Figure 1.
Response of small cell lung cancer cells to Hsp90 inhibition.
A, MTT assay of H69 cells treated with geldanamycin for 48 h; B, MTT assay of H69 cells treated with radicicol for 48 h; C, MTT assay of H187 small cell lung cancer cells with geldanamycin for 12, 36 and 60 h; D, MTT assay of H889 cells treated with geldanamycin for 48 h; E, MTT assay of U87MG glioblastoma cells treated with geldanamycin for 48 h; F, H69 cells treated with geldanamycin for 48 h and assayed for viable (○) and total (•) cell counts using a Vi-cell XR cell viability analyzer. Error bars show the mean ± SE of three replicates.
Figure 2.
Proliferation after withdrawal of Hsp90 inhibitors.
Cells were treated for 48 h with the indicated concentration of inhibitor, which was then removed. Viable cell counts were then determined at the indicated times after inhibitor removal. A, H69 cells treated with geldanamycin; B, H187 cells treated with geldanamycin; C, H69 cells treated with 17-AAG. D, H69 cells treated with 100 nM geldanamycin for 4, 12 or 24 h. E, H69 cells were treated for 48 h with 100 nM geldanamycin in the absence or presence of 100 µM Z-VAD-FMK, a general caspase inhibitor. Z-VAD-FMK treatment was started 1 h before the addition of geldanamycin. F, U87MG cells treated with geldanamycin.
Figure 3.
BrdU incorporation and client protein responses in Hsp90 inhibitor-treated cells.
A. H69 cells were treated for 48 h with 100 nM geldanamycin. Geldanamycin was then removed. Six or eight days after drug removal, 25,000 treated cells (GA) per well were plated in ninety-six well plates along with the same number of untreated H69 cells for comparison (no GA). Cells were allowed to uptake BrdU for 16 h and BrdU incorporation was then assayed as described in Materials and Methods. Results were normalized to the values for untreated cells. Data shown are the mean ± SD of six replicates. B. Cells were treated for 48 h with geldanamycin. Total cell lysates were then collected and analyzed by Western blotting using antibodies to the indicated proteins. The bottom panel shows a blot stained for total protein with amido black prior to antibody incubation. Equal amounts of DMSO were added to cells for each treatment, except in the leftmost lane, where DMSO was omitted.
Figure 4.
Senescence markers in Hsp90-inhibitor-treated small cell lung cancer cells.
A. H69 cells were treated with DMSO alone or 100 nM geldanamycin for 48 h. Geldanamycin was then removed. Eight days later cells were allowed to settle onto poly-L-lysine-coated coverslips, fixed with paraformaldehyde and photographed under phase contrast using a 63X objective lens. B. SAHF formation after treatment of cells with Hsp90 inhibitors. H69 cells were treated with DMSO or 100 nM geldanamycin for 48 h. Cells were then fixed, stained with DAPI and examined by fluorescence microscopy. C. H69 cells were treated with DMSO (control) or 100 nM geldanamycin. DMSO and geldnamycin were then removed on Day 0. Total cell lysates were collected on the indicated days after geldanamycin removal and analyzed for γH2AX and H2AX levels by Western blotting. The graph shows densitometry for Western blot analyses of three biological replicates. Data were normalized to the vehicle-treated control cells and are shown as the mean ± SE.
Figure 5.
Properties of variant H69 cells that recover growth after Hsp90 inhibitor withdrawal.
A. Photographs of H69 and H69/41d cells under phase microscopy, at low (top panels) and high (bottom panels) magnification; B. H69 and H69/41d cells were treated with 100 nM geldanamycin for 48 h. Geldanamycin was then removed and numbers of viable cells were assessed at the indicated times after drug removal. C. H69 and H69/41d cells were treated with 5 µM radicicol for 48 h. Radicicol was then removed and numbers of viable cells were assessed at the indicated times after drug removal. D. MTT assay of H69/41d cells treated for 48 h with geldanamycin.
Figure 6.
Client protein degradation and SAHF induction in H69/41d cells.
H69 and H69/41d cells were treated for 48 h with geldanamycin or DMSO as vehicle control. A. Total cell lysates were collected after 48 h treatment and analyzed by Western blotting as in Figure 3. B. DMSO-treated cells were stained for SAHF 2 days after removing the DMSO. Geldanamycin-treated cells were stained for SAHF 2, 4 and 6 days after drug removal. Representative fluorescence microscopy images of H69 and H69/41d cell nuclei, six days after removal of geldanamycin, are shown. C. SAHF in H69 (light gray) and H69/41d cells (dark gray) were counted at the indicated times after drug removal. Data are expressed as the percent of nuclei that are SAHF positive. Bars show the mean percentage ± SE. D. Total cell extracts of untreated H69 and H69/41d cells were analyzed for expression of p21 protein by Western blotting.