Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Co-introduction of anti-DNA ligase IV antibody reduces DNA end-joining frequency in normal diploid fibroblasts but does not affect FA cells. (A) End-joining frequency of cohesive-ended DNA was determined in HT1080 cells (black bars) and normal HDFs (white bars) in the presence of no antibody (N), in the presence of anti-DNA ligase IV antibody (L), in the presence of anti-Fancd2 antibody (D), and in the presence of both anti-DNA ligase IV and anti-Fancd2 antibodies (L + D). In all cases, antibody treatment significantly reduced plasmid end-joining levels compared to those observed in cells not treated with antibody, P < 0.0001, χ2-test. (B) End-joining frequency of cohesive-ended DNA (black bars) and blunt-ended DNA (white bars) was determined in patient-derived FA-C cells in the absence of antibody (−) and in the presence of anti-DNA ligase IV antibody (+).

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques: Plasmid Preparation, Derivative Assay

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Immunocytochemical staining for FANCD2. A: Fibroadenoma showing strong (+++) nuclear staining (N) and moderate (++) cytoplasmic staining (C) of the tumor cells. B: Fibroadenoma at higher magnification showing stronger nuclear staining of the myoepithelial cells (M) than the surrounding epithelial cells (E) of the tumor. C and D: Invasive carcinoma showing strong (+++) cytoplasmic staining (arrows) of the carcinoma cells at lower (C) and higher (D) magnification. E: invasive carcinoma showing no nuclear (N) or cytoplasmic staining (C) of the carcinoma cells. F: invasive carcinoma showing borderline (1–5%) cytoplasmic staining (arrow) of the carcinoma cells. G and H: Serial sections of invasive carcinoma incubated with (G) anti-FANCD2 and (H) anti-FANCD2 pre-incubated with immunizing peptide showing (G) strong (+++) cytoplasmic staining (arrows) and (H) no staining (−), respectively. Magnification: ×220 A and C; ×550 B and D–H. Scale bar: 50 μm (A and C); 20 μm (B and D–H).

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining, Incubation

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Nuclear and Cytoplasmic Staining for FANCD2 in Both Benign and Malignant Lesions

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Nuclear and Cytoplasmic Staining of Human Cell Lines for FANCD2

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Immunoblotting for FANCD2 in nuclear and cytoplasmic fractions of cultured cell lines. A: Lysates of breast cell lines SVE3, Huma 121, MCF-7, and MDA-MB-157 were fractionated into nuclear and cytoplasmic extracts and Western blotted for FANCD2 (using H300 anti-rabbit antibody). MMC treatment (+MMC) was at 50 nmol/L for 18 hours before the preparation of lysates. Nuclear (N) extracts show the presence of TATA binding protein (TBP) while cytoplasmic (C) extracts show the presence of tubulin, with no detectable cross-contamination. Immortalized normal (SVE3) and immortalized benign (Huma 121) cell lines both express immunodetectable FANCD2-S (non-monoubiquitylated; 155 kDa) and FANCD2-L (monoubiquitylated; 162 kDa) in the nucleus (irrespective of MMC-treatment), while only FANCD2-S is detectable in the cytoplasm. In the low invasive malignant cell line MCF-7, only the non-ubiquitylated FANCD2 (155 kDa) is immunodetectable and this is confined to the cytoplasm of these cells. The high invasive malignant cell line MDA-MB-157 exhibits no immunodetectable FANCD2 in either fraction. The arrows indicate the position of the marker proteins for FANCD2-L, FANCD2-S, tubulin, and TBP. B: PD20 cells (FA-D2 complementation group) and PD20-3-15 cells (PD20 functionally complemented with human chromosome 3p) were treated with 50 nmol/L MMC for 18 hours before the preparation of lysates. PD20 exhibits no immunodetectable FANCD2 protein. In PD20-3-15, both FANCD2-S (non-monoubiquitylated; 155 kDa) and FANCD2-L (monoubiquitylated; 162 kDa) are present in the nucleus, while only FANCD2-S is detectable in the cytoplasm.

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Western Blot, Cell Culture, Binding Assay, Marker

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: 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).

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Expressing, Western Blot, Transduction, Marker, Inhibition, Plasmid Preparation, Functional Assay

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Association of Immunocytochemical Staining for Cytoplasmic FANCD2 with Other Tumor Variables

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Association of immunocytochemical staining of the primary carcinoma for cytoplasmic FANCD2 with overall survival of patients. A: The cumulative proportion of patients surviving as a fraction of the total at 12 monthly intervals after presentation for patients with carcinomas classified as follows: (a) +++, strong staining (——– ; 100% = 5 patients); (b) ++, moderate staining (------; 100% = 36 patients); (c) +, intermediate staining (........ ; 100% = 55 patients); (d) +/−, borderline staining (– – –); 100% = 96 patients) and (e) −, negative staining (- — - —, 100% = 118 patients) for FANCD2. There were 5 censored observations in a (4 dead of other causes (doc); cumulative proportion surviving (cps) = 100%; median survival time (mst) > 204 months); 31 in b (14 doc; cps = 84%; mst >216 months); 52 in c (13 doc; cps = 94%; mst >216 months); 65 in d (17 doc; cps = 64%; mst >228 months); and 15 in e (12 doc; cps = 4%; mst = 46.8 months). The cumulative proportions surviving ± 95% CIs were (a) 1.00 ± 0.0, (b) 0.91 ± 0.05, (c) 0.94 ± 0.03, (d) 0.75 ± 0.05, and (e) 0.33 ± 0.04, after 5 years; (a) 1.00 ± 0.0, (b) 0.84 ± 0.07, (c) 0.94 ± 0.03, (d) 0.69 ± 0.05, and (e) 0.16 ± 0.03, after 10 years; (a) 1.00 ± 0.0, (b) 0.84 ± 0.07, (c) 0.94 ± 0.03, (d) 0.64 ± 0.05, and (e) 0.04 ± 0.03, after 15 years; and (a) 1.00 ± 0.0, (b) 0.84 ± 0.07, (c) 0.94 ± 0.03, (d) 0.64 ± 0.05, and (e) 0.04 ± 0.03, after 20 years. The five curves were significantly different (Wilcoxon statistic χ2 = 125.83, 4 d.f., P < 0.0001), and significantly different in the successive pairwise combinations of c with d (χ2 = 13.85, 1 d.f., P < 0.0001) and d with e (χ2 = 53.29, 1 d.f., P < 0.0001) but not b with c (χ2 = 2.03, 1 d.f., P = 0.155) nor a with b (χ2 = 0.63, 1 d.f., P = 0.43). The malignant grade of the 5 strong staining carcinomas (+++) (a) was confirmed as grade I (2 patients), II (2 patients), and III (1 patient). B: The cumulative proportion of patients surviving as a fraction of the total at 12 month intervals after presentation with carcinomas classified as (a) positive staining using a 1% cut off (——– ; 100% = 192 patients) and (b) −, negative staining (----- ; 100% = 118 patients) for FANCD2. There were 153 censored observations in a (48 doc; cps = 77%; mst >228 months) and 15 in b (12 doc; cps = 4%; mst = 46.8 months). The cumulative proportions surviving ± 95% CIs were (a) 0.84 ± 0.03, (b) 0.33 ± 0.04, after 5 years; (a) 0.80 ± 0.03, (b) 0.16 ± 0.03, after 10 years; (a) 0.77 ± 0.03, (b) 0.04 ± 0.03, after 15 years; and (a) 0.77 ± 0.03, (b) 0.04 ± 0.03, after 20 years. The two lines were significantly different (Wilcoxon statistic χ2 = 117.14, 1 d.f., P < 0.0001).

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: Staining, Negative Staining

Journal:

Article Title: Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

doi: 10.2353/ajpath.2010.090779

Figure Lengend Snippet: Summary of the Cox’s Proportional Hazards Model for Cancer-Related Deaths

Article Snippet: Immunofluorescent staining of MCF-7 and T47D cell lines was conducted with rabbit (H300; sc-28194, Santa Cruz) and goat (E19; sc-23584, Santa Cruz) polyclonal anti-FANCD2 at dilutions of 1:10 on neutral buffered formalin or methanol-fixed cultures grown in multiwell Lab-Tek 8 chambered slides (177445, Nunc Scientific, Roskilde, Denmark) and bound antibodies were detected with the relevant fluorescein isothiocyanate-labeled second antibodies at 1:50 dilution, as described previously.

Techniques: