Effects of hyperthermia on p53 protein expression and activity
Authors: Jun Guan
George Iliakis
Dennis B Leeper
Elena Stavridi
Journal: Journal of Cellular Physiology (2002)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: p53 responses; heat shock; DNA damage; hyperthermic exposures; protein levels
Affiliations: Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania, USA.

Abstract:  Although p53 responses after DNA damage have been investigated extensively, p53 responses after heat shock, which exerts cytotoxic action by mechanisms other than direct induction of DNA damage, are less well characterized. We investigated, therefore, the effect of hyperthermic exposures on the levels and DNA-binding activity of p53. Experiments were carried out with U2OS and ML-1 cells, known to express wild-type p53 protein. Although heating at 41 degrees C for up to 6 h had only a small effect on p53 levels or DNA binding activity, exposure to temperatures between 42.5 and 45.5 degrees C caused an immediate decrease in protein levels that was associated with a reduction in DNA binding activity. This observation is compatible with a high lability of p53 to heat shock, or heat sensitivity of the pathway regulating p53 levels in non-stressed cells. When cells were heated to 42.5 degrees C and returned to normal temperatures, a strong p53 response associated with an increase in protein levels and DNA binding activity was observed, suggesting the production of p53-inducing cellular damage. At higher temperatures, however, this response was compromised in an exposure-time-dependent manner. The increase in DNA binding activity was more heat sensitive than the increase in p53 levels and was inhibited at lower temperatures and shorter exposure times. Thus, the pathway of p53 activation is itself heat sensitive and compromised at high levels of exposure. Compared to p53 activation after exposure to ionizing radiation, heat-induced activation is rapid and short lived. When cells were exposed to combined heat and radiation, the response observed approximated that of cells exposed to heat alone. Wortmannin at 10 microM inhibited p53 activation for up to 2 h after heat shock suggesting the involvement of wortmannin-sensitive kinases, such as DNA-PK and ATM. Heat shock causes phosphorylation of p53 at Serine-15, but there is no correlation between phosphorylation at this site and activation of the protein. The results in aggregate indicate p53 activation in the absence of DNA damage by a heat-sensitive mechanism operating with faster kinetics than radiation-induced p53 activation. The former response may induce pathways preventing other stimuli from activating p53, as heat-induced activation of p53 is dominant over activation of p53 by DNA damage in combined-treatment experiments. These observations suggest means for abrogating p53 induction after DNA damage with the purpose of potentiating response and enhancing cell killing. 

Retracted: No

PubPeer comments:
 Figure 1. More similar and different than you would expect. https: imgur.com BzJxVNn , 
 Figure 1A and 2A. more similar and different than you would expoect. https: imgur.com zZfNTZZ , 
 Dear Coenonympha Dorus: This is Jun Guan, the first author of this paper. First, I would like to thank you for the scrutinizing the results of our paper and to apologize for any confusion. Here some clarifications for the issues raised. For your Comment #1: I noticed that you put red boxes and blue boxes on the bands of WB and DB, respectively, at 37 oC at 12h point in Figure 1 B and C. You commented that there were u201cMore similar and different than you would expectu201d. Here is why: The highlighted bands of each control circled in the red and blue box were obtained from different experiments and this is why they are similar but not identical. Each experiment had its own controls and for each experiment I used a different X-ray film, which explains some of the variation you see. The entire rows of WB bands or DB bands at 12 h in Figure 1B or 1C were generated on different films - not the individual columns of bands. It is better to compare the entire row of WB or DB bands between Figure 1 B and C. For your Comment #2: I noticed that in Figure 1 A and 2A you placed blue boxes and red boxes on the bands of WB and DB, respectively, at 37 oC at the 4 h point. You commented that there were u201cMore similar and different than you would expectu201d. The highlighted bands of each control circled are clearly not the same bands. They were obtained from different experiments. Each experiment had its own control, which was exposed to a different X-ray film. The entire row of WB bands or DB bands at the 4h point was taken from a different film, not the individual columns of bands. It is better to compare the entire row of WB or DB bands between Figure 1 A and 2A. Thanks again. I shall be very happy to answer any further questions you may have. Sincerely, Jun Guan , 
 #3 The point is that the signal inside the blue boxes is very similar, yet the accompanying signal inside the red boxes is different. , 
 Figure 1 continued. Much more similar than you would expect. https: imgur.com q5yfWJO , 
 #5 Dear Hydrophis Spiralis and Agabus Unguicularis: This is Jun Guan, the first author of this paper. I have performed myself the experiments you indicate. Thank you two for Hydrophisu2019 comment:  The point is that the signal inside the blue boxes is very similar, yet the accompanying signal inside the red boxes is different and Agabusu2019 comment: u201cFigure 1 continued. Much more similar than you would expect.u201d You are right! We noticed this issue before sending out our work for publication. As I mentioned in my previous comment, the highlighted bands of each control circled in the red and blue boxes were obtained in different experiments. Since WB (Western Blot) and DB (DNA binding) assays are semi-quantitative, and we did not obtain standard curves, we could not generate absolute values for all samples. For the qualitative analysis we discuss in the paper, we compare the relative band intensities within each experiment to arrive to conclusions (i.e. the conclusions were drawn from the relative differences of signals followed by comparison of the different treatment groups with the control group in each row from the same X-ray film). I hope this addresses the point you raised. Thanks again. I shall be very happy to answer any further questions you may have. Sincerely, Jun Guan ,