Cycloheximide- and puromycin-induced heat resistance: different effects on cytoplasmic and nuclear luciferases

Abstract

Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation.

Fig 1.

Effect of CHX or PUR on luciferase inactivation in situ. O23 cells transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were heated at 43°C in the absence (circles) or presence of either CHX at 20 μg/mL (triangles) or PUR at 100 μg/mL (squares) 30 minutes before and during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock

Fig 2.

Time kinetics of CHX-mediated protection of luciferase against heat inactivation. O23 cells transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were incubated with CHX (20 μg/mL) for 0–120 minutes before heating for 20 minutes at 43°C with the drug present during heating. Luciferase activity is shown as a percentage of the activity before heat shock. The closed symbols indicate the remaining luciferase activity after a heat treatment at 43°C for 20 minutes of non-CHX pretreated controls

Fig 3.

Effect of CHX or PUR on luciferase inactivation in vitro. Recombinant luciferase was diluted in buffer, without translation inhibitor (circles) or with CHX (20 μg/mL) (triangles) or PUR (100 μg/mL) (squares). Luciferase was heated at 42°C and luciferase activity was measured. Luciferase activity is shown as a percentage of the starting activity

Fig 4.

Effect of CHX on luciferase inactivation in cells cotransfected with Hsp70 O23 cells were transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were either cotransfected with pCMV70 (Hsp70: squares) or pSP64 (control: circles). The cells were heated at 43°C in the absence (closed symbols) or presence (open symbols) of CHX (20 μg/mL) added 30 minutes before and present during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock

Fig 5.

Titration of Hsp70 expression and luciferase inactivation: effects of CHX. O23 cells were transiently transfected with pRSVLL/V and cotransfected with increasing quantities of pCMV70. (A) Western analysis of Hsp70 expression after transfection with the indicated plasmid concentrations. (B) Luciferase activity after a heat treatment of cells at 43°C for 20 minutes is plotted as function of Hsp70 expression. This was measured by densitometric analysis of the Western blots in panel A and expressed in arbitrary units (a.u.). Cells were heated either in the absence (closed symbols) or presence (open symbols) of CHX (20 μg/mL) added 30 minutes before and present during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock

Fig 6.

Effect of CHX on luciferase inactivation in thermotolerant O23 cells transiently transfected with pRSVnlsLL/V (nuc-luciferase: A) or pRSVLL/V (cyt-luciferase: B) were made thermotolerant by a preheat shock of 20 min at 44°C, followed by a 16-hour period of recovery at 37°C (TT: triangles) or left untreated (control: circles). The cells were heated at 43°C in the absence (closed symbols) or presence (open symbols) of CHX (20 μg/mL) added 30 minutes before and present during heat shock. Luciferase activity is shown as a percentage of the activity before heat shock

Fig 7.

Time kinetics of CHX-mediated protection of luciferase against heat inactivation in Hsp70 overexpressing and in thermotolerant cells. O23 cells transiently transfected with pRSVLL/V (cyt-luciferase) were either cotransfected with pCMV70 (Hsp70: squares) or pSP64 (control: circles). Also, pSP64-cotransfected cells were made thermotolerant by a priming heat shock of 20 minutes at 44°C, followed by a 16-hour period of recovery at 37°C (TT: triangles). CHX (20 μg/mL) was added 0–120 minutes before heating for 20 minutes at 43°C with the drug present during heating. Luciferase activity is shown as a percentage of the activity before heat shock. The closed symbols indicate the remaining luciferase activity after a heat treatment at 43°C for 20 minutes of non-CHX pretreated controls