From "Click" to "Fenton" chemistry for 5-bromo-2'-deoxyuridine determination.

Ascorbic acid (AA) and copper have been increasingly employed in flow cytometry (FCM) and high content analysis (HCA) since the introduction of "click chemistry" as a non-destructive alternative to classical 5-bromo-2'-deoxyuridine (BrdU) immunodetection for DNA synthesis and proliferation assays. Mixtures of ascorbate and catalytic copper, under certain experimental conditions, act as oxidizing agent, catalyzing the formation of reactive hydroxyl radicals through hydrogen peroxides decomposition via Fenton reaction. We developed a procedure for BrdU incorporation detection based on the use of AA and cupric ions as DNA damaging agents. Optimal DNA damaging conditions were identified and found to provide results comparable with "click" 5-ethynyl-deoxyuridine (EdU) cycloaddition approach and classical BrdU immunodetection. Scavenger agents were found to prevent hydroxyl-induced DNA damages, providing the proof-of-concept for the use of this procedure for DNA denaturation prior to BrdU detection. We demonstrated hydroxyl radicals' reaction to be readily applicable to HCA and FCM assays, for both classical BrdU immunostaining and EdU cycloaddition procedure. This technique was successfully employed for BrdU pulse-chase experiments and in multiparametric immunofluorescence assays for the simultaneous detection of labile phosphoproteins in intact cells. The use of AA/Cu prior to immunodetection for BrdU incorporation assays is a viable alternative to chemical/physical DNA denaturing agents (acids or heat), since it allows preservation of labile epitopes such as phosphoproteins, and over enzymatic agents (digestion with DNases) for its lower cost.

5-(2-amino-pyrimidin-4-yl)-1H-pyrrole and 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives as new classes of selective and orally available Polo-like kinase 1 inhibitors.

The discovery and characterization of two new chemical classes of potent and selective Polo-like kinase 1 (PLK1) inhibitors is reported. For the most interesting compounds, we discuss the biological activities, crystal structures and preliminary pharmacokinetic parameters. The more advanced compounds inhibit PLK1 in the enzymatic assay at the nM level and exhibit good activity in cell proliferation on A2780 cells. Furthermore, these compounds showed high levels of selectivity on a panel of unrelated kinases, as well as against PLK2 and PLK3 isoforms. Additionally, the compounds show acceptable oral bioavailability in mice making these inhibitors suitable candidates for further in vivo activity studies.

Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation.

In vivo imaging of apoptosis in a preclinical setting in anticancer drug development could provide remarkable advantages in terms of translational medicine. So far, several imaging technologies with different probes have been used to achieve this goal. Here we describe a bioluminescence imaging approach that uses a new formulation of Z-DEVD-aminoluciferin, a caspase 3/7 substrate, to monitor in vivo apoptosis in tumor cells engineered to express luciferase. Upon apoptosis induction, Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase generating measurable light. Thus, the activation of caspase 3/7 can be measured by quantifying the bioluminescent signal. Using this approach, we have been able to monitor caspase-3 activation and subsequent apoptosis induction after camptothecin and temozolomide treatment on xenograft mouse models of colon cancer and glioblastoma, respectively. Treated mice showed more than 2-fold induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D: -luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, we confirmed that it is possible to follow non-invasively tumor growth inhibition and induction of apoptosis after treatment in the same animal over time. Moreover, here we have proved that following early apoptosis induction by caspase 3 activation is a good biomarker that accurately predicts tumor growth inhibition by anti-cancer drugs in engineered colon cancer and glioblastoma cell lines and in their respective mouse xenograft models.

Neuroinflammation, body temperature and behavioural changes in CD1 male mice undergoing acute restraint stress: An exploratory study.

BACKGROUND: Animal models used to study pathologies requiring rehabilitation therapy, such as cardiovascular and neurologic disorders or oncologic disease, must be as refined and translationally relevant as possible. Sometimes, however, experimental procedures such as those involving restraint may generate undesired effects which may act as a source of bias. However, the extent to which potentially confounding effects derive from such routine procedures is currently unknown. Our study was therefore aimed at exploring possible undesirable effects of acute restraint stress, whereby animals were exposed to a brightly lit enclosed chamber (R&L) similar to those that are commonly used for substance injection. We hypothesised that this would induce a range of unwanted physiological alterations [such as neuroinflammatory response and changes in body weight and in brown adipose tissue (BAT)] and behavioural modification, and that these might be mitigated via the use of non-aversive handling methods: Tunnel Handling (NAH-T) and Mechanoceptive Handling (NAH-M)) as compared to standard Tail Handling (TH). METHODS: Two indicators of physiological alterations and three potentially stress sensitive behavioural parameters were assessed. Physiological alterations were recorded via body weight changes and assessing the temperature of Brown Adipose Tissue (BAT) using infra-red thermography (IRT), and at the end of the experiment we determined the concentration of cytokines CXCL12 and CCL2 in bone marrow (BM) and activated microglia in the brain. Nest complexity scoring, automated home-cage behaviour analysis (HCS) and Elevated Plus Maze testing (EPM) were used to detect any behavioural alterations. Recordings were made before and after a 15-minute period of R&L in groups of mice handled via TH, NAH-T or NAH-M. RESULTS: BAT temperature significantly decreased in all handling groups following R&L regardless of handling method. There was a difference, at the limit of significance (p = 0.06), in CXCL12 BM content among groups. CXCL12 content in BM of NAH-T animals was similar to that found in Sentinels, the less stressed group of animals. After R&L, mice undergoing NAH-T and NAH-M showed improved body-weight maintenance compared to those exposed to TH. Mice handled via NAH-M spent a significantly longer time on the open arms of the EPM. The HCS results showed that in all mice, regardless of handling method, R&L resulted in a significant reduction in walking and rearing, but not in total distance travelled. All mice also groomed more. No difference among the groups was found in Nest Score, in CCL2 BM content or in brain activated microglia. CONCLUSIONS: Stress induced by a common restraint procedure caused metabolic and behavioural changes that might increase the risk of unexpected bias. In particular, the significant decrease in BAT temperature could affect the important metabolic pathways controlled by this tissue. R&L lowered the normal frequency of walking and rearing, increased grooming and probably carried a risk of low-grade neuro-inflammation. Some of the observed alterations can be mitigated by Non-aversive handlings.

Miniaturizing bromodeoxyuridine incorporation enables the usage of flow cytometry for cell cycle analysis of adherent tissue culture cells for high throughput screening.

Analysis of cell cycle progression by 5-bromo-2'-deoxyuridine (BrdU) incorporation is commonly used for evaluating the mode of action of anticancer drugs, but usually requires a high number of cells and large amounts of monoclonal antibodies. In addition, manual sample handling is not suitable for high throughput. To circumvent these limitations, we have developed a miniaturized method to measure BrdU incorporation into DNA directly in 96-wells plates. Adherent cells were grown in 96-well plates in the absence or presence of compounds of interest. After BrdU pulse labeling or pulse chase, cells were harvested, transferred to polymerase chain reaction (PCR) V-bottom plates, and fixed by adding methanol. DNA denaturation was performed directly in the plates by heat using a PCR thermocycler. BrdU incorporation was detected by indirect immunocytochemical staining, and cellular DNA was counterstained using propidium iodide. Samples were acquired by a BD FACSCalibur with BD Multiwells Auto sampler or BD HTS. We defined a dynamic range of the optimal cell number, for which cells maintained exponential growth up to 72 h. The assay was robust up to 30,000 cells per well. BrdU dot plots of cell cycle phases showed an excellent separation of cell populations, and DNA histograms showed a low coefficient of variation. Thermal denaturation was suitable for 96-well plates to detect BrdU incorporation with a good signal-to-noise ratio, and cluster analysis allowed fingerprint readouts for drug sensitivity and mechanism of action as exemplified for paclitaxel and doxorubicin. This method provided rapid high-throughput BrdU/DNA content analysis with high accuracy and reproducibility, accompanied by a reduction in reagent consumption. A critical step was identified as the standardization of DNA denaturation using a PCR thermocycler. Here,we show some applications of this method for cell cycle studies in drug discovery.

Thieno[3,2-c]pyrazoles: a novel class of Aurora inhibitors with favorable antitumor activity.

A novel series of 3-amino-1H-thieno[3,2-c]pyrazole derivatives demonstrating high potency in inhibiting Aurora kinases was developed. Here we describe the synthesis and a preliminary structure-activity relationship, which led to the discovery of a representative compound (38), which showed low nanomolar inhibitory activity in the anti-proliferation assay and was able to block the cell cycle in HCT-116 cell line. This compound demonstrated favorable pharmacokinetic properties and good efficacy in the HL-60 xenograft tumor model.

A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining.

Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments.

PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer.

PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition-related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status-dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser(10) is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers.

PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity.

PURPOSE: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy. EXPERIMENTAL DESIGN: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer. RESULTS: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632. CONCLUSIONS: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.

CyTOF Mass Cytometry for Click Proliferation Assays.

Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S-phase determination in cell cycle applications, combining well-established click chemistry with a novel iodo-deoxyuridine (IdU) azide derivative and a cross-reactive anti-IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU-based assays for complex and multiparametric experiments. It provides a feasible cost-effective approach for detecting ethynyl-labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.

FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients.

BACKGROUND: Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro. METHODS: Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT-qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients' prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR. RESULTS: A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response. CONCLUSIONS: FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.

1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: identification of a potent Aurora kinase inhibitor with a favorable antitumor kinase inhibition profile.

The optimization of a series of 5-phenylacetyl 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole derivatives toward the inhibition of Aurora kinases led to the identification of compound 9d. This is a potent inhibitor of Aurora kinases that also shows low nanomolar potency against additional anticancer kinase targets. Based on its high antiproliferative activity on different cancer cell lines, favorable chemico-physical and pharmacokinetic properties, and high efficacy in in vivo tumor models, compound 9d was ultimately selected for further development.

Multiparametric cell cycle analysis by automated microscopy.

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.

Interpreting cell cycle effects of drugs: the case of melphalan.

Multiple effects usually occur in the cell cycle, during and after the exposure to a drug, while treated cells flowing through the cycle encounter G1, S and G2M checkpoints. We developed a simulation tool connecting the microscopic level of the cellular response in G1, S and G2M with the experimental data of growth inhibition and flow cytometry. We found that multiple-often not intuitive-combinations of cytostatic and cytotoxic effects can be in keeping with the observations. This multiplicity of interpretation can be strongly reduced by considering together data with different methods, ideally reaching a reconstruction of the underlying cell cycle perturbations. Here, we propose an experimental plan including a time course of DNA flow cytometry and absolute cell count measurements with several drug concentrations and a limited number of flow cytometric DNA-Bromodeoxyuridine and TUNEL analyses, coupled with computer simulation. We showed its use in the attempt to define the complete time course of the effects of melphalan on three cancer cell lines. After drug treatment, each subset of cells experienced blocks and lethality in all phases of the cell cycle, but the dynamics was different, the differences being strongly dose-dependent. Our approach allows a better appreciation of the complexity of the cell cycle phenomena associated with drug treatment. It is expected that such level of understanding of the time- and dose-dependence of the cytostatic and cytotoxic effects of a drug might support rational therapeutic design.

Potent and selective Aurora inhibitors identified by the expansion of a novel scaffold for protein kinase inhibition.

Potent and selective Aurora kinase inhibitors were identified from the combinatorial expansion of the 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole bi-cycle, a novel and versatile scaffold designed to target the ATP pocket of protein kinases. The most potent compound reported in this study had an IC(50) of 0.027 microM in the enzymatic assay for Aur-A inhibition and IC(50)s between 0.05 microM and 0.5 microM for the inhibition of proliferation of different tumor cell lines.

Application of click chemistry conditions for 5-bromo-2'-deoxyuridine determination through Fenton and related reactions.

Mixtures of ascorbate and copper used in certain click chemistry experimental conditions act as oxidizing agents, catalyzing the formation of reactive oxygen species through Fenton and related reactions. Hydroxyl radicals act as chemical nucleases, introducing DNA strand breaks that can be exploited for BrdU immunostaining in place of acid denaturation. This procedure is readily applicable to high content analysis and flow cytometry assays, and provides results comparable to click chemistry EdU cycloaddition and classical BrdU immunodetection. Importantly, this approach allows preservation of labile epitopes such as phosphoproteins. This unit describes an optimized method that successfully employs Fenton chemistry for simultaneous detection of phosphoproteins and BrdU in intact cells.

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining.

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry.

A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds.

The identification of drugs capable of reactivating γ-globin to ameliorate ß-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/ß globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing ß-globin (ß-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and ß-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of ß-hemoglobinopathies.

Highly multiplexed phenotypic imaging for cell proliferation studies.

The application of multiplexed imaging technologies in phenotypic drug discovery (PDD) enables profiling of complex cellular perturbations in response to drug treatment. High-content analysis (HCA) is among the most pursued approaches in PDD, with a proven capability to identify compounds with a given cellular mechanism of action (MOA), as well as to unveil unexpected drug cellular activities. The ability of fluorescent image-based cytometric techniques to dissect the phenotypic heterogeneity of cell populations depends on the degree of multiplexing achievable. At present, most high-content assays employ up to four cellular markers separately detected in distinct fluorescence channels. We explored the possibility to increase HCA multiplexing through analysis of multiple proliferation markers in the same fluorescence channel by taking advantage of the different timing of antigen appearance during the cell cycle, or differential intracellular localization. Simultaneous analysis of DAPI staining and five immunofluorescence markers (BrdU incorporation, active caspase-3, phospho-histone H3, phospho-S6, and Ki-67) resulted in the first six-marker high-content assay readily applicable to compound MOA studies. This approach allows detection of rare cell subpopulations, unveiling a high degree of phenotypic heterogeneity in exponentially growing cell cultures and variability in the individual cell response to antiproliferative drugs.