Figure 7
Figure 7. 17-AAG enhances DNA cross-linker–induced cytotoxicity and chromosome abnormalities. (A) HeLa cells were treated with various concentrations of cisplatin (CDDP) alone or with 250 nM 17-AAG for 14 hours. RPMI8226 cells were treated with various concentrations of cisplatin (CDDP) alone or with 1 μM 17-AAG for 20 hours. Cells were washed and then incubated in drug-free culture medium. After 72 hours from the time of initial drug application, cell survival was colorimetrically determined. Data represent means ± SD from triplicate measurements. (B) FANCA-deficient (GM6914) and complemented (GM6914/FANCA) cells were treated with various concentrations of MMC alone or with 250 nM 17-AAG for 14 hours. Cells were washed and incubated in drug-free culture medium. After 72 hours from the time of initial drug application, cell survival was colorimetrically measured. (C-D) FANCA-deficient (GM6914) and complemented (GM6914/FANCA) cells were treated with MMC (0, 3, 10, 30 nM) and 250 nM 17-AAG for 14 hours. Cells were washed and then incubated in drug-free culture medium to complete a total of 40 hours from the time of initial drug application. Apoptotic cells were detected by TUNEL staining. In each sample, at least 400 cells were examined at original magnification ×100, and percentages of apoptotic cells were determined. Data represent means ± SD from triplicate measurements. TUNEL stainings of GM6914/FANCA cells treated with 10 nM MMC alone (MMC) or with 250 nM 17-AAG (MMC + 17-AAG) are shown in panel D. Images were obtained on an Olympus AX70 microscope equipped with UPlanApo 10×/0.40 NA and WH 10×/22 lenses (Olympus) using a PXL charge-coupled device camera (model CH1; Photometrics). (E) HeLa cells were treated with 100 nM MMC or 2 μM cisplatin (CDDP) with or without 250 nM 17-AAG for 24 hours. Cells in metaphase were assessed as aberrant if they presented chromatid breaks. Data represent means ± SD from 3 independent experiments. Arrowheads indicate chromatid breaks. Images were obtained on a Leica DM3000 microscope equipped with HI PLAN 100x/1.25 NA and HC PLAN 10x/22 lenses (Leica, Bensheim, Germany) using a DFC280 digital camera (Leica). (F) A model illustrating how Hsp90 regulates intracellular stability and trafficking of FANCA. FANCA shuttles between the cytoplasm and the nucleus. Cytoplasmic FANCA, newly synthesized and exported from the nucleus, is folded into proper conformation required for nuclear entry by interacting with the Hsp90-based multichaperone complex.1,2 Hsp90 is probably recycled to form a complex with FANCA in the cytoplasm. The 17-AAG–mediated inhibition of the chaperone cycle promotes proteasomal degradation of FANCA, at least in part, through Hsp70-mediated association with CHIP. In the nucleus, FANCA, -B, -C, -E, -F, -G, -L, and -M are assembled into a multisubunit complex (FA core complex) that is required for FANCD2 activation.

17-AAG enhances DNA cross-linker–induced cytotoxicity and chromosome abnormalities. (A) HeLa cells were treated with various concentrations of cisplatin (CDDP) alone or with 250 nM 17-AAG for 14 hours. RPMI8226 cells were treated with various concentrations of cisplatin (CDDP) alone or with 1 μM 17-AAG for 20 hours. Cells were washed and then incubated in drug-free culture medium. After 72 hours from the time of initial drug application, cell survival was colorimetrically determined. Data represent means ± SD from triplicate measurements. (B) FANCA-deficient (GM6914) and complemented (GM6914/FANCA) cells were treated with various concentrations of MMC alone or with 250 nM 17-AAG for 14 hours. Cells were washed and incubated in drug-free culture medium. After 72 hours from the time of initial drug application, cell survival was colorimetrically measured. (C-D) FANCA-deficient (GM6914) and complemented (GM6914/FANCA) cells were treated with MMC (0, 3, 10, 30 nM) and 250 nM 17-AAG for 14 hours. Cells were washed and then incubated in drug-free culture medium to complete a total of 40 hours from the time of initial drug application. Apoptotic cells were detected by TUNEL staining. In each sample, at least 400 cells were examined at original magnification ×100, and percentages of apoptotic cells were determined. Data represent means ± SD from triplicate measurements. TUNEL stainings of GM6914/FANCA cells treated with 10 nM MMC alone (MMC) or with 250 nM 17-AAG (MMC + 17-AAG) are shown in panel D. Images were obtained on an Olympus AX70 microscope equipped with UPlanApo 10×/0.40 NA and WH 10×/22 lenses (Olympus) using a PXL charge-coupled device camera (model CH1; Photometrics). (E) HeLa cells were treated with 100 nM MMC or 2 μM cisplatin (CDDP) with or without 250 nM 17-AAG for 24 hours. Cells in metaphase were assessed as aberrant if they presented chromatid breaks. Data represent means ± SD from 3 independent experiments. Arrowheads indicate chromatid breaks. Images were obtained on a Leica DM3000 microscope equipped with HI PLAN 100x/1.25 NA and HC PLAN 10x/22 lenses (Leica, Bensheim, Germany) using a DFC280 digital camera (Leica). (F) A model illustrating how Hsp90 regulates intracellular stability and trafficking of FANCA. FANCA shuttles between the cytoplasm and the nucleus. Cytoplasmic FANCA, newly synthesized and exported from the nucleus, is folded into proper conformation required for nuclear entry by interacting with the Hsp90-based multichaperone complex.1,2  Hsp90 is probably recycled to form a complex with FANCA in the cytoplasm. The 17-AAG–mediated inhibition of the chaperone cycle promotes proteasomal degradation of FANCA, at least in part, through Hsp70-mediated association with CHIP. In the nucleus, FANCA, -B, -C, -E, -F, -G, -L, and -M are assembled into a multisubunit complex (FA core complex) that is required for FANCD2 activation.

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