Novel use of the fluorescent dye 5‐(and‐6)‐chloromethyl SNARF‐1 acetate for the measurement of intracellular glutathione in leukemic cells and primary lymphocytes

Abstract

Glutathione (GSH) plays an important role in protecting cells against injury, particularly during oxidative stress. Alterations in GSH metabolism are becoming the focus of attention in many diseases such as cancer, neurodegeneration, and AIDS. As such, a rapid assessment of GSH levels in a clinical setting is of increasing importance. We tested the efficacy of the thiol-labeling fluorescent dye CM-SNARF in its ability to measure variations in GSH concentration using a visible-light flow cytometer. GSH levels in I83, Jurkat, and primary lymphocytes were depleted with buthionine sulfoximine (BSO) or diamide, or increased with N-acetylcysteine (NAC). Following each treatment, cells were divided and either labeled with CM-SNARF followed by flow cytometry analysis, or assayed for GSH using a biochemical method. BSO treatment caused a maximal 87-90% decrease in GSH and 68-76% decrease in fluorescence units. Diamide depleted GSH 91-95%, corresponding to a fluorescence decrease of 85-88%. NAC treatment increased GSH levels 27% and fluorescence 12-19%. The overall correlation (R 2 ) between mean GSH concentration and mean fluorescence was 0.80-0.88. CM-SNARF can be used to semi-quantitatively and rapidly determine intracellular variations in GSH concentration in the range of 10-150 nmoles GSH/mg protein. ' 2007International Society for Analytical Cytology Key terms glutathione (GSH); flow cytometry; 5-(and-6)-chloromethyl SNARF-1 acetate; buthionine sulfoximine (BSO); diamide; primary lymphocytes; N-acetylcysteine GLUTATHIONE (GSH) is a major intracellular antioxidant and plays an important role in several cellular processes including the maintenance of redox potential, amino acid transport, and the protection of cells from damage by free radicals, peroxides, and toxins (1). Disturbances within the GSH system, resulting in decreased cellular GSH, have been documented in many conditions including Alzheimer's (2,3) and Parkinson's (4) diseases, schizophrenia (5-7), and AIDS (8). Conversely, increased tumor GSH can be associated with chemotherapy drug resistance (9-13). Some reports have shown that peripheral erythrocyte GSH levels and glutathione-S-transferase (GST) activity positively correlate with tumor levels and activity and chemotherapeutic response (14), while other reports have shown that increasing GSH levels via the administration of thiol compounds can help to diminish the level of GSH depletion associated with the use of some chemotherapeutic agents, thus offering a cytoprotective effect (15,16). Collectively, these reports suggest that the clinical measurement of GSH concentration should prove useful in disease diagnosis and monitoring, especially in terms of cancer management where patient GSH levels may influence the choice and dosing of chemotherapy regimens. As a result, the ability to rapidly measure GSH concentrations in clinical samples is of increasing importance.

Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism

“…Flow cytometric studies using chloromethyl SNARF-1 acetate (CM-SNARF, a dye that forms covalent adducts with intracellular reduced thiols) (17) along with ALDH1 activity demonstrates that breast cancer stem cells (ALHD1+) derived from SUM159 mouse xenografts, have more total reduced thiols than non-stem cells (ALDH-) (Figure 6C). Furthermore the treatments with BSO, shown to deplete >90% of total GSH (8), revealed that both the fraction of reduced thiols representing GSH (BSO sensitive) as well as the fraction of thiols that remains following GSH depletion were greater in ALDH1+ stem cells, relative to the bulk tumor cell population (Figure 6C).…”

“…1 × 10 6 SUM159 cells were resuspended in ALDH buffer containing 10 µM SNARF-1 and ALDH ± DEAB for 30 minutes at 37°C. (17) After incubation cells were resuspended in ALDH buffer and analyzed as before additionally using the Argon 561 laser with 610/20 band pass filter to determine SNARF-1 mean fluorescent intensity on a minimum of 5000 ALDH positive cells.…”

Median Effect Dose and Combination Index Analysis of Cytotoxic Drugs Using Flow Cytometry

“…Chloromethyl SNARF-1 acetate is similar to CM-FDA but exhibits red fluorescence when excited with 488 nm blue laser. Thus it can be used when the green fluorescence channel is needed for other purpose and a UV or violet illumination line is not available (Hamilton et al, 2007). Carboxi-fluorescein-succinimidylester (CFSE) is converted to fluorescent compound by intracellular esterases but covalently binds amino groups of proteins and is completely retained within cells, even after damage of cell membrane (Fujioka et al, 1994;Li et al, 2003).…”

Targeting unique oxidative metabolism of clonogenic multiple myeloma cells for therapy

“…To measure GSH, we estimated the pool of total thiols in the cell and the amount inhibited by L-buthionine sulfoximine (B2515, Sigma), which blocks the activity of GCL, the enzyme responsible for GSH production as previously described [189]. Briefly, cells…”

“…We measured the amount of cellular thiols qualitatively in the CD138 low vs. bulk tumor cells. To estimate GSH, we utilized the SNARF-1 probe which reacts with thiols inducing a change in fluorescence of the dye [189]. HMCLs were treated with and without BSO to reduce cellular GSH levels followed by co-staining with SNARF-1 and APC-CD138 antibody.…”