1 Division of Pharmaceutics, College of Pharmacy, University of Texas at Austin, Austin 78712
2 United States Army Medical Research Detachment, Brooks Air Force Base 78235
3 United States Army Institute of Surgical Research, Fort Sam Houston, Texas 78234
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ABSTRACT |
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cDNA arrays; retinal cell culture; ARPE-19 cells; heat shock protein; thermal injury
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INTRODUCTION |
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Although larger external burns are one of the most common forms of accidental injury, and heat is often used in conjunction with radiation for control of cancerous tissue (16), very little is known about the gene response of cells to thermal injury (23, 38). In the case of thermal treatment of cancer, heat is often used in conjunction with radiation therapy for control of cancerous tissue; however, the effectiveness of secondary heat applications is limited by the induction of thermotolerance from the initial treatment (17). The induction of thermotolerance, evident by the heat shock response, has been shown to provide temporary protection against ensuing episodes of injurious stimuli (8). Analysis of an Arrhenius plot for the inactivation of keratinocytes from short exposure (seconds) to high temperatures (5060°C) indicated that the absorbing species or the injury mechanism may be somewhat different from injury induced in the most studied range (4050°C over minutes) (6). Above a certain threshold of heating all cells die from exposure to short-duration thermal injury, yet they express large amounts of heat shock protein (HSP) and cytokines before doing so. Human keratinocytes exposed for 1 s at 58°C synthesized the cytokines interleukin-8 (IL-8) as well as mRNA for tumor necrosis factor- and IL-1
and exhibited an intense heat shock response. Depending on the intensity of the heating, the initial form of cell death was apoptotic but quickly became accidental cell death as the intensity of heating increased (33, 47). Understanding how cells respond to a sublethal, survivable thermal injury may provide a means by which to enhance protective cellular responses and increase survival. Although the heat shock response is the best studied response to thermal injury and can perhaps be explained as an adaptive, protective response to heat and stress (51), other adaptive responses may be involved. The progression of the heat shock response is usually detected by radiolabeling or by antibody detection of proteins in polyacrylamide gels after electrophoresis. Recent developments, particularly in gene expression profiling with macro- and microarrays, have greatly increased the efficiency of gene expression analysis. Arrays allow for the parallel analysis of many genes from the same sample, revealing the complex interplay of genes invoked at any given time. Here we have examined the genetic response of 588 known genes to thermal injury. In addition to the well-described HSP response, genes involved in a variety of other metabolic pathways were altered. Although most genes exhibited no alteration in expression by 1 h after thermal injury, a few were mildly suppressed. By 4 h posttreatment a greater than twofold elevation was observed for some genes, including growth arrest/repair, DNA binding, and heat shock and other stress response proteins. Of particular interest were the heat shock proteins HSP40, HSP70, and HSP86, the 45-kDa and 153-kDa growth arrest and DNA-damage-inducible genes, and genes for DNA-binding and repair proteins. The translation of protein product from the induced mRNA expression was confirmed for HSP70 and HSP40.
By observing the gene response to thermal stress in an in vitro model, we may better understand the benefits and targets of drug treatment for secondary damage associated with thermal injury. Gene expression profiling may be applied to both drug discovery and development to define the response of specific cell or tissue systems to a drug, or to evaluate a drugs mechanism of action. As we gather more knowledge of how and why genes function, we can use this knowledge to systematically find targets and complementary drugs for enhancing treatment.
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METHODS |
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Thermal injury experiments.
For survival experiments the cells were seeded on 13-mm Thermanox coverslips in 24-well plates, and for gene expression profiling they were cultured in 100-mm culture dishes. Thermal injury experiments were carried out after the cells reached confluence by dipping the cultured cells in HEPES-buffered saline heated to 55°C for the appropriate time. To determine the optimal time of exposure for inflicting a sublethal thermal injury, preliminary experiments using increasing times of heating (09 s) with a constant temperature of 55°C were carried out with cells grown on coverslips. The percentage of viable cells at 24 h postheating was determined for each experiment, using mitochondrial redox activity as an indicator. The optimal time and temperature for producing a 25% decrease in redox activity was chosen. Although this level of injury did not lead to morphological changes in the cells, it produced both a decline in metabolic activity and induced a genetic response that triggers early response and stress response genes.
For gene expression profiling, cells seeded in 100-mm petri dishes were dipped for 3 s, then returned to medium and the incubator for the appropriate recovery period. RNA and protein were isolated from cells 1, 4, 8, and 24 h after heating. Control cells were treated in a manner identical to that for heated cells, excluding the heating step, and were isolated concurrently with the 1-h and 24-h thermally injured cells. In preliminary experiments (data not shown), control cells were isolated at every time point until the reproducibility of control samples between time points was verified. The gene expression experiment was repeated five times.
Cell viability.
Cell viability after thermal injury was assessed using Alamar Blue (Biosource International, Camarillo, CA), which is converted to a fluorescing compound in amounts proportional to the number of viable cells (24). The viability measurements were taken at 24 h to determine that a recoverable sublethal injury was inflicted. Under these conditions, good quality RNA as judged by gel electrophoresis, a necessity for performing gene expression analysis, could be obtained. The cells were incubated for 2 h at 37°C with culture medium containing 10% Alamar Blue according to the manufacturers instructions. After incubation, fluorescence was measured at 530-nm excitation and 590-nm emission (Cytofluor 2350; Millipore, Bedford, MA).
RNA isolation.
RNA isolation was performed following the protocol of the Molecular Research Center (Cincinnati, OH) for TRI Reagent. RNA yield was determined spectrophotometrically, and the quantity and quality of the RNA were estimated from absorption at 260 nm and 280 nm and gel electrophoresis on a 0.9% agarose gel containing 1:10,000 SYBR Gold nucleic acid stain (Molecular Probes, Eugene, OR). Only undegraded RNA free of genomic DNA contamination was used. Isolated RNA was solubilized in deionized H2O and stored at -20°C until 33P labeling.
cDNA production.
Two micrograms of total RNA was heated to 70°C for 5 min, then immediately frozen in liquid nitrogen and lyophilized. The lyophilized RNA was combined with a master mix containing 2 µg oligo dT (1 µg/µl; Research Genetics, Huntsville, AL), 6 µl of 5x first-strand buffer, 3 µl 0.1 M DTT (GIBCO-BRL, Grand Island, NY), 3 µl of 5 mM dNTPs mixture, 1 µl RNase inhibitor (5Prime3Prime, Inc.), 1.5 µl SuperScript II RT (200 U/µl, GIBCO-BRL), and 1 µl [33P]dCTP (10 mCi/mmol; Amersham Pharmacia, Arlington Heights, IL) and then incubated at 39°C. The reaction was stopped by the addition of 4 µl EDTA (0.5 M, Ambion). Hydrolysis of the RNA after reverse transcription was carried out at 68°C with 4 µl 1 M NaOH for 20 min. Unbound nucleotides were separated from the labeled probe by passage through a Centri-Spin 20 column (Princeton Separations, Adelphi, NJ).
Hybridization and phosphor imaging.
Blocking of nonspecific binding was performed in 5 ml MicroHyb (Research Genetics) with 1.0 µg/ml poly-dA (Research Genetics) and 1.0 µg/ml COT-1 DNA (GIBCO-BRL) at 42°C for 2 h. The purified probe was hybridized to an Atlas human cDNA expression array (no. 7740-1; Clontech, Palo Alto, CA) membrane at 42°C for 1824 h in a 35 x 150-mm roller bottle (Roller Oven, Hybaid). Washes were performed twice at 50°C in 20 ml of 2x SSC and 1% SDS for 20 min and once at 25°C in 100 ml of 0.5x SSC and 1% SDS for 15 min. The membranes were then placed on moistened Whatman filter paper and wrapped with plastic wrap before exposing to super resolution screens (Packard Instrument, Meridian, CT). Following exposure to the phosphor-imaging screens (14 days), the distribution and intensity of radioactivity were determined by scanning the screens with a phosphor imager (Cyclone, Packard Instrument). The images were digitally acquired using OptiQuant software (v. 3.0, Packard Instrument), and the autoradiographs were analyzed for changes in gene expression using AtlasImage software (v. 1.5, Clontech).
Data analysis.
For analysis of gene expression, the ratios of the integrated optical density (IOD) for each cDNA target were compared between heated and control cells. Cluster analysis was then performed with software originally from Stanford University, courtesy of Dr. Michael Eisen (http://rana.lbl.gov/EisenSoftware.htm).
To determine significance, two separate array membranes were hybridized with the same control sample. Then, the average and standard deviation of IOD ratios between the two were used to indicate the level of difference necessary to claim a significant difference. A scatter plot of the two control IOD values illustrates the distribution along a line with the slope of the average intensity (Fig. 1). With an average and standard deviation of 1.14 ± 0.32, an IOD ratio of at least 2.10 represents a significant difference (P = 0.01).
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Gel electrophoresis, total protein determination and western blotting.
Ten microliters, corresponding to 10 µg of protein from each treatment, was run on NuPage 412% Bis-Tris Gels (NOVEX) for 1 h at 125 mA. Additionally, 5 µl of MultiMark Multicolored Standard (NOVEX) was run on each gel as the molecular weight marker. The protein was then transferred to a polyvinylidene difluoride membrane (PVDF, NOVEX) for 5 h at 95 mA (Semi-Dry Blotter II; Kem En Tec, Flander, NJ). After transferring the protein, 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) fluorescent stain was utilized for total protein quantification, following the procedure outlined by Alba et al. (2). MDPF fluorescently tagged all protein, and a relative total protein ratio was calculated by dividing the fluorescence value for each sample by that of the control. The ratio was then used to normalize for any variability in the amount of protein from sample to sample. The blots were hydrated in a 50% methanol/water solution for 10 min and then washed twice in 10 mM sodium borate, pH 9.5, for 5 min each time. After the second wash, the blots were incubated for 10 min in 10 ml borate buffer containing 50 µl (0.50%) of 35 mM MDPF in DMSO. The blot was then rinsed briefly in borate buffer before visualizing under an ultraviolet light source. The total fluorescence for each sample was calculated using Gel-Pro Analyzer 2.0 software (Media Cybernetics, Silver Spring, MD).
Following total protein quantification, the membranes were hydrated in a 50% methanol/water solution for 10 min. The membranes were then incubated three times, for 15 min each, in a blocking solution consisting of 0.2% I-Block (Tropix, Bedford, MA), 0.1% Tween-20 (Sigma, St. Louis, MO), and 0.1% thimerosal (Sigma) in PBS (Sigma). The blots were then incubated with a primary antibody for 2 h. Rabbit anti-human HSP40 (1:10,000 dilution) and mouse anti-human HSP70 (1:1,000 dilution) were purchased from StressGen Biotechnologies (Victoria, British Columbia, Canada). After the initial hybridization in primary antibody, the blots were washed another three times in blocking solution for 15 min each. A 1:4,000 dilution of alkaline phosphatase-labeled secondary antibody (goat anti-rabbit from Zymed, San Francisco, CA; and goat anti-mouse from Tropix) was then added, and the blots were incubated for another 2 h. Finally, the blots were washed three times in PBS, 15 min each, before staining. The blots were stained in a 10% solution of nitroblue tetrazolium 5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP, Zymed), until the bands developed. Quantitative analysis was performed using Gel Pro Analyzer 2.0 software on blots scanned into the computer.
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RESULTS |
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By 8 h, the extent of upregulation for several HSPs, as well as GADD genes, had begun to decrease, whereas upregulation of c-src-kinase and chaperonin increased. The message for glutathione S-transferase pi, which had not been significantly altered before, was significantly suppressed at 8 h. DNA-repair proteins and the HSPs at 70 kDa, 86 kDa, and 60 kDa were still elevated. By 24 h, however, most of the early upregulated genes had either fallen back to background levels, or were minimally elevated. An elevation of proliferation-associated gene, melanoma differentiation associated protein 6 (mda-6), transcription and RNA polymerase elongation factor subunits, and DNA-binding protein TAX became apparent. On the other hand, the message for glia maturation factor-ß was 20 times lower at 24 h. Other genes significantly downregulated include glutathione S-transferase pi, IL-1ß, TDGF1, VEGF receptor 1 precursor, and IL-5
receptor.
The temporal pattern of expression for these genes is more easily recognized through clustering (Fig. 4). Analysis of the most highly regulated subset of genes would suggest which cellular functions are most important for repair of injured cells. The clustered genes were grouped into dendrograms containing sets of genes that possess similar functions. Groupings within each dendrogram show which genes are regulated in a similar manner over the 24-h time course. For each dendrogram, the average fold change in regulation for all genes in the set was also calculated at each time point. The plots of the average fold change over time indicate the trends in regulation for each set of functionally related genes.
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DISCUSSION |
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We have observed that for gene expression profiling following a potentially lethal insult, care must be taken to ensure that the RNA is representative of injured cells, but not dead cells. An overly severe injury results in RNA that is too degraded to be useful in gene expression profiling. The quality of RNA from cells heated up to 7 s at 55°C remained undegraded, as shown by agarose gel electrophoresis.
Another major advantage to array profiling, unachievable by other techniques, is the ability to assess expression of genes not yet known to be involved in a biological process (20). Gene clustering applications can then be utilized to group genes by their patterns of expression over time or after specific treatments (12). By grouping genes together in this manner, genes that are interdependent and function together can be visualized. Emerging techniques such as clustering allow analysis of large amounts of data generated from an experiment.
The set of genes presented in this report is from a representative experiment. We have performed this experiment five times with similar results, but considerable variability was observed between experiments, resulting in variability in ratios and number of genes up- or downregulated. This may have resulted from the nature of the injury, using different lots of arrays, or other unknown variables. However, many of the same genes were consistently altered and, more importantly, show similar patterns of gene expression in response to thermal injury. It is imperative that the "fold change" in gene expression is interpreted in relative terms as an indication of trends in expression, not as concrete numbers. Again, the ratios of fold change are not quantitative, but qualitative, and therefore can change with each experiment. Much of the variability may be due to the nature of the injury itself and the difficulty in inflicting a reproducible thermal injury. Furthermore, it is only logical to expect many different modes of response to any single type of insult. Therefore, although the set of genes induced by thermal injury may fluctuate between experiments, they must all be involved in the repair process, with the most essential genes induced more consistently and presumably to a greater extent.
Of 588 known human genes analyzed in this experiment, 120 exhibited a greater than twofold alteration in expression after sublethal thermal injury of ARPE-19 cells heated for 3 s at 55°C. As expected, the HSPs were highly elevated in response to thermal injury. Messages for the 70-kDa and 40-kDa HSPs were the two most highly elevated following injury. These proteins are also elevated in response to oxidative stress and may indicate one pathway by which thermal injury causes secondary damage (22, 26). The heat shock response, first described by Rotissa (39), is one of the best understood forms of induction of gene expression. Trimerization of the heat shock transcription factor (HSTF) yields a configuration that binds favorably to the heat shock response element and enhances transcription of RNA for several HSPs (35, 45). Constitutively expressed HSPs are postulated to act as molecular chaperones involved in protein folding, assembly, and transport. During stress, induced HSPs probably participate in an attempt by the cell to restore denatured proteins to their active configuration and therefore prevent the accumulation of aggregated, misfolded, and damaged proteins (15, 18, 34).
The presence of elevated message does not always ensure translation into product. One example of this is the disparity between the presence of mRNA for both HSP40 and HSP70 in control cells and the translation of protein product for HSP40 only. Message for HSP70 was present in control cells and peaked at 4 h, but protein was not detected until 4 h after heating, with a 33-fold elevation by 24 h (Fig. 5). On the other hand, HSP40 protein was present in the control but was elevated only sevenfold by 24 h. This indicated that HSP70 was translationally activated, whereas activation of constitutively expressed HSP40 may be regulated by a different mechanism (36).
The protective characteristics of HSPs are also being studied for therapeutic purposes. Aside from the ability to establish a state of thermotolerance following sublethal thermal injury, the induction of HSPs has been implicated in protection against ischemic damage (7, 26). The ability of HSPs to protect against stresses other than heat implies that the mechanism of induction is not specific for thermal insult or that there is more than one mechanism. In addition to heat damage, a variety of other insults such as trauma, ischemia, heavy metal, and alcohol can produce similar responses in cells. Other artificial means of induction, such as the utilization of prostaglandin A1 (3, 13), herbimycin A (18, 36), and sodium arsenite (27), are being explored for possible pharmacological application. By observing the specific pathways of induction and the resulting downstream changes in cellular function and expression, the mechanism of protection may be elucidated.
The DNA-binding protein TAX and the 153-kDa GADD gene were also highly elevated. The GADD stress response genes have generally been reported to be induced by ionizing and ultraviolet radiation or alkylating agents and are proposed to be regulated by distinct kinase-mediated pathways (9). Genes previously described as affecting growth and differentiation, including the retinoic acid binding protein (43), placenta growth factor, mda-6 gene (21), and the protein tyrosine kinase c-src (14) were highly elevated. Both the proliferation-associated gene (pag) (37) and neurotrophin-4 (49, 50) additionally have protective roles in reaction to oxidative or ischemic damage. The elevation of IL-12, which leads to the production of interferon- and which is largely induced by bacterial infection, supports the manifestation of a general immune response resulting from thermal injury (10, 48).
One aspect of visible laser light injuries to the retina is that in most instances, the laser beam produces a Gaussian distribution of energy and does not produce a homogeneous population of sublethally injured cells. Lethal thermal injury occurs immediately to the RPE cell layer in the center of injury zones, whereas sublethal injury occurs to a subset of cells on the periphery of the injury (19, 32). Evaluation of the response of those small number of cells at the periphery is therefore difficult, but they are the relevant cells for staging the healing response and preventing additional expansion of the injury site. The dipping method of heating was therefore employed in this study to inflict a reproducible high-intensity, short-duration thermal injury as a model for the damage on the burn perimeter. Furthermore, during the early stages of the injury and prior to the onset of wound healing, sublethally injured cells may either undergo accidental cell death or apoptosis (47). However, factors regulating these processes are unclear. Thermal damage induced by laser irradiation will have similar effects on other tissue types, and this type of tissue response is necessary to assess as new applications of lasers are found in medicine.
The cellular response to sublethal thermal insult has been highly conserved to promote both cellular and organism survival. All cells live within relatively narrow tolerances for heat and when subjected to elevated temperature adaptively respond to protect themselves. The cells may survive up to some point, but then may either die or recover. In multicellular organisms, the evolution of thermal homeostasis was an important advance for survival. Because the brain precisely regulates temperature, optimizing function over a very narrow range, a form of thermal insult occurs naturally in the form of fever, with prostaglandins elevating temperature as a physiological response to invasion by foreign substances (13, 41). The RPE cell may also have adapted to tolerate somewhat higher temperature levels by the fact that photon energy is converted to heat at intracellular sites of melanin accumulation.
Interestingly, many of the genes upregulated by heat shock have also recently been reported to be upregulated during normal aging, indicating the accumulation of denatured protein with age and its acceleration by heating (25). Although the entire significance of discovered alterations in gene expression is not yet clear, by observing the changes in metabolic processes due to thermal stress, we may better understand the benefits and targets of drug treatment for secondary damage associated with thermal injury. Based on gene expression profile data, potential therapeutics will be chosen depending on their ability to restore the genetic responses to a more normal pattern of expression or to reduce deleterious genetic responses to thermal injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. D. Bowman, US Army Institute of Surgical Research, 3400 Rawley E. Chambers Ave., Bldg. 3611, Fort Sam Houston, TX 78234-6315 (E-mail: phillip.bowman{at}amedd.army.mil).
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REFERENCES |
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