Apoptosis in an Interleukin-2-dependent Cytotoxic T Lymphocyte Cell Line Is Associated with Intracellular Acidification

doi:10.1074/jbc.270.7.3203

Apoptosis in an Interleukin-2-dependent Cytotoxic T Lymphocyte Cell Line Is Associated with Intracellular Acidification

ROLE OF THE Na/H-ANTIPORT (*)

Jinfang Li and
Alan Eastman(§)

From the Department of Pharmacology and Toxicology and the Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire 03755

^§ To whom correspondence should be addressed:
Dept. of Pharmacology and Toxicology, 7650 Remsen, Dartmouth Medical School, Hanover, NH 03755.
Tel.: 603-650-1667; Fax: 603-650-1129.

Abstract

Apoptosis is a form of cell death associated with DNA fragmentation and chromatin condensation. We recently established that intracellular acidification occurred during apoptosis following cytotoxic insult. The current studies were designed to determine whether intracellular acidification was more generally associated with apoptosis, specifically in a model of growth factor withdrawal. Upon withdrawal of interleukin-2, CTLL-2 cells accumulated in the G₁ phase of the cell cycle and started to fragment their DNA around 12 h concurrent with both decreased pH and increased Ca. Chelation of Ca did not inhibit DNA digestion, whereas incubation with a calcium ionophore prevented both acidification and DNA digestion. Hence, acidification rather than increased Ca was associated with apoptosis. The acidified cells represented a discrete population up to 0.7 pH units below normal. The extent of acidification depended upon the extracellular pH; above pH 6.3, intracellular pH was significantly below extracellular pH, whereas below pH 6.3, the cells still regulated their pH. Inhibition of the Na/H-antiport prevented the apoptotic cells from regulating their intracellular pH under these acidic conditions. These results demonstrate that apoptotic cells retain a functional antiport but that its set-point has changed. Many survival factors are known to phosphorylate and activate the antiport, hence apoptosis is likely to be associated with dephosphorylation. Although acidification always occurred during apoptosis, maintaining intracellular pH above 7.2 did not prevent apoptosis, suggesting that an acid pH is not essential for apoptosis. We hypothesize that other critical regulators of apoptosis must be subject to dephosphorylation.

Footnotes

↵* This study was supported by National Institutes of Health Grant CA50224. Flow cytometry was performed in the Fannie E. Rippel Flow Cytometry Laboratory, supported in part by a Cancer Center Core Grant CA23108. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

IL-2

interleukin-2

AM

acetoxymethyl ester

EIPA

ethylisopropylamiloride

pH

extracellular pH

pH

intracellular pH

TPA

12-O-tetradecanoylphorbol-13-acetate

Mes

2-(N-morpholino)ethanesulfonic acid.
- Received December 19, 1994.