Combined Targeting of the Glutathione and Thioredoxin Antioxidant Systems in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is characterized by increased generation of reactive oxygen species that can cause lethal oxidative stress. Here, we evaluated the combined inhibition of the glutathione and thioredoxin antioxidant systems in preclinical models of pancreatic ductal adenocarcinoma, using buthionine sulfoximine (BSO) that targets glutathione synthesis, and auranofin that targets thioredoxin recycling. BSO potentiated the cytotoxicity of auranofin and induced lethal oxidative stress in primary pancreatic cancer cells.

An Iodinated DAPI-Based Reagent for Mass Cytometry.

Multiparametric single-cell analysis has seen dramatic improvements with the introduction of mass cytometry (MC) and imaging mass cytometry (IMC ). These technologies expanded the number of biomarkers that can be identified simultaneously by using heavy-isotope-tagged antibody reagents. Small-molecule probes bearing heavy isotopes are emerging as additional useful functional reporters of cellular features.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells.

Mobilization of Leukemic Cells Using Plerixafor as Part of a Myeloablative Preparative Regimen for Patients with Acute Myelogenous Leukemia Undergoing Allografting: Assessment of Safety and Tolerability.

Allogeneic hematopoietic cell transplantation (HCT) is potentially curative for acute myelogenous leukemia (AML); however, a major cause of treatment failure is disease relapse. The purpose of this single-center Phase I study was to determine the safety and tolerability of administration of the CXCR4 inhibitor plerixafor (Mozobil; Sanofi Genzyme) along with myeloablative conditioning in patients with AML undergoing allogeneic HCT. The rationale was that plerixafor may mobilize leukemic stem cells, making them more susceptible to the conditioning chemotherapy (registered at ClinicalTrials.

A phase I trial of the aurora kinase inhibitor, ENMD-2076, in patients with relapsed or refractory acute myeloid leukemia or chronic myelomonocytic leukemia.

ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase, as well as c-Kit, FLT3 and VEGFR2. A phase I study was conducted to determine the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and toxicities of ENMD-2076 in patients with acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Patients received escalating doses of ENMD-2076 administered orally daily [225 mg (n = 7), 375 mg (n = 6), 325 mg (n = 9), or 275 mg (n = 5)].

A phase I trial of ANG1/2-Tie2 inhibitor trebaninib (AMG386) and temsirolimus in advanced solid tumors (PJC008/NCI♯9041).

Background: There is crosstalk between the ANG-Tie2 and the PI3K/Akt/mTOR pathways. Combined ANG1/2 and mTOR blockade may have additive anti-cancer activity. The combination of trebananib, an inhibitor of ANG1/2-Tie2 interaction, with temsirolimus was evaluated in patients with advanced solid tumors to determine tolerability, maximum tolerated dose (MTD), and preliminary antitumor activity.

The study of epigenetic mechanisms based on the analysis of histone modification patterns by flow cytometry.

Epigenetic regulation of genes involved in cell growth, survival, or differentiation through histone modifications is an important determinant of cancer development and outcome. The basic science of epigenetics uses analytical tools that, although powerful, are not well suited to the analysis of heterogeneous cell populations found in human cancers, or for monitoring the effects of drugs designed to modulate epigenetic mechanisms in patients. To address this, we selected three clinically relevant histone marks (H3K27me3, H3K9ac, and H3K9me2), modulated their expression levels by in vitro treatments to generate high and low expressing control cells, and tested the relative sensitivity of candidate antibodies to detect the differences in expression levels by flow cytoametry using a range of sample preparation techniques.

Glycogen synthase kinase-3 inhibition sensitizes pancreatic cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines.

Cytometry of intracellular signaling: from laboratory bench to clinical application.

The analysis of signaling pathways based on combinations of phospho-specific antibodies is now a well-recognized flow cytometry technique. Despite its wide-ranging potential in the fields of biology, industry, and medicine, it has been relatively slow to gain widespread use, and is often considered to be technically challenging. In this chapter, we detail protocols developed in our laboratory for monitoring signaling pathways in blood samples based on combinations of phospho-specific antibodies.

Tenofovir selectively regulates production of inflammatory cytokines and shifts the IL-12/IL-10 balance in human primary cells.

Objectives: In this study, we aimed to investigate the possible immune modulatory effects of HIV nucleoside reverse transcriptase inhibitors during secondary infections and inflammation, focusing on inflammatory cytokine responses and the interleukin (IL)-12/IL-10 balance.

Methods: We investigated the in vitro effect of tenofovir and zidovudine (AZT) on production of proinflammatory cytokines in monocytes and human peripheral blood mononuclear cells (PBMCs). Stimulation panels included Toll-Like receptor (TLR) ligands; the inflammation mediator tumor necrosis factor-α; and the pathogens cytomegalovirus, Neisseria meningitides, Escherichia coli, and Streptococcus pneumoniae.

Potential use of cetrimonium bromide as an apoptosis-promoting anticancer agent for head and neck cancer.

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines.

Whole blood processing for measurement of signaling proteins by flow cytometry.

Signal transduction pathways link external stimuli with cellular responses, which normally regulate cell proliferation, death, and differentiation. The study of signal transduction was revolutionized through the development of phospho-specific antibodies that recognize proteins only when they are phosphorylated at specific sites. As discussed by Nolan and co-workers (UNIT ), one of the unique features of flow cytometry is its ability to perform correlated measurements of multiple phosphorylation states at the single cell level.

Cyproheptadine displays preclinical activity in myeloma and leukemia.

D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine.

Pharmacodynamic monitoring of molecular-targeted agents in the peripheral blood of leukemia patients using flow cytometry.

The introduction of specific, molecular-targeted drugs is radically changing cancer treatment. Pharmacodynamics, which measures drug effects on the host, is key during early-phase clinical trials of novel agents to determine the relations between drug dose and target inhibition as well as measure the downstream effects of target inhibition on the cancer. In this article, we describe the application of flow cytometry to the pharmacodynamic monitoring of molecular-targeted agents in leukemia patients.

Benzethonium chloride: a novel anticancer agent identified by using a cell-based small-molecule screen.

Purpose: This study aims to identify a novel therapeutic agent for head and neck cancer and to evaluate its antitumor efficacy.

Experimental Design: A cell-based and phenotype-driven high-throughput screening of approximately 2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal cancer) and GM05757 (primary normal human fibroblast) lines. The "hit" compound was assayed for efficacy in combination with standard therapeutics on a panel of human cancer cell lines.

Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients.

Objective: The phosphorylation state of the S6 ribosomal protein was measured in the peripheral blasts of 19 newly diagnosed patients with acute leukemia.

Methods: We employed a flow cytometry protocol that enabled correlated measurement of pS6, phosphorylation of extracellular signal-regulated kinase (pERK), and cluster differentiation surface markers. Baseline levels of pS6 in leukemic blasts were compared with those found when the samples were activated using stem cell factor, or exposed to rapamycin, LY294002, or the mitogen-activated protein kinase inhibitor U0126.

Disruption of the endoplasmic reticulum and increases in cytoplasmic calcium are early events in cell death induced by the natural triterpenoid Asiatic acid.

Triterpenoids are a novel class of compounds being investigated as potential therapeutic agents for the treatment of prostate cancer and other malignancies. Asiatic acid (AA) is a member of the ursane family of triterpenoids and has anticancer activity, but its mechanism of action is not completely understood. To investigate its mechanism of action, PPC-1 prostate cancer cells were treated with AA at increasing concentrations and times.

Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells.

Background: We previously reported a flow cytometry technique to monitor pharmacodynamic effects of the raf kinase inhibitor BAY 43-9006 based on the ability of phorbol ester (PMA) to phosphorylate extracellular-regulated kinase (ERK) in peripheral blood (Chow et al., Cytometry 2001;46:72-78). In this article, we describe its application to phase I trials of BAY 43-9006 in solid tumor and AML/MDS patients.

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations.

Background: Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis.