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. 2020 Jan 17;9(1):238. doi: 10.3390/cells9010238

Figure 2.

Figure 2

Low homologous recombination (HR) capacity is compatible with efficient replication fork protection and resistance to PARP1 inhibition and mitomycin C (MMC) in TNBCs. (A) Immunoblot detection of BRCA1, BRCA2, ATR, FANCD2, PARP1, CHK1 and RAD51 derived from total cell extracts of exponentially growing MCF7 and MDA-MB-231/BR/SA cells. The MDA-MB-231 showed a slightly increased expression of CHK1 (1.49 ± 0.1) and a further increase in BR (3.7 ± 0.1) and SA (4.3 ± 0.3) compared to MCF7 cells. The same pattern is observed for the expression of RAD51 with a significant increase of 1.64 ± 0.3 in MDA-MB-231, 2.4 ± 0.5 and 2.6 ± 0.3 in BR and SA compared to MCF7 cells. ß-Actin was used as the loading control. Data are presented as mean ± SEM. Immunoblot signals were detected and quantified by a LiCor system. (B–D) Repair of open and replication-associated DNA double-strand break (DSB) as a measure of HR capacity, determined by plasmid-reconstruction assay and analyzed by FACS. Cells were transiently transfected with the pDRGFP construct (C) or DR-ori-GFP plus the ori-activating MSCV-N EBNA1 construct (D) for 24 h. The number of GFP-expressing cells was normalized to the absolute HR capacity of MCF7. A significantly lower HR capacity for frank DSB was found in all TNBCs compared to MCF7 cells, with 0.12 ± 0.008 for MDA-MB-231, 0.23 ± 0.04 for BR, and 0.45 ± 0.07 for SA. The same pattern was observed for DSBs adjacent to a DNA replication origin, with 0.17 ± 0.03 for MDA-MB-231, 0.27 ± 0.03 for BR, and 0.75 ± 0.05 for SA compared to MCF7 cells. (E) Mean length of DNA fibers in MCF7 and MDA-MB-231/BR/SA cells. The cells were sequentially labelled with CldU and IdU for 30 min and treated with HU between both labels for 4 h. DNA was spread on slides, fixed and incorporated nucleotides were detected by immunofluorescence. Although all cell lines showed a shortening of the CldU tract, MCF7 showed the most pronounced shortening with 0.8 ± 0.004, followed by MDA-MB-231 and BR with 0.89 ± 0.003 and 0.89 ± 0.001. SA showed only a minimal shortening of the CldU tract with 0.93 ± 0.006. The length of the DNA fibers was measured with the Image J software and calculated relative to the absolute length of the untreated controls. (F) Cellular survival after treatment with olaparib (left) or MMC (right) in MCF7 and MDA-MB-231/BR/SA cells. The cells were seeded 24-h prior treatment with olaparib or MMC for 5 days or 1 h, fixed after 14 days, and the number of colonies was counted. Shown are means from three independent experiments ± SEM. Asterisks represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n.s. not significant; Student’s t-test).