Uncoupling of oxidative phosphorylation does not induce thermotolerance in cultured Chinese hamster cells

Abstract

Two uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), were tested for their ability to modify the survival of cultured Chinese hamster ovary (CHO) and Chinese hamster V79 cells treated with hyperthermia. The uncouplers were used under conditions that inhibit oxidative ATP synthesis, as judged from measurements of cellular ATP levels. Incubation of CHO cells in glucose-free Hanks″ balanced salt solution (HBSS) containing 1 mM DNP for 1 h at 37°C followed by reincubation at 37°C in complete growth medium for 3 or 16 h, showed no substantial changes in the 45°C heat survival curve as compared to heated cells not exposed to DNP. Thus, DNP treatment of CHO cells did not induce thermotolerance.

Carbonyl cyanide m-chlorophenylhydrazone (CCCP), tested under similar experimental conditions, did alter cellular heat resistance. The major change in the 45°C survival curve of CHO cells pretreated with CCCP was an increase in the width of the shoulder: the D_q value increased from 14 min to 24 min, for the control and CCCP-treated cells respectively. The D₀ value did not change appreciably. In contrast, heat-induced thermotolerance (10 min, 45°C + 16 h, 37°C) was characterized primarily by an increase in the D₀ parameter from 4 min (unheated cells) to 17 min. Similar results were observed with CCCP-treated V79 cells.

The data demonstrate that heat resistance induced by 1.2 μm CCCP was manifest as an increased cellular capacity to accumulate and/or repair hyperthermia damage, rather than an induction of thermotolerance, and that this effect probably was not related to the action of CCCP as an uncoupler of oxidative phosphorylation.

Key Words:

• Carbonyl cyanide m-chlorophenylhydrazone
• 2,4-dinitrophenol
• cell survival
• 45°C hyperthermia
• Chinese hamster cells