Intracellular Alkalinization Suppresses Lovastatin-induced Apoptosis in HL-60 Cells through the Inactivation of a pH-dependent Endonuclease

doi:10.1074/jbc.270.11.6235

Intracellular Alkalinization Suppresses Lovastatin-induced Apoptosis in HL-60 Cells through the Inactivation of a pH-dependent Endonuclease (*)

From the (1)Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid and
the (2)Instituto López Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain

^§ To whom correspondence should be addressed:
Centro de Investigaciones Biológicas, C.S.I.C., C/Velázquez, 144, 28006 Madrid, Spain.
Tel.: 34-1-5611800 (ext. 4302); Fax: 34-1-5627518.

Abstract

Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na⁺/H⁺ antiporter induces a raise in pH which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pH which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pH was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na⁺/H⁺ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOVinduced intracellular acidification was not due to a complete inhibition of the Na⁺/H⁺ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na⁺/H⁺ antiporter with a marked increase in pH. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na⁺/H⁺ antiporter and the suppression of apoptosis.

Footnotes

↵* This work was supported by Grant PM92-0003 from the Dirección General de Investigación Científica y Técnica (DGICYT). 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:

MVA

mevalonic acid

G protein

guanine nucleotide-binding protein

LOV

lovastatin

CHX

cycloheximide

PMA

phorbol 12-myristate 13-acetate

PKC

protein kinase C

EIPA

5-N-ethyl-N-isopropylamiloride

BCECF

2′,7′-bis(carboxyethyl)-5carboxyfluorescein

H7

1-(5-isoquinolinesulfonyl)-2-methyl-piperazine

bp

base pair(s).
- Received June 10, 1994.
- Revision received January 4, 1995.