PMC

In response to MMS, human cells induce a signaling pathway that culminates in BRCA1/BARD1 downregulation. This prevents the unwanted assembly of the HR machinery at the early stage of the MMS-induced DNA damage response. At the second stage, recovery and assembly of HR proteins ensure the repair of DSBs generated by replication blocks.

A) Schematic view of the deletion constructs used in this study. B) HeLa cells were transfected with various expression constructs for BRCA1 and 2 days post-transfection, cells were treated with 200 µM MMS and harvested at the indicated times for immunoblotting to detect either endogenous BRCA1 or mutant forms using anti-BRCA1 or anti-GFP respectively. β-actin immunodetection was used as a loading control.

A) Immunostaining of BRCA1 in HeLa cells treated with 200 µM MMS. Cells were harvested at 3 and 6 hrs or changed to MMS free medium for the later times. The nuclei were counterstained with DAPI. B) Top left, immunoblotting of BRCA1 and apoptosis markers, PARP-1 and Caspase-3, in HeLa cells treated as indicated above. Bottom left, BRCA1 band intensity was quantified and data are expressed as percentage of untreated cells. Right, immunoblotting for PARP-1 and Caspase-3 following treatment with high dose of UVC (100 J/m²).

A) Top, BRCA1 expression in HeLa cells treated with UVC (30 J/m²) was detected by immunoblotting after harvesting at the indicated times. Bottom, immunostaining of BRCA1 at 4 hrs post-treatment. DNA was counterstained with DAPI. B) BRCA1 levels in HeLa cells treated with IR (10 Gy) for the indicated times. C) BRCA1 levels in HeLa cells treated with the DNA alkylating agent, methylmethanesulfonate (MMS, 200 µM) for the indicated times. D) BRCA1 levels in various cell types treated with 200 µM MMS for the indicated times. All immunoblottings were conducted using total cell extracts. β-actin was detected to ensure equal protein loading.

A) Immunodetection of various BRCA1-associated and DNA damage/repair proteins following treatment of HeLa cells with 200 µM MMS. HeLa cells were treated with 200 µM MMS and harvested at the indicated times for immunoblotting. The star indicates the specific protein band detected with a given antibody. B) Foci formation of HR proteins following MMS treatment. HeLa cells were treated with 200 µM MMS and harvested at the indicated times for immunostaining. Bottom, cells with more than 10 foci were counted and the data are presented as percentage of cells with foci under each condition. The values represent the average ± SD of three independent experiments.

A) U2OS cells were pre-treated with IR (10 Gy) for 12 hrs and then with or without 200 µM MMS for 6 hrs and harvested for immunostaining. Bottom, cells with more than 10 foci were counted and the data are presented as percentage of cells with foci under each condition. The values represent the average ± SD of three independent experiments. B) Immunoblotting detection of BRCA1, BARD1 and Rad51 in HeLa cells pre-treated with IR (10 Gy) for 12 hrs and then left untreated or exposed to 200 µM MMS for 3 and 6 hrs.

A) Synchronized HeLa cells, using a thymidine double block (TDB) method, were treated with 200 µM MMS for 3 hrs at various time points post-release. Cell cycle analysis (top panel) and immunoblotting (low panel) were conducted at the indicated time points. B) Downregulation of BRCA1 during cell cycle progression in primary cells. Top, human primary fibroblasts were synchronized in G0/G1 by contact inhibition and were released by replating at low density. Bottom, following UVC (30 J/m²) treatment for the last 2 hrs, cell were harvested at the indicated times for immunoblotting. β-actin immunodetection was used as loading control.

A) HeLa cells were incubated with 20 µg/ml of cycloheximide alone or with 200 µM MMS (with or without cycloheximide) and harvested at the indicated times for immunoblotting. B) HeLa cells were pre-treated with 20 µM proteasome inhibitor MG132 for 30 min and then incubated with MMS in the presence of the inhibitor and harvested at 6 hrs for immunoblotting. C) Detection of BRCA1 ubiquitination following DNA damage in HEK293T cells. Following MMS treatment for 3 hrs, cell extracts were used for immunoprecipitation with an anti-BRCA1 antibody. A non-related polyclonal antibody was used as a control. The immunoprecipitates were used for immunoblotting using anti-BRCA1 or anti-ubiquitin antibodies. Densitometry quantification was conducted on BRCA1 and ubiquitin and the ratio ubiquitin/BRCA1 is shown.

A) HeLa cells were treated for 6 hrs with IR (10 Gy) or 200 µM MMS (with or without pretreatment with MG132). Chromatin from control or treated cells was prepared as described in and proteins were detected by western blotting. Histones were stained with coomassie blue to ensure equal loading. B) HeLa cells were treated as in panel A and harvested for immunostaining (left panel). Cells with more than 10 foci were counted and the data are presented as percentage of cells with foci under each condition (right panel). The values represent the average ± SD of three independent experiments. C) HeLa cells were treated as in A except that a permeabilization step was added before fixation and immunostaining (left panel). Cells with more than 10 foci were counted and the data are presented as percentage of cells with foci under each condition (right panel). The values represent the average ± SD of three independent experiments.

A) Immunoblotting detection of BRCA1 in HeLa cells treated with 10 Gy IR or 200 µM MMS. B) BRCA1 downregulation is not blocked by caffeine. Immunoblotting detection of BRCA1 in HeLa cells pre-treated with 10 mM caffeine for 30 min prior to 200 µM MMS treatment for 6 hrs. C) The downregulation of BRCA1 is not prevented by the ATM inhibitor (KU-55933). Immunoblotting detection of BRCA1 in HeLa cells pre-treated with 10 µM KU-55933 for 30 min prior to 200 µM MMS treatment for 6 hrs. γH2AX and pChk2 detection were used as controls to confirm inhibition of ATM and/or ATR kinases. D) BRCA1 is downregulated in ATM-deficient human fibroblasts. Cells were treated with 200 µM MMS treatment for 6 hrs and harvested for immunoblotting. E) Depletion of ATR by RNAi does not prevent BRCA1 downregulation by MMS. Left, immunodetection of ATR following shRNA constructs transfection and puromycin selection. Right, ATR-depleted cells were treated with 200 µM MMS and harvested at the indicated times for immunoblotting. F) BRCA1 is downregulated in DNA-PKcs deficient cells. Glioblastoma DNA-PKcs proficient (MO59K) or deficient (MO59J) were treated with 200 µM MMS and harvested at the indicated times for immunoblotting.