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First published online 24 October 2006
doi: 10.1242/jcs.03263

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DNA damage responses in progeroid syndromes arise from defective maturation of prelamin A

Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA

View larger version (16K): [in a new window]   Fig. 1. Early replication arrest of HGPS and RD cells. The replication assay was performed with [methyl-3H] thymidine labeling as described in Materials and Methods. Symbols represent radioactivity values as follows: , BJ cells; , HGPS cells; , RD cells. The values were calculated from three independent experiments. Error bars represent standard deviations.

View larger version (43K): [in a new window]   Fig. 2. Activation of ATM and ATR in HGPS and RD cells. (A) CPT treatment was done by incubating the cells with 4 µM camptothecin for 1 hour. Cells with or without treatment were fixed with cold methanol (–20°C) followed by immunofluorescence microscopy with ATM antibody staining. Blue, DAPI; red, ATM. (B) UV treatment was performed by irradiating the cells with 20 J/m2 UV. Two hours after the treatment, the cells with or without treatment were fixed and stained with ATR antibody for immunofluorescence microscopy. Blue, DAPI; red, ATR. (C) HeLa cells grown on coverslips were transfected with the plasmid for expressing progerin (LA50) or empty parent vector. Twenty-four hours after transfection, the cells were irradiated with 20 J/m2 UV or mock treated. After additional 2-hour culture, the cells were processed for immunofluorescence microscopy. Blue, DAPI; green or red, ATR. Photomicrographs were taken at 63x magnification. Bar, 50 µm.

View larger version (24K): [in a new window]   Fig. 3. Phosphorylation of Chk1, Chk2 and p53 in HGPS and RD cells. Western blotting was performed as described in Materials and Methods. The phosphorylation status of p53 was determined with p53 Ser-15 phosphorylation-specific antibody. The total p53 was probed as the loading control to ensure that the same amounts of p53 were loaded for BJ, RD and HGPS cells. In the right panel, ß-actin was probed to ensure that similar amounts of proteins were loaded for the three cell lines.

View larger version (28K): [in a new window]   Fig. 4. Restoration of replication activity in HGPS cells by inactivation of ATM and ATR. (A) Western blotting shows the knockdown of ATR and ATM in BJ and HGPS cells by RNAi. ß-actin was used as sample loading control. (B) The replication assay was performed with [methyl-3H]-thymidine labeling as described in Materials and Methods. BJ cells and HGPS cells used were at passage 12. The asterisk indicates a significant difference, P<0.05.

View larger version (17K): [in a new window]   Fig. 5. FTI treatment of HGPS and RD cells. (A) Improvement of nuclear shapes of HGPS and RD cells by FTI treatment. The patient cells were treated with FTI and prepared for immunofluorescence microscopy as described in Materials and Methods. The nuclei were stained with DAPI. Misshapen nuclei are indicated by arrows. Photomicrographs were taken at 63x magnification. Bar, 50 µm. (B) FTI treatment blocked farnesylation of prelamin A, but did not change the production of -H2AX, a molecular marker for DSBs. In the left panel, BJ and HGPS cells were lysed in 2x SDS gel loading buffer and probed with the antibody directly against prelamin A. In the right panel, HGPS and RD cells were lysed and probed with the antibody for -H2AX. ß-actin was loaded to ensure similar amounts of samples were used for each pair of experimental groups. (C) Comet assays were performed with HGPS cells in the presence and absence of FTI as described in Materials and Methods. DSBs of 50 randomly chosen cells were quantitated as the percentage of DNA in tail. (D) Phosphorylation of Chk1 and Chk2 in HGPS cells with or without FTI treatment. The FTI treatment of BJ and HGPS and western blotting were performed as described in Materials and Methods. The phosphorylation statuses of Chk1 at Ser-345 and Chk2 at Thr-68 were determined with corresponding antibodies. ß-actin was probed as a loading control. (E) Accumulation of -H2AX in cells expressing prelamin A mutants. HeLa cells were transfected with plasmid pEGFP (empty parent vector), pEGFP-LA50 or pEGFP-LA50-SSIM, followed by western blot analysis. GAPDH was probed as a loading control.