse blood, showing specificity of mouse CD45 (middle) (TIF)Click here for additional data file.


GNAQ or GNA11 carry Q209X or R183X activating mutations and are present with ~90% frequency in human uveal melanomas. Forced expression of GNAQ/11Q209L in melanocytes is sufficient to drive metastatic melanoma in immune-compromised mice. No known drugs directly target these oncogenic G proteins. Ric-8A is the molecular chaperone that selectively folds Gαq/i/13 subunits. Targeting Ric-8A serves as a rational, yet unexplored approach to reduce the functional abundance of oncogenic Gαq/11 in order to blunt cancer signaling. Here, using mouse melanocyte cell graft tumorigenesis models, we determined that Ric-8A genetic ablation attenuated the abundance and melanoma-driving potential of Gαq-Q209L. A new conditional Ric-8AFlox/Flox; Rosa-CreER+/− mouse strain was derived and used as a tissue source to culture an immortalized, tamoxifen-inducible Ric-8A knockout melanocyte cell line that required 12-O-tetradecanoylphorbol-13-acetate  for growth. The cell line failed to grow tumors when grafted into immune-compromised mice regardless of Ric-8A expression. Stable expression of human GNAQQ209L, but not GNAQWT in the cell line promoted TPA-independent cell proliferation, and upon cell grafting in mice, the initiation and robust growth of darkly-pigmented melanoma tumors. Deletion of Ric-8A in GNAQQ209L cells restored TPA-dependent growth, reduced Gαq-Q209L below detectable levels and completely mitigated tumorigenesis from primary or secondary cell line grafts. Interestingly, TPA treatment of cultured GNAQQ209L cells or host animals grafted with GNAQQ209L cells also sharply reduced Gαq-Q209L abundance and tumorigenic capacity. Finally, tumorigenesis initiated from GNAQQ209L cell grafts, followed by host mouse systemic tamoxifen treatment to delete Ric-8A in the grafted cells completely abrogated GNAQQ209L-driven tumor progression unless a stable human RIC-8A transgene was used to rescue the floxed Ric-8A alleles. Our work defines two new rational targets that may be developed as potential uveal melanoma therapies through reduction of Gαq/11-Q209L oncoprotein abundance: (1) Ric-8A inhibition and (2) phorbol ester treatment.The heterotrimeric G protein α subunit oncogenes  Owing to limited responses to systemic chemotherapies, metastatic forms of the cancer have a poor prognosis with a median survival rate of 12 months, accounting for ~5% of all lethal melanoma cases.GNAQ or GNA11 activating mutations induce dermal hyperpigmentation, and are frequently found in cutaneous benign blue nevi and a small subset of melanomas, indicating that overactive Gαq/11 signaling may also be important during priming events of dermal melanocyte neoplasms.16 Studies using genetic or xenograft mouse models demonstrated that melanocyte-specific GNAQ or 11 Q209L expression promoted invasive and metastatic melanoma.18 Moreover, massive metastatic cutaneous melanomas were induced when the Gq/11-coupled GPCRs, mGluR1 or mGluR5 were ectopically expressed from mouse melanocyte-specific promoters.21 Both mGluRs 1 and 5 have reasonably high basal ability to activate Gq/11 in the absence of agonist.23 The emerging evidence is quite convincing that aberrant stimulation of Gαq/11 signaling pathways by hyperactive GPCRs or oncogenic GNAQ/11 mutations, contributes to the development of various melanocyte neoplasms including cellular transformation and uveal melanoma.9Interestingly, 25 Existing small-molecule Gαq inhibitors exhibit efficacy to inhibit wild-type Gαq or Gq, and some related G proteins, but fail to attenuate Gαq-Q209L, or provide limited inhibition of Gαq-R183C-dependent signaling in cultured cells.28 These inhibitors may ultimately prove useful to block pathogenic, hyperactive GPCR signaling, but the inability to inhibit oncogenic Gαq/11 directly has prompted us to explore an alternative means to block oncogenic Gα subunits by targeting the highly-substrate-specific molecular chaperones, Ric-8A or Ric-8B that act collectively to maintain the proper abundances of all heterotrimeric G protein α subunits.31 Studies using cell-free protein translation/folding systems demonstrated that Ric-8A directly participates in the biosynthetic folding of oncogenic Gαq-Q209L and Gαq/i/13.32 When these G proteins are produced in the absence Ric-8A, they are mis-folded and rapidly degraded, accounting for massive ~90–95% reductions in membrane-associated G protein levels.29There are no current drugs that directly target oncogenic Gαq/11 proteins, although encouraging developmental efforts are underway.Ric-8A blocked GNAQQ209L-driven melanocyte transformation and melanoma pathogenesis using cell graft mouse tumor models. A new C57Bl6J mouse with floxed Ric-8A alleles was derived for the work that permitted conditional Ric-8A deletion. Primary melanocytes were cultured from this strain and used to create immortalized murine melanocyte cell lines that stably expressed human GNAQWT or oncogenic GNAQQ209L. The GNAQQ209L but not the GNAQWT melanocyte cell line exhibited phorbol ester-independent proliferation in culture; a feature associated with melanocyte transformation, and one that was abolished by Ric-8A deletion.33 When the GNAQQ209L melanocyte cell line was grafted into immune-compromised mice, pigmented melanoma tumors grew robustly. No tumors formed from GNAQWT melanocyte cell line grafts. Deletion of Ric-8A in culture before GNAQQ209L cell grafting completely abrogated tumor growth. Ric-8AFlox/Flox; GNAQQ209L murine melanoma cell lines were cultured ex vivo from primary tumor explants and secondary tumor formation from these cells was also blocked by in vitro Ric-8A deletion. GNAQQ209L melanocyte cell grafts were then permitted to initiate tumorigenesis, followed by host mouse tamoxifen treatment to delete floxed Ric-8A in the grafted melanocytes. Systemic tamoxifen treatment specifically abrogated GNAQQ209L-driven tumorigenesis from Ric-8AFlox/Flox melanocytes.Here, we conducted a proof-of-concept investigation demonstrating that genetic ablation of GNAQQ209L melanocytes in the presence of phorbol ester led to a dramatic decrease in Gαq-Q209L oncoprotein levels. Accordingly, phorbol ester-pre-cultured GNAQQ209L melanocytes completely failed to form melanoma tumors when grafted into mice, and systemic phorbol ester treatment of host mice grafted with GNAQQ209L melanoma cells suppressed tumor initiation and progression. In sum, our study has identified Ric-8A inhibition and phorbol ester (over)stimulation of protein kinase C (PKC) as two new rationale means to attenuate Gαq-Q209L oncoprotein levels. Successful future development of therapeutics against these new targets could provide wanted therapies for GNAQ/11-driven uveal melanoma, and perhaps additional oncogenic G protein-influenced cancers.We also made an unexpected observation that culture of Ric-8A causes embryonic lethality due to severe gastrulation defects.34 We created a C57Bl/6 J mouse strain with potential for conditional Ric-8A knockout using Ric-8A gene-targeted embryonic stem cell lines available from the Knockout Mouse Project   were cultured from our Ric-8AFlox/Flox mice and infected with a Cre-NLS-expressing lentivirus. PCR analysis of MEF genomic DNA revealed efficient Cre-mediated deletion of Ric-8A exon 5 (32Germline deletion of mouse SD70793) . Ric-8A A exon 5 . ImmunobA exon 5 .32Ric-8A deletion on Gα subunit abundances in melanocytes and melanocyte transformation induced by oncogenic Gαq-Q209L, we first created and characterized an immortalized melanocyte cell line with inducible Ric-8A knockout potential. Primary melanocytes were isolated from Ric-8AFlox/Flox; Rosa-CreER+/− neonatal mice and immortalized by serial passaging in medium containing 12-O-tetradecanoylphorbol-13-acetate  and cholera toxin (CTX).37 The Ric-8AFlox/Flox; Rosa-CreER+/− melanocyte cell line exhibited TPA- and CTX-dependent growth, a shared characteristic with the non-tumorigenic mouse melanocyte cell line, Melan-a  activation of Cre recombinase, decreased Ric-8A abundance and caused concomitant decreases in the levels of G protein subunits folded by Ric-8A . y Ric-8A . Gαs levRic-8A deletion had no effect on the requirements of TPA or CTX for melanocyte cell line growth, but did modestly enhance the cell proliferation rate (Ric-8A null cell types.39 We hypothesize that Ric-8A deletion and the consequent decreases in G protein α subunit abundances (Gαq/i/13 classes) release a modest cell proliferation damper conferred normally by homeostatic G protein signaling.ion rate . EnhanceRic-8AFlox/Flox; Rosa-CreER+/− melanocyte cell line require continuous signaling stimulus of PKC  and Gαs (CTX) for in vitro proliferation  melanocyte cell lines (5 × 106 cells each) were grafted subcutaneously into the rear flanks of immune-compromised NSG mice. GNAQQ209L cells pre-cultured in the absence of TPA formed tumors that could first be measured beneath the skin after an ~28-day latency. The tumors continued to grow for an additional ~30 days until the mice were killed and the heavily melanin-pigmented tumors were excised and weighed. The GNAQQ209L tumors were roughly 0.6–2.0 cm in diameter and often oblong and/or multi-lobed. No tumors were formed from GNAQWT or GFP cell grafts . The GFP-only melanocyte cell line had no tumorigenic capacity regardless of Ric-8A expression or deletion  tumors from ex vivo in standard melanocyte culture medium lacking TPA to derive two independent GNAQQ209L melanoma cell lines, with which to determine the effect of Ric-8A deletion on secondary tumor progression. The melanoma cell lines exhibited TPA-independent growth and acquired TPA dependence after Ric-8A deletion, both characteristics of the parental GNAQQ209L melanocyte cell line used to generate the primary tumors  melanocyte cell line was transduced with a human RIC-8A cDNA or control lentivirus and selected for stable expression of the transgene. 4OHT treatment of both melanocyte cell lines in culture induced deletion of the floxed, mouse Ric-8A alleles, which resulted in greatly reduced Gαq-Q209L and Gαq/11 levels in the cell line lacking the RIC-8A transgene. Gαq-Q209L remained at normal levels in the counterpart RIC-8A transgene expressing cell line, despite 4OHT treatment  injected with tamoxifen q.a.d. to induce floxed Ric-8A deletion in the grafted cells. Solvent injections were performed similarly for control, Ric-8AFlox/Flox-grafted mice . In vivo tamoxifen treatment effectively ablated tumorigenesis driven by GNAQQ209L in the Ric-8AFlox/Flox background, but not in the background in which the floxed Ric-8A alleles were rescued by expression of the RIC-8A cDNA transgene  melanoma cell line was generated from an excised primary tumor in GNAQQ209L melanocyte cell line in in vitro responsiveness to phorbol ester-mediated Gαq-Q209L oncoprotein level reduction . Blunting the abundance of client oncoproteins through inhibition of the chaperones that fold them is an active strategy in cancer therapeutic development, with Hsp90 inhibitors providing a prominent precedent.Ric-8A knockout potential was derived and used as a source to create immortalized melanocyte cell lines from neonatal dermal explants. The parental melanocyte cell line was made to stably express human GNAQWT or GNAQQ209L. Induced Ric-8A deletion in the cultured melanocyte cell lines effectively reduced Gαq-Q209L protein levels and had no obvious cytotoxic effects. In fact, Ric-8A deletion imparted a modest in vitro growth advantage to cultured melanocytes and melanoma cell lines. This effect has been observed for other Ric-8A-null cell types and suggests that the net effect of endogenous G protein signaling is to provide a cell proliferation brake.39 However, this in vitro growth enhancement provided no tumorigenic propensity when Ric-8A-null cells lacking a driver oncogene were grafted into immune-compromised mice. Ric-8A deletion completely blocked melanoma tumorigenesis of the grafted GNAQQ209L melanocyte cell line and secondarily-derived GNAQQ209L melanoma cell lines.For this work, a new C57Bl/6 J transgenic mouse strain with inducible Ric-8A deletion/inhibition-mediated reduction of endogenous G proteins would potentially mitigate all of these processes. An additional observation made with Ric-8A null cells may be specifically relevant to a potential uveal melanoma therapy; Gαq/11-Q209L-mediated uveal melanoma progression involves constitutive stimulation of Rho/Rac guanine nucleotide exchange factors (GEFs) that regulate actin polymerization, which is thought to contribute the signal that activates YAP as the main signaling driver of the cancer.45Ric-8A-null mouse embryonic stem cells and Ric-8A shRNAi-treated MEFs have substantial deficiencies in polymerized actin levels (F-actin) that may be attributable to reductions in the G proteins (Gα12/13/q/11) primarily responsible for stimulating Rho-signaling pathways.39 So, in addition to inhibited Ric-8A resulting in reduced Gαq/11-Q209L oncoprotein levels, there could be additional attenuation of the YAP pathway at the level of reduced actin polymerization.A potential complication of any approach to genetically disrupt or inhibit Ric-8A is the reduction in endogenous G protein levels that will occur. On the other hand, if Ric-8A inhibition could be directed to tumor cells, the effect on endogenous G proteins may provide added therapeutic efficacy of tumor inhibition. G protein signaling imparts important advantages within the tumor microenvironment including pro-migratory properties of cancer cells, secretion of angiogenic factors that promote tumor vascularization, responsiveness to host growth factors and usurpation of the immune system. in vitro.46 Evidence from many organismal systems showed that Ric-8 orthologs are required to maintain proper G protein abundances.31 Ric-8 acts as a chaperone during biosynthesis to facilitate Gα subunit protein folding. G proteins produced in the absence of Ric-8 are mis-folded and rapidly degraded.32 It is not clear whether GEF and chaperoning activities are one and the same, or whether Ric-8 is a multi-functional protein that acts during biosynthetic G protein folding and later facilitates Gα GTP binding to evoke signaling outputs. The fact that Ric-8A deletion suppresses the oncogenic action of Gαq-Q209L indicates that the chaperoning activity is either the authentic cellular activity or is more penetrant than GEF activity. The Gαq/11-Q209L oncoprotein has greatly impaired GTP hydrolysis activity that renders it constitutively active, therefore bypassing the need of a GEF for sustained signaling output.Our findings help clarify a current controversy regarding Ric-8 cellular activity. Ric-8A was initially characterized as a GEF that stimulates Gα subunit guanine nucleotide exchange GNAQ/11Q209L drives the majority of uveal melanomas, yet the oncogene is far less prevalent in cutaneous melanomas.9 Deciphering this difference will provide useful insight toward the development of melanoma therapies. We hypothesize that uveal melanocytes possess a survival privilege stemming from an innate ability to tolerate a higher ‘dose' of Gαq/11 signaling, or as others have suggested, ocular melanocytes are compartmentally shielded from attack by the host immune system.47 Cutaneous melanocytes may be more sensitive to Gαq/11 signaling and have a capacity to downregulate GNAQ/11Q209L transcript levels or Gαq/11-Q209L protein levels, or otherwise enter into an apoptotic cell death program.18 Constitutively-active GNAQ/11 mutations are in fact highly prevalent in benign melanocytic neoplasms and can cause skin hyperpigmentation.18 These pre-cancerous dermal lesions could require a lower threshold of Gαq/11 signaling to develop, yet the higher level necessary to drive cellular transformation may not be well tolerated by dermal melanocytes. There are many clear examples in which modestly elevated Gq/11 signaling induces proliferation in specific tissues or cultured cell lines, but chronic Gq/11-coupled GPCR agonist treatment(s) or constitutively-active mutant overexpression induces apoptosis.52A key remaining question is to understand why GNAQQ209L cutaneous murine melanocyte or melanoma cell lines were treated in culture with a phorbol ester, Gαq-Q209L protein abundance, but not wild-type Gαq/11 abundance was reduced dramatically, an effect on the order of that which occurred when Ric-8A is deleted. The TPA-treated GNAQQ209L cell line did not form tumors when grafted, despite efficient production of the GNAQQ209L-IRES-GFP transcript as visualized via the efficient GFP fluorescent signal  PKC-activated feedback reduction of Gαq/11 oncoprotein abundance or (2) inducement of cancer cell apoptosis. PKC activators, including phorbol esters, inhibited growth of various non-uveal melanoma cell lines and tumors through inducement of cell-cycle arrest or apoptosis.56 Wild-type PKC rescue of loss-of-function PKC alleles in human tumor cells inhibited tumorigenesis, showing that PKC is a tumor suppressor.57 Our demonstration in GNAQQ209L cell line is highly encouraging. Our ongoing and future work involves active trials to reduce Gαq/11-Q209L oncoproteins through Ric-8A inhibition or phorbol ester inhibitory feedback as two new potential strategies to treat GNAQ/11Q209L melanoma.Our results raise the intriguing prospect that phorbol esters may be an effective way to mitigate uveal melanoma oncogenesis. Phorbol esters directly activate PKC and are commonly thought of as tumor promoters. Yet for Ric-8A knockout-first allele targeted mouse embryonic stem cell (ES) lines were purchased from Knockout Mouse Project (KOMP) Repository (Project ID CSD70793) at UC Davis. Microinjection and implantation of Ric-8A-Neo targeted ES cells was carried out by the Gene Targeting and Transgenic Mouse core facility at the University of Rochester Medical Center. Chimeras were mated with C57Bl/6 J mice . One out of four chimeras carried a germline copy of the Ric-8A-Neo allele. Mice harboring the Ric-8A Neo allele were crossed with FLPer mice (Jax Lab stock # 009086) to generate Ric-8A floxed allele progeny. Ric-8A homofloxed mice that express tamoxifen-inducible-Cre (CreER) recombinase driven by the ubiquitous Rosa promoter were generated upon breeding to R26-Cre-ERT2 mice (Jax Lab stock # 008463). Mice were handled and maintained in accordance with University of Rochester Institutional Animal Care and Use committee.The Ric-8AFlox/Flox embryos and cultured in DMEM containing 10% FBS.58 Spontaneously immortalized melanocyte cell lines were generated from epidermal cell explants isolated from 3-day old neonates as described.36 In detail, cell explants were co-cultured in melanocyte medium  with XB2 keratinocyte feeder cells  that had been mitotically-inactivated with mitomycin C. Growth of melanocytes was selectively promoted by TPA and CTX supplementation.37 After passage 4, XB2 feeder cells were eliminated and melanocytes were passaged past the senescence phase until colonies began to grow. Spontaneously immortalized, feeder cell-free, melanocyte cell line growth was confirmed by examination of cells by microscopy for the presence of melanin pigment granules.MEFs were isolated from day E13.5 m final concentration in melanocyte medium was used to inhibit proteasome-mediated degradation in GNAQQ209L-melanocyte or -melanoma cell line culture. The MG132-treated cells were washed with phosphate-buffered saline with protease inhibitor mixture  and scraped from the culture dish. Cells were pelleted at 1000 g for 5 min. Pelleted cells were lysed in the sample buffer containing protease inhibitors mixture and 10 μm MG132, boiled at 95 °C for 5 min and centrifuged at 1 50 000 g for 5 min. The resulting supernatants were used for western blot analyses.MG132 (in DMSO) at 10 μg for 5 min. The 500-g supernatant was centrifuged at 1 50 000 g for 45 min. The resulting crude membrane pellet was solubilized in sample buffer with protease inhibitor mixture and processed for western blot analyses.To prepare crude membranes, cultured cells were washed and scraped in phosphate-buffered saline with protease inhibitor mixture. The cells were lysed by nitrogen cavitation using a Parr bomb . The lysate was clarified by centrifugation at 500 59 using the forward primer: 5′-CGCGCGCCATGGATGCCAAAAAAAAAGAGGAAGGTGTCCAATTTACTGACCGTACACC-3′, encoding the N-terminal nuclear localization signal, MPKKKRK, and the reverse primer: 5′-CGCGCGGTCGACCTAATCGCCATCTTCCAGCAGGCG-3′ and inserted into the Gateway Entry Vector, pENTR4.60 A bicistronic pENTR4-IRES-GFP vector was created by excision of the IRES-GFP cassette from pIRES2-GFP (gift from Dr David I Yule) with SalI-XbaI and ligation into these sites of pENTR4. The coding sequences of human GNAQWT or GNAQQ209L were PCR amplified from the corresponding pcDNA3.1 constructs (cDNA Resource Center) using linker-based oligonucleotides and inserted 5′ of the IRES-GFP cassette in pENTR4-IRES-GFP. Lentiviral donor expression vectors were created using the Gateway LR Clonase Enzyme kit  to recombine the Cre-NLS-IRES-GFP, and GNAQWT- or GNAQQ209L-IRES-GFP modules from the respective pENTR4 entry vectors into the CMV promoter Gateway destination vectors, pLenti-Hygro or Puro.61The coding sequence of Cre recombinase was amplified from pENTR4-CreERT2GNAQQ209L and control melanocyte cell lines using a Qiagen DNAeasy Blood and Tissue kit . The following human GNAQ-specific primer pair was used, forward primer: 5′-GCGCGCGCGTACGCGCATGACTCTGGAGTCCATCATG-3′ and reverse primer: 5′-CGCGCGCAATTGTTAGACCAGATTGTACTCCTTCAG-3′ with the program: 40 cycles of 94 °C for 1 min, 56 °C for 45 s, 72 °C for 1 min 30 s, followed by a 5-min extension phase at 72 °C. Total RNA was isolated from GNAQQ209L and control melanocyte cell lines that had been treated with or without TPA for 11 days. Total RNA (400 ng) was reverse transcribed using the GNAQQ209L-specific antisense primer, 5′-CCATTTTCTTCTCTCTGACCTTAGGCCCCCTACATCGACC-3′ with a Superscript III Reverse transcriptase kit . cDNA was subjected to nested PCR using the human GNAQQ209L transcript-specific primer pair: forward primer 5′-GCGCGCGCGTACGCGCATGACTCTGGAGTCCATCATG-3′ and reverse primer 5′- GGTAGGCAGGGTCAGCTACGC-3′ with the program: 40 cycles of 94 °C for 1 min, 53 °C for 45 s, 72 °C for 1 min 30 s, followed by a 5-min extension phase at 72 °C. PCR products were obtained after the indicated number of amplification cycles and resolved on ethidium bromide-stained 1% Agarose gel and visualized by UV transillumination.PCR was performed using 100 ng of genomic DNA extracted from GNAQWT or -GNAQQ209L-IRES-GFP donor vectors as follows: 4 × 106 HEK293T cells were seeded in 10-cm diameter culture dishes in DMEM containing 10% FBS. After 24 h, cells were transfected with 4 μg of pLenti construct, 2.5 μg of pMDLg/pRRE, 2.5 μg of pRSV-Rev and 2 μg of pSVS/pMD2.g using Lipofectamine 2000 (Thermo Fisher).62 Lentiviral-containing media overlaying the transfected cells was harvested at 48 h, filtered through a 0.45-μm syringe filter and supplemented with 8 μg/ml of polybrene . Immortalized mouse melanocytes were infected with 1:1 ratio of viral medium: fresh melanocyte medium for 48 h. Transduced melanocytes were selected with 200 μg/ml and then 100 μg/ml hygromycin B over 72 h and maintained in melanocyte medium containing 50 μg/ml hygromycin B (Thermo Fisher). The GNAQWT and GFP stable melanocyte cell lines were continuously cultured in the presence of TPA. The GNAQQ209L melanocyte cell line was cultured in the absence of TPA in order to maintain Gαq-Q209L abundance.Lentiviruses were prepared from the pLenti-Cre-NLS, pLenti-IRES-GFP and pLenti-Ric-8AFlox/Flox MEFs were infected with Cre-NLS-lentivirus for 48 h, selected with 100 μg/ml hygromycin B for an additional 48 h to achieve Ric-8A knockout. Cell lysates were harvested for quantitative western blot analyses and mRNA isolation. Deletion of Ric-8A in cultured Ric-8AFlox/Flox; Rosa-CreER+/- melanocyte cell lines was achieved by treating the cells in culture with 500 nm 4OHT (Sigma-Aldrich) for 5 days or by infection with Cre-NLS-lentivirus for 48 h followed by selection with 2 μg/ml puromycin (Sigma-Aldrich).Prkdcscid Il2rgtm1Wjl/SzJ) mice were from the Jackson laboratory (Stock #005557). For the subcutaneous cell graft tumor model, live melanocytes were suspended by brief trypsinization, washed extensively in melanocyte medium and counted using a hemacytometer after Trypan blue dye exclusion. NSG host mice were injected subcutaneously in each hind flank with 5 × 106 cells of the GFP-, GNAQWT- or GNAQQ209L-melanocyte cell lines in RPMI base medium, or 1 × 106GNAQQ209L tumor-derived melanoma cell lines for the secondary tumor growth progression studies. In vivo deletion of Ric-8A in melanocyte cell lines grafted into NSG mice was achieved by treating host animals with 10–15 i.p. injections of 1 mg tamoxifen or vehicle over a course of 20 days. Tamoxifen was dissolved in ethanol at 100 mg/ml followed by 1:10 dilution in sterile corn oil. The TPA treatment of host mice was performed by mixing melanocytes in a suspension of RPMI base medium and 200 nm TPA or vehicle before cell grafting. At day 1 post graft, one i.p. injection of 200 μg TPA  or vehicle was performed, followed by i.p. injection of 4 μg TPA or vehicle, q.a.d, over days 5–12 post cell grafting. Injection sites were monitored thrice weekly for tumor growth. Tumor size was measured using calipers and volume was calculated using the formula: length × width2/2.64 Mice were killed when measured tumors reached ~1000 mm3 and the tumors were isolated from the adhered tissue.Eight-week-old female NSG  for 5–10 min at 37 °C. The resulting cell suspension was washed twice and cultured in fresh melanocyte medium.Subcutaneous 29 DAPI-stained images of fixed cells were captured using Metamorph software  with UPLSAPO × 10 (n.a. 0.35) objective on an Olympus IX70 microscope and an ORCA-ER digital camera . Phase-contrast images were captured with a Nikon Eclipse TS100 inverted microscope , × 10 objective (n.a. 0.25) using an attached Canon EOS Rebel XSI camera .Melanocyte cell lines were fixed with 4% paraformaldehyde and stained with DAPI nuclear stain as described.29 Gαo [S214]65 and Gαi1/2 [B084]66 antisera were described previously. Rabbit polyclonal Gαq/11 [C-19] and Gα13 [H-300] antisera were purchased from Santa Cruz . Monocolonal α-tubulin and γ-tubulin were from Sigma-Aldrich. The antibody against the Gαq-Q209L oncoprotein was purchased from NewEast Biosciences, King of Prussia, PA, USA [26328].Rabbit polyclonal Ric-8A [1184],t-tests were performed as indicated using GraphPad Prism . P-values <0.05 were regarded as significant.Error bars throughout are the mean±s.e.m. Two-tailed Student's


There are approximately 100 publicly available ovarian cancer cell lines but their cellular and molecular characteristics are largely undescribed. We have characterized 39 ovarian cancer cell lines under uniform conditions for growth characteristics, mRNA/microRNA expression, exon sequencing, drug response for clinically-relevant therapeutics and collated all available information on the original clinical features and site of origin. We tested for statistical associations between the cellular and molecular features of the lines and clinical features. Of the 39 ovarian cancer cell lines, 14 were assigned as high-grade serous, four serous-type, one low-grade serous and 20 non-serous type. Three morphological subtypes: Epithelial (n = 21), Round (n = 7) and Spindle (n = 12) were identified that showed distinct biological and molecular characteristics, including overexpression of cell movement and migration-associated genes in the Spindle subtype. Comparison with the original clinical data showed association of the spindle-like tumours with metastasis, advanced stage, suboptimal debulking and poor prognosis. In addition, the expression profiles of Spindle, Round and Epithelial morphologies clustered with the previously described C1-stromal, C5-mesenchymal and C4 ovarian subtype expression profiles respectively. Comprehensive profiling of 39 ovarian cancer cell lines under controlled, uniform conditions demonstrates clinically relevant cellular and genomic characteristics. This data provides a rational basis for selecting models to develop specific treatment approaches for histological and molecular subtypes of ovarian cancer.Epithelial ovarian cancer is a highly heterogeneous disease and remains the most lethal gynaecological malignancy in the Western world. Therapeutic approaches need to account for inter-patient and intra-tumoural heterogeneity and detailed characterization of  Epithelial ovarian cancer is the most lethal gynaecological malignancy in the Western world and advanced disease remains incurable for the majority of patients. Despite testing of diverse treatment strategies and new cytotoxic agents, optimal primary therapy and survival rates have not substantially changed since the introduction of platinum and taxane treatment TP53 mutation (96%), homologous recombination DNA repair defects (∼50%), CCNE1 amplification and genomic instability TP53 wildtype and show frequently activating RAS pathway mutations PIK3CA , and ARID1A mutations  Recent insights have indicated that ovarian cancer is a collective term for invasive pelvic cancers that are derived from different tissues with distinct histological and epidemiological features. The five major histiotypes have been shown to have specific genetic profiles and should be treated as distinct diseases. High-grade serous (HGS) carcinoma represents 80% of ovarian cancers and is defined by Furthermore, large studies have identified several molecular subtypes of HGS based on gene and microRNA expression It is imperative that experimentally tractable in vitro models, such as cell lines, accurately represent the different histological and molecular subtypes in order to test new subtype-specific treatment strategies. Worldwide, there are about 100 ovarian cancer cell lines generated as described in literature in vitro studies. We identified histological as well as morphological subtypes which associate with clinical pathological characteristics of ovarian carcinomas as well as prognosis. Furthermore, the morphological subtypes associate with the molecular subtypes identified by Tothill et al. in vitro therapeutic studies.We here describe an extensive and uniform characterization of a collection of 39 ovarian cancer cell lines commonly used for Cell lines studied were CAOV3, CAOV4, ES-2, OV-90, OVCAR3, TOV-112D, TOV-21G, UWB1.289, UWB1.289+BRCA1 (purchased from ATCC), 59M, A2780, A2780CIS, A2780ADR, ‘COLO720E’, COV318, COV362, COV362.4, COV413A, COV413B, COV504, COV644, OAW28, OAW42, OV56, OV7, OV17R, PEA1, PEA2, PEO1, PEO4, PEO14, PEO16, PEO23, SKOV3 , 2774, A2780, HOC7, SKOV3, SKOV6 , IGROV1 . All cell lines were initially cultured using the medium and supplements as recommended by the suppliers. In contrast to other studies, we cultured all cell lines under the same culture conditions to avoid biases due to varying concentrations of supplements within different media (e.g. growth factors) or different percentages of serum. All cell lines were cultured in RPMI-1640 glutamax, Gibco/Invitrogen supplemented with 10% FCS , 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamycin. All experiments and nucleic acid isolations were performed after culturing the cell lines for at least one month using these standard conditions.BRCA1 2594delC mutation. UWB1.289+BRCA1 is stable transfected with a pcDNA3 plasmid containing a BRCA1 insert and was maintained with 200 µg/ml Geneticin (G418) to maintain selection for transfected cells. A2780ADR and A2780CIS were challenged once a month with 100 nM Doxorubicin and 1 µM Cisplatin respectively.UWB1.289 is a BRCA1-null cell line from woman with a germline The PowerPlex 16 System (Promega) was used according to the manufacturer's protocol using an ABI PRISM 3100 to generate an STR (Short tandem repeat) fingerprint of each cell line to determine unique identity and/or lack of co-culture contamination. In addition, the STR profile was checked before and during culturing of the cells to ensure that cross-contamination or erroneous substitution did not occur.The MTT colorimetric assay, which measures metabolic activity of viable cells, was used to generate growth curves and determine the chemosensitivity of the cell lines.First, growth curves were generated in duplicate to determine the optimal number of cells to use for the drug sensitivity assay. This was done to avoid growth inhibition due to seeding not enough cells or depletion of the medium after seeding too many cells. The highest number of cells showing continuing exponential growth after five days was selected for the drug response MTT assays , File S1Response curves were generated for Carboplatin, Cisplatin, Oxaliplatin, Doxorubicin and 5-Fluorouracil , Paclitaxel , Docetaxel , and Gemcitabin . Cell viability was assessed in quadruplicate using the MTT assay after a five day exposure to 18 concentrations of the compound. Phoenix WinNonLin 1.1 software (Pharsight) was used to fit a dose response curve and to calculate the 50% growth inhibition values (GI50) with error and 95% confidence intervals see also .The NucleoSpin kit (Machery-Nagel) was used according to the manufacturer's protocol to isolate DNA from cells harvested with trypsin and washed with PBS.For the isolation of total RNA (including microRNAs), exponentially growing cells were directly lysed in culture flasks with RNA-Bee to avoid changes in expression due to harvesting or washing. Total RNA was isolated from lysates as previously described For microRNA expression analysis, TaqMan Array Human MicroRNA A fluidic Cards v2.0 (Applied Biosystems) containing qRT-PCR assays to quantify 381 unique microRNAs were used according to the manufacturer's protocol. The expression data was normalized using the median Ct of all measured microRNAs as described by Vandesompele et al. Gene expression was measured using the GeneChip Human Exon 1.0 ST Array (Affymetrix) according to manufacturer's protocol see also . The acqFile S1).SNaPshot analysis was performed as described previously Processing of samples for sequencing on the SOLiD5500 sequencing platform (Life Technologies) was performed on a Sciclone NGS liquid handling robot (Perkin Elmer), based on the protocol for manual library preparation Data were mapped to the reference genome (GRCh37/hg19) using BWA and SNP and indel calling was done using a custom analysis pipeline. Based on the COSMIC database For the analysis of gene amplification, robust Z-scores were calculated per exon as described by Iglewicz and Hoaglin The SOLiD Exon sequencing has been deposited in the European Nucleotide Archive (PRJEB5114).The coding sequences of TP53, BRCA1, BRCA2, PTEN, PIK3CA, EGFR and APC genes were amplified and sequenced using the Tam-Seq method and the Fluidigm Access Array 48.48  according to manufacturer's protocol as described previously Microsatellite instability was determined using the MSI Analysis System Version 1.2 (Promega) according to the manufacturer's protocol and as performed routinely electrophoresis using an ABI PRISM 3100. MSI is determined on five mononucleotide repeat markers .The cells were harvested and stained with properly titrated fluorochrome-conjugated monoclonal antibodies  and the possibly contaminated cell line ‘COLO720E’ were left out of these analyses. Statistical analyses were done using nonparametric methods within the STATA statistical package, release 12.0 . P-values were two-sided and P<0.05 was considered statistically significant.Statistical analyses were performed on the 33 cell lines that were generated from unique ovarian cancer patient material. Duplicates (SKOV3 and A2780), BRB array tools (v4.3.1) was used to select genes and microRNAs differentially expressed between the three morphological subtypes. First, microRNAs with >80% missing values were filtered out. Then missing values that were not flagged due to low quality were replaced with the minimal value over all microRNAs and samples followed by 2log normalization. Within BRB, the 50% most variable microRNAs and mRNAs were selected and the class comparison algorithm was used to calculate the permutation p-value after 10.000 permutations. P<0.05 was considered significant. Hierarchical clustering was done on median centred mRNA and microRNA expression data using Eisen Gene cluster 3.0. For the GSE9891 data set multiple probes per gene were averaged which most closely resembles the approach taken by the exon arrays (i.e. the probe set summarization within the Affymetrix Expression Console software). Next, the cell line gene expression data and the GSE9891 data (excluding the LMP samples) were median centred separately to correct for differences between the platforms used and subsequently combined before the hierarchical clustering. Identification of biological function of the morphology related genes was performed using IPA .The measured number of repeats for each of the 16 STR loci as well as reference STR profiles from public databanks and literature are given in The concordance between the peaks of the reference profiles and the profiles generated in this study was 100% for 22 cell lines, 90–99% for ten cell lines and 79–89% for four cell lines. For three cell lines, there are no references available .One cell line, ‘COLO720E’, showed only 4% concordance with the STR profiles published by Korch et al. The available literature information on the origin of the 39 cell lines is summarized in The origin , time  and whether the patient had received platinum-based chemotherapy before culture was known in 32, 21 and 25 cell lines respectively. The tissue-originated cell lines were mostly from platinum-naive patients  and primary disease  whereas the ascites-originated cell lines were more often platinum-treated  and cultured after relapse or clinical resistance .Microsatellite instability for all five markers studied was observed for: 2774, both SKOV3 sources, IGROV1, TOV21G and ‘COLO720E’ . A2780ADAlthough the numbers are very small, the four MSI cell lines showed significantly less concordance of their STR profile to the reference STR profile  and more mutations than the 29 non-MSI cell lines .Three morphological phenotypes were distinguished based on two independent observations of morphological and growth characteristics during culturing from low-density up to 70/80% confluence, i.e. cell shape, cell size and growth pattern during culturing as well as proliferation rate. There was an association between morphology and the origin of the 33 unique cell lines, 74% of Epithelial cell lines originated from ascites (14/19), all four Round cell lines were culture from tissue, while Spindle cell lines originated equally from ascites, tissue or pleural effusion  (Fisher's exact p = 0.002). Although not significant, Epithelial cell lines were more often of serous origin (83%) compared to Round (33%) and Spindle (56%) cell lines .Interestingly, the morphological subtype was significantly associated with prior platinum-based chemotherapy .The expression of CD44, CD24, EpCAM and luminal cytokeratin's are given in high/CD24− expression was not observed in the four Round cell lines and in only 6/20 Epithelial cell lines.The stem cell marker CD44 was expressed in 31/33 of the cell lines and was associated with morphology . Interestingly, 6/9 Spindle cell lines showed high CD44 expression combined with absent CD24 expression which has been suggested as a characteristic of stem cell populations. By contrast, CD44The epithelial markers EpCAM and luminal Cytokeratin's were expressed in most Epithelial cell lines (19/20 and 18/20 respectively) but only in one Round cell line (1/4). All Spindle cell lines showed absent or very low expression of EpCAM but 5/9 did express luminal Cytokeratin's. EpCAM and luminal Cytokeratin's expression was associated with morphology  as well as serous versus non-serous histology .The mutation status of 53 genes was determined with three techniques: SNaPshot, SOLiD and/or Tam-Seq exon sequencing. Discordant data between these three techniques were checked manually using the raw sequence data. ERBB2 or CCNE1 amplification showed the highest expression of these genes. For CCNE1 this association was significant  showed a two log range between sensitive and resistant cell lines . NotablyBRCA1 and BRCA2 have been associated with response to platinum drugs. However, there was no clear association between response to platinum and mutation status or expression of BRCA1 and BRCA2 in our experiments (data not shown). Approximately 50% of the BRCA mutations observed were not known to be clinically relevant  and 1–10 microRNAs (p<0.01). We designated cell lines as putative HGS or endometrioid/clear cell cell lines based on four criteria. Criteria for the putative HGS origin were: 1) HGS origin with TP53 mutation and no MSI, or 2) serous origin with TP53 mutation and amplification of CCNE1, MYC or TPX2. Criteria for putative endometrioid/clear cell origin were: 1) endometrioid and/or clear cell origin, or 2) ARID1A mutation.Using these criteria, we deduced the putative histological subtype for the 39 cell lines as 20 non-serous .From the non-serous cell lines, group 1 see also , containFrom the serous cell lines, group 3 contains seven cell lines described as high-grade serous (HGS) in origin and all, except PEO16, show a TP53 mutation as expected for HGS tumours. PEO16 does show a BRCA2 mutation and a TP53 mutation might have been missed since it was sequenced with one technique, although with a reasonable coverage (mean coverage  = 113). In addition, we identified seven other putative HGS cell lines (Group 4) that were previously described as (low-grade) serous or unknown histology that all show a TP53 mutation combined with an amplification of CCNE1, MYC or TPX2 which are characteristics of the HGS subtype.The seven remaining putative serous cell lines, Group 5, were described to be of serous origin and six show a TP53 mutation but no additional HGS characteristics. OAW42 shows no TP53 mutation but a PIK3CA and a BRCA1 mutation and both SKOV3 cell lines are MSI which is more often seen in the mucinous and endometrioid subtypes Interestingly, the putative histological subtypes  were significantly associated with the morphological phenotypes. Twelve out of 13 HGS cell lines have an Epithelial morphology and one Spindle, 4/6 serous cell lines have Epithelial and two Spindle morphology and for the 13 Clear cell/Endometrioid/Mixed cell lines three have Epithelial, four Round and six have a Spindle morphology (Fisher's exact p = 0.002).Four molecular subtypes were defined by unsupervised clustering of the expression of the 80% most variable genes (n = 17609) and microRNAs (n = 177). The 80% most variable genes and microRNAs were selected by ranking based on their variability in expression across the 26 unique cell lines (excluding the seven PEO/PEA cell lines that were not profiled).The four clusters identified associated with specific cell line characteristics . The putWe selected 1141 genes and 18 microRNAs differentially expressed between the three morphological subtypes (permutation derived p<0.05) . HierarcTwo cell lines did not cluster with their morphological subtype, SKOV3 clusters with Spindle instead of Epithelial (as is SKOV3 ECACC) and COV413A with Epithelial instead of Round.We used the expression data for 267 malignant ovarian carcinomas (GSE9891) We also observed that the Spindle cell lines clustered with the C1  tumours, the Round cell lines with the C5-mesenchymal subtype and the Epithelial cell lines with the C4 subtype . The cluWorldwide about 100 ovarian cancer cell lines are available and since 1990, a total of over 2000 papers have been published using these cell lines as models for ovarian cancer. Within this study we provide an extensive characterization of 39 of these cell lines on cellular and molecular level. The major strength of this study is that the analyses were all uniformly performed and therefore provide a rigorous comparison between the expression signatures of the cell lines. This uniformity was achieved by culturing all cell lines with the same medium and culture conditions, using the first passages after stable growth on the standardized culture conditions of the cell lines for all cellular and molecular analyses, and performing the molecular analysis of all cell lines simultaneously within one batch. We identified that the ‘COLO720E’ cell line did not match the reference short-tandem repeat (STR) profile and was excluded from further analyses.The DNA mismatch repair system (MMR) is involved in repair of errors in these STR that occur during DNA synthesis. Therefore, defective MMR is associated with a large variability in the length of these repeats which is called micro-satellite instability (MSI). Indeed, the STR profiles of the MSI cell lines showed a significant lower concordance with their reference STR profiles. For instance, IGROV1 is one of the MSI cell lines that has been suggested by Korch et al. to have a variable STR profile with multiple peaks which may indicate cross-contamination Furthermore, MSI cell lines showed significantly more mutations per cell line then the non-MSI cell lines (median of 13 vs 2). Similarly, MSI colorectal cancer was shown to have ten times more non-synonymous mutations per tumour compared to non-MSI colorectal cancer in vitro experiments using these lines and are vital for a rational experimental approach for future studies to develop subtype specific treatment approaches.To be able to develop and test subtype-specific treatments in the correct model, it is imperative to know which histological subtype each cell line represents. To identify HGS cell line models, Domcke et al. analysed genomic data for 47 ovarian cancer cell lines from the CCLE collection in vitro morphological subtypes . These three morphological phenotypes were previously observed in multiple types of cancer cell lines including cervical, gastric, prostate, colorectal, lung and glioblastoma in vitro Round morphology has been associated with the side population that shares characteristics with tumour stem cells of cervical and gastric tumour cell lines Based on differences in morphological and growth characteristics, we identified three in vitro morphological phenotype to a clinical phenotype. We performed an in silico analysis to determine whether the three in vitro identified morphological subtypes associate with clinical characteristics including prognosis as well as the molecular subtypes identified within ovarian carcinomas These morphological subtypes are observed in cell lines from multiple tumour types and seem to associate with characteristics of putative clinical importance. It is therefore important to translate the The 152 genes over-expressed in the Spindle-like cluster  associatECM remodelling as well as EMT have been associated with invasion and metastasis and thus a clinically more aggressive subtype. Indeed, the Spindle-like tumour cluster showed a significant enrichment for metastases as arrayed site, higher stage, sub-optimal debulking, and decreased progression-free survival. The increased expression of cell movement and invasion-associated genes could explain the metastasis, higher stage and worse prognosis observed.We also found that the morphological tumour clusters were associated with the six molecular subtypes C1-C6 identified by Tothill et al. in vitro morphological phenotypes resemble the clinical molecular subtypes and could be used to select relevant cell lines for tumour subtype specific studies.Closer examination of the clustering showed that the Spindle cell lines have a very similar gene expression pattern as seen for the C1  tumours, the Round cell lines resembled the C5-mesenchymal subtype and the Epithelial cell lines the C4 subtype . This coin vitro morphology genes with the CLOVAR subtype signature of 100 genes Interestingly, 61/152 genes overexpressed in the Spindle-like cluster  overlap The tumour microenvironment is also involved in multidrug resistance through a range of putative mechanisms, e.g. by decreasing drug concentrations through increased interstitial fluid pressure, 3D structure or by activating cellular pathways through ECM-Integrin binding, the so-called cell adhesion-mediated drug resistance (CAM-DR) This relation between mesenchymal subtype and therapy resistance was not observed for the Spindle cell lines. Possibly since within cell lines the cross-talk between the mesenchymal tumour cells and stroma is limited to the autonomously produced ECM and could therefore not fully mimic the clinical impact of the tumour microenvironment.Although Spindle morphology itself was not associated with taxane response, one of the Spindle cell line characteristics, i.e. high CD44 expression, did associate with response to taxanes. The microtubules targeted by taxanes are indeed very important in cell movement which could explain why CD44-high Spindle cell lines were more sensitive. The literature suggests that CD44 high tumour cells display increased self-renewal, migration and therapy resistance and are suggested to be cancer initiating cells (CIC) In summary, our study provides a uniformly generated data resource for a panel of 39 ovarian cancer cell lines. We identified histological as well as morphological subtypes which were associated with clinical pathological and molecular characteristics of ovarian carcinomas and prognosis. Therefore, this study is of considerable research value to those looking for better defined model systems with consistency to valid clinical phenotypes.Figure S1Representative images of six examples of each of the three morphological subtypes to illustrate differences in cell shape, size and growth pattern between the morphological subtypes .(TIF)Click here for additional data file.Figure S2CCNE1 amplification in three categories: A. no , B. medium  and C. high amplification . The Y-axes represents the number of median absolute deviation (MADs) from the median coverage (Z-score), the maximum is given above each graph, each bar in the histogram represents an exon of the gene, blue bars are exons with >1.5 MAD above the median. D. CCNE1 mRNA expression (y-axes) relative to yes or no gene amplification (x-axes) (Mann-Whitney p<.001).(TIF)Click here for additional data file.Figure S3Unsupervised clustering of the 26 unique ovarian cancer cell lines using the 80% most variable expressed mRNAs and microRNAs.Morphology: E Epithelial, R Round, S Spindle, Histology & Putative Histology: S serous, HGS high-grade serous, LGS low-grade serous, E endometrioid, C clear cell, Mx mixed, M mucinous, Time & treatment: P primary disease, PU untreated primary disease, RU untreated relapsed disease, RP platinum treated relapsed disease, U untreated, (R)O (relapsed) disease that received other treatment, CRP at clinical resistance and platinum treated. Protein markers: bright red no expression , light red low expression , light green expression , bright green high expression , grey not determined. Median response: of eight therapeutics green to red scale sensitive to resistant. Doubling time: green less than one day, yellow 1–2days, orange >2days. MSI microsatellite instability. # mutations: total number of mutations. Gene amplification: orange amplified (2–3x SD above the median), red highly amplified (>3x SD above the median). Heatmap: Red colour high expression, Green colour low expression.(TIF)Click here for additional data file.Table S1Background information on the origin of the cell lines based on literature.(XLSX)Click here for additional data file.Table S2Normalized expression data for 384 microRNAs in all 39 cell lines.(XLSX)Click here for additional data file.Table S3Short tandem repeat profiles of 39 cell lines measured with the PowerPlex 16 system (in bold) compared to the reference STR profiles from the cell line banks ECACC and ATCC  and STR profiles measured by Korch et al. (in regular font). See Korch et al. nd not determined.(XLSX)Click here for additional data file.Table S4Overview table of all mutations detected in the 39 cell lines measured by exon sequencing (SOLID and Tam-Seq), Sanger sequencing and SNApSHOT analysis.(XLSX)Click here for additional data file.Table S5Morphological subtype associated genes (n = 1141) and microRNAs (n = 18). The table includes the corresponding cluster  as well as whether the gene is an extracellular matrix component according to Hynes et al. (XLSX)Click here for additional data file.File S1Supplemental methods.(DOCX)Click here for additional data file.


Streptomyces are prolific producers of majority of bioactive secondary metabolites which are used in pharmaceutical industry as effective drugs against life threatening diseases. The cytotoxic activity of the pure compound 1, 2- benzene dicarboxylic acid, mono 2- ethylhexyl ester (DMEHE) from marine derived actinomycete Streptomyces sp. VITSJK8 was investigated against mouse embryonic fibroblast (NIH 3T3) and human keratinocyte (HaCaT) normal cell lines, human hepatocellular liver carcinoma (HepG 2) and human breast adenocarcinoma (MCF-7) cell lines by using MTT assay. The compound DMEHE exhibited IC 50 values of 42, 100, 250 and 500 µg/ ml against HepG2, MCF-7, HaCaT and NIH 3T3 cell lines, respectively. The effect of DMEHE on the growth of cancer cell lines was expressed as the % of viability. Cell viability was recorded as 67.7%, 78.14%, 82.23% and 96. 11% in HepG2, MCF-7, HaCaT and NIH 3T3 cells, respectively. The results of the study conclude that the bioactive compound isolated from the potential isolate Streptomyces sp. VITSJK8 exhibited cytotoxic activity against HepG2 and MCF- 7 cancer cell lines and low toxicity against normal HaCaT and NIH 3T3 cell lines.Marine  Marine microorganisms continue to be a potential source of bioactive secondary metabolites. 70% of earth surface is covered with water and it has been estimated that only 1% of microorganisms has been cultured and identified . Marine Streptomyces are saprophytic gram positive bacteria which have the ability to produce vast number of structurally and functionally wide variety of antibiotics with clinical and industrial importance and are responsible for the production of about half of the discovered bioactive compounds  with a molecular formula of C16H22O4  medium (tryptone yeast extract) at 4 °C  medium and the pure isolate of VITSJK8 was maintained on international  at 4 °C .Streptomyces fradiae (accession - JF9153041). Based on the molecular taxonomy and phylogeny, the isolate was identified as Streptomyces species and designated as Streptomyces sp. VITSJK8.The morphological characteristics of the active isolate showed an abundant matured spore with pink color which was observed in ISP 1 with an incubation time of 7-9 days. The isolate VITSJK8 was able to grow at 28- 32 °C, pH 7.0 and it could tolerate 2% NaCl and produced brown pigment on ISP-7 medium. The 16S rRNA partial gene sequencing of actinomycetes isolate VITSJK8 yielded 837 nucleotides (GenBank accession number KF289838). The isolate using NCBI sequence database showed maximum (99%) similarity with 16S rRNA secondary structure and restriction sites analysisThe secondary structure and the restriction sites in the 16S rRNA sequence of the isolate VITSJK8 were predicted using online softwares Genebee and NEB Cutter (version 2.0) respectively.Extraction, purification and identification of bioactive compoundStreptomyces sp. VITSJK8 in 50 ml of ISP1 medium and kept in a shaker at 120 rpm for 3 days. Furthermore, the seeds were inoculated in 1l of ISP1 medium and kept in a shaker at 120 rpm for 9 days. After the fermentation period, the culture was harvested and the secondary metabolites were extracted using ethyl acetate (EA) as solvent (1:1 v/v). The solvent layer was concentrated using rotary evaporator to give crude extract (. The yield of EA extract was found to be 130 µg per1000 ml of the culture broth. The crude suspension (2 g) was chromatographed over silica gel column , and eluted with chloroform: methanol (98:2) at a constant flow rate. The active fractions were collected and concentrated.The seed was prepared by inoculation of one mature colony of  extract . The yieSpectroscopic analysis of the purified compound1H and 13C nuclear magnetic resonance spectra (NMR) (Bruker Advance III 500 mHz A V 500) was used to exploit the atomic structure of the compound  and λ max was measured. The key functional groups present in the compound were determined by Fourier transform infrared spectroscopy (FTIR) (Thermo Nicolet-330 USA). Gas chromatography– mass spectroscopy (GC- MS) was used by injecting (1 µl) of the sample with the injection temperature at 220 °C into the spectrometer equipped with HP-5 capillary column. Proton (1H) and carbon (13C) compound . The strAnalysis of cytotoxic activity of 1, 2-Benzene dicarboxylic acid, mono (2-ethylhexyl) ester Streptomyces sp. VITSJK8 was investigated for the cytotoxic activity against NIH 3T3, HaCaT, Hep G2 and MCF 7 cells by the method of MTT  assay.The compound DMEHE isolated from The cell lines were obtained from the American type culture collection . Cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 100 mg/ l penicillin . The cells were maintained under 97% humidity in a biological incubator at 37 °C in a 5% of carbon dioxide atmosphere. The cell numbers were determined with a hemocytometer and the viability was assessed using the MTT assay described earlier .The principle of the assay is based on the reduction of soluble yellow tetrazolium salt to insoluble purple formazan crystals by metabolically active cells. Only live cells are able to take up the tetrazolium salt. The enzyme  present in the mitochondria of the live cells is able to convert internalized tetrazolium salt to formazan crystals, which are purple in colour. Then the cells were lysed and dissolved in DMSO solution. The intensity of the color developed was measured using ELISA reader  at 570 nm. 5 cells/ well. After 24 h incubation, cells were washed twice with 100 µl of serum- free medium and starved for an hour at 37 °C. After starvation, cells were treated with different concentrations of the compound DMEHE (6- 500 µg/ml) with three replicates of each concentration for 24 h. At the end of the treatment period the medium was refreshed with serum- free medium containing MTT (0.5 mg/ml). The microtiter plates were incubated for four hours in dark at 37 °C. The MTT containing medium was then discarded and the cells were washed with PBS (200 µl). The formazan crystals formed in the cells were dissolved with 100 µl of DMSO and the rate of the color developed was measured. Spectrophotometric absorbance of the purple blue formazan dye was measured in a microplate reader  at 570 nm. IC50 values were determined by calculating the % of cell viability. Percentage of cell viability was calculated by using formula: % viability= * 100. Data are reported as mean IC50 values± S.D from three independent experiments. The graph was plotted with cell viability against various concentrations of the compound.For the MTT assay the NIH 3T3, HepG2, HaCaT and MCF-7 cells were plated separately in 96 well plates at a concentration of 1×10Structural elucidation of the compound16H22O4. UV- visible absorption spectra of DMEHE in methyl alcohol showed λ max of 222, FT-IR (cm-1) spectra at 3130,1404 corresponds to C-C, 2875.86, 858-704 corresponds to C-H, 1298, 1124 corresponds to C-N and 948 indicates the presence of O- H groups in the compound. The 1H- NMR spectrum  was obtained with 0.8- 0.85 , 1.2- 1.4 , 3.8  and 7.56- 7.74 . The 13C-NMR spectrum was observed with 170.1 (COOH), 168.6 (C=O), 133.8 (Ar-c), 132.9 (Ar-c), 132.8 (Ar-c), 132.5 (Ar-c), 128.4 (Ar-c), 128.0 (Ar-c), 66.9 (CH2), 38.9 (CH), 31.9 (CH2), 29.3 (CH2), 23.7 (CH2), 23.0 (CH2), 14.1(CH3) and 11.6 (CH3). The 3D–structures of the compound  and the molecular formula was determined as Ccompound  was modeCytotoxic activity of 1, 2-Benzene dicarboxylic acid, mono (2-ethylhexyl) esterIn vitro cytotoxicity assays of bioactive lead compound (DMEHE) extracted from Streptomyces sp. VITSJK8, against the cell lines revealed that DMEHE exhibited cytotoxic activities on HepG2 and MCF-7 cancer cell lines and it showed less cytotoxic activity on normal cell lines, NIH 3T3 and HaCaT by MTT assay in 96 well plates. The MTT assay is commonly used to determine mitochondrial reductive function and hence, is a good indicator of cell death or inhibition of growth. The different concentrations of DMEHE tested against normal cell lines exhibited less toxicity with IC50> 500 µg/ ml against NIH 3T3 and IC50> 250 µg/ ml against HaCaT cell line. DMEHE exhibited a cytotoxic activity on cancer cell lines. Among the cell lines, HepG2 was more susceptible and the cell viability was decreased to 68%, followed by MCF-7 (78%), HaCaT (83%) and NIH 3T3 cells (96%) by MTT assay. The results showed that the DMEHE exhibited cytotoxicity on all the cancerous cells in a concentration- dependent manner. Each cell line was treated with increasing concentrations (6- 250 µg/ ml) of DMEHE for 24 h. Untreated cells were used as a negative control. HepG2 was found to be more susceptible followed by MCF-7, HaCaT and NIH 3T3 cells . DMEHE showed potential cytotoxic activity against HepG2 and MCF- 7 cell lines. It showed less cytotoxic activity on HaCaT and NIH 3T3 normal cell lines. The observed IC50 values suggest that DMEHE was less toxic to normal cell lines (NIH3T 3 and HaCaT) when compared to the malignant cell lines (HepG 2 and MCF- 7). Streptomyces sp. is considered as potentially important source of novel bioactive anticancer compounds and capable of producing chemically diverse compounds without any side effects for variety of clinical applications (Streptomyces VITSVK5 spp. exhibited cytotoxic activity on HEP 2 and Hep G2 cell lines with IC50 values of 2.8 and 8.3 µg/ ml, respectively, as compared to Vero cell line 22.6 µg/ ml (Streptomyces sp. nov. WH26 have been shown to exhibit potent cytotoxicity against several cancer cell lines. It was reported that Naphthomycin A showed IC50 values of 9.2 ± 0.8, 15.2 ± 1.1, 7.9 ± 1.4 and 20.8 ± 1.6 l M against A549, HeLa, BEL-7402 and HT-29 cell lines respectively (Streptomyces showed high cytotoxic activity against cancer cell lines and less cytotoxicity towards normal cell lines.In this study, the bioactive actinomyceteal metabolite DMEHE extracted from ications . The ant6 µg/ ml . Naphthoectively . The aboThe HepG2 cell line was found to be more sensitive to DMEHE after 24 h of incubation by MTT assay. The differences in sensitivity might be due to the characteristics of each cell line and their origin . The red the increased incidence of drug resistance by many tumor cells, there is a need to explore microbial-derived secondary metabolites as anticancer drugs with specific antitumor activity to circumvent the demand for anticancer chemo-therapeutic agents (Streptomyces sp. VITSJK8 lead to the identification of bioactive compound 1, 2- benzene dicarboxylic acid, mono 2- ethylhexyl ester which exhibited potential cytotoxic activity against HepG 2 and MCF- 7 cancer cell lines. Streptomyces sp.VITSJK8 can be explored further as a source of novel metabolites with cytotoxic activity.Cytotoxic mechanism of natural bioactive compounds, interfere with basic cellular functions such as cell cycle, apoptosis, inflammation, angiogenesis, invasion and metastasis. . Many anc agents . Severalc agents . Natural