High-throughput RNAi screening in vitro: From cell lines to primary cells

ABSTRACT In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity.

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In Vitro High-Throughput RNAi Screening to Accelerate the Process of Target Identification and Drug Development

Methods in Molecular Biology - High-Throughput RNAi Screening ◽

10.1007/978-1-4939-6337-9_11 ◽

2016 ◽

pp. 137-149 ◽

Cited By ~ 1

Author(s):

Hongwei Yin ◽

Michelle Kassner

Keyword(s):

Drug Development ◽

High Throughput ◽

Target Identification ◽

Rnai Screening

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Characterization Of FLT3/ITD Resistant Cells and Identification Of Novel Therapeutic Targets Utilizing High Throughput Drug Screening

Blood ◽

10.1182/blood.v122.21.2522.2522 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2522-2522

Author(s):

Katherine Tarlock ◽

C. Anthony Blau ◽

Timothy Martins ◽

Soheil Meshinchi

Keyword(s):

Cell Lines ◽

High Throughput ◽

Drug Screening ◽

Therapeutic Options ◽

High Throughput Drug Screening ◽

Mutational Profiling ◽

Flt3 Inhibitors ◽

Resistant Cells

Abstract The overall survival (OS) of pediatric acute myeloid leukemia (AML) exceeds 60%, however for high risk (HR) patients, including high allelic ratio FLT3/ITD+, survival remains poor. FLT3/ITD is one of the first genomic alterations in AML to be exploited for therapeutic benefit as it has greater sensitivity to the pro-apoptotic effects of FLT3-inhibitors. Children’s Oncology Group (COG) phase III AML trial AAML1031 is investigating the role of sorafenib in combination with chemotherapy in HR FLT3/ITD+ patients. In vitro and in vivo studies indicate that resistance to FLT3-inhibitors can develop through varying mechanisms including up-regulation of FLT3 receptor, acquisition of secondary mutations, or activation of alternate survival mechanisms leading to apoptotic escape. For FLT3/ITD+ patients who relapse despite treatment with FLT3-inhibitors, there are often no therapeutic options and survival is very poor. In evaluation of therapeutic options for those who relapse on sorafenib, we developed an in vitro resistance model using the FLT3/ITD+ cell line MV4-11. Resistance was induced thru long-term exposure to incrementally increasing doses of sorafenib. Two distinct cell lines with resistance at 10 and 100 fold above the IC50 of naïve MV4-11 were generated for experimental evaluation. Genotypic and phenotypic characterization of the resistant cells was conducted by multidimensional flow cytometry (MDF), conventional karyotyping, and mutational profiling. MDF revealed an overall similar immunophenotype, however the resistant cells were significantly more homogeneous for expression of HLA-DR and had significantly higher CD11b expression compared to their naïve counterparts. CD135 expression was minimally increased in the resistant cells. In comparison of the karyotypes, the resistant cells were a more homogenous population with emergence of one dominant clone and disappearance of a number of pre-existing sub-clones. Mutational profiling by Sanger sequencing revealed a novel N841Y mutation in activation loop, an area implicated in TKI-resistance. Using a high throughput drug screening assay, we explored sensitivity profiles of the naïve and resistant MV4-11 cells to 163 oncology agents, including 45 FDA approved and 118 investigational agents that target a number of key pathways regulating cell growth, differentiation, and survival. The naïve MV4-11s had a sorafenib IC50 of 1.3 nM (published 1-5nM) and resistant cells had IC50 of approximately 2-log folds above the naïve, which was consistent to what we had seen in our lab-based validations. We initially assessed whether resistance to sorafenib induces cross-resistance to other TKIs. Agents in the panel with previously demonstrated efficacy for FLT3/ITD included quizartinib (AC-220), tandutinib, ponatinib, sunitinib, and midostaurin, and in all cases sorafenib-resistant cells were also more resistant to these agents. We then examined whether we could identify agents with efficacy in the resistant cells. We identified 5 novel agents to which the resistant cells retained sensitivity. Two bcl-2 inhibitors tested maintained sensitivity in the resistant cells with IC50s in the 20-100nM range. In addition, YM-155, a survivin inhibitor, also maintained sensitivity in the resistant cells with IC50s of approximately 25-50nM across the cell lines. Survivin over-expression is associated with AML stem progenitor cells and decreased OS in adults, and transcription regulation has been linked to the FLT3/STAT5 pathway. Two CRM inhibitors, a novel class of agents which inhibit nuclear export to restore tumor suppressor function, also maintained sensitivity in the resistant cell lines with an approximate 3-fold increase in IC50 from 12nM in the naïve to 32-40nM in the resistant cells. Experience with the use of directed therapy to target specific somatic events has provided evidence that leukemic evolution can continue under this selection pressure and therapeutic options for patients with emergent disease is often insufficient. Using the high throughput drug assay in a FLT3/ITD+ cell line as an in vitro model for sorafenib-resistant FLT3/ITD patients, we identified classes of targeted agents that maintain sensitivity in resistant cells. Further validation of the targets in specimens from those with resistance to such TKIs can inform on the class of agents that can be used to treat or prevent refractory disease FLT3/ITD+ patients. Disclosures: No relevant conflicts of interest to declare.

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Proliferation Inhibition and Apoptosis Induction Of Imatinib Resistance Chronic Myeloid Leukemia Cells By Down-Regulated PPP2R5C

Blood ◽

10.1182/blood.v122.21.5158.5158 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5158-5158

Author(s):

Qi Shen ◽

Sichu Liu ◽

Yu Chen ◽

Lijian Yang ◽

Shaohua Chen ◽

...

Keyword(s):

Gene Expression ◽

Chronic Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Primary Cells ◽

Healthy Individuals ◽

Hoechst 33258 ◽

Imatinib Resistance ◽

Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. The prognosis in CML improved markedly after introduction of abl tyrosine kinase inhibitors (TKI), still a lot of CML patients die due to abl mutation related drug resistance and the blast crisis, moreover, de novo or secondary TKI-resistance is a significant problem in CML. The aim of the study is to down-regulate the PPP2R5C gene expression in imatinib-sensitive or imatinib-resistant chronic myeloid leukemia (CML) cell lines: K562, K562R (imatinib resistance without abl gene mutation), 32D-Bcr-Abl WT (imatinib sensitive, murine CML cell lines with wild type abl gene) and 32D-Bcr-Abl T315I (imatinib resistance, with abl gene T315I mutation) and primary cells from CML patients by RNA interference, thereby inhibit the CML cells proliferation and induce apoptosis. PPP2R5C-siRNAs numbered 799 or 991 were obtained by chemosynthesis. Non-silencing siRNA control (SC)-treated, mock-transfected, untreated cells were used as controls. PPP2R5C expression in mRNA levels from CML cells were analyzed after siRNAs delivered by nucleofection using the real-time quantitative PCR. The PPP2R5C protein levels were analyzed by Western blotting. Cell proliferation in vitro was assayed by the cell count kit-8 method after treatment. The morphology and the percentage of apoptosis were revealed by Hoechst 33258 stain and flow cytometry (FCM). Bone marrow mononuclear cells (BM-MNCs) from healthy individuals were transferred by PPP2R5C-siRNA-991. BFU-E, CFU-Meg and CFU-GM were performed from PPP2R5C-siRNA-991 treated BM-MNCs by methyl cellulose semi-solid culturing method, to estimate the role of differentiation and proliferation in BM-MNCs after PPP2R5C-siRNA transfection. The results showed that both PPP2R5C-siRNA-799 and PPP2R5C-siRNA-991 took best silencing results after nucleofection in all of four cells and primary cells from CML patients. The reduction about 2 to 7 folds in PPP2R5C mRNA level was observed in PPP2R5C-siRNA799 or PPP2R5C-siRNA991 treated cells. And PPP2R5C protein expression inhibition rate reached 38.08%-55.26% at 48 or 72 h after treatment. The proliferation rates of PPP2R5C-siRNA-799 or 991 treated CML cells were significantly decreased at 72 h (P < 0.05). PPP2R5C-siRNA-799 or 991 treated CML cells lines showed a significantly increase in AnnexinV/PI-positive cells (apoptosis) (P < 0.05), similar results in the morphological changes of apoptosis were found by Hoechst 33258 staining test. PPP2R5C gene mRNA expression levels in BM-MNCs from healthy individuals were significantly lower than that in K562 cells (P < 0.05), and the expression level was not significant changed after PPP2R5C-siRNA-991 transfection. The formation of BFU-E, CFU-Meg and CFU-GM from BM-MNCs showed no significant difference between PPP2R5C-siRNA-991 treatment and MOCK control group (P > 0.05). In conclusions, suppression of PPP2R5C by RNA interference could inhibit the proliferation and induce the apoptosis effectively in CML cells either in imatinib sensitive or imatinib resistance cell lines, while no significant effect of PPP2R5C-siRNA on the proliferation and differentiation of BM-MNCs in vitro, suggesting that PPP2R5C-siRNA might specially target on the CML cells. Down-regulating the PPP2R5C gene expression might be considered as a new target therapeutic strategy in CML, especially in imatinib-resistant CML. Disclosures: Li: This work was supported by Grants from National Natural Science Foundation of China (30871091 and 91129720), the Collaborated grant for HK-Macao-TW of Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (2012B0506: Research Funding.

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Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

Oxidative Medicine and Cellular Longevity ◽

10.1155/2017/5061639 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

M. R. Ricciardi ◽

R. Licchetta ◽

S. Mirabilii ◽

M. Scarpari ◽

A. Parroni ◽

...

Keyword(s):

Cell Lines ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Leukemic Cell ◽

Bioactive Compound ◽

Primary Cells ◽

Peripheral Blood Mononuclear ◽

Normal Peripheral Blood ◽

Leukemic Cell Lines

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

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Activation of the Unfolded Protein Response with the First-in-Class P97 Inhibitor CB-5083 Induces Stable Disease Regression and Overcomes Ara-C Resistance in AML

Blood ◽

10.1182/blood.v126.23.1350.1350 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1350-1350

Author(s):

Steffan T. Nawrocki ◽

Yingchun Han ◽

Ronan LE Moigne ◽

Valeria Visconte ◽

Bartlomiej Przychodzen ◽

...

Keyword(s):

Unfolded Protein Response ◽

Board Of Directors ◽

Cell Lines ◽

Primary Cells ◽

Advisory Committees ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Abstract Acute myeloid leukemia (AML) therapy has remained relatively unchanged for more than 40 years with the majority of patients not achieving long-term remission when treated with currently available agents. Novel strategies are urgently needed to improve outcomes. The constitutive dysregulation of protein synthesis/turnover contributes to disease progression and drug resistance in many forms of cancer including AML. p97 (VCP) is a master regulator of protein turnover that has been implicated in oncogenesis and malignant pathogenesis. CB-5083 is a first-in-class selective and potent orally available inhibitor of p97 that in currently being evaluated in phase I clinical trials in patients with multiple myeloma and advanced solid tumors. To assess the potential benefit of p97 inhibition as a novel approach for AML therapy, we investigated the efficacy, pharmacodynamics (PD), and pharmacokinetics (PK) of CB-5083 in a panel of human AML cell lines with diverse genetic backgrounds, primary AML specimens from both newly diagnosed and relapsed/refractory patients, and xenograft mouse models of AML. In vitro treatment with CB-5083 potently diminished the viability of AML cell lines (n = 7) and primary CD34+ blasts obtained from patients (n = 10) with IC50s significantly below 1 µM (range 200 - 700 nM) in all lines and specimens evaluated to date. Diminished viability was associated with reduced clonogenic survival and increased apoptosis in AML cell lines and primary blasts. In contrast to many conventional and experimental drugs that are less active against primary AML cells than established AML cell lines, primary cells exhibited sensitivity to CB-5083 that was similar to cell lines. Additionally, CB-5083 was highly active in 3 different cell line models of cytarabine resistance and primary cells from refractory AML patients. This suggests that CB-5083 may be effective for patients who are relapsed/refractory to conventional therapy. In vitro PD analyses demonstrated that CB-5083 rapidly triggered the accumulation of ubiquitin-conjugated proteins, activated the unfolded protein response (UPR), disrupted STAT5 signaling, reduced levels of key STAT5 targets including BCL-xL and PIM-2, and induced apoptosis. The pro-apoptotic effects of CB-5083 were associated with activation of the endoplasmic reticulum (ER) resident initiator caspase-4 and induction of the BH3-only protein NOXA, which has been previously demonstrated to be an important mediator of cell death induced by other agents that disrupt protein homeostasis. RNA sequencing (RNASeq) gene ontology (GO) analyses of MV4-11 and MOLM-13 AML cells following treatment with CB-5083 demonstrated that short-term treatment (6h) caused significant increases in multiple regulators of the unfolded protein response, protein biosynthesis, and other ubiquitin-related pathways (p<0.001). Results were confirmed by qRT-PCR. The in vivo anti-leukemic activity of CB-5083 was investigated in two different xenograft mouse models of AML: the FLT3-ITD+ MV4-11 cell line and APML HL-60 cells. Oral administration of CB-5083 (once daily, 4 days on, 3 days off) was well tolerated and induced disease regression in both xenograft models (p<0.01). In vivo PD studies demonstrated that administration of CB-5083 led to reduced AML cell proliferation (PCNA), to the induction of apoptosis (active caspase-3), and pathway inhibition as evidenced by poly-ubiquitin accumulation and elevated expression of CHOP, GRP78, and NOXA. PK-PD analyses demonstrated a correlation between the kinetics of the in vivo PD effects and drug exposure. Our collective preclinical data demonstrate that p97 inhibition is a very effective novel anti-leukemic strategy and support clinical investigation of CB-5083 in patients with relapsed/refractory AML. Disclosures LE Moigne: Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Anderson:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.

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Performance of high-throughput CometChip assay using primary human hepatocytes: a comparison of DNA damage responses with in vitro human hepatoma cell lines

Archives of Toxicology ◽

10.1007/s00204-020-02736-z ◽

2020 ◽

Vol 94 (6) ◽

pp. 2207-2224 ◽

Cited By ~ 3

Author(s):

Ji-Eun Seo ◽

Qiangen Wu ◽

Matthew Bryant ◽

Lijun Ren ◽

Qiang Shi ◽

...

Keyword(s):

Dna Damage ◽

Cell Lines ◽

High Throughput ◽

Hepatoma Cell ◽

Human Hepatocytes ◽

Human Hepatoma ◽

Human Hepatoma Cell ◽

Hepatoma Cell Lines ◽

Dna Damage Responses

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Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630318803749 ◽

2018 ◽

Vol 24 (1) ◽

pp. 28-40 ◽

Cited By ~ 2

Author(s):

Kelli M. Wilson ◽

Lesley A. Mathews-Griner ◽

Tara Williamson ◽

Rajarshi Guha ◽

Lu Chen ◽

...

Keyword(s):

Cell Lines ◽

High Throughput ◽

Drug Screening ◽

Kinase Inhibitors ◽

Proteasome Inhibitors ◽

Drug Efficacy ◽

Driver Genes ◽

Human Gbm

Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.

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Phosphodiesterase (PDE) inhibitors reduce in vitro proliferation of prostate primary cells but do not interfere with growth of prostate carcinoma cell lines

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.14604 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 14604-14604 ◽

Cited By ~ 1

Author(s):

D. G. Pernkopf ◽

G. Untergasser ◽

P. Berger ◽

E. Plas

Keyword(s):

Cell Lines ◽

Prostate Carcinoma ◽

Carcinoma Cell ◽

Polyclonal Antiserum ◽

Primary Cells ◽

In Vitro Proliferation ◽

Carcinoma Cell Lines ◽

Prostate Carcinoma Cell ◽

High Concentrations

14604 Background: PDE 5 is highly expressed in cavernosal and prostatic tissue. The mechanism of PDE-5 inhibitors on cavernosal tissue is well established but the effects of PDE-5 inhibitors on prostatic cells are unknown. The aim of this study was to analyze in vitro effects of PDE-5 inhibitors on prostate primary cells, fibroblasts (PrSC), basal epithelial cells (PrEC) and prostate cancer cell lines. Methods: Cultivated PrEC and PrSC, immortalized BPH cells (BPH 1), androgen dependent (LNCaP) and androgen independent (PC3) prostate carcinoma cell lines were exposed to increasing concentrations of commercially available PDE 5 inhibitors Sildenafil (Sil), Tadalafil (Tad), Vardenafil (Var). After incubation for 3 days cell viability was determined by a WST-1 assay (Roche-Biochemicals). Cells were evaluated morphologically by invert-light microscopy. PDE-5 protein concentrations were determined by western blot analyses and tissue distribution of PDE-5 by immunohistochemistry (IHC) with a polyclonal antiserum. Results: None of the PDE-5 inhibitors induced cell proliferation. Significant decreases in proliferation and viability were observed at high concentrations (1 mg/ml) of all substances in PrSC and PrEC. In PrSC, proliferation rate decreased to 37.7% in Sil, to 16.9% in Tad and to 63.7% for Var as compared to controls. In PrEC, proliferation decreased to 72.7%, 21.6% and 84.4% for Sil, Tad and Var, respectively. At 0.1 mg/ml only Tad reduced proliferation significantly to 57.4%. Moreover, Tad induced neuroendocrine-like morphology in some PrEC. High protein concentrations of PDE 5 were observed in PrEC, low concentrations in PrSC but none in cancer cells. Conclusions: Sil, Tad and Var inhibit proliferation of prostate primary cells in vitro. Tad showed highest inhibition. Tumor cells were insensitive to PDE-5 inhibitors, due to the lack of PDE-5 protein. It seems unlikely that any of these substances increases proliferation of prostate carcinoma. Tad induced neuroendocrine-like morphology in some basal PrEC indicating effects on cellular differentiation. No significant financial relationships to disclose.

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