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. 2010 Jun;176(6):2935–2947. doi: 10.2353/ajpath.2010.090779

Figure 3.

Figure 3

Fanconi anemia-like phenotype in malignant breast cancer cell lines that lack FANCD2 expression. A: Western blots of high invasive malignant cell lines MDA-MB-157 and MDA-MB-231. They lack immunodetectable FANCD2 in whole cell extracts (cells treated with 50 nmol/L MMC for 18 hours before preparation of lysates). Transduction of a human cDNA for FANCD2 into MDA-MB-157 restores the expression of both isoforms of FANCD2. Loading has been normalized to that of β-actin and the arrows indicate the position of the marker proteins for FANCD2-L, FANCD2-S, and β-actin. B: Growth inhibition by MMC is shown as the mean of three independent experiments for each of the indicated cell lines. The mean percentages (±SD) of cells after exposure to MMC for 72 hours, relative to the untreated cells are shown. MDA-MB-157 and MB-MB-231 exhibit a response to MMC that is similar to that shown by the Fanconi anemia patient FA-D2 cell line PD20. The MMC-response of MDA-MB-157 cells transduced with an expression vector for FANCD2 (+FANCD2), is very similar to PD20-3-15, indicating functional complementation of MDA-MB-157 by the wild-type FANCD2 cDNA. C: Induction of chromosomal aberrations in MDA-MB-157 and MDA-MB-157+FANCD2 by MMC. Chromosomal aberrations were scored by metaphase analysis in cells exposed to MMC for 24 hours before the addition of colcemid for 2 hours. For each cell line/treatment, 20 metaphases were scored for chromosomal aberrations using the criteria described by Scott et al.35 Chromatid breaks or exchanges involve single arms of metaphase chromosomes, while chromosome breaks or exchanges involve both arms. No chromosomal aberrations were observed in untreated MDA-MB-157 cells while 2 chromosome exchanges (a ring chromosome and a dicentric) were observed in untreated MDA-MB-157+FANCD2 cells. On treatment with MMC, the level of aberrations in MDA-MB-157 increased to a mean of 3.5 chromosomal aberrations per cell. On average, 2.1 chromatid breaks, 1.3 chromatid exchanges and 0.1 chromosome breaks per cell were observed, with all of the chromatid exchanges being complex arrangements (exchanges involving more than two chromosomes), typical of those seen in FA patient cells following MMC treatment.44 In contrast, MDA-MB-157+FANCD2 cells exhibited a much more modest increase in chromosomal aberrations with MMC (0.4 per cell; 5 chromatid breaks, 2 chromatid exchanges and a single chromosome exchange in the 20 metaphases scored).