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Research Papers

# Dynamic Effect of Heat Shock Pretreatment on Apoptotic Responses to $TNF-α$ in Liver Cells

[+] Author and Article Information
Sihong Wang

Department of Biomedical Engineering, The City College of the City University of New York, New York, NY 10031

Pohun C. Chen, Francois Berthiaume, Mehmet Toner

Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Burn Hospital, and Harvard Medical School, Boston, MA 02114

Arul Jayaraman

Department of Chemical Engineering, Texas A&M University, College Station, TX 77843

Martin L. Yarmush1

Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Burn Hospital, and Harvard Medical School, Boston, MA 02114ireis@sbi.org

1

Corresponding author.

J Biomech Eng 131(7), 071003 (Jun 05, 2009) (6 pages) doi:10.1115/1.3118768 History: Received July 13, 2008; Revised February 25, 2009; Published June 05, 2009

## Abstract

The heat shock (HS) response is a protective mechanism for cells to protect themselves against subsequent lethal stress. HS upregulated heat shock protein (HSP) expression reduced apoptosis following tumor necrosis factor-$α$ ($TNF-α$) stimulation. However, vector-mediated overexpression of HSP70 failed to provide similar protection but rather sensitized cells to $TNF-α$ induced apoptosis. This may be due to the fact that the kinetics of vector-mediated HSP overexpression is totally different from that of HSP upregulation by HS. We hypothesized that the response depends on the timing of $TNF-α$ challenge relative to HSP expression dynamics after HS. Therefore, we investigated the correlation between the dynamic change of HSP expression and the levels of apoptosis induced by $TNF-α$ after HS. Hepatoma cells were subjected to mild heat shock at $42°C$ for 2 h followed by varied recovery times and then treated with $TNF-α$ to induce apoptosis. The results from quantitative apoptosis assays using the TUNEL reaction reveal an optimal HS protection window centered around 5 h post-HS against $TNF-α$ induced apoptosis. In addition, we found a window extending up to 2 h after HS where HS sensitized cells to $TNF-α$ stress. Importantly, the correlation between apoptosis and HSP expression kinetics demonstrates that both high levels of HSPs and proper timing between HS and $TNF-α$ stress were critical for optimal protection. Our study establishes a dynamic experimental model for further investigation of HS as a potential clinical approach to target tissue survival or death.

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## Figures

Figure 1

Apoptosis measured by TUNEL technology at the end of 18 h TNF-α stimulation after 2 h HS with various recovery times. The normalization using Eq. 1 drove the apoptosis level of control samples without TNF-α stimulation to 0 (negative control) and with TNF-α to 1 (positive control). Data shown are mean ± SD, n=7. “*” indicates statistical significance (p<0.01) compared with the positive control.

Figure 2

Quantified HSP expressions at various recovery times after HS. (A) HSP70 and HSP25 with HSP70 controls (18 h TNF-α stimulation without HS is 4.2±0.78 and no treatment 3.6±0.16). (B) HSP90 with controls of no TNF-α without HS (16.64±1.93) and 18 h TNF-α stimulation without HS (16.13±2.22). Data shown are mean ± SD, n=8.

Figure 3

Representative FACS dot plots for Annexin V and PI labeling of (A) 0 h, (B) 8 h, (C) 12 h, and (D) 24 h TNF-α stimulation using green (FL1) and red (FL3) fluorescence channels. The magnitude of cell density is represented in a decreasing order by red (highest)→yellow→green→blue (lowest). (E) kinetics of TNF-α induced early stage apoptosis from quantified Annexin V and PI staining FACS results. Data shown are mean ±SD. Percentage of apoptotic cells is defined as the cell number of region E4 over the total cell number, and percentage of apoptotic+dead cells is the sum of regions E2 and E4 cells divided by the total cell population. Curve fitting using Eq. 2 provides the following two kinetic functions, %apoptotic cells=52–61.07 exp(−t/9.42) and %apoptotic+dead cells=62–69.71 exp(−t/12.24), where t is the TNF-α stimulation time in hours.

Figure 4

Correlation study between HSP expression and TNF-α induced apoptosis (A) overlap of HSP70/HSP25 expression kinetics and 8 h shifted apoptosis curve and (B) correlation coefficient plot of HSP expression versus apoptosis data from Fig. 4 at the recovery times of (a) 8 h, (b) 12 h, (c) 14 h, (d) 15 h, and (e) 24 h

Figure 5

Percentage of early stage apoptosis plus dead cells based on Annexin V and PI staining using FACS. “*” indicates statistical significance (p<0.05) comparing H2R0 with H2R5 at TNF-α stimulation times of 15 h and 24 h.

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