In this review, we discuss the current knowledge of Treg biology and their potential for cell-based immunotherapy in allogeneic stem cell transplantation.Growing evidence suggests that cellular adoptive immunotherapy is becoming an attractive though challenging approach in regulating tumor immunity and alloresponses in clinical transplantation. Naturally arising CD4+CD25+Foxp3+ regulatory T cells (Treg) have emerged as a key component in this regard. Over the last decade, a large body of evidence from preclinical models has demonstrated their crucial role in auto- and tumor immunity and has opened the door to their “first-in-man” clinical application. Initial studies in clinical allogeneic stem cell transplantation are very encouraging and may pave the way for other applications. Further improvements in Treg  Treg ca (Foxp3) , 10.in vitro via specific stimulation conditions or induced in the peripheral lymphoid organs in vivo, with transforming growth factor β (TGFβ) playing a pivotal role [Induced or adaptive Treg (iTreg) can be generated from naïve T cells tal role –13.Foxp3 gene  results in the development of severe autoimmune disorders as can be observed in the “scurfy” mouse mutant [As demonstrated a decade ago, the transcription factor Foxp3 is indispensable for both nTreg and iTreg development and suppressive function , 14, 15.e mutant , 17.Foxp3 [Foxp3 locus, the so-called Treg-specific demethylation region (TSDR), is completely demethylated in nTreg but fully methylated in Tcon and iTreg [Recent studies suggest that nTreg are more stable compared with iTreg. This is related to their different DNA methylation profiles and to other epigenetic regulations of Foxp3 –21. In pnd iTreg . The TSDnd iTreg , 24.in vitro differentiated human iTreg might not be stable phenotypically and functionally, implying that in vivo transfer of iTreg for therapeutic purposes may give unexpected results and should be considered with caution [ Thus, due to unstable Foxp3 expression,  caution .In vitro, nTreg were shown to inhibit the activation of effector CD4+CD25− T cells predominantly by cell-cell contact dependent mechanisms. nTreg express on their surface important molecules for their suppressive function such as cytotoxic-T-lymphocyte-associated-antigen-4 (CTLA-4), membrane-bound TGFβ latency-associated peptide (LAP), glucocorticoid induced tumour necrosis factor receptor (GITR), CD4-related lymphocyte-activation-gene-3 (LAG-3), galectin-1, and CD39. Moreover, after activation, human nTreg were shown to be able to directly kill CD4+ and CD8+ T cells via the secretion of perforin and granzyme B. The role of regulatory cytokines such as IL-10, TGFβ, and, more recently, IL-35 in nTreg-mediated suppression of immune pathologies has mainly been described in in vivo experimental models [Significant progress has been made over the last few years in delineating the mechanism of suppression exerted by nTreg . Numerouin vitro and in vivo and for their peripheral maintenance and competitive fitness [Also, IL-2 is crucial for nTreg homeostasis, as these cells are highly dependent on exogenous IL-2 for growth  fitness , 28. Mor fitness .In addition to directly affecting effector T cell function, nTreg can modulate the maturation and/or function of dendritic cells required for effector T cell activation .ex vivo culture or in vivo by specific stimulation will help to devise novel therapeutic strategies to control untoward immune responses.To summarize, Treg are a specialized subpopulation of T cells that suppress the activation, expansion, and function of other T cells, thereby maintaining homeostasis through a fine balance between reactivity to foreign and self-antigens. More importantly, strategies to expand this population by In the allogeneic stem cell transplantation setting, preclinical models demonstrated (as discussed below) that graft-versus-host disease (GVHD) prevention and transplantation tolerance require tipping the balance in favour of Treg against effector T cells.In vitro analysis of splenic Tcon on day 5 from different experimental groups for their phenotype, activation markers, and cytokine production has shown that Treg do not inhibit activation and functional maturation of Tcon.A number of groups have demonstrated that in aggressive murine GVHD models, where bone marrow and GVHD-inducing Tcon were transplanted across complete major histocompatibility complex (MHC) class I and II barriers, lethal acute GVHD was prevented by donor Treg, if cotransplanted at 1 : 1 ratio with Tcon , 5, 29. In two independent tumor models, including the leukemia line A20, which infiltrates the bone marrow, and BCL1 lymphoma, which primarily invades liver and spleen, Treg did not interfere with the GVT activity of Tcon but permitted the elimination of tumors from all these compartments, resulting in long-term survival of the hosts.The presence of Treg therefore does not abrogate the activation of Tcon or their cytolytic response. The investigators hypothesized that the significant decrease in Tcon proliferation by Treg controlled acute GVHD, which generally requires a strong alloreactive response. However, this decrease is not complete, and the remaining Tcon are sufficient to effectively mount a GVT effect . Additional studies  demonstrin vitro against donor antigen-presenting cells (APCs). When a third-party bone marrow was cotransplanted, host preactivated Treg preferentially augmented donor chimerism, as assessed 2 weeks after transplantation (most notably at lower Treg to effector T cell ratios). Treg have been shown to mediate transplantation tolerance in murine models of skin, solid organ transplantation –34, and,in vitro polyclonal T and B cell stimulations. In another murine MHC-mismatched bone marrow transplantation (BMT) model , cotransUnlike generalized immunosuppressive regimens, Treg are long-lived and functional in a dominant and antigen-specific manner. Thus, therapeutic infusion of Treg has the potential to induce long-term donor-specific tolerance without impeding desired immune responses to pathogens and tumors in transplant patients. There is accumulating evidence that Treg may also modulate host T cell activity against tumor-associated antigens, thereby facilitating tumor escape from immunological control. Treg were shown to be expanded in murine tumor models . MoreoveSeveral reports have demonstrated that Treg are expanded in patients with solid tumors –45, B cein vitro inhibitory effect of lenalidomide on Treg [In CLL, a direct correlation between higher Treg numbers and more aggressive clinical-biological features of the disease, as well as with disease progression, has been described . Moreove on Treg  and suggP = 0.013, at median follow-up of 32 months). Interestingly, Treg frequencies were not influenced after chemotherapy with novel antimyeloma agents  nor autologous stem cell transplantation reduced Treg significantly.In a recent study in newly diagnosed MM patients , increasAlternatively, suppressive Treg might actually play a beneficial role in Hodgkin lymphoma (HL), which is characterized by a chronic background inflammatory response, important for the proliferation and survival of Hodgkin-Reed-Sternberg (HRS) cells. The frequency of Foxp3+ T cells was determined in lymphoma-afflicted lymph nodes (LNs). Low frequencies of Foxp3+ cells and high frequencies of cytotoxic T/NK lymphocytes (CTLs) in the reactive background of LNs correlated with poor overall survival , 60. HowAs already outlined, a correlation of increased Treg with greater disease burden and poorer overall survival has been reported. Treg are able to recognize tumor-associated self-antigens and to control natural T cell responses against various cancer antigens, which may explain the failure of many cancer vaccines . In addiin vitro on the response to the MM tumor vaccine [γ, decreased levels of Treg, and, most significantly, an increased capacity of vaccine stimulated T cells to lyse autologous MM cells. Hence, lenalidomide may create an ideal platform for myeloma-specific immunotherapy that acts synergistically with therapeutic vaccination with DC/MM fusions to induce myeloma-specific immunity. A recent phase I study  demonstr vaccine . StimulaIn conclusion, therapeutic vaccination in conjunction with Treg depletion as a mean to augment vaccine response remains an area of further investigation.Given the striking results in murine GVHD and bone marrow graft rejection models, the ready availability of donor Treg together with the known and transient risk period for adverse consequences from alloreactive T cells and the high degree of morbidity and mortality associated with allogeneic stem cell transplantation (SCT), it is not surprising that GVHD prevention has emerged as the first clinical application for human Treg.in vitro expanded Treg (average 64% Foxp3+ after expansion) derived from partially HLA-matched third-party umbilical cord blood units were used in 23 patients undergoing double-cord blood transplantation. Treg were administered on day +1 post-transplant (fresh) (doses ranged from 1 × 105 to 3 × 106/kg) and additionally on day +15 (3 × 106/kg) in 13 patients, using cryopreserved Treg expanded from the same cord blood unit. The rates of GVHD and infectious complications were compared with those from 108 historical controls. Importantly, no increase in opportunistic infections and no Treg-related acute toxicities were observed. The authors did report a reduced incidence of grades II–IV acute GVHD  in the trial group, but efficacy for the prevention of GVHD could not be demonstrated definitely in this phase I safety and feasibility trial, as standard pharmacologic prophylaxis was coadministered (cyclosporine A/mycophenolate mofetil or sirolimus/mycophenolate mofetil). In the “first-in-man” clinical trial , in vitr6 cells/kg (>50% Foxp3+) were administered after the cessation of pharmacologic GVHD prophylaxis (within a year after SCT). After an observation period of 8 weeks, additional Tcon were administered at the same dose to promote GVL activity. No Treg-related acute toxicity was observed; neither GVHD nor opportunistic infections or early disease relapses occurred after Treg transfusion, despite the absence of pharmacologic immunosuppression. By design, this trial was not suited to prove the efficacy of Treg for the prevention of GVHD because of the low patient number and the lack of control group.In another small phase I safety and feasibility trial , Edingern = 24 with 2 × 106/kg, n = 4 with 4 × 106/kg) on day −4, which was followed by transfer of a highly purified CD34+ stem cell graft together with Tcon. Patients did not receive any prophylactic immunosuppression. For safety reasons of this pilot trial, the first group of 4 patients received only 25% Tcon compared to Treg. Because none of these first 4 patients developed acute GVHD, Tcon were then escalated to 50% of the Treg dose. Rapid and stable engraftment was seen, and, surprisingly, only 2 of 26 evaluable patients developed grades II–IV acute GVHD, while the majority of patients remained free of clinically relevant GVHD. At a median follow-up of 11.2 months, no patient developed chronic GVHD. When compared to a dataset of 152 patients receiving haploidentical SCT without Treg transfer, this approach promoted lymphoid reconstitution and improved immunity to opportunistic pathogens. These first clinical data suggest that donor Treg infusion prevents acute GVHD after allogeneic SCT in humans, because lethal GVHD would have otherwise been expected in all patients after the administration of such high donor Tcon numbers in the absence of pharmacologic immunosuppression. Thus, these results are highly encouraging and now demand confirmation in randomized controlled multicenter trials.A more recent study  demonstrClinical trials exploring the efficacy of Treg for the treatment of GVHD are much more challenging than prevention trials, and therefore it is not surprising that evidence is still sparse. in vitro expansion, cell production may often be too slow for patients with severe and rapidly progressive disease. High Treg numbers and maximum Treg purity would be required to avoid aggravation of GVHD by contaminating Tcon. Since Treg have to be isolated from the stem cell donor and require two to three weeks in vitro expanded donor Treg  contributed to the amelioration of chronic GVHD in a single patient and allowed mycophenolate mofetil (MMF) withdrawal and a reduction in steroids. A second patient treated with higher Treg numbers  for treatment-resistant acute GVHD had no benefit. The last Treg infusion for that patient contained only 40% Foxp3+ cells, and it is questionable whether such a cell product should have been administered in a life-threatening disease caused by donor Tcon.In a recent anecdotal report  it was sin vitro expanded Treg (>95% Foxp3+) in a small number of patients with treatment-resistant acute GVHD and found that these cells survive in vivo and may ameliorate severe acute gastrointestinal GVHD. Clinical trials are clearly warranted to test the therapeutic potential of donor Treg infusion for ongoing GVHD.Edinger and Hoffmann  have alsex vivo expanded Treg is associated with potential risks. One issue to be investigated in future studies is whether adoptive immunotherapy with Treg compromises general immunity, blunting responses to infectious agents.Like all therapies, clinical use of In animal models, it has been demonstrated that Treg not only prevent GVHD but also enhance immune reconstitution after bone marrow plus Tcon transplant, by preventing GVHD-induced damage of the thymus and secondary lymphoid organs, thus allowing protection against lethal cytomegalovirus (CMV) infection .Limited safety data have been obtained from initial clinical trials. As described above, the phase I clinical trial by Brunstein et al.  reportedIn the clinical trial by di Ianni et al. , Treg inA significant challenge for the use of nTreg in the clinic is the difficulty in isolating sufficient numbers of Treg for clinical application, since circulating numbers of Treg in the peripheral blood are limited (5%–10% of CD4+ T cells) , 71. CurThe use of granulocyte-colony-stimulating-factor (G-CSF) may also be considered to increase Treg mobilization into the peripheral blood for potential extraction during leukapheresis . Under sin vivo is critical for their GVHD suppressive activity [ex-vivo manipulations, thus rendering the therapy more clinically applicable. It has been recently shown that expansion of adoptively transferred Treg activity . This woin vivo Treg expansion.Recent data suggests that T cell depletion protocols used to induce tolerance in clinical transplantation, for example, by using total lymphoid irradiation (TLI) and antithymocyte globulin (discussed below) as conditioning regimen , 79, altin vitro to selectively expand or preferentially preserve Treg over Tcon [in vivo SCT models support the observation of Treg-supportive effects of rapamycin [Rapamycin, a small molecule inhibitor of the Akt/mTOR pathway used for the prophylaxis and treatment of GVHD, has been shown ver Tcon , thus atapamycin , includiapamycin  and infiapamycin . in vivo homeostasis and expansion of Treg are highly dependent on IL-2. Thus, IL-2 is also investigated as a putative agent for selective expansion of Treg [As stressed before,  of Treg . Recentl of Treg . Adminisin vivo expansion of nTreg, enhanced conversion of iTreg by IL-2, and inhibition of effector T cells [Also, the combination of rapamycin plus IL-2 appears to be more effective than rapamycin alone in the prevention or suppression of GVHD by  T cells .in vivo that suppress mixed lymphocyte culture in patients undergoing allogeneic SCT [It has also been shown that rabbit-derived anti-T lymphocyte immunoglobulin (ATG) is a potent inducer of iTreg. Lopez et al.  and Fengneic SCT . These rin vivo will improve both the safety and efficacy of Treg therapy. Since Foxp3 is the master regulator of Treg function, alterations in Foxp3 expression or activity are likely involved in converting Treg to Tcon. Foxp3 expression is modulated by DNA methylation; therefore, administration of selective demethylating agents may enhance Treg function and fidelity in vivo [The majority of adoptively transferred Treg maintain their suppressive activity, but a minority of cells lose Foxp3 expression and can differentiate into Tcon . It is c in vivo .In preclinical studies, Azacitidine (AzaC) treatment of allotransplanted mice mitigates deleterious GVHD while preserving beneficial GVT effect, by peripheral conversion of alloreactive effector T cells into Foxp3+ Treg and epigenetic modulation of genes downstream of Foxp3 required for the suppressor function of Treg . Thus, tin vitro and in vivo [It is likely that different immunosuppressive and immunomodulatory agents will be more or less permissive for Treg development and function. For example, cyclosporine A (CsA) was shown to inhibit Treg function. Reduced suppressor function of CsA-exposed Treg was IL-2 dependent and correlated with a decreased number of Foxp3+ T cells both  in vivo . On the in vitro [Interestingly, the role of lenalidomide in the modulation of Treg remains unresolved. As discussed above, lenalidomide was shown to inhibit the proliferation and function of Treg in vitro . Idler ein vitro  studied in vitro , as discin vitro . Howeverin vitro . NotablyRaja et al.  have shoin vitro studies that lenalidomide inhibits Treg suggest that the in vivo effects of lenalidomide might be a result of the microenvironment on the immune cells. Thus, the data may be interpreted such that, once lenalidomide induces effector immune responses in vivo, there is a negative feedback induced by transformation of the T helper cells into Treg to maintain the immune homeostasis [The conflicting but compelling data from eostasis .Building on extensive research in Treg biology and preclinical testing of Treg therapeutic efficacy over the past decade, we are now at the point of evaluating the safety and efficacy of Treg therapy in humans. SCT seems a clinical setting suited to prove the efficacy of adoptive Treg therapies and may pave the way for further applications.As discussed in our review, initial studies in clinical allogeneic SCT are very encouraging and have demonstrated that Treg-based clinical studies are feasible and do not result in toxicity. Human Treg infusion appears to suppress GVHD risk of Tcon while promoting enhanced immune reconstitution and decreasing the incidence of infectious complications. In patients with malignant diseases, a direct correlation between higher Treg numbers and more aggressive clinical-biological features, as well as with disease progression, has been described in several types of tumors. Elimination or inhibition of Treg might be particularly useful in the context of therapeutic vaccination against tumor-associated antigens, as stressed above. ex vivo or in vivo expansion technologies and the use of costimulatory novel compounds to get higher cell yields, as detailed above, will “unlock the power” of Treg and facilitate the broader exploration of Treg therapies, for example, for the treatment of active GVHD or the prevention of graft rejection after solid organ transplantation. Finally, further improvements in Treg


Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF) was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.Alpha- ( Primaryexposure .α-particle exposure  were commonly expressed between the two radiation types. The remainder of the responding cytokines were unique to α-particle exposed cells and included IL-17, IL-2 MIP-1B, and VEGF.To further assess if similar cytokine responses were obtained for an alternative cell type, the effects of radiation exposure were determined in a circulating cell type, THP-1, which maybe more suitable for biomarker discovery. As was observed in the A549 cells, significantly modulated cytokines in this cell line varied with radiation type. However, unlike the A549 cells, the human monocytic cells displayed a greater number of upregulated cytokines, potentially because of the innate properties of monocytic cells as an inflammatory cell-type. In THP-1 cells, three of the seven statistically significant cytokines modulated after α-particle-induced cytokine secretions between A549 and THP-1 with the exception of VEGF. VEGF was modulated in both A549 and THP-1cells; however, its response was shown to be bidirectional, and this was validated at lower doses of radiation. It was observed that in THP-1 cells, VEGF expression was significantly upregulated, while in A549 cells, expression of this growth factor was downregulated. These modulations were observed to be dose responsive and specifically induced by α-particle insult, whereas X-irradiated cells showed no statistically significant changes in expression of VEGF in either cell type. VEGF's biological role as a vascular growth factor has been shown to be somewhat dichotomous, as it is needed for novel tissue vascularization but may promote tumour survival when dysregulated pathologically . It is α-particle radiation was shown to elicit specific cell-type secretory responses. A comparison of the responses with X-ray showed a limited number of comparable cytokines. Responding proinflammatory cytokines proteins were broadly related to interleukin family of proteins and growth factors. A number of the secreted proteins which were observed to be expressed following α-particle exposure have been shown to have clear links to neoplastic transformation and tumour promotion. Although in this study, no unique biomarkers of α-particle exposure were definitively identified, VEGF emerged as a candidate for further studies, as it was modulated exclusively by α-particle insult.To summarize,


The identification of a small number of monoclonal antibody (mAb) producing candidate cell lines from the large number of clones generated post transfection is one of the bottlenecks of cell line development. Clone numbers are reduced significantly during initial medium exchange and static scale up stages but significant numbers can still progress to evaluation in shaking cultures. This is often carried out in shake flasks where the number of clones that can be evaluated may be restricted due to resource limitations.The Duetz Microflask system is a microtitre plate based system which uses ‘Sandwich Covers’ to convert the individual wells of 24 well plates into individual ‘mini reactors’. The ‘Sandwich Covers’ consist of a stainless steel cover, a 0.2µm filter, microfibre inlays and a flexible silicone sealing layer to ensure adequate oxygen transfer rates to individual wells.This work describes the use of the Duetz Microflask system to evaluate cell lines in culture prior to scale up to production shake flasks. We have compared the growth and rank order of performance for a number of cell lines in the Duetz Microflask system with data obtained from shake flasks and demonstrated this system can be used successfully to identify candidate cell lines for further progression.A series of preliminary experiments was completed to determine suitable volumes and operating conditions to minimise evaporation. Unless otherwise stated these were: Fill volume – 1.5mL, Shaker speed – 200rpm, Humidity – 80%.After initial transfection a typical cell line development process would involve plating cell lines to multiwell plates followed by several static media exchange, scale up and selection steps. These are used to reduce the number of cell lines carried forward to larger scale static T-flasks and eventually to shake flasks for further evaluation and selection. Although the multiwell plate steps are amenable to automation the manual handling aspects of culturing multiple cell lines in shake flasks can be resource limiting and therefore the ability to improve cell line selection using shaking multiwell plates offers significant time line and resource advantages.The performance of a series of 28 mAb producing clones in hydrolysate containing media in Duetz Microflasks was compared with standard shake flask evaluation data.2 = 0.46. However this improved significantly (R2 = 0.92) when a further set of Duetz Microflasks (‘Late’ Duetz) was run in parallel to the flasks.The rank order of titre performance of these clones in ‘Early’ Duetz Microflasks (ie inoculated before repeated shake flask subculture) was compared to that in shake flasks inoculated after the cultures had been subcultured for 6 passages. There were some differences in the rank orders resulting in a correlation RThis data indicates that although Duetz Microflasks can be used at an early screening stage to identify high producing clones the correlation with subsequent performance is improved after the cell lines are adapted to shaking growth conditions (ie after repeated subculture in shake flasks).Figure Similar results were obtained using a hydrolysate feed fed batch process in which the titre rank order and the response to feed in Duetz Microflasks was similar to that in shake flasks. Thus Figure 2 = 0.34) but was significantly improved after Duetz Microflasks were inoculated with cells previously subcultured in shake flasks (R2 = 0.73).A similar experiment was carried out with a different mAb producing CHO cell line in a chemically defined fed batch process. The standard (bioreactor and SF model) process involves a series of feeds during the fermentation. This multiple feed strategy was found to be impractical at the Microflask scale and so a compromise ‘single feed’ model was used in Duetz Microflasks. The correlation of rank order between ‘Early’ Duetz Microflasks and shake flasks was poor  this data has also demonstrated that screening in Duetz Microflasks inoculated directly from static cultures or after minimal shake flask subculturing can be used to differentiate between cell lines that are high and low performers in conventional screening systems. We have used this system to reduce the number of cell lines carried forward to shake flask screening by approximately 70%.The simplicity of this system combined with the standard multiwell plate footprint facilitates automation and potentially enables large numbers of cell lines to be screened simultaneously and we are currently incorporating this system into our automated cell line generation process.


To date there are a limited number of well-characterized models available. In this study, nine immortal liposarcoma cell lines were evaluated for tumor-forming ability, stem cell- and differentiation potential, and metastatic potential, with the aim to generate a well-characterized liposarcoma cell line panel. Detailed stem cell and differentiation marker analyses were also performed. Five of the liposarcoma cell lines were tumorigenic, forming tumors in mice. Interestingly, tumor-forming ability correlated with high proliferative capacity in vitro. All the cell lines underwent adipocytic differentiation, but the degree varied. Surprisingly, the expression of stem cell and differentiation markers did not correlate well with function. Overall, the panel contains cell lines suited for in vivo analyses , for testing novel drugs targeting cancer stem cells (LPS141) and for studying tumor progression and metastasis (T449 and T778).Liposarcoma cell lines represent  Liposarcoma is categorized into three main subtypes; well-differentiated/dedifferentiated liposarcomas (WD/DDLPSs), myxoid/round-cell liposarcomas, and undifferentiated high-grade pleomorphic liposarcomas (reviewed ). WDLPSsSW872 (undifferentiated LPS) and SA-4 (classified as “liposarcoma”) were both purchased from ATCC. LISA-2, generated from a metastasis of a poorly differentiated liposarcoma , was proCellular proliferation rates were determined by live cell imaging . More specifically, an equal number of cells were plated in 96-well format and phase contrast photographs were taken automatically every second hour for the duration of the experiment. The data was presented as cell confluence over time.Adipocytic differentiation and oil red O staining was performed as described in . hMSCs wμm) were counted using GelCount .One thousand single cells were plated in methocult  supplemented with stem cell medium ; 20 ng/mL basic fibroblast growth factor; 20 ng/mL epidermal growth factor ; as recommended by Stem Cell Technology. Uniform colonies  were performed by flow cytometry, as described previously.http://www.fdu.no/). 1 × 106 cells were injected subcutaneously into both flanks of locally bred NOD/SCID IL2R-gamma-0 (NOD/SCID) mice. Cell viability was confirmed prior to injection.Animal experiments were performed according to protocols approved by the National Animal Research Authority in compliance with the European Convention of the Protection of Vertebrates Used for Scientific Purposes  through which the cells can migrate. For invasion assays, similar chambers covered with matrigel were used (BD Biosciences). Chemoattractant was RPMI-1640 containing 10% FBS. Cells were incubated in a humidified incubator at 37°C for 22 hours. Nonmigratory/noninvasive cells were removed by “swabbing” and migratory/invasive cells were fixed and stained with Hemacolor  and counted.We evaluated the proliferative capacity of the LPS cell lines by live cell imaging. LPS141, SA-4, T778 and SW872 displayed relatively high proliferative capacity and LISA-2, T449, GOT-3, FU-DDLS-1, and T1000 displayed lower proliferative capacity .6 cells from each line subcutaneously into both flanks of 3 NOD SCID mice. Experiments were repeated if no tumors were obtained. LPS141, SA-4, T778, SW872, and LISA-2 all formed tumors by 6 months . TBP was chosen as a calibrator for the cell lines since TBP was expressed at similar levels in all the cells lines, while GAPDH expression varied greatly between the different LPS cell lines. GAPDH was chosen as a calibrator for the hMSC differentiation experiment since GAPDH was expressed at equivalent levels throughout differentiation while TBP was expressed at very low levels in primary hMSC. Only LISA-2 appeared well differentiated by oil red O staining during logarithmic growth under standard cell culture conditions  , while LLPS141, T778, and FU-DDLS-1 displayed high capacity to migrate during 22-hour incubation in Boyden Chambers. SW872, LISA-2, T449, and GOT-3 were also migratory, although to a lesser degree. SA-4 and T1000 displayed low ability to migrate . LPS141 http://www.liposarcom/aresearch.org/) have characterized 9 immortal LPS cell lines in detail  were able to undergo induced adipocytic differentiation, while only a subset of the cell lines analyzed in the latter study underwent adipocytic differentiation. LPS141, which is the only cell line included in both studies, did not undergo induced differentiation in the hands of Peng et al. It is possible that these observed differences are due to the use of different adipocytic differentiation media in the two studies.The stemness and differentiation potential of the LPS cell lines were determined by a number of complementary analyses. LISA-2 was the only cell line staining positive for oil red O and expressing high levels of Re-expression of the embryonic stem cell and pluripotency factors OCT4, SOX2, and NANOG has been associated with poorly differentiated and aggressive cancers, such as high-grade breast cancer, glioblastomas, and bladder carcinomas . Only LPhigh population. However, the activity can be rescued or increased by xenotransplantation or serum-free culturing [highCD133high [highCD133high subpopulation in LPS141, the only line harboring more than 0.2%  Aldefluorhigh cells, displays CSC characteristics.Cell surface antigen expression is frequently used as CSC markers. LPS CSCs may arise from hMSCs and we determined the expression of CD90, CD105, CD73, and CD44, all expressed on MSCs –32. We aulturing . Since Lhigh and Aldefluorhigh cells. T449 is slow growing, nontumorigenic, noninvasive and able to undergo differentiation, while T778 (from the same patient) is fast growing, tumorigenic, invasive and displays resistance to differentiation and the two lines may serve as models for studying tumor progression and recurrence. T1000 and GOT-3 are the least aggressive cell lines in the panel and suitable models for studying suspected LPS oncogenes by overexpression. We observed a correlation between high proliferation rate and tumor-forming ability in  vivo, but there was surprisingly little correlation between the outcome of the different stem cell and differentiation assays, and between the functional assays and the expression of markers.In conclusion, the characterization of the LPS cell lines presented here will support further research on this serious orphan disease and benefit researchers when choosing cell lines for their experimental and preclinical studies. More specifically, the LPS141 is a candidate cell line for testing novel drugs targeting CSCs, displaying tumorigenic and invasive traits, expressing high levels of the pluripotency factors OCT4, SOX2 and NANOG as well as harboring CD133


The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2′-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels.Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins.In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients. Aberrant DNA methylation patterns are associated with various human diseases It was also discovered that microRNAs (miRNAs), endogenous non-coding RNAs with 19–25 nucleotides in size, play important roles in various cellular processes including cellular growth, differentiation and apoptosis Genetic changes such as mutation or deletion are resulting in permanent loss of gene expression while epigenetic changes are often reversible The 5-aza-2′-deoxycytidine , has recently been approved by the Food and Drug Administration (FDA) for the treatment of patients with Myelodysplastic Syndromes (MDS) and leukemia Pan-omics approaches at multiple molecular levels, after DAC treatment for solid tumors, are promising in opening new mechanistic insights in this area of cancer biology. These approaches enable to synoptically probe the transcriptome and the proteome to epigenetic changes in order to understand the complete phenotype of treated cells. Therefore we used DAC in this study as a tool to predict early and late effects of DAC on different breast cancer cell lines using pan-omics approaches.An optimal treatment dose screening for DAC was established based on the viability, toxicity, apoptosis and methylation alterations of candidate TSGs, for six breast cancer cell lines MDA-MB231 and a br2 nM, ∼5 days), then cells were cultured up to 10 passages at “drug holiday” condition. Simultaneous extraction of DNA, RNA, miRNA and proteins was performed according to a previously published protocols st, 3rd, 5th, 7th and 10th passages) at “drug holiday” condition. Differentially expressed genes, miRNAs and proteins, after treatment and at follow-up passages, were measured relative to the untreated samples. Microarray expression profiling of mRNAs and miRNAs were conducted using the Human Genome 133 Plus 2.0 GeneChips and Affymetrix GeneChipR miRNA array v1.0, respectively using protocols recommended by the manufacturer (Affymetrix). In order to avoid random fluctuations in mRNAs/miRNAs expression, after log transformation and Robust Multi-array Analysis (RMA) normalization, differentially expressed mRNAs/miRNAs were defined by applying three stringent filtering criteria  using Partek Genomics Suite software v6.5 . All mRNA microarray data compiled for this study is made publicly available on GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE28968 and miRNA microarray data under accession number GSE28969. To validate the microarray findings, quantitative real-time (qRT) PCR was performed for 28 candidate genes and 15 candidate miRNAs.After finding an optimal dose, three selected cell lines  were treated with the optimal dose of DAC . The protein spots of interest were excised from the gels and analyzed by capillary liquid chromatography tandem MS (LC-MS-MS). Protein identification was done using TurboSequest software The proteomic profile was investigated using two-dimensional gel electrophoresis (2DE) based on the previously published method 1 to 104 nM on seven different breast cell lines (six breast cancers and a control cell line) based on the cell viability, cytotoxicity, apoptosis and DNA methylation.In the present study, we have first identified the optimal, effective dosage of DAC by investigating the impact of concentrations in a range of 101 to 104 nM) elevates the cytotoxicity and reduces the ratio of the cell viability in all cell lines. The results of these two measurements, cell viability and cytotoxicity, are inversely correlated (as expected). Two concentrations, 101 and 102 nM DAC, showed viability higher than at EC50 for all studied cell lines and they revealed lower cytotoxicity compared to the other concentrations.The MultiTox-Glo Multiplex Cytotoxicity assay measured the relative number of live and dead cells in cell populations after treatment with increasing doses of DAC and could thus determine cell viability and cytotoxicity . Increas3 and 104 nM of drug were significantly higher than the lower dosages (P = 0.001) while the mean activities of the caspase-3/7 were similar for both 101 and 102 nM concentrations of DAC.Two cellular caspases (caspase-3 and caspase-7) play a crucial role in apoptosis and are activated in a sequential cascade of cleavages from their inactive forms. To investigate the extent of apoptosis after exposure to different concentration of DAC, the enzymatic activity of caspase-3/7 was measured . These cBMP6, BRCA1, CST6, CDKN2A, CDKN1A and TIMP3) to assess the effective DAC dose range. We analyzed the methylation proportion of these six breast cancer candidate TSGs in all seven cell lines before and after treatment at increasing doses. For all studied genes one amplicon containing CpG rich islands (number of CpG sites higher than 20) was analyzed (P<0.01)   . This da2 nM of DAC was identified to be the most favorable because of the less cytotoxicity and good efficiency in demethylation of DNA. This dose of DAC was chosen for the further study on the early and late effects of the treatment using pan-omics assays for the MDA-MB231 (highly aggressive) and SKBR3 (non-aggressive) cell lines together with HB2 as a control cell line. The terms “aggressive” and “non-aggressive” cell lines refer to the in vitro phenotype of the cell lines as indicated by a previously published paper Based on the results obtained from studies of the cell viability, cytotoxicity, apoptosis and DNA methylation profiling, the concentration 10P<0.05, >2 fold change; Gene expression analysis using microarray chips was assessed before treatment, after ∼5 days of treatment and at five follow-up passages at “drug holiday” condition. Dysregulated genes in at least in one of the analyzed passages of each cell line were considered as “union genes”. The common up/down-regulated genes in different cell lines were considered as “intersection genes”. Annotated lists were created for the total of 991 union genes for HB2, 431 union genes for MDA-MB231 and 2406 union genes for SKBR3 cell line  was up-regulated after treatment in both cancer cell lines, while JUP (a proliferation and oncogenesis marker) was up-regulated in MDA-MB231 and down-regulated in SKBR3  upon treatment was significantly up-regulated in all three cell lines. This is of some importance, since CDKN1A (P21) plays a regulatory role in DNA replication, DNA damage repair and has strong tumor suppressor activity. GJA1 (CX43) was up-regulated in HB2 while down-regulated in MDA-MB231 and SKBR3. This is an important finding since GJA1 is a TSG which is associated with suppression of metastasis GJA1 down-regulation might be strong activation of IL8 in cancerous cell lines that has been also reported previously as a suppressor of GJA1VEGFR was over-expressed in SKBR3 and HB2. This gene was down-regulated in the MDA-MB231. Since the VEGFR gene is augmenting the cell proliferation and oncogenesis, it can play an oncogenic role in carcinogenesis IL6 was very strongly expressed in all passages of three studied cell lines as both early and late effect of DAC treatment. IL6 is a multifunctional regulator of immune and inflammatory responses, and an increase of its expression has been detected in multiple epithelial tumors including breast cancer IL6 is involved in the regulation of the cell proliferation, survival, and metabolism. Additionally, high expression level of IL6 is correlated with a poor clinical prognosis which can reflect its oncogenic role IL6 in cancer is still controversial, as dichotomous roles for IL6 in both tumor-promoting and -suppressive activities have been reported There were 78 intersection genes which were differentially expressed in all three cell lines (treated and follow up passages in comparison to untreated for each cell line respectively) . Based oThe GO enrichment analysis was assessed for three cell lines based on the list of significantly expressed union genes in order to group them in the functional hierarchy . The enrCDKN1A, PTEN, CST6 BRCA1 and RASSF1) were differentially expressed after treatment (It is known that TSGs are hypermethylated in different tumor types as an early phenomenon during carcinogenesis reatment . In HB2 reatment .CDKN1A (P21) gene showed significant hypomethylation and activation in all three studied cell lines. CDKN1A was previously reported as a hypermethylated TSG in breast cancerous tissue PTEN is an essential component of TP53 gene that regulates p53 function through keeping Akt inactive and making Mdm2 incapable of translocation into the cell nucleus PTEN gene in breast cancer patients lacking TP53 mutations CST6) is more frequently observed in metastatic lesions than in primary cancers CST6 to be significantly hypermethylated in breast tumors as compared to the matched normal tissue CST6 was significantly reactivated after treatment in MDA-MB231. Two important TSGs, BRCA1 and RASSF1, which were hypermethylated in breast tumors, The RAB family genes, ETSA, CXCL1, CXCL2, CXCL3, ERBB2, MAFF, MERTK, MYC and PDGFB were differentially expressed after treatment  in HB2, MDA-MB231 and SKBR3 cell lines, respectively  which were differentially expressed in two cancer cell lines ; Table 2In total 23 intersection miRNAs were differentially expressed in all three studied cell lines . PathwayNumerous reports demonstrated that miRNAs contribute to the development and progression of cancer by acting as oncogenes or tumor suppressor genes CYPB1 and acts as a tumor suppressor that is suppressed in breast cancer As down-regulated oncosuppressor miRNAs, we can mention in both cancerous cell lines the lower-expression of two members of let-7 family, let-7f and let-7g. The expression of several members of let-7 family including let-7f and let-7g, was previously reported to be down-regulated in breast cancer samples with either lymph node metastasis or distance metastases As oncomiRs, several miRNAs were differentially expressed after treatment . InteresPTEN, PDCD4, TPM1, and MASPIN has been implicated in the acquisition of invasiveness and in promoting tumor metastatic properties in breast cancer P<0.001) after treatment. As another important example, miR-27a plays an important role in breast cancer by suppressing the expression of the transcription factors ZBTB10/RINZF, and subsequently increasing several angiogenic molecules, such as Survivin, VEGF and VEGFR1From oncomiR group, miR-21 has been reported as a potential oncogene which promotes cell survival and proliferation P value, theoretical pI and spot volumes are summarized in Proteins from the three selected cell lines were analyzed by 2D gel electrophoresis. We could detect a total of 27 intersection significantly up/down-regulated proteins  . PrincipIn total, 18 candidate spots were excised and submitted to LC-MS-MS analyses for the protein identification. Identified proteins, their gene IDs, theoretical molecular weights (kDa), number of identified peptides and the percentage of amino acid coverage are shown in p53 pathway, suggesting their possible role in tumorigenesis Notably, in the spot 1543 (SKBR3 cell line) we could identify four different isoforms of 14-3-3 protein which were confirmed with two different replicates referring to the same spot; their calculated molecular masses or pI values deviated from the observed ones . This highly conserved protein family, is a large family of 25–30 kDa acidic proteins and has seven homologous isoforms in mammalian cells which are involved in a variety of biological interactions, cell-cycle progression, apoptosis, and mitogenic signaling, such as the ATM-Interestingly, five differentially expressed genes detected by transcriptomic analysis were also identified by mass spectrometry at the proteome level, which indicated similar expression trends at both, the transcriptome and proteome . The reshttp://pantherdb.org) and ResNet® 7  database. The analyzed data showed that 28 out of 41 detected proteins/isoforms in the three analyzed cell lines are linked to neoplasms, metastasis or carcinogenesis  and significantly down-regulated in the non-aggressive cell line (SKBR3) (PDCD4 and are considered to be possible suppressors of this gene PDCD4 in the same cell line (P<0.001)  . Two miRP<0.001) . This fill lines , which iCXCR4 as one of 19 known human G protein–coupled chemokine receptors is specifically implicated in cancer metastasis and HIV-1 infection CXCR4 has been associated with more than 23 cancer types promoting the metastasis, angiogenesis, tumor growth and poor prognosis of patients. Over-expression of CXCR4 has also been shown in ∼10% of the tumors CXCR4 as a therapeutic target against solid tumors CXCR4 was significantly down-regulated in all scheduled follow-ups after demethylation treatment in MDA-MB231  for HB2  and MDA-MB231 . In SKBR3 (ERBB2/HER2 positive cell line), ERBB2 showed a steady down-regulation after treatment  receptor family of tyrosine kinases. Amplification and/or over-expression of this gene have been reported in numerous cancers, including breast and ovarian tumors ERBB2 down-regulation, we analyzed the expression profile of negative upstream regulators (BRCA1 and MYC) and a miRNA (miR-125b) showed significant up-regulation in the treated cells. From these three negative regulators, expression of two (MYC and miR-125b) were inversely correlated with ERBB2 expression (P<0.001)  (ERBB2 which can contribute to the cell proliferation and breast neoplasm The estrogen receptor (ER), progesterone receptor (PR) and ERBB2/HER2 markers are used as prognosis indicators in breast cancer to stratify patients for appropriately targeted therapies reatment . The ERBgulators . From 21P<0.001) . MiR-125(P<0.05) . Pdia6 iIL6, TGFB1, VEGFA, SERPINB5, FGF2, SFN, ERBB2, RAD51, CSF2, COL18A1, MDK and TNFSF10) and four proteins detected proteomically  were mostly up-regulated after DAC treatment  in many nonendocrine tumors CDKN2A (P16-INK4A) gene in breast cancer tissues as well as in the circulating cell free DNA (cff DNA) of patients P16-INK4A was not transcriptionally active and after treatment with DAC showed no significant change in the expression level in both cancer cell lines (P16-INK4A has homozygote deletion (c.1_471 del 471) in MDA-MB231 cell line ll lines . It is rP16-INK4A promoter (P<0.05) after treatment with DAC  and 3′UTR. The sequencing result showed two different mutations/polymorphisms, a heterozygote transition mutation in 5′UTR (c. 81 G>A) and a homozygote transversion mutation in 3′UTR (c.771 G>C) of the gene 3A and -3B (de novo methyltransferases), two key enzymes involved in DNA methylation, that are frequently up-regulated in solid tumors and associated with the poor prognosis. In lung squamous cell carcinomas, elevated Dnmt1 expression has been reported as a poor prognosis factor. Moreover, elevated expressions of both Dnmt1 and Dnmt3B have been shown to be correlated with hypermethylation of TSG promoters DNMT3A and -3B and is inversely correlated to their expression in lung cancer tissues DNMT3A expression in SKBR3  with enforced expression of miR-29b in breast cancer might have a synergistic hypomethylation effects that may result in a better disease response in breast cancer along with more robust gene re-expressions especially for the epigenetically silenced TSGs.The miRNA (miR)-29 family  has intriguing complementarities to 3′-UTRs of DNA methyltransferase  The aberrant DNA methylations of genes/miRNAs which are involved in malignant phenotypes provide interesting targets for chemotherapeutic intervention. To the best of our knowledge, the study presented here is the first pan-omics approach to synoptically identify epigenetic, transcriptome and proteome-wide alterations after effective demethylation treatment with DAC in the context of breast cancer.The results of our synoptic pan-omics analysis provide a comprehensive view of DAC treatment defined by stable or transient early and late systematic effects in the model of cancer specific changes. We investigated DAC targets in depth  and identified new pathways as well as biological interactions at the molecular levels that may correlate with particular steps in breast neoplasm including cell proliferation, cell/tissue invasion, oncogenesis, angiogenesis, apoptosis, neoplasm metastasis and senescence.In total, SKBR3 (non-aggressive breast cancer cell line) showed more significant changes in the gene expression and proteomics levels than MDA-MB231 (highly aggressive breast cancer cell line) does. This finding highlights that the epigenetic treatment of hypermethylation genes during carcinogenesis, might have a more advantageous effect when cells are still less aggressive.Additionally, along with the activation of several epigenetically suppressed TSGs, we also showed significant down-regulation of some important miRNAs with oncogenic functions in breast cancer cell lines (e.g. miR-21) as well as over-expression of some miRNAs with tumor suppressor functions (e.g. miR-155) that highlights the potential of a miRNA-based therapy in breast cancer. In the present study, there is a limitation of lacking inclusion of a luminal cell line to be compared with two studied basal like breast cancer subtypes MDA-MB231 (the triple negative receptor subtype) and SKBR3 (the Her2-associated subtype).The presented approach might become a useful model for other human solid tumor malignancies, alone or in combination with other treatments such as enforced targeted therapies for miR-29b and/or IL6 (IL6 or IL6 receptor antagonists).Dataset S1Complete materials and method:Eligibility criteria and study designCell lines and culture conditionsDAC treatmentsMultiplex quantification of cell viability and cytotoxicity protease activitiesQuantification of caspase-3 and caspase-7activitiesSimultaneous isolation of DNA, RNA miRNA and proteinsMethylation quantification of candidate tumor suppressor genes (TSGs) using thymidine-specific cleavage mass array on MALDI-TOF silico-chipMicroarray analysis and qRT-PCR validationsP16-INK4A geneMutation screening of the Proteomic profilingLiquid Chromatography - Mass Spectrometry and Liquid Chromatography - Tandem Mass Spectrometry (LC-MS-MS)Gene ontology enrichmentIn silico prediction of miRNA targetsCell signaling and pathway analysis(PDF)Click here for additional data file.Dataset S2MRNA Expression Profiles.PCA analysis of studied cell lines K-means cluster analysis of three cell lines based on the Euclidean distance and variance within different treatment passagesThe number of significant up/down-regulated genes within different passagesHeatmaps of significant up/down-regulated oncogenesPathway analysis of oncogenes that are linked to breast neoplasms and metastasisHeatmaps of significant up/down-regulated tumor suppressor genesPathway analysis of tumor suppressor genes that are linked to breast neoplasms and metastasisExpression level of six cancer related genes after treatment using qPCR(PDF)Click here for additional data file.Dataset S3MicroRNA Expression Profiles.PCA analysis of studied cell lines K-means cluster analysis of three cell lines based on the Euclidean distance and variance within different treatment passagesThe number of significant up/down-regulated miRNAs within different passagesPathway analysis of miRNAs that are linked to neoplasms, metastasis or carcinogenesisHeatmaps of significant up/down-regulated union miRNAsExpression level of important cancer related miRNAs using qPCR(PDF)Click here for additional data file.Dataset S4Proteomic Profiles.Spot picking gels for significant up/down-regulated intersection proteinsExpression profile of candidate spots within different treatment passagesPathway analysis of proteins/isoforms in that are linked to neoplasms, metastasis or carcinogenesisExpression profiles of five genes/proteins that were detected similarly in both transcriptomic and proteomic analysis(PDF)Click here for additional data file.Dataset S5List of significantly up/down-regulated genes/miRNAs as early or late effects of the treatment in three cell lines.(XLS)Click here for additional data file.


Survivin and CDK1. We evaluated expression of Survivin and CDK1 in glioblastoma and analyzed the effects of M4N combined or not with temozolomide and radiation on cell lines and primary cultures of GBM.Glioblastoma (GBM), one of the most malignant human neoplasms, responds poorly to current treatment modalities, being temozolomide (TMZ) the mostly used drug for treatment. Tetra-O-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependent of Sp1 transcription factors, such as survivin-spliced variants and CDK1 mRNA expression in glioblastoma tumor samples and cell lines. Cell proliferation was measured by XTT assay, and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyzes using different schedules of administration  were made based in Chou-Talalay method on GBM cell lines and primary cultures. Gamma radiation for clonogenic survival used the doses of 2, 4, and 6 Gy.RT-PCR assays were performed to determinate survivin-spliced variants and CDK1 gene were expressed in GBM samples (n=16) and cell lines (n=6), except the survivin-2B variant that was expressed exclusively in GBM cell lines. M4N decreased cell proliferation separately and synergistically with TMZ, besides enhancement of radiation effects, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased mitotic index and arrested the cell cycle in G2/M phase. M4N treatment down-regulated CDK1 gene and survivin and survivin-ΔEx3 variants, while the survivin-2B variant was up-regulated.All Our results suggest a potential clinical application of M4N in combination with TMZ in GB treatment.CAPES and FAPESP (process number 2009/50118-2).


Aberrant deregulation of some Receptor Tyrosine Kinases (RTKs) signalling underlies diverse facets of tumor pathobiology providing an attractive target for cancer therapy. Searching for novel cancer-associated RTKs is an important issue to discover new therapeutic opportunities. For this reason, we investigated the contribution of Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) to human cancer.in vitro and in vivo biological assays.The level of ROR1 protein expression, phosphorylation and cellular growth response to RNAi-mediated ROR1 knockdown was evaluated by an integrated screening in a panel of 43 cancer cell lines. ROR1 auto-kinase activity and transphosphorylation were determined by biochemical assays. Functional consequences of ROR1 silencing were evaluated by several MET oncogene. ROR1 phosphorylation was abrogated by MET inhibition, indicating MET dependent transphosphorylation of ROR1. Silencing of ROR1 in HS746T and NCI-H1993 cells impaired cellular proliferation, growth and migration in vitro and induced a dramatic inhibition of tumorigenesis in vivo.We demonstrated that although ROR1 is expressed in approximately 75% of the screened cancer cell lines, only gastric carcinoma cells (HS746T) and non-small cell lung carcinoma cells (NCI-H1993) exhibit high levels of ROR1 tyrosine phosphorylation and experience growth inhibition upon ROR1 suppression. Biochemical assays revealed that ROR1 is a pseudokinase lacking autocatalytic activity. Intriguingly, the two phospho-ROR1 positive cell lines both exhibited amplification and constitutive activation of the MET oncogene.Our data show that ROR1 is a pseudokinase functionally transphosphorylated by MET RTK, suggesting a critical role for ROR1 in malignant phenotypes sustained by the AIRC special program Molecular Clinical Oncology “5x1000” and AIRC grants.


Listeria monocytogenes. Here, we examined the relationship of LLO toxicity to its antigenicity and immunogenicity. LLO offered to antigen presenting cells (APC) as a soluble protein, was presented to CD4 T cells at picomolar to femtomolar concentrations- doses 3000–7000-fold lower than free peptide. This presentation required a dose of LLO below the cytotoxic level. Mutations of two key tryptophan residues reduced LLO toxicity by 10–100-fold but had no effect on its presentation to CD4 T cells. Thus there was a clear dissociation between the cytotoxic properties of LLO and its very high antigenicity. Presentation of LLO to CD8 T cells was not as robust as that seen in CD4 T cells, but still occurred in the nanomolar range. APC rapidly bound and internalized LLO, then disrupted endosomal compartments within 4 hours of treatment, allowing endosomal contents to access the cytosol. LLO was also immunogenic after in vivo administration into mice. Our results demonstrate the strength of LLO as an immunogen to both CD4 and CD8 T cells.The presentation of microbial protein antigens by Major Histocompatibility Complex (MHC) molecules is essential for the development of acquired immunity to infections. However, most biochemical studies of antigen processing and presentation deal with a few relatively inert non-microbial model antigens. The bacterial pore-forming toxin listeriolysin O (LLO) is paradoxical in that it is cytotoxic at nanomolar concentrations as well as being the source of dominant CD4 and CD8 T cell epitopes following infection with  L. monocytogenes infection, yet it is a highly cytotoxic protein. LLO, a member of the cholesterol-dependent cytolysin (CDC) family of bacterial toxins L. monocytogenes requires LLO to escape the phagosome and survive in infected cells. LLO-deficient L. monocytogenes (Δhly) are not pathogenic and are poorly immunogenic even at high doses L. monocytogenes engineered to have uncontrolled LLO activity are less virulent because they destroy their protective host niche LLO is an immunological enigma: it is both a major virulence determinant and a major immunogen following L. monocytogenes a CD4 and CD8 T cell response is directed to LLO During infection with in vivo immune responses. The parameters of LLO binding, uptake, and catabolism by APC, as well as breakdown of endosomal vesicles were included in this evaluation.In this work, we attempt to explain these apparently contradictory issues regarding LLO antigenicity and immunogenicity focusing on its effects on APC and the T cell response. We found LLO to be one of the strongest generators of CD4 T cell responses we have tested. It elicited CD4 T cell responses at unprecedented [fM]/[pM] levels, approximately 3000–7000 times more efficiently than the responses to the cognate peptide. LLO was also presented to CD8 T cells. Importantly, the immunogenic and cytotoxic activities of LLO were distinct because LLO mutants having much reduced cytotoxicity were processed and presented equivalently to the wild-type protein and elicited We examined the antigenicity of soluble LLO (referred to as LLOWT) as well as of LLO having two key tryptophans mutagenized to alanines The concentration of LLO required to induce cell death in DCs and macrophages was examined. Bone marrow-derived DCs (BMDC)  and boneThe mechanisms of cell death induced by LLO are not entirely understood. LLO is an endosomolytic agent that can cause cell death from within a cell by releasing intracellular stores of granzymes The experiments only examine the processing and presentation of LLO independent of the costimulatory activity of the APC. For this, T cell hybridomas were used since their responses are quantitatively related to the amount of peptide-MHC complexes presented by the APC. CD4 T cell hybridomas were generated against the 190–201 segment of LLO + and CD11c+ cells (data not shown). The response of T cell hybridomas was initially detected at the 0.1–10 pM range, peaked between 10–100 pM, and dropped at 1–10 nM. The drop corresponded to the doses that induced cytolytic and apoptotic effects on APC and T cells (L. monocytogenes at multiplicities of infection (MOI) ranging from 0.1–5  . There w T cells  [22]. Thom 0.1–5 . The enhom 0.1–5 . LLO preterparts , furtherLLOW492A and LLOWW were presented by either BMDC  or BMM (We wanted to determine if LLO protein could enhance the presentation of an ectopic antigenic specificity. A construct was generated that contained the 45–65 segment of the hen-egg white lysozyme (HEL) protein fused to the amino terminus of LLO. There was a ∼1000-fold enhancement in presentation of LLO-HEL(45–65) compared to presentation of the HEL(48–62) peptide . The res125Iodine and its binding, uptake, and catabolism by APC was evaluated. Radiolabeling did not affect the hemolytic activity of LLOWT, LLOW492A, or LLOWW (data not shown). All LLO preparations bound to BMM incubated on ice, at the levels shown in 125I bound to BMM at ∼0.297% of input. As noted the mutation of tryptophans reduced, but did not prevent LLO from binding to cells. LLO-125I was internalized rapidly by BMM  peptide presented by the class I MHC protein H-2Kd was examined L. monocytogenes-infected DC at MOI ranging from 0.1 to 5  were emulsified in incomplete Freund's adjuvant (oil∶water emulsion) and injected into the footpads of C57BL/6 mice. An IFNγ and IL-2 was elicited with 250 picomoles per injection from both protein and peptide immunizations . The aveWe have shown that purified recombinant LLO was processed and presented to CD4 T cell hybridomas at picomolar antigen concentrations. The ability of LLO to be presented to CD4 T cells at such low concentrations was not a peculiarity of the LLO peptides that were selected for processing. Fusion of an HEL(45–65) peptide to the amino terminus of the LLO protein led to similar enhancement in presentation. We also found presentation of LLO to CD8 T cell hybridomas, albeit at nanomolar levels. The enhanced presentation of LLO was independent of its cytotoxic qualities. Mutants of LLO that caused reduced cytolysis and apoptosis had equivalent enhancement in presentation to the wild-type protein.The relationship between immunogenicity and cytolysis explains previous studies in which LLO was shown to be an ineffective immunogen or an inhibitor of T cell activation LLO was internalized very rapidly by APC. Likewise, processing and catabolism was rapid, presentation occurred by 15 min after uptake, with maximal presentation at about 2 hrs. These early times indicate presentation by class II-MHC molecules before there was any signs of cytotoxicity or escape of contents into the cytosol. After 2 hours, and peaking at 4 hours of LLO treatment, there was release of vesicular dextran into the cytosol of cells. The detection of cytosolic dextran coincided with the earliest markers of apoptosis that were detectable on BMM or BMDC. Peak BMM/BMDC apoptosis occurred at ∼6 hours post-LLO treatment. Catabolic products of LLO were detected within 30 minutes of treatment, and LLO was maximally degraded by ∼6 hours of treatment.We hypothesize that three of LLO's important biological activities: 1) high affinity membrane binding, 2) pH dependence, and 3) rapid catabolism contribute to its antigenicity. The immunogenicity of LLO to both CD4 and CD8 T cells can be maintained despite mutations that reduce the pre-pore to pore transition, and by extension toxicity (LLOW492A and LLOWW). There are residues of CDCs/LLO responsible for high-affinity cholesterol binding (Loop containing regions (LC) 1–3) −/−Tlr4 and −/−Myd88 cells indicate that the TLR pathway did not play a role in the enhanced antigenicity of LLO in vitro. Another member of the CDC family, intermedilysin, has been shown to bind the complement regulatory protein CD59 The interactions of LLO with plasma membrane proteins are not entirely known. We have not excluded the possibility of receptor-mediated entry. Although LLO has been shown to trigger TLR4 signaling, binding affinity and direct interaction between LLO and TLR4 has not been determined An issue with CDC field is their transition from a membrane-associated CDC to the formation of a functional pore. Based on our current understanding, CDCs should bind to membranes loosely, form a pre-pore complex, and at sufficient CDC density form the final functional pore L. monocytogenesFinally, our data indicate that LLO potency as an antigen did not depend on intrinsic properties of the peptide segment being presented rather than on its handling by the APC: a peptide attached to it was highly antigenic indicating that LLO is a potent delivery agent for ectopic proteins. LLO-fusion proteins have been tested as DNA vaccines, as chemical conjugates with ectopic antigens, or as antigens carried by recombinant in vivoL. monocytogenesOur findings need to be placed in the context of the natural infection in which LLO is strong donor of peptides both to the class I and class II-MHC pathway. How LLO becomes accessible to phagocytes during the natural infection needs to be evaluated. Several avenues for presentation by class II molecules are apparent: i) in the early vesicular compartments of APC, LLO is released and can be handled like the soluble LLO protein examined here; indeed LLO in vesicles can be targeted by specific antibodies indicating its availability These experiments were approved by the Division of Comparative Medicine (DCM) of Washington University School of Medicine  accreditation number A3381-01). All experiments were performed under institutional (DCM) guidelines and all efforts were made to minimize suffering. The institutional (DCM) approval number for these studies was protocol number 20110150.The pET29b expression vector containing the LLO sequence was kindly provided by Dr. Daniel Portnoy  3 colony-forming units of L. monocytogenes EGD strain. Seven days following infection, mice were sacrificed, spleens were removed, and single-cell suspensions were generated. Isolated spleen cells were cultured with LLO(190–201) or LLO(91–99) peptide for 3 days then fused to BW5147 CD4+ or CD8+ to generate stable hybridomas using a previously described protocol Mice (C57BL/6 or BALB/c) were infected intraperitoneally with 102 unless otherwise specified. BMM were prepared as described previously 4 or 105 APC, 5×104 T cell hybridomas, and antigen for ∼16 hr. Culture supernatants were tested for IL-2 production by CTLL-2 proliferation assay using 3H-thymidine incorporation. For fixation experiments, BMM (105) were allowed to internalize antigen for 15–120 min., cells were washed 3 times in DMEM, fixed with 2% paraformaldehyde  in phosphate buffered saline pH 7.4 for 15 min. at 25°C, washed 3 times, then incubated with 0.2 M lysine (Sigma-Aldrich) for 30 min. at 25°C. Then, cells were washed 3 times with DMEM/10% FCS and T cell hybridomas (5×104) in DMEM/10% FCS were added for ∼16 hrs. T cell activation was determined by CTLL-2 assay. All antigen presentation assays were performed in 200-µL final volume in 96-well tissue culture coated flat-bottom plates . All data plotting and statistical calculations were performed with Prism 5.0 .All cell culture work was performed in DMEM  containing 10% (v/v) fetal calf serum (Thermo Fisher Scientific), 37°C and 5% COin vivo responses to LLO, mice were immunized with 250 pmol of LLOWT, LLOWW or LLO(190–201) in 1∶1 oil∶saline emulsion  in the footpads. Seven days later, mice were sacrificed and popliteal lymph nodes were isolated and dispersed. Cells were plated on cytokine capture antibody pre-coated Multiscreen-IP plates  with or without recall antigen (LLOWW at 10 nM) for 16 hours. ELISpot assay was then used to determine the number of interferon-γ (IFNγ) and interleukin-2 (IL-2) producing cells . ELISpot assay was performed following the manufacturer's specifications (BD Biosciences). ELISpot plates were scanned and spots were enumerated using CTL-ImmunoSpot S5 Analyzer .To determine 125I using the chloramine-T method 5 BMM were incubated with ∼150 ng of LLO or HEL (specific activity ∼7×103 CPM/ng protein) in 200 µL of DMEM/1% FCS in 1.7 mL siliconized tubes for 30 min. on ice. Cells were spun through oil, and radioactivity was counted in the supernatant versus cellular fraction . For uptake experiments, 2×106 BMM were incubated with 50 ng LLO or HEL (specific activity ∼2–5×103 CPM/ng of protein) in 1 mL DMEM/10% FCS for 15 min at 25°C. Cells were centrifuged and washed twice with DMEM/10% FCS and plated in 1 mL volume for 0–60 min. at 37°C. At different, times cells were removed and spun through oil to remove free radioactivity. For catabolism experiments, the same protocol was followed except that at the appropriate times, cells and media were harvested separately and adjusted to 10% trichloroacetic acid  to precipitate protein. TCA precipitate was centrifuged for 30 min. at 10,000 RCF, and the amount of radioactivity in the pellet (proteins) and supernatant  was determined. The amount of radioactivity in all fractions was determined using a gamma counter .LLO and HEL were radiolabeled with 5 BMM adhered to 12-mm glass coverslips were treated with 0.5 mg/mL FITC-dextran (Life Technologies) in DMEM/1% FCS for 30 minutes. Cells were then washed 3 times with DMEM/1% FCS and incubated with LLO in DMEM/1% FCS or left untreated for 4 hr. Following incubation, cells were washed with 1 mL PBS 3 times, fixed with 0.5 mL of 2% PFA in PBS, washed 3 times with 1 mL DMEM/10% FCS, washed 3 times with 1 mL PBS, then mounted to a slide with Prolong Gold. Images were acquired using either an Olympus BX51  with a 100W mercury source or a Zeiss Axiovert 100M microscope with a LSM510 source . On the BX51, a UPlanFl 60×/1.25 objective was used. On the Axiovert 100M either a 40×/1.3 FLUAR or a Plan Apochromat 63×/1.4 objective was used. Spot Advanced  was used to acquire epiluminescence images on the BX51. LSM510 Version 3.2  software was used to acquire/analyze images and determine colocalization of LLOWW with MHC II on the Axiovert 100M/LSM510. Quantification of pixel density and particle size was performed with ImageJ . Images were thresholded using the default setting then the analyze particle feature was used to calculate pixel number and size of particles in multiple fields. Microscopy images were resized and adjusted for brightness and contrast using Adobe Photoshop CS4 .For FITC-dextran treatment, 10


A set of 132 DH lines derived from three European landraces and 106 elite flint (EF) lines were genotyped for 56,110 single nucleotide polymorphism (SNP) markers and evaluated in field trials at five locations in Germany in 2010 for several agronomic traits. In addition, the landraces were compared with synthetic populations produced by intermating DH lines derived from the respective landrace. Our objectives were to (1) evaluate the phenotypic and molecular diversity captured within DH lines derived from European landraces, (2) assess the breeding potential (usefulness) of DH lines derived from landraces to broaden the genetic base of the EF germplasm, and (3) compare the performance of each landrace with the synthetic population produced from the respective DH lines. Large genotypic variances among DH lines derived from landraces allowed the identification of DH lines with grain yields comparable to those of EF lines. Selected DH lines may thus be introgressed into elite germplasm without impairing its yield level. Large genetic distances of the DH lines to the EF lines demonstrated the potential of DH lines derived from landraces to broaden the genetic base of the EF germplasm. The comparison of landraces with their respective synthetic population showed no yield improvement and no reduction of phenotypic diversity. Owing to the low population structure and rapid decrease of linkage disequilibrium within populations of DH lines derived from landraces, these would be an ideal tool for association mapping. Altogether, the DH technology opens new opportunities for characterizing and utilizing the genetic diversity present in gene bank accessions of maize.Landraces are valuable genetic resources for broadening the genetic base of elite germplasm in maize. Extensive exploitation of landraces has been hampered by their genetic heterogeneity and heavy genetic load. These limitations may be overcome by the  Zea mays L.) was introduced into Europe over two distinct paths. A first introduction of Caribbean Flint maize in Spain by Colombus in 1493 was followed by introductions of Northern Flints from North America to North-Western Europe during the 16th century ex situ, in gene banks.According to molecular evidence, maize  technique ad libitum and evaluated with any desired degree of precision in replicated trials, whereas landraces in any generation represent a conglomerate of highly diverse, unique and non-reproducible individuals. This enables maintenance and efficient selection of the most promising genotypes for further use in breeding.Inbreeding uncovers the genetic load and can be used to purge landraces from lethal or detrimental alleles in-vivo DH technique to produce fixed lines from European and tropical landraces showed a generally lower testcross performances but also a high improvement potential of such materials per se performance of a broad set of DH lines derived from landraces would be of equal interest with regard to their introgression into modern breeding material. In addition to an evaluation of the phenotypic diversity captured in these lines, it would allow selection of agronomic traits relevant for line maintenance and hybrid seed production First studies using the Bugard, Gelber Badischer and Schindelmeiser with EF lines in field trials and marker assays revealed substantial phenotypic and molecular variation within the populations of DH lines, supporting their potential to broaden the genetic base of the EF germplasm.The goals of this study were to (1) evaluate the phenotypic and molecular diversity captured within DH lines derived from European landraces and compare them with the diversity of EF lines, (2) assess the breeding potential (usefulness) of DH lines derived from landraces to broaden the genetic base of the EF germplasm, and (3) compare the performance of each landrace with the synthetic population produced from their respective DH lines. The comparison of DH lines derived from the landraces P<0.05) were observed among the means of the populations of DH lines from landraces and the EF lines for all morphological and agronomic traits except female flowering (Bugard (DH-BU) had on average a low emergence rate, high leaf chlorosis, low early growth rate and short anthesis-silking interval. They had short plants with intermediate ear height, well developed ear shanks and almost no husk flag leaves. The plants carried short broad ears with 10.8 kernel rows and 16.4 kernels per row on average, with a tendency to develop a second seed setting ear. The DH lines derived from the landrace Gelber Badischer (DH-GB) had a high emergence rate, low leaf chlorosis, high early growth rates and an intermediate anthesis-silking interval. The tall plants with high ear insertion also had long ear shanks and husk flag leaves. Their ears were long and slender, with 8.4 kernel rows and 20.3 kernels per row. The DH-GB lines showed the highest mean for 100 kernel weight among all populations. The DH lines derived from the landrace Schindelmeiser (DH-SC) showed values intermediate to those of DH-BU and DH-GB for most of the traits. Exceptions were ear height, ear shank and ear dry matter content, where DH-SC lines had lower means than DH-BU and DH-GB. In contrast, the DH-SC lines showed higher means for anthesis-silking interval and kernel rows. The EF lines had on average 22% higher grain yield (55.2 g plant−1) than the DH lines derived from landraces (43.0 g plant−1). Grain yield and number of kernel rows were the only traits for which the EF lines significantly (P<0.05) outperformed all three populations of DH lines. The founder lines EP1, F2, F7 and DK105 had a grain yield of 49.5, 38.9, 47.2 and 55.0 g plant−1, respectively, and were thus below the average grain yield of the EF lines.Significant differences  associated with all yield components , with thThe average pair-wise phenotypic distance was 5.30 among the DH-BU lines, 4.93 among the DH-GB lines, 5.22 among the DH-SC lines, and 4.44 among the EF lines . AverageP<0.05) for all populations and traits (40%U) of the populations of DH lines derived from landraces surpassed the mean of the EF lines. Important exceptions were grain yield for all populations of the DH lines and the number of kernel rows for DH-GB lines, as none of them had more than 10 kernel rows. The usefulness criterion at a selected fraction of α = 10% (10%U) of the populations of DH lines reached in many instances the usefulness of the EF lines. The top 10% DH lines (DH10%) selected based on index performance across all three DH line populations had an average grain yield of 54.05 g plant−1 while the top 10% EF lines (EF10%) selected according to the same index had an average grain yield of 69.43 g plant−1.Estimates of genotypic variance were significant (d traits  and 2. IMean pair-wise modified Rogers' distance (dW) between lines calculated from 24,572 SNP markers was highest in DH-BU (0.52), and lowest in DH-SC (0.45) . The ranwiH) was similar for the EF and DH-BU materials, whereas DH-GB and DH-SC showed lower values (wH) was 0.233 and the total genetic diversity among all flint lines (tH) was 0.312, resulting in a mean genetic differentiation (STG) of 0.253. Linkage disequilibrium (LD) decreased below the threshold of r2 = 0.1 within less than 0.8 Mbp in the populations of DH lines derived from landraces but only after 3.8 Mbp within the EF lines  for most of the traits, with the exception of emergence rate, leaf chlorosis score, kernels per row and 100 kernel weight  and systematic differences were observed for female flowering, anthesis-silking interval, ear height, ear diameter, number of kernel rows, kernel dry matter content, number of kernels per plant, number of barren stalks and incidence of common smut (P<0.05) from their respective source landraces for plant height, ear shank and husk flag leaves score. Estimates of residual variance among individuals decreased significantly (P<0.05) in the synthetic population produced from the DH-BU lines compared to Bugard for plant height. Similarly, the same trend was observed in the synthetic population produced from the DH-GB lines compared to Gelber Badischer for number of kernel rows. Increased estimates of residual variance among individuals were observed for husk flag leaves in all synthetics (data not shown). No significant differences between estimates of residual variance among individuals were observed for the remaining traits.In the comparison of landraces with their corresponding synthetic population, significant (mon smut . No imprin vivo double haploid (DH) techniques would be appropriate methods to produce fixed lines and purge the genetic load present in landraces. According to a roughly ten-fold lower efficiency of DH production when landraces are used as source population in comparison to elite crosses , and following the reports on the development of first cycle inbred lines Unlocking the genetic diversity present in the huge collections of maize landraces accessions by developing homozygous lines from these highly heterogeneous populations in vivo DH technique could be raised because of the segregation distortion observed in DH lines produced by in vitro anther culture in vivo maternal DH production technique showed no systematic effect on phenotypic and allelic distribution e.g., plant height, husk flag leaves) might reflect random drift due to the small number of DH lines (DH-BU and DH-GB) extracted from landraces. In contrast, systematic effects  suggested loss of specific alleles during the DH production or by selection during line multiplication.Legitimate concerns on the selective neutrality of the Although improved seed set, reduced number of barren stalks, reduced anthesis-silking interval and lower incidence of common smut might suggest some positive purging of detrimental alleles wiH  markers wiH of the populations of DH lines were comparable to values reported for European landraces in previous studies As indicated by the estimates of genotypic variances  and 2 anHwi , both thwd values within the populations of DH lines compared to the one observed within the EF material  were compared with commercial hybrids per se performance under stress conditions will also result in higher-yielding hybrids The strongest phenotypic difference between the EF lines and the populations of DH lines derived from landraces was observed for grain yield . With onU(α) showed that the generally lower performance level of the populations of DH lines was largely compensated by large estimates of genotypic variance  present in the landraces can be removed prior to introgression into EF matrerials by selecting superior lines among the DH lines derived from landraces. Moreover, the yield gap can be substantially reduced because the top DH10% lines reached nearly the mean grain yield performance of the EF lines. Similarly, a selection of the best performing DH lines reduced the yield gap between commercial hybrids and testcrosses of DH lines derived from Gelber Badischer and Schindelmeiser by 50% 10% lines with EF will still result in lower means of the progenies than crosses between top EF10% lines, because the grain yield of the selected DH10% lines remained far below the performance of the top EF10%. Yet, the usefulness of crosses does not only depend on the mean but also on the variance among their offspring. The higher phenotypic and genetic distances observed between DH lines derived from landraces and the EF lines, on one hand, and those obtained among the EF lines . Second, we also need to take into account some ascertainment bias of the SNP chip towards higher diversity among Lancaster germplasm than among European flint, even after the exclusion of the marker set developed by Syngenta Owing to the large estimates of genotypic variance among the DH lines derived from landraces, individual lines with superior performance for agronomic and morphological traits can be selected and introgressed into the elite material. As suggested by the high phenotypic and genetic distance between the DH lines and the EF lines, the generally lower grain yield and testcross performance of DH lines derived from landraces might be well compensated by a large genotypic variance for these trait in the progenies of crosses with EF lines Bugard , Gelber Badischer , and Schindelmeiser  by a proprietary in-vivo haploid induction technique similar to the one described by Röber et al. Bugard, Gelber Badischer, and Schindelmeiser were maintained by KWS SAAT AG. Testcross performance of the DH lines derived from the landraces Gelber Badischer and Schindelmeiser were already reported in a previous study i.e., F2, F7, DK105 and EP1 were included in the EF material. Lines F2 and F7 are derived from the French landrace Lacaune, DK105 from the German landrace Gelber Badischer and EP1 from the Spanish landrace LizargaroteA set of 132 DH lines was produced by KWS SAAT AG  from the European maize landraces Bugard, Gelber Badischer and Schindelmeiser were multiplied in the winter nursery in Chile during the season 2009/2010 by sowing 200 plants and pollinating each row with bulked pollen from the other rows. Synthetic populations of Bugard (SYN-BU), Gelber Badischer (SYN-GB) and Schindelmeiser (SYN-SC) were produced by intermating all DH lines of the respective population. For this purpose, pollen of five  or ten (SYN-SC) DH lines was bulked to pollinate one ear of each of the remaining DH lines from the respective landrace. This procedure was repeated until all lines were used once in a pollen bulk. Equal numbers of seeds per pollinated ear were bulked to ensure an equal contribution of each DH line to the synthetics.The landraces Genomic DNA from the inbred lines was extracted from pooled leaf tissue samples of five seedlings per genotype using the CTAB method Wd) Wd were determined among the lines of the four groups, as well as between the EF lines and individual populations of the DH lines. Nei's total genetic diversity (tH) was estimated over all loci and lines wiH) was computed for each individual population and averaged to obtain the mean genetic diversity within populations (wH). Overall genetic differentiation (STG) was calculated as 1-(wH/tH), and population-wise genetic differentiation (STiG) as 1-(wiH/tH). Linkage disequilibrium (LD) was calculated within each population as r2 values between all pairs of loci for each chromosome r2 values were binned according to the distance between markers in steps of 0.05 Mbp and averaged over chromosomes. The threshold of r2 below which LD was considered non-significant was set to 0.1 Number of population-specific SNPs and pair-wise modified Rogers' distances , is considered optimal for maize cultivation, whereas Oberer Lindenhof, located at an altitude of 700 m a.s.l. on the Swabian Alb, is a marginal environment for maize growing due to low average temperature (6.6°C). The plants at Oberer Lindenhof were thus harvested already at the eight-leaf stage. Both locations were amended with fertilizer according to usual cultivation practice (150 kg N ha−1). In Hohenheim , the trials were conducted on a conventionally fertilized field (150 kg N ha−1) and on a nitrogen deficient one (0 kg N ha−1), where only P and K fertilization was kept at optimum. The location Kleinhohenheim , adjacent to Hohenheim, was cultivated according to organic farming directives and amended with 50 Mg ha−1 organic manure of undetermined N-availability.The 256 elite inbred lines and 132 DH lines derived from landraces were divided into two sets of 200 entries each. Twelve inbred lines were common to both sets to allow for a combined analysis and adjust for potential differences between the experimental sets. The two sets were evaluated in separate but adjacent field trials laid out as a 20-by-10 alpha design with two replications −2 was determined two to four times, depending on the location, between the four-leaf and eight-leaf stage by a non-destructive phenotyping platform described in detail by Montes et al. −1 and 100 kernel weight in g were determined from bulked seeds of the primary and secondary ears (as far as present) of the five plants.Sixteen traits were evaluated on a plot basis for all lines. Emergence was determined as the ratio in percent of emerged plants to sown seeds per plot before thinning. Leaf chlorosis was scored between the four-leaf and six-leaf stage on a 1 (no chlorosis) to 9 (severe chlorosis) scale. Fresh above-ground biomass in g mBugard, Gelber Badischer and Schindelmeiser and the corresponding three synthetic populations produced from the intermated DH lines were evaluated in a randomized complete block design with three replications. The trials were conducted in fields adjacent to Experiment 1 in the same environments except Oberer Lindenhof. Four-row plots of 3 m  or 4 m (Kleinhohenheim) length with 0.75 m between rows were overplanted and later thinned to a final plant density of 9 plants m−2.The three landraces Ustilago maydis) and barren stalks was recorded on these 20 plants. The two center rows were harvested at maturity with a combine to determine fresh grain yield. A grain sample of ∼500 g was dried at 60°C to a constant weight to determine kernel dry matter content and 100 kernel weight. Grain yield was calculated for a final dry matter content of 85%. Five ears each were harvested by hand from the center of the two remaining outer rows, to measure ear length and diameter, kernel rows, and number of kernels per row.Emergence, leaf chlorosis, relative growth rate, female and male flowering, and anthesis-silking interval were determined on a plot basis as in Experiment 1. Plant and ear height, ear shank and husk flag leaves scores were determined on 10 plants from each of the two center rows. Occurrence of common smut  were calculated within populations on an entry-mean basis, according to Hallauer et al. For analysis of the phenotypic data, DH-BU lines, DH-GB lines, DH-SC lines, EF lines and EL lines were considered as five populations. The following model was employed to estimate variance components in Experiment 1:μ, ip, and ijg as fixed effects in G(α), as well as the usefulness criterion U(α), of the populations of DH and elite lines. The parameter U(α) combines the estimate of the population mean and ΔG(α) and allows a comparison between populations, with regard to the prospects to identify individuals or lines with superior performance. The parameters ΔG(α) and U(α) were computed following Prigge et al. α = 10% (10%U) and 40% (40%U), corresponding to a selection intensity of i = 1.76 and 0.97, respectively. Response from selection was computed as ΔG(α) = i(α)ghσ, where i(α) is the selection intensity, h the square root of the heritability, and gσ the genotypic standard deviation U(α) = μ±ΔG(α), where μ is the mean of the respective set of lines. The sign of ΔG(α) was chosen depending on whether higher values of the trait expression were regarded as positive or negative. The best 10% DH lines across the three landraces (DH10%) and EF lines (EF10%) were selected according to the index IP = 2×ear dry matter content+grain yield, commonly used by maize breeders in Central Europe. Within each breeding group, phenotypic correlations (pr) between grain yield and the remaining traits were determined as Pearson's correlation coefficient and the significance level was Bonferroni-corrected to account for multiple comparisons among populations. Pair-wise Euclidean distances (ED) were calculated among the EF inbred lines and DH lines derived from landraces from the adjusted entry means of the flint genotypes for the sixteen traits evaluated in Experiment 1, centered to mean zero and scaled to unit variance. Minimum, maximum and average of ED were calculated within each population, as well as between the EF lines and individual populations of DH lines.For calculation of the adjusted means of the lines, best linear unbiased estimates (BLUEs) were computed by considering μ is the overall mean, ip the effect of the entry i (landraces and synthetic populations), je the effect of environment j, ijpe the interaction between landrace or synthetic population i and environment j, jkr the effect of replication k within environment j, and ijkε the residual. All effects except μ and ip were considered as random in μ is the overall mean, ig the effect of the landrace i , jt the effect of the population type j (source landrace vs. synthetic population), ke the effect of environment k, ijgt, ikge, jkte, ijkand gte the interactions among landrace i, population type j and environment k, klr the effect of replication l within environment k, and ijklε the residual. The parameter μ, ig and jt were considered as fixed in In Experiment 2, the following model was used in a first step to estimate adjusted means of and test for differences between the six populations:All calculations were performed within the R-environment Table S1List of genotypes belonging to the elite flint (EF) and further elite lines (EL) populations.(XLSX)Click here for additional data file.