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. 2000 Jul;5(3):173–180. doi: 10.1379/1466-1268(2000)005<0173:eoahia>2.0.co;2

Expression of antisense hsp70 is a major determining factor in heat-induced cell death of P-19 carcinoma cells

Robert N Nishimura 1,3, Donaldson Santos 1, Linda Esmaili 1, Su-Ting Fu 1, Barney E Dwyer 2
PMCID: PMC312883  PMID: 11005375

Abstract

Overexpressed heat shock protein 70 (Hsp70) is known to be associated with thermoprotection in a number of cell lines and transgenic animals. We hypothesized that because overexpression of Hsp70 protects cells from lethal heat stress, inhibition of expression should make cells susceptible to heat stress. The model used for this study was a stably transfected P-19 carcinoma cell line expressing antisense hsp70 under the control of the hsp70b promoter. The results showed marked inhibition of Hsp70 expression after heat shock correlated with heat-induced cell death. Hsp90 and Hsc70 protein expression were not affected by the antisense construct. Unexpectedly, heme oxygenase (HO-1), another highly inducible heat shock protein, was not induced after heat shock in the antisense hsp70 cell line. Heat shock transcription factor-1 (HSF-1) was in a highly phosphorylated state in the antisense cell line before and after heat shock. This was in contrast to the untransfected control P-19 cells where HSF-1 was primarily highly phosphorylated after heat shock. A control cell line expressing only the vector, pMAMneo, without the antisense construct also showed partial loss of Hsp70 induction but not increased cell death after heat shock. The findings support the role of Hsp70 in thermoresistance.

INTRODUCTION

In all organisms studied, heat shock proteins (Hsps) have been found to provide thermoresistance (Morimoto et al 1990). In addition the expression of these proteins has been found to protect cells from other types of stress including toxins, chemicals, and oxidative stress (Parsell and Lindquist 1994). In the nervous system Hsps have been found to protect cells from severe heat stress (Amin et al 1996; Fink et al 1997) and medically related and more complex conditions such as hypoxia-ischemia in stroke models (Amin et al 1996; Plumier et al 1997; Rajdev et al 1997; Yenari et al 1998). The role of the highly inducible Hsp70 in the nervous system has been associated with survival from hypoxia-ischemia, but until recently it was not known whether the expression of the protein indicated a reaction to injury or predicted survival of the injured cells (Nishimura and Dwyer 1997). Only recently direct evidence that Hsp70 protects neurons from hypoxia-ischemia in stroke models has emerged. These studies have included transfecting Hsp70 into neurons in areas of subsequent hypoxic-ischemic stress (Yenari et al 1998) and overexpressing Hsp70 in cells and mimicking hypoxic-ischemic conditions in tissue culture (Amin et al 1996). The strongest evidence that Hsp70 protects cells in hypoxia-ischemia comes from data produced in transgenic Hsp70 mice (Plumier et al 1997; Rajdev et al 1997). Evidence from these transgenic mice subjected to cerebral hypoxia-ischemia have shown that constitutive overexpression of Hsp70 correlated with neuronal survival following the hypoxia-ischemia.

Though these CNS models have shown that overexpression of Hsp70 in cells and transgenic animals was associated with cellular protection in hypoxia-ischemia, several questions still arise. Because all of the models overexpress Hsp70 constitutively, it is unknown whether Hsp70 overexpression influences the expression of other proteins or cellular function. In one transgenic model it appears that Hsp70 overexpression does not influence the expression of Hsp27 or Hsp90 (Marber et al 1995). However, it is unclear whether the overexpression influences expression of other Hsps under conditions of stress or how protein synthesis might be affected by the presence of Hsp70 in unstressed or stressful conditions.

A different method for investigating the function and importance of Hsp70 studied in this paper is to inhibit selectively the synthesis of Hsp70 by transfecting antisense hsp70 DNA under the control of a heat shock promoter. Without the synthesis of this protein after heat stress we hypothesized that the antisense hsp70-expressing cell line would be more sensitive to heat stress. We also proposed to study the effect of the transfected gene on other Hsp synthesis and general protein synthesis. The model used to study the effects of Hsp70 in the nervous system, the P-19 carcinoma cell line, was chosen because these cells can differentiate into a neuronal morphology after retinoic acid exposure and express glutamate receptors (MacPherson et al 1997) and GABA receptors (Reynolds et al 1996). Studies characterizing an antisense hsp70 DNA transfected P-19 cell line response to heat stress are presented. Experiments include investigations of the expression of heat shock transcription factor-1 (HSF-1), protein synthesis, and cell viability after heat shock.

MATERIALS AND METHODS

Cell line and materials

P-19 cells from ATCC (Manassus, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1 by weight) medium supplemented with 5% calf serum. P-19 cells were chosen for these experiments because of their ability to differentiate into nervous sytem cell types such as neurons and glial cells. Cells were grown in 6-well dishes (CoStar, Corning, NY, USA) until 50–70% confluent and were then used for transfection experiments and later biochemical studies. Cells for gel shift assay and RNA extraction were grown to confluence in 75-cm2 flasks (CoStar).

Antisense HSP70 construct and transfection

Rat hsp70 cDNA was obtained from the laboratory of Dr Frank Sharp (Longo et al 1993). An antisense construct consisting of 513 bp (−9 to 504) covering the start site was subcloned in the reverse direction into the SalI site of the pD3SX vector (StressGen, British Columbia, Canada) and under the control the the human hsp70B (hHsp70B) promoter (Fig 1). The hHsp70B promoter consists of bases −270 to 113 of the hHsp70B gene (Voellmy et al 1985). This construct was cotransfected into P-19 cells with the pMAMneo expression vector (Clontech, Palo Alto, CA, USA) that includes a neomycin selection gene. A control line was transfected with the pMAMneo construct alone. Transfection was performed as per Lipofectin protocol (Gibco BRL, Rockville, MD, USA). Selection of cell lines began 48 hours after the transfection using G418 (100 μg/mL medium). Selection of 2 cell lines for study were chosen after serial screenings with increasingly higher concentrations of G418 (200–400 μg/mL medium). Antisense cell lines were selected for their inability to express Hsp70 protein 4 hours after heat stress of 45°C for 20 minutes in a circulating water bath.

Fig 1.

Fig 1.

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Schematic diagram of the pD3SX vector (StressGen) containing the rat antisense hsp70 construct. The antisense hsp70 construct is downstream of the hsp70B promoter. No mammalian selection gene is contained in the vector. Reproduced with modifications by the kind permission of StressGen, Vancouver, Canada

Protein analysis and western immunoblotting

Cells from experiments were heat stressed in a circulating water bath as noted in the legends. Polyacrylamide gel electrophoresis and transblotting of proteins to Immobilon-P membrane (Millipore, Bedford, MA, USA) were performed as previous described (Nishimura et al 1988, 1991; Dwyer et al 1995). Cultured cells were prepared for sodium dodecyl sulfate (SDS) gel electrophoresis by precipitating total cellular proteins in 10% trichloroacetic acid (TCA). Samples were solubilized in Laemmli's sample buffer and boiled for 3 minutes. Equal amounts of protein samples were run on 12% polyacrylamide slab gels with 4% stacking gels on a minigel apparatus (Bio-Rad, Hercules, CA, USA). After a 1-hour run at constant voltage (60 V for 15 minutes, then 100 V for the remainder of the run), the proteins were transferred to Immobilon-P membrane (Towbin et al 1979). Western blots were performed as previously described except that ECL (Amersham, Buckinghamshire, UK) was used as a developer (Nishimura et al 1988, 1991). Antibodies for these studies were mouse monoclonal C-92 for Hsp70 (courtesy of W. Welch, University of California, San Francisco, CA, USA), rabbit polyclonal anti-rat heme oxygenase (HO)-1 (Dwyer et al 1995), rabbit polyclonal anti-mouse HSF-1 (developed in our laboratory from recombinant mouse HSF-1), mouse monoclonal anti-Hsp 90 (Affinity, Golden, CO, USA), and rabbit polyclonal anti-Hsc70 (Sigma, St. Louis, MO, USA). All antibodies were used at 1:1000 or 1:2000 dilution.

For radiolabeling, P-19 cells were changed to methionine-deficient DMEM/Ham's F-12 (1:1). Cells were then labeled with [35S]methionine (30 μCi/mL medium) and immediately subjected to experimental control or heat shock conditions as described. Four hours after the experimental condition, labeled cells were TCA precipitated and solubilized in Laemmli's sample buffer. Gel electrophoresis was performed as noted above. Equal counts (cpms) were added to each lane. After the run, gels were dried and exposed to X-OMAT AR film (Kodak).

Mobility gel shift assay

The gel shift assay was performed per a published protocol (Mosser et al 1988). Whole cell extracts were prepared by several freeze-thaw cycles of treated cells (Mosser et al 1988). The extracts were then frozen at −80°C until use.

Cell death

Cell viability was assessed by uptake of propidium iodide (1 μg/mL medium). Dead cells showed red nuclear fluorescence. Live cells were assessed by the Live/Dead Assay (Molecular Probes, Eugene, OR, USA). Manually counted cells, 3 fields of 100 cells or more of 3 separate samples, were averaged and presented as % dead cells of total counted cells.

RNA extraction and Northern blots

Total RNA was extracted from cells using TRIzol Reagent (Life Technologies-GibcoBRL, Rockville, MD, USA) per protocol. Northern blots were done as previously described (Nishimura et al 1991). Hybridization of [32P]ATP-labeled riboprobe was done as previously described (Nishimura et al 1991).

Statistics

Data from samples of radiolabeling and cell viability experiments were counted and analyzed by one-way ANOVA and post hoc Tukey's test.

RESULTS

The immediate goal of this work was to establish a P-19 cell line that inhibited Hsp70 when heat stressed. Figure 2 demonstrates that after heat shock, cell line, P-19#1 (transfected with antisense hsp70) did not express Hsp70. This was in comparison with cell line, P-19#3, (transfected with the pMAMneo vector only) that expressed Hsp70 but less than the untransfected cell line, P-19 (Fig 2). The untransfected control P-19 cells also synthesized Hsp70 constitutively. This was not noted in the control P-19#1 or P-19#3 cells. Next, as a measure of cellular metabolism the cell lines were examined for protein synthesis by radiolabeling with [35S]methionine. Autoradiographic evidence for altered patterns of protein synthesis in each cell line is evident in both control and heat shocked cells (Fig 3A,B). Several prominent radiolabeled bands appear at approximately 40, 55, 66, and 80 kDa in the P-19#1 cell line. After heat shock, the P-19#1 cells appear to have fewer prominent bands and no incorporation of labeled methionine at 70 kDa, likely Hsp70 and 30 kDa, approximately the molecular mass of HO-1 (Fig 3B). In contrast, the untransfected P-19 cells showed marked induction of both Hsp70 and HO-1 (Fig 3B). Because of the differences in protein synthesis and the possibility that other Hsps might be affected by the transfections, Western immunoblots were performed for Hsp90, HO-1, and Hsc70. Examination after heat shock showed no significant differences between heat-stressed vs control cells of P-19#1 and P-19#3 cell lines for Hsp90 and Hsc70 (Fig 4). However, while HO-1 was increased an estimated 4-fold in P-19 heat shock vs control untransfected cells, neither of the transfected cell lines, P-19#1 or P-19#3, showed induction of HO-1. Both cell lines also showed decreased expression of HO-1 in nonheat stress conditions, approximately 30% less than control P-19 cells. Figure 5 shows incorporation of [35S]methionine into samples from the same experiment as Figure 3. Protein synthesis in Hsp70-deficient cells was significantly inhibited after heat shock (P < 0.001) when compared with the heat shocked P-19 and P-19#3 cell lines. Because Hsps have a promoter containing the concensus heat shock response element, we investigated whether the lack of Hsp70 synthesis and decreased HO-1 expression were secondary to altered expression of HSF-1. Western blot analysis demonstrated that HSF-1 was in a hyperphosphorylated state in P-19#1 and P-19#3 unstressed and heat-stressed cells (Fig 6). Only a minimal shift or increase in molecular mass was noted in P-19#3 cells and less shift was noted in P-19#1 cells after heat shock. Those findings were in contrast with P-19 cells where the expected shift from a low molecular mass to a higher molecular mass of HSF-1 was evident after heat shock. A gel shift mobility assay (Fig 7) for HSF-1 binding showed that P-19#3 and P-19#1 heat shock cellular extracts had little binding to oligonucleotides containing the heat shock element consensus sequence (HSE). Increasing the amount of cellular extract in the assay did not change HSF-1 binding in heat shocked samples of P-19#1 or P-19#3 (not shown). Those were in contrast with the untransfected heat shocked P-19 cells that showed moderate binding to the HSE oligonucleotides. Northern blot analysis showed a marked decrease of Hsp70 mRNA in both P-19#1 and P-19#3 cell lines when compared with untransfected P-19 heat shocked cells (Fig 8). Finally we studied cell survival after heat stress in all 3 cell lines. Figure 9 demonstrates that the Hsp70-deficient cells were significantly more sensitive to heat stress than either P-19 and P-19#3 cell lines.

Fig 2.

Fig 2.

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Western immunoblot of Hsp70 in P-19, P-19#1, and P-19#3 cell lines. Cells were heat stressed at 45°C for 20 minutes and then harvested after 4 hours at 37°C for SDS gels, blotted, and probed for Hsp70 immunoreactivity. Control unheated cells (C) were compared with heat-stressed cells (H). Each lane represents 20 μg of total cellular protein

Fig 3.

Fig 3.

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Autoradiograph of [35S]methionine-labeled proteins from the transfected cell lines and control run on a 12% SDS polyacrylamide gel. P-19, P-19#1, and P-19#3 cells were run in triplicate samples. (A) Unheated control cells. (B) Heat shocked at 45°C for 20 minutes and then harvested after 4 hours and run on SDS gels. Equal cpms were added to each lane. Molecular mass standards are noted in the right margin. The arrows in the left margin indicate Hsp70 (68 kDa) and HO-1 (32 kDa)

Fig 4.

Fig 4.

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Western immunoblot of P-19, P-19#1, and P-19#3 cell lines probed for Hsc70, Hsp90, and HO-1. Control unheated cells (C) were compared with heat-stressed cells (H) at 45°C for 20 minutes. Cells were harvested for SDS gels 4 hours after heat treatment. This was 1 of 3 similar experiments showing the same findings. Each lane represents 20 μg of total cellular protein

Fig 5.

Fig 5.

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Total [35S]methionine incorporation (cpms) into proteins derived from the same experiment described in Figure 3. Cells were heat treated at 45°C for 20 minutes, incubated at 37°C for 4 hours, then TCA precipitated. TCA-precipitable protein was represented as cpm/μg total cellular protein. The incorporation rate was linear for 4 hours (not shown). Each bar denotes n = 3 with SEM. *P < 0.001 of heat shocked #1 cells compared with heat shocked control P-19 and P-19#3 cells

Fig 6.

Fig 6.

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Western immunoblot of P-19, P-19#1, and P-19#3 cell lines probed for HSF-1. Control unheated cells (C) were compared with heat-stressed cells (H) at 45°C for 20 minutes. Cells were harvested for SDS gels 1 hour after heat treatment. This was 1 of 3 similar experiments showing the same findings. Each lane represent 20 μg of total cellular protein. The arrow indicates the higher molecular mass form of highly phosphorylated HSF-1. The double arrow indicates the lower molecular mass of the unphosphorylated HSF-1

Fig 7.

Fig 7.

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Gel shift mobility assay for HSF-1 binding in heat shocked P-19, P-19#1, and P-19#3 cell lines. Control unheated cells (C) were compared with heat-stressed cells (H) at 45°C for 20 minutes. All cells were harvested for whole cell extracts 1 hour after heat shock. Each lane represents 20 μg of sample whole cell extract protein used in the assay

Fig 8.

Fig 8.

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Northern blot analysis for Hsp70 mRNA in the P-19, P-19#1, and P-19#3 cell lines. Control unheated cells (C) were compared with heat-stressed cells (H) at 45°C for 20 minutes. Total cellular RNA was harvested 1 hour after heat shock. Three micrograms of total cellular RNA was loaded into lanes 1 and 2. Ten micrograms of total cellular RNA was added into lanes 3–6

Fig 9.

Fig 9.

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Cell viability 24 hours after heat stress in transfected cell lines. Heat shock at 45°C for 30 minutes was performed on cells plated in 6-well dishes at 80–90% confluence. Cell counts were obtained 24 hours after heat shock and are representative of 3 separate experiments. Each bar represents n = 3 with SEM. *P < 0.001 of heat shocked P-19#1 cells compared with heat shocked control P-19 and P-19#3 cells

The transfected cells demonstrate only short and few processes after retinoic acid induction, indicating modest differentiation into a neuronal phenotype (not shown). Because of that finding we did not present separate experiments using the neuronal phenotype in this work. Future experiments will study whether the inability to synthesize Hsp70 was associated with phenotypic neuronal differentiation.

DISCUSSION

The experiments demonstrated that Hsp70 was inhibited by stable transfection of antisense hsp70 DNA. Previous experiments in this laboratory using antisense hsp70 DNA controlled by a constitutive promoter failed or led to partial and/or transient transfection. We reasoned that the failure might have been on the basis of the very high copy number of induced Hsp70 mRNA that could not be accommodated by the constitutively expressed antisense Hsp70 RNA. The difference with the present antisense cell line is that the antisense construct was controlled by the same promoter as the hsp70 gene, the heat shock promoter. Though the copy number of antisense genes was not measured, we believe that there were enough active antisense copies to inhibit even the greatest induction of Hsp70 when HSF-1 was activated. This newly developed antisense cell line has shown no induction of Hsp70 by heat stress on repeated experiments. Also of note was the decreased synthesis of Hsp70 in P-19#3 cells after heat shock. The vector, pMAMneo, does not contain an antisense construct, and it is unclear why constitutive synthesis of Hsp70 was decreased in the control P-19#3 cells as well as the heat-stressed cells. The pMAMneo construct expresses the neo gene protein, which is an aminophosphotransferase with a molecular mass of 25 kDa that acts by phosphorylating and inactivating neomycin (Davies and Smith 1978). Whatever the cause of the hyperphosphorylated HSF-1 in P-19#1 and P-19#3 cells, it is possible that the phosphorylated state of HSF-1 is inhibited from translocating into the nucleus and thus disrupting Hsp70 synthesis.

Radiolabeling experiments showed that Hsp70 was not synthesized by heat shock in the P-19#1 cell line. In addition, the radiolabeling of HO-1 was decreased in both transfected cell lines P-19#1 and P-19#3, and the accumulation by Western blot analysis (Fig 4) was also decreased in control and heat shocked cells. Those findings are difficult to explain because HO-1 and Hsp70 cDNA have little homology. It raises the question of whether HO-1 was regulated by and directly correlated with the synthesis of Hsp70. As mentioned previously, the hyperphosphorylated HSF-1 may be inhibited from translocating into the nucleus, and thus HO-1 and Hsp70 synthesis would be inhibited. In previous studies it has been suggested that Hsp70 associated with ribosomes and promoted translational tolerance (Peterson and Mitchell 1981; Mizzen and Welch 1988). This was supported by data in Figure 5 that demonstrated that the lack of Hsp70 was associated with a significant generalized decrease of proteins synthesized after heat shock in P-19#1 but not in P-19#3 cells. The same heat stress was not associated with significant decreases in protein synthesis in control untransfected P-19 cells. The present study did not investigate the relationship of Hsp70 synthesis with ribosomes or protein synthesis initiation factors. The transfection of the vectors changed the baseline protein synthesis pattern from control untransfected cells (Fig 3A). This finding may have important implications for experiments proposing gene therapy in animals or humans. It will be important to monitor the effects of transgene expression on overall protein expression.

Hsp70 synthesis is largely under the control of HSF-1 binding with the HSE of the heat shock promoter (Morimoto et al 1990). Hsp70 is a molecular chaperone for HSF-1, acting as a negative regulator of HSF-1 transcriptional activity (Dwyer et al 1991; Shi et al 1998). The present study found that detectable HSF-1 was decreased in transfected antisense hsp70 and pMAMneo vector transfected cells. HSF-1 was also in a hyperphosphorylated state in P-19#1 cells and less so in P-19#3 cells in nonstress conditions. The shift to a higher molecular mass by phosphorylation after heat shock was less evident than in untransfected P-19 cells (Fig 6). The explanation for the apparent decreased synthesis and hyperphosphorylation of HSF-1 expression in these cells is unknown. A recent study showed that phosphorylation of HSF-1 was inhibited by overexpression of Hsp70 by activating protein phosphatase and inhibiting protein kinase C (PKC) activity (Ding et al 1998). The opposite effect may be true, and underexpression of Hsp70 might lead to phosphorylation of HSF-1 through higher PKC activity. This might explain the findings in the antisense hsp70 cell line but does not explain the findings in the P-19#3 cell line. It is unclear how phosphorylation of HSF-1 influences transcription of Hsp70, but it appears that inducible phosphorylation is not necessary for DNA-binding activity of HSF-1 trimers (Jurivich et al 1992). Further experiments will be needed to explain these findings, including examining the signal transduction pathways. With decreased total HSF-1 phosphorylation it was expected that the gel shift and Hsp70 mRNA expression were decreased in the P-19#1 and P-19#3 cell lines (Figs 7 and 8). One explanation for the lack of Hsp70 mRNA in P-19#1 cells is that the activity of the enzyme RNase H recognizes the double-stranded RNA Hsp70 hybrids and rapidly degrades both messages (Crooke 1993). Another mechanism for the antisense effect is by translational arrest. The antisense message becomes bound specifically to the Hsp70 mRNA and prevents its translation by ribosomes (Probst and Skutella 1996). The lack of message by Northern blot analysis would favor increased degradation by RNase H activity in our present study. However, the decrease in transcription of Hsp70 mRNA may also be a factor because HSF-1 is decreased.

The effect of the transfections were monitored by looking at the expression of other Hsps under control and heat shock conditions. As expected, Hsp90 and Hsc70 were unaffected after heat shock in all 3 cell lines (Fig 4). A transgenic Hsp70 mouse model also found no increase in Hsp90 or Hsc70 synthesis (Marber et al 1995). However, the expected induction of HO-1 after heat shock was absent in both transfected cell lines, P-19#1 and P-19#3, when compared with P-19 control cells (Fig 4). The mechanism for decreased HO-1 expression is unknown but is likely related to the expression and phosphorylation of HSF-1 in both cell lines.

The focus of our studies has been to study whether Hsp70 was responsible for thermoprotection and possibly protection from other types of stress. This study indicated that the absence of Hsp70 in cells was associated with significant cell death, indicating the necessity for Hsp70 for heat stress protection (Fig 9). These findings are not surprising in view of the multiple studies overexpressing Hsp70 that have shown cellular protection from heat and other types of stress (Li et al 1991; Uney et al 1993; Mestril et al 1994; Samali and Kotter 1996; Kiang et al 1998). In the nervous system, similar findings have been found in Hsp70 transfected neuronal cultures and in Hsp70 transgenic animals (Amin et al 1996; Plumier et al 1997; Rajdev et al 1997; Yenari et al 1998). However, all studies overexpressing Hsp70 did not exclude the possible role of other major Hsps in cellular protection. It is likely that other Hsps contribute to thermoprotection and that the lack of induction of these proteins lead to cell death. Hsp90 is unlikely to be thermoprotective in P-19 cells because it is not heat inducible and the P-19#1 cells died despite no change in the relative amount of Hsp90. HO-1 induction was not increased after heat shock in both P-19#1 and P-19#3 cell lines. HO–1-deficient fibroblast and liver cells showed increased sensitivity to oxidative stress (Poss and Tonegawa 1997). This is in contrast to work in hippocampal slices where inhibition of HO-1 activity using metalloporphyrin derivatives resulted in neuroprotection from traumatic injury (Panizzon et al 1996). The finding in the latter study may be related to the inhibition of HO-2 as well as HO-1 induced by the inhibitors. It is also possible that the combination of the lack of induction of HO-1 and the absence of Hsp70 contributed to heat shock induced cell death in the P-19#1 cell line. Studies are now ongoing to investigate the role of another Hsp, Hsp27, in thermoprotection in the absence of Hsp70. Also planned are experiments to study the association of decreased phenotypic neuronal differentiation with the inhibition of Hsp70 synthesis.

Acknowledgments

We acknowledge the assistance of Sharon Dabby, Xiong Lin, and Alfred Behruti for their important contributions to this study. We acknowledge the support of the Research Service of the Department for Veterans Affairs for this study. The authors also acknowledge StressGen, Vancouver, Canada, for permission to use the template for Figure 1.

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