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J Thorac Cardiovasc Surg 2003;126:748-754
© 2003 The American Association for Thoracic Surgery

General thoracic surgery

Cell death induced by down-regulation of heat shock protein 70 in lung cancer cell lines is p53-independent and does not require DNA cleavage

Steffen Frese, MDa,*, Manuela Schaper, BSca, Jan-Rasmus Kuster, MDa, Daniela Mieschera, Marja Jäättelä, MD, PhDb, Thomas Buehler, PhDc, Ralph A. Schmid, MDa

a From the Division of General Thoracic Surgery, University Hospital, Berne, Switzerland
b Apoptosis Laboratory, Danish Cancer Society, Copenhagen, Denmark
c Novartis, Basel, Switzerland

Received for publication November 1, 2001; accepted for publication January 21, 2003.

* Address for reprints: Steffen Frese, MD, Department of Clinical Research, Murtenstrasse 35 Room C807, CH-3010, Berne, Switzerland
steffen.frese{at}web.de


   Abstract
Top
 Abstract
Material and methods
 Results
 Discussion
 References
 
OBJECTIVE: The inducible form of heat shock protein 70 is known to be overexpressed in tumors and seems to be necessary for the survival of tumor cells via an unknown mechanism. We therefore evaluated whether selective depletion of heat shock protein 70 induces cell death in lung cancer cells.

METHODS: An adenovirus expressing antisense heat shock protein 70 and an adenovirus with ß-galactosidase were used for transduction of the lung cancer cell lines A549, NCI-H358, LXF-289, LOU-NH91, normal human bronchial epithelial cells, and normal lung fibroblasts IMR90. Cell death was determined by morphology, propidium iodide uptake, and trypan blue staining; DNA cleavage was assessed by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Expression of heat shock protein 70, heat shock cognate 70, and phosphorylated p53 was determined by Western blot analysis.

RESULTS: Transduction of lung cancer cells with adenovirus expressing antisense heat shock protein 70 but not with adenovirus with ß-galactosidase resulted in extensive cell death after 96 hours (A549: 53.2 ± 9.44% versus 12.9 ± 6.6%; NCI-H358: 48.4 ± 7.2% versus 25.2 ± 1.4%; LXF-289: 58.8 ± 6.5% versus 24.7 ± 5.4%; LOU-NH91: 82.5 ± 1.8% versus 38.5 ± 2.6%). In contrast, adenovirus expressing antisense heat shock protein 70 showed much less cytotoxicity in normal human bronchial epithelial cells (16.0 ± 0.5% versus 17.1 ± 7.3%) and in normal lung fibroblasts IMR90 (17.2 ± 3.6% versus 8.2 ± 1.6%). After treatment with adenovirus expressing antisense heat shock protein 70, transactivation of p53 in A549 but not in NCI-H358, a cell line deleted for p53 has been observed. Furthermore, 22.0 ± 3.0% of A549 cells treated for adenovirus expressing antisense heat shock protein 70 stained positive with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling versus 10.2 ± 4.0% treated with control virus. This effect but not cell death itself was blocked by treatment with 10 µmol/L zVAD-fmk, a broad caspase inhibitor.

CONCLUSIONS: Selective down-regulation of heat shock protein 70 induces cell death in lung cancer but not in normal lung cells. The demonstrated effect is p53-independent and does not require DNA cleavage. The data suggest that gene transfer of antisense heat shock protein 70 might be useful in developing new strategies for the treatment of lung cancer.

Lung cancer is the leading cause of cancer death in the United States among both men and women. The projected number of new cases of lung cancer in 1999 in the United States is 171,600, with more than 158,000 people dying from the disease during the same year. In fact, more people die from lung cancer than from breast, colorectal, prostate, and ovarian malignancies combined.1 Eighty percent of all newly diagnosed lung cancers is non–small cell lung cancer (NSCLC) and approximately two thirds of patients have inoperable, advanced disease. The 5-year survival for patients with stage IIIb cancer is approximately 5% and 1% for patients with stage IV cancer.

Current protocols for therapy of NSCLC include surgery and radiotherapy and chemotherapy.2 Administration of radiotherapy or chemotherapy leads to the induction of programmed cell death or apoptosis. The effectiveness of the nonsurgical treatment modalities is limited by side effects and by resistance of tumor cells to undergo cell death. Resistance of tumor cells may be explained by a failure to activate the apoptotic machinery. The underlying mechanisms are not fully understood yet, but it is believed that some proteins (for example Bcl-2, survivin, or c-FLIP) play a role as intracellular inhibitors of apoptosis.3 Similar effects have been shown for the heat shock protein (Hsp) 70.4,5 Furthermore, it has been demonstrated that Hsp70 is overexpressed in a wide range of tumors, whereas selective depletion of Hsp70 can induce apoptosis in tumor cells.6

We therefore assessed in the present study (1) whether selective down-regulation of Hsp70 is able to induce cell death in lung cancer but not in normal cells and (2) whether this type of cell death requires DNA cleavage or depends on p53, a protein that is often mutated in lung cancer.


   Material and methods
Top
 Abstract
 Material and methods
Results
 Discussion
 References
 
Reagents
Bradford reagent, the secondary goat anti-mouse, the monoclonal anti-{alpha}-tubulin antibody, and 5-bromoindolyl ß-D-galactopyranoside were purchased from Sigma Chemical Co (St Louis, Mo). The protease inhibitor cocktail was produced by Boehringer (Mannheim, Germany). Culture media and penicillin/streptomycin were purchased from Life Technologies (Paisley, UK); fetal calf serum and trypan blue solution were obtained from Biochrom (Berlin, Germany). The antibodies for Hsp70 and heat shock cognate (Hsc) 70 were from Stressgene (Victoria, Canada), and phosphorylated p53 was from Oncogene (Boston, Mass). The reagents for enhanced chemoluminescence (ECL) were produced by Amersham Pharmacia Biotech (Buckinghamshire, UK).

Tumor cell lines and cell culture
The human lung cancer cell lines A549 (nonspecified lung carcinoma) and NCI-H358 (bronchoalveolar carcinoma; both cell lines from American Type Culture Collection, Manassa, Va), LXF-289 (adenocarcinoma) and LOU-NH91 (squamous cell carcinoma; both cell lines from DSMZ, Braunschweig, Germany), and normal lung fibroblasts IMR90 (American Type Culture Collection) were cultured at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics. Normal human bronchial epithelial (NHBE) cells were obtained from Biowhittaker (Walkersville, Md). The cells were maintained in media recommended by the supplier and used within 2 to 4 passages.

Transduction with adenovirus, staining for ß-galactosidase
Cells were plated at 5 x 104 cells/well in 24-well plates and allowed to attach overnight. At the day of transduction the virus was diluted in medium containing 0.5% fetal calf serum at the indicated concentrations (800 to 1600 multiplicity of infection). After removing culture medium 200 µL of virus was added to each well. The plates were gently rocked and incubated for 2 hours with rocking every 30 minutes. Subsequently the virus was removed and cells were incubated with normal culture medium. The viruses used for this study were: adenovirus expressing Hsp70 (Ad.asHsp70) harboring a part of human Hsp70 sequence in antisense orientation as described elsewhere6 and adenovirus with ß-galactosidase (Ad.ß-gal) containing the ß-galactosidase gene kindly provided by Novartis Inc (Basel, Switzerland). Both viruses are deleted for the whole E1 and the majority of E3 region and were propagated in 293 or S8 cells.

To determine transduction efficiency, staining for ß-galactosidase was performed 24 hours after transduction with Ad.ß-gal. Cells were washed with phosphate-buffered saline (PBS), then fixed with 0.25% glutaraldehyde for 15 minutes, washed again with PBS 3 times, and staining solution (0.4 mg/mL 5-bromoindolyl ß-D-galactopyranoside, 2 mmol/L MgCl2, 5 mmol/L K4Fe(CN)6, K3Fe(CN)6 solved in PBS) was added. After an incubation period of 24 hours at 37°C, the stain was analyzed.

Trypan blue staining, prodidium iodide uptake
Trypan blue staining and propidium iodide uptake were used to measure the viability of cells. After the indicated time, cells were harvested by trypsinization. For trypan blue staining, cells were centrifuged and resuspended in 100 µL PBS; 100 µL of 0.5% trypan blue solution was added and probes were incubated for 5 minutes at 37°C. The percentage of dead blue cells was determined by using a hemocytometer and by counting a minimum of 100 cells per sample. Because NHBE cells were clumping when stained with trypan blue, propidium iodide uptake was used to determine cell death. After harvesting the cells, propidium iodide was added to a final concentration of 10 µg/mL and samples were immediately analyzed by FACScan (Becton Dickinson, San Jose, Calif). A minimum of 1 x 104 cells per sample was analyzed. All experiments were performed in triplicate.

Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining
DNA cleavage was assessed by a terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) kit (Roche, Mannheim, Germany) following the manufacturer’s instructions. In brief, cells were harvested, fixed for 30 minutes at room temperature with 4% paraformaldehyde, washed with PBS, then permeabilized with buffer containing 0.1% Triton X-100 and 0.1% sodium citrate for 2 minutes on ice and washed again with PBS. Cells were than incubated with 50 µL reaction mixture and incubated for 1 hour at 37°C. Percentage of TUNEL positive cells was determined by flow cytometry using a FACScan and analyzing a minimum of 1 x 104 cells per sample.

Western blot
Western blot was performed as described elsewhere.7 In brief, cells were harvested and then lysed for 30 minutes on ice in a buffer containing 20 mmol/L HEPES, pH 7.6; 120 mmol/L NaCl; 0.2 mmol/L ethylenediaminetetraacetic acid; 1% Triton X-100; and protease inhibitors. Lysates were centrifuged at 14,000 rpm for 15 minutes, and supernatants were collected. Protein concentration was determined with Bradford reagent. Proteins were separated by denaturating gel electrophoresis. The proteins were transferred to nitrocellulose membranes and incubated with blocking buffer (20 mmol/L Tris-HCL, pH 7.5; 500 mmol/L NaCl; 0.05% Tween20, 5% milk powder). After washing with buffer containing 20 mmol/L Tris-HCl (pH 7.5), 500 mmol/L NaCl, and 0.05% Tween20, blots were incubated with the primary antibodies diluted in blocking solution. After a second wash the blots were incubated with horseradish peroxidase–coupled secondary antibody, washed again, and developed with ECL.

For stripping, membranes were incubated for 30 minutes at 50°C in a buffer containing 62.5 mmol/L Tris-HCl (pH 6.7), 2% sodium dodecyl sulfate, 100 mmol/L ß-meraptoethanol. Then the blots were washed and blocked again.

Statistics
Data are expressed as mean ± SD. Statistical significance was determined using one-way analysis of variance followed by Bonferroni’s t test. Values of P < .05 were considered significant.


   Results
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 Abstract
 Material and methods
 Results
Discussion
 References
 
Expression of Hsp70 in lung cancer and normal lung cells
Expression level of Hsp70 was determined by Western blot. The experiment revealed that Hsp70 is highly expressed in 3 out of 4 lung cancer cells lines. In normal lung fibroblasts, IMR90, there was only a faint band for the expression of Hsp70 detectable. Interestingly, NHBE cells showed an Hsp70 expression comparable to the expression of the 3 lung cancer cell lines A549, NCI-H358, and LXF-289 (Figure 1).



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  		Figure 1. Expression of Hsp70 in the lung cancer cell lines A549, NCI-H358, LXF-289, and LOU-NH91, normal lung fibroblasts IMR90, and normal human bronchial epithelial (NHBE) cells. Expression of Hsp70 was assessed by Western blot. To show equal amounts of protein the blot was stripped and reincubated with an antibody against {alpha}-tubulin.

 
Selective depletion of hsp70 by Ad.asHsp70
Treatment of lung cancer cells with Ad.asHsp70 leads to down-regulation of Hsp70, whereas cellular levels of the structurally similar heat shock protein Hsc70 remains unchanged. To show this, A549 and NHBE cells were transduced with Ad.asHsp70 and lysed at the indicated time points. Levels of heat shock proteins were visualized by Western blot using monoclonal antibodies specifically recognizing Hsp70 and Hsc70 (Figure 2).



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  		Figure 2. Selective down-regulation of Hsp70 by Ad.asHsp70. A549 and NHBE cells were transduced with Ad.asHsp70 and lysed at the indicated time points. Expression of Hsp70 was determined by Western blot; the same blot was stripped and developed with an anti-Hsc70 antibody.

 
Morphologic changes after treatment with Ad.asHsp70
Transduction of A549 lung cancer cells with Ad.asHsp70 resulted in morphological changes by rounding up and loosing surface attachment, although A549 transduced with the control virus Ad.ß-gal remained morphologically unchanged (Figure 3, C). Furthermore, normal lung fibroblasts IMR90 (Figure 3, A) and normal human bronchial epithelial cells (Figure 3, B) transduced with Ad.asHsp70 or Ad.ß-gal retained their normal morphology. However, Ad.asHsp70-treated NHBE cells showed a markedly reduced cell number compared with cells transduced with Ad.ß-gal, suggesting that Ad.asHsp70 does not induce cell death but suppresses proliferation in these primary normal lung cells.



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  		Figure 3. Transduction efficiency and morphologic changes in IMR90 (A), NHBE (B), and A549 (C) after treatment with Ad.asHsp70 and Ad.ß-gal. To detect transduction efficiency, cells were stained 24 hours after transduction with Ad.ß-gal for ß-galactosidase (A through C, first column). Changes in morphology 96 hours after transduction were determined by light microscopy (A through C, columns 2 and 3). To assess caspase dependence, A549 in addition to Ad.asHsp70 were treated with 10 µmol/L zVAD, a caspase inhibitor. After 48 hours of incubation medium was changed and fresh zVAD was added (C, column 4).

 
As a control, ß-galactosidase staining is shown for NHBE, A549, and IMR90 cell lines. After treatment with Ad.ß-gal using a multiplicity of infection of 800 for A549 and IMR90 transduction, efficiency was 90% to 100% for both cell lines. Similar experiments have been performed with NCI-H358, LXF-289, LOU-NH91, and IMR90 cell lines (data not shown). NHBE cells used to demonstrate the toxicity of Ad.asHsp70 to normal lung epithelial cells has been transduced with an MOI of 1600, the highest for cancer cells.

Cell death after treatment with Ad.asHsp70
Cell death was assessed by trypan blue exclusion or propidium uptake 96 hours after transduction. According to the changes in morphology, lung cancer cells transduced with Ad.asHsp70, in comparison with those transduced with Ad.ß-gal, showed extensive cell death (mean ± SD: A549: 53.2 ± 9.44% versus 12.9 ± 6.6%; NCI-H358: 48.4 ± 7.2% versus 25.2 ± 1.4%; LXF-289: 58.8 ± 6.5% versus 24.7 ± 5.4%; LOU-NH91: 82.5 ± 1.8% versus 38.5 ± 2.6%; P < .001 for Ad.asHsp70-treated cells compared with nontreated cells and P > .05 for Ad.ß-gal-treated cells versus nontreated cells). In contrast, depletion of Hsp70 by Ad.asHsp70 revealed no significant cytotoxicity in normal lung fibroblasts IMR90 (17.2 ± 3.6% versus 8.2 ± 1.6%) or in primary human bronchial epithelial cells (16.0 ± 0.5% versus 17.1 ± 7.3%; Figure 4).



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  		Figure 4. Viability of lung cancer cells, lung fibroblasts, and NHBE cells after transduction with Ad.asHsp70 and Ad.ß-gal. Cells were transduced with Ad.asHsp70 or Ad.ß-gal, achieving 90% to 100% transduction efficiency; 96 hours after transduction, cells were stained with trypan blue. zVAD was used in a concentration of 10 µmol/L, changing medium after 48 hours. For NHBE cells, cell death was determined by staining with propidium iodide followed by FACScan analysis. Experiments were performed in triplicates. *P < .001 and **P < .01 (mean ± SD) compared with nontreated cells.

 
Independence of Ad.asHsp70-induced cell death
To determine whether cell death induced by selective depletion of Hsp70 depends on p53, we compared 2 cell lines, A549 with a functioning p538 and NCI-H358, which are homozygous-deficient for this protein.9 Using an antibody that recognizes activated p53 phosphorylated on serine 15,10 we noted that Ad.asHsp70 but not Ad.ß-gal activated p53 in A549 (Figure 5). Although no activation of p53 was observed in NCI-H358 (Figure 4), both cell lines showed similar rates of cell death after treatment with Ad.asHsp70 (Figure 4).



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  		Figure 5. Cell death induced by down-regulation of Hsp70 does not depend on p53. A549 and NCI-H358 cells were treated as indicated. Virus-treated cells were lysed 96 hours after transduction. Activated p53 phosphorylated on serine 15 was determined by Western blot. To confirm equal protein levels the same blot was stripped and developed with an anti-{alpha}-tubulin antibody.

 
Relation of Ad.asHsp70-induced cell death to DNA cleavage
Treatment of A549 cells with Ad.asHsp70 induced DNA cleavage (22.0 ± 3.0% versus 0.6 ± 0.6% in nontreated cells, P < .001), which was determined by TUNEL assay. This is 2-fold more than that measured for A549 transduced with the control virus Ad.ß-gal (10.2 ± 4.0%; Figure 6) . Cotreatment of Ad.asHsp70-transduced cells with 10 µmol/L zVAD, a broad caspase inhibitor, decreased the amount of cleaved DNA to the level of cells transduced with Ad.ß-gal (11.9 ± 3.4%; Figure 6). However, 10 µmol/L zVAD did not affect cell death induced by Ad.asHsp70 (Figures 2, D and 3; P < .01 for Ad.asHsp70 + zVAD–treated versus nontreated cells). Therefore, DNA cleavage is not essential for cell death induced by down-regulation of Hsp70 in lung cancer cells.



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  		Figure 6. DNA cleavage is not essential for cell death induced by depletion of Hsp70. A549 cells were transduced with Ad.asHsp70 or Ad.ß-gal as indicated. zVAD was used in a concentration of 10 µmol/L; fresh zVAD was added after 48 hours. Cells were harvested after 96 hours, and TUNEL staining was performed followed by flow cytometry. *P < .001 versus Ad.ß-Gal (mean ± SD of 4 independent experiments).

 

   Discussion
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 Abstract
 Material and methods
 Results
 Discussion
References
 
In the present study we demonstrated that adenovirus-mediated selective down-regulation of Hsp70 induces cell death in lung cancer cells but not in normal lung fibroblasts or in primary normal human epithelial cells. Hsp70 belongs to the family of heat shock proteins, a phylogenetically conserved family of proteins. Heat shock proteins act as molecular chaperones protecting cells from toxic (eg, misfolded or aggregated) proteins derived in stress situations.4 It has been shown by several groups that increased expression of heat shock proteins correlates with enhanced resistance to apoptosis induced by a wide range of stimuli.5 These findings led to the hypothesis that heat shock proteins may be involved in tumorigenesis. Indeed, increased expression of heat shock proteins was demonstrated for a wide range of tumors and has been correlated with poor prognosis.3,4 However, others could not find such a correlation.11 In NSCLC increased expression of Hsp70 was demonstrated in dysplastic lesions and stage I tumors, suggesting an involvement of Hsp70 in early carcinogenesis.12 Recently, some of the molecular mechanisms for protection of cells by heat shock proteins have been identified. It was shown that Hsp70 is able to prevent activation of caspases, the execution proteases in programmed cell death. Namely, Hsp70 acts on the level of caspase-9, involving the mitochondrial pathway of apoptosis.13

Based on this background, it was hypothesized that down-regulation of Hsp70 would render tumor cells susceptible to proapoptotic stimuli. This was demonstrated by Robertson and colleagues,14 who showed synergistic effects of Hsp70 antisense oligomers and proteasome inhibitors. Surprisingly, 2 other studies demonstrated that depletion of Hsp70 by antisense alone is sufficient to induce cell death in tumor cells. Kaur and colleagues15 induced cell death in human oral cancer cells using Hsp70 antisense oligonucleotides, whereas Nylandsted and colleagues6 treated human breast carcinoma cells with an adenovirus expressing an Hsp70 sequence in antisense orientation. The same virus was used in our study to evaluate whether lung cancer cells undergo cell death when Hsp70 is down-regulated. We could demonstrate that the lung cancer cell lines used, but not normal lung fibroblasts or normal human bronchial epithelial cells, underwent cell death. Therefore, these data strongly support the idea that down-regulation of Hsp70 induces tumor-specific cell death. However, when comparing the data of expression of Hsp70 in the cancer and normal lung cells with the rate of cell death induced by down-regulation of Hsp70, there was no correlation between expression level and the extent of cell death. One might speculate from this whether in certain types of cancer cells different cellular factors are necessary for survival. The data also suggest that overexpression of Hsp70 alone does not account for the decision to be a cancer cell or not.

Why and how tumor cells die from selective depletion of Hsp70 is not known yet. Nylandsted and colleagues6 suggested that down-regulation of Hsp70 induces a specific form of apoptosis or programmed cell death that is caspase-independent and cannot be blocked by overexpression of Bcl-2.

Our data indicate that cell death induced by down-regulation of Hsp70 is not p53-dependent. p53 is a tumor suppressor gene that is frequently mutated in human cancer. A nonfunctional p53 gene can render tumor cells resistant to proapoptotic stimuli.16 Consequently, there is a need for antitumor strategies that are p53-independent, as we have shown in the present study for selective depletion of Hsp70. Also important, by demonstrating the difference in activity of p53 in lung cancer cells treated with Ad.asHsp70 or with control virus Ad.ß-gal, these data give some more evidence that the observed cell death after treatment with Ad.asHsp70 is due to depletion of Hsp70 and not an artifact of the adenovirus itself.

In addition to p53, we examined whether tumor-selective cell death induced by Hsp70 depletion depends on nuclear changes—for example, DNA fragmentation caused either by activation of caspase-activated DNase (CAD)17 or by activation of mitochondrial apoptosis-inducing factor (AIF).18 Although activation of CAD requires activation of caspases, mitochondrial AIF acts in a caspase-independent manner. Therefore, if cell death induced by down-regulation of Hsp70 is a form of caspase-independent apoptosis,6 mitochondrial AIF would be most likely a downstream target. This hypothesis is supported by data from Ravagnan and colleagues19 showing an interaction between Hsp70 and mitochondrial AIF and demonstrating that Hsp70 is able to block mitochondrial AIF. Our own findings do not confirm the idea that mitochondrial AIF is involved in this form of cell death. In contrast, our results suggest that DNA fragmentation itself is not essential, which might indicate that cytosolic changes are more important for cell death induced by depletion of Hsp70. Similar data showing that DNA damage is only of minor importance for cell death have been demonstrated for ultraviolet-induced apoptosis.20 Remarkably, Nylandsted and colleagues6 conclude from morphologic evaluation of nuclear changes by electron microscopy that cell death induced by down-regulation of Hsp70 is a form of apoptosis. Because we hypothesized a minor importance for nuclear changes, our data do not support the idea that down-regulation of Hsp70 initiates apoptosis. Which kind of cell death is based on down-regulation of Hsp70 and how it is mediated on cellular level needs further evaluation.

In conclusion, the present study demonstrates that adenovirus-mediated selective depletion of Hsp70 can efficiently induce cell death in lung cancer cells but not in normal lung fibroblasts. Furthermore, it shows that this effect does not depend on p53 and DNA fragmentation. Therefore, it might help to develop new therapeutic p53-independent strategies for the treatment of lung cancer. Further in vivo studies in our group will prove whether cell death induced by depletion of Hsp70 will also efficiently work in primary culture of lung cancer derived from patient material. Preliminary results from these experiments show that it is difficult to achieve high transduction efficiency. Encouraged by other promising studies using adenovirus-mediated gene transfer,21,22 we hope to solve the problems of transduction efficiency to obtain more preclinical data for the induction of cell death by antisense Hsp70 treatment in lung cancer.


   Footnotes
 
Supported by grants from the Bernensis Cancer League, Berne, Switzerland (BKL 61), and Novartis Foundation, Basel, Switzerland (00B33).


   References
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 Abstract
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 References
 

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