Abstract
The intracellular distribution of glutathione (GSH) was measured by a quantitative image cytometry method, using the sulphydryl-reactive agent mercury orange. This readily forms fluorescent adducts with GSH and other non-protein sulphydryls (NPSH), but reacts much more slowly with protein sulphydryls. Under optimum staining conditions mean integrated mercury orange fluorescence per cell was closely correlated with a standard biochemical assay for GSH. Use of the DNA dye DAPI as a counterstain allowed measurement of nuclear NPSH. The mean nuclear-cytoplasmic ratio was 0.57 +/- 0.05. Isolation of nuclei under aqueous conditions resulted in the loss of approximately 90% of mercury orange fluorescence, compared with nuclear fluorescence from intact cells, suggesting that background labelling of protein sulphydryls or other macromolecules is low. Depletion of GSH with N-ethylmaleimide or diethylmaleate decreased mercury orange fluorescence in the nucleus and cytoplasm to a similar extent. In contrast, mercury orange fluorescence in the nucleus was much more resistant to DL-buthionine-S,R-sulphoximine (BSO) depletion than that in the cytoplasm. This finding is compatible with a distinct pool of GSH in the nucleus that is comparatively resistant to BSO depletion. Alternatively, the retention of fluorescence in the nucleus following GSH depletion by BSO treatment might be due to accumulation of cysteine. These findings have implications for cancer treatment since the level of NPSH in the nucleus might be a more important determinant of resistance to DNA-damaging agents than that in cytoplasm. The image cytometry method described here is quantitative, allows a measure of tumour cell heterogeneity and can be applied to small biopsy samples obtained by fine-needle aspiration. Thus it appears suitable for prospective clinical studies in cancer patients, and for monitoring the effects of GSH-depleting agents used as adjuncts to cancer chemotherapy or radiotherapy.
Highlights
Smar, The intracellular distribution of glutathione (GSH) was measured by a quantitative image cvtometry method, using the sulphydryl-reactive agent mercury orange
In order to protect the nuclear structures from damage (Sandstrom and Marklund 1990) and to participate to DNA synthesis (Thelander and Reichard, 1979), GSH must be present in the nucleoplasm, but little is known about the nuclear GSH content, and the published values for the nuclear-cytoplasmic distribution of GSH vary according to the techniques used (Edgren and Revesz, 1987; Tirmenstein and Reed, 1988; Britten et al, 1991; Jevtovic-Todorovic and Guenthner, 1992)
The human breast cancer cell line MCF-7 and the mouse lung fibrosarcoma cell line KHT-c were grown in ax-MEM supplemented with 10% fetal bovine serum (FBS) and the rat mammary carcinoma Mat B in a-minium essential medium (a-MEM) supplemented with 10% FBS, 1% L-glutamine 200 mM (Gibco BRL, Grand Island, NY, USA) and 1% hypoxanthine (Gibco)
Summary
All cell lines used were grown in 25 cm Corning flasks in a humidified 5% carbon dioxide/air incubator at 37C. Mouse mammary carcinosarcoma EMT-6 cells were grown as a. M Thofas etal monolayer culture in a-minium essential medium (a-MEM) supplemented with 5% fetal bovine serum (FBS) (PA Biologials, Sydney, Australia). Chinese hamster ovary cells were maintained as spinner cultures in cz-MEM supplemented with 5% FBS. The human breast cancer cell line MCF-7 and the mouse lung fibrosarcoma cell line KHT-c were grown in ax-MEM supplemented with 10% FBS and the rat mammary carcinoma Mat B in a-MEM supplemented with 10% FBS, 1% L-glutamine 200 mM (Gibco BRL, Grand Island, NY, USA) and 1% hypoxanthine (Gibco). Cells were removed from the monolayer using 0.05% trypsin and 0.53 mM EDTA (Gibco) for 5 min at 37C and resuspended in a-MEM supplemented with 5% or 10% of FBS at a final concentration of 1 x 10' cells ml-'. Before mercury orange staining the slides were thawed for 1 h and unwrapped
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