Microarray analysis of cellular thermotolerance.
OBJECTIVE
Previously, we have shown that a 43°C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45°C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70.


METHODS
Murine embryonic fibroblast cells with and without Hsp70 (MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43°C for 30 minutes in a constant temperature water bath. After 3 hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a twofold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P < 0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology.


RESULTS
While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance.


CONCLUSIONS
The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation.
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
Josh T
JT
Beckham
Gerald J
GJ
Wilmink
Susan R
SR
Opalenik
Mark A
MA
Mackanos
Alex A
AA
Abraham
Keiko
K
Takahashi
Chris H
CH
Contag
Takamune
T
Takahashi
E Duco
ED
Jansen
D000818:Animals; D001833:Body Temperature Regulation / Q000235:genetics*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D005347:Fibroblasts / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D018869:Heat-Shock Response / Q000235:genetics*; D006358:Hot Temperature / Q000009:adverse effects*; D051379:Mice; D046228:Microarray Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018840:HSP70 Heat-Shock Proteins