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.

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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, MD, Manuela Schaper, BSc, Jan-Rasmus Kuster, MD, Daniela Miescher, Marja Jäättelä, MD, PhD, Thomas Buehler, PhD, Ralph A Schmid, MD The Journal of Thoracic and Cardiovascular Surgery Volume 126, Issue 3, Pages 748-754 (September 2003) DOI: 10.1016/S0022-5223(03)00703-7

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 α-tubulin. The Journal of Thoracic and Cardiovascular Surgery 2003 126, 748-754DOI: (10.1016/S0022-5223(03)00703-7)

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. The Journal of Thoracic and Cardiovascular Surgery 2003 126, 748-754DOI: (10.1016/S0022-5223(03)00703-7)

Figure 3 Transduction efficiency and morphologic changes in IMR90 (A), NHBE (B), and A549 (C) after treatment with Ad.asHsp70 and Ad.β-gal. To detect transduction efficiency, cells were stained 24 hours after transduction with Ad.β-gal for β-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). The Journal of Thoracic and Cardiovascular Surgery 2003 126, 748-754DOI: (10.1016/S0022-5223(03)00703-7)

Figure 4 Viability of lung cancer cells, lung fibroblasts, and NHBE cells after transduction with Ad.asHsp70 and Ad.β-gal. Cells were transduced with Ad.asHsp70 or Ad.β-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. The Journal of Thoracic and Cardiovascular Surgery 2003 126, 748-754DOI: (10.1016/S0022-5223(03)00703-7)

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-α-tubulin antibody. The Journal of Thoracic and Cardiovascular Surgery 2003 126, 748-754DOI: (10.1016/S0022-5223(03)00703-7)

Figure 6 DNA cleavage is not essential for cell death induced by depletion of Hsp70. A549 cells were transduced with Ad.asHsp70 or Ad.β-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.β-Gal (mean ± SD of 4 independent experiments). The Journal of Thoracic and Cardiovascular Surgery 2003 126, 748-754DOI: (10.1016/S0022-5223(03)00703-7)