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Similar Actions of Glucocorticoids and Calcium on the Regulation of Apoptosis in S49 Cells

Department of Pharmacology, University of North Carolina Chapel Hill, North Carolina 27599
Department of Physiology, University of North Carolina Chapel Hill, North Carolina 27599
Department of Lineberger Comprehensive Cancer Research Center Cell Biology, Program, University of North Carolina Chapel Hill, North Carolina 27599

Address requests for reprints to: Dr. Lu-Ann M. Caron-Leslie, Department of Physiology, CB#7545 460 Medical Science Research Building, University of North Carolina, Chapel Hill, North Carolina 27599–7545.

Abstract

Glucocorticoid-induced lymphocyte cell death is a programmed process which is thought to involve the calcium-dependent degradation of DNA into multiples of 180 basepairs, characteristic of internucleosomal degradation. We have used the glucocorticoid- sensitive mouse lymphoma cell line S49.1 [wild-type (wt)] and the glucocorticoid-resistant cell line S49.22r (nt^–) to evaluate the role of both glucocorticoid receptors and calcium in the regulation of internucleosomal DNA degradation and expression of calcium-dependent deoxyribonuclease activity. DNA was isolated from untreated (control) and dexamethasone (dex)-treated viable cells and analyzed for internucleosomal DNA degradation by agarose gel electrophoresis, followed by ethidium bromide staining. Glucocorticoid treatment resulted in substantial internucleosomal DNA degradation in wt cells, but not in nt^– cells. This effect was inhibited by coincubation of cells with dex and the glucocorticoid receptor antagonist RU486. In contrast to the glucocorticoid response, administration of either of two calcium ionophores, ionomycin or A23187, produced internucleosomal degradation of DNA in both wt and nt^– cells, although the latter were less sensitive to ionophore treatment. Interestingly, A23187 treatment also resulted in a loss of cell viability in HeLa S₃ cells, a cell line that does not exhibit glucocorticoid-induced apoptosis. No internucleosomal DNA degradation was detected in HeLa S₃ cells killed by A23187. To determine whether similar nucleases are associated with this internucleosomal DNA degradation resulting from both glucocorticoid and calcium ionophore treatment, 0.3 M NaCI nuclear protein extracts were prepared from control and treated cells and analyzed for protein composition or nuclease activity. To assay for nuclease activity, nuclear extracts were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels impregnated with [³²P]DNA. Nuclease activity was detected by removal of sodium dodecyl sulfate from the gel, activation with calcium, and subsequent visualization of the loss of [³²P]DNA by autoradiography. Dex treatment of wt cells resulted in the appearance of several proteins within the mol wt range of 12–18 kDa, only one of which (16–18 kDa) exhibited calcium- dependent nuclease activity. The appearance of these proteins in nuclear extracts was inhibited by coincubation of glucocorticoid-treated cells with RU 486. Glucocorticoid treatment did not result in the appearance of nuclease activity in nuclear extracts from nt^– cells. Interestingly, A23187 or ionomycin treatment resulted in an increase in activity of the 16- to 18-kDa nuclease in both wt and nt^– cells. These findings indicate that both glucocorticoid receptors and calcium may share common features in the regulation of apoptosis in lymphoid cells.

FOOTNOTES

This work was supported by NIH Grant DK-32078.

Received for publication March 26, 1991. Revision received June 3, 1991. Accepted for publication June 3, 1991.

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