Quantitative flow cytometry reveals a hierarchy of glucocorticoid effect on cell surface mouse mammary tumor virus gp52.
A flow cytometry protocol with CM mouse mammary tumor cells (Mm5mt/C1) was utilized to provide a fluorescence measurement of hormone-mediated changes in mouse mammary tumor virus (MMTV) cell surface envelope glycoprotein (gp52 CSA). Standards permitted gp52-specific fluorescence intensity to be measured as molecules of equivalent soluble fluorescein (MESF). The feasibility of using MESF determinations to reflect hormone-modulated changes in continuously infected cells was tested. A panel of five glucocorticoids having differing affinity for the glucocorticoid receptor were tested in 60 h treatments at dosages ranging from 10(-6) M to 10(-8) M. Determinations of MESF, as a measure of gp52 CSA, were highest with 10(-6) M treatments (36.7-44.5 x 10(-6) MESF). At lower dosages, MESF determinations were lower but showed a clear hierarchy of glucocorticoid effect. At 10(-8) M treatments, determinations of MESF x 10(-6) demonstrated the following glucocorticoid hierarchy: triamcinolone acetonide (TA) (33.7 +/- 1.6) > dexamethasone (DEX) (26.1 +/- 1.7) > prednisolone (8.0 +/- 0.3) > triamcinolone (6.6 +/- 0.4) > hydrocortisone (6.4 +/- 0.4) > control (2.4 +/- 0.1). The MESF-derived respective fold increases over control for this hierarchy were: 13.87, 10.74, 3.31, 2.71, and 2.65. The ability of TA to enhance gp52 CSA was 1.3-fold greater than DEX. 10-fold higher levels of steroid controls did not significantly elevate MESF levels. Findings argue that dosage, duration of treatment and relative affinity of glucocorticoids for receptor are reflected in MESF determinations of changing gp52 levels. Therefore, this new measure of effect may be useful in studying hormonal influence on viral and cellular regulatory systems in chronically infected cells.
Ritzi
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Department of Microbiology and Immunology School of Medicine, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
D000287:Administration, Topical; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000957:Antigens, Viral, Tumor / Q000032:analysis / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D019793:Fluorescein; D005452:Fluoresceins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005938:Glucocorticoids / Q000032:analysis / Q000494:pharmacology*; D006854:Hydrocortisone / Q000378:metabolism / Q000494:pharmacology; D008325:Mammary Neoplasms, Experimental / Q000473:pathology; D051379:Mice; D008809:Mice, Inbred C3H; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000893:Anti-Inflammatory Agents; D000957:Antigens, Viral, Tumor; D005452:Fluoresceins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; C040612:glycoprotein 52 antigen, Mouse mammary tumor virus; D003907:Dexamethasone; D019793:Fluorescein; D006854:Hydrocortisone