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Volume 130, Issue 7, Pages 2145-2154 (June 2006)
Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options Eike Gallmeier, Eric S. Calhoun, Carlo Rago, Jonathan R. Brody, Steven C. Cunningham, Tomas Hucl, Myriam Gorospe, Manu Kohli, Christoph Lengauer, Scott E. Kern Gastroenterology Volume 130, Issue 7, Pages (June 2006) DOI: /j.gastro Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 1 Structural and functional demonstration of FANCG and FANCC gene disruption. (A) Targeting scheme. OF + OR primer pair outside the deleted sequence; IF + IR primer pair inside the deleted sequence; SF + SR and SF2 + SR2 screening primer pairs; LHA + RHA left and right homology arms. (B) PCR detecting the wt and recombinant alleles. (C) Reverse-transcription PCR detecting the wt and a truncated (ΔEx) mRNA transcript. (D) Western blot detecting FANCG and FANCC protein. FANCC was immunoprecipitated (ip) before Western blotting. (E) Western blot detecting monoubiquitinated (l) and nonmonoubiquitinated (s) FANCD2 protein. (F) Analysis of 50 metaphases of each clone for chromosomal aberrations 48 hours after treatment with DEB. Breaks were scored as 1 and radials and rings were scored as 2 aberrations. ■, % of cells with aberrations; □, aberrations per 50 cells. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 2 Cell-cycle analysis 48 hours after treatment with MMC. (A) Representative cell-cycle profiles. (B) Graphic representation of the fraction of cells in G1, S, or G2/M for all clones tested. Arrows indicate the lowest doses at which a G2/M arrest of more than 40% of the cells was observed. , G1; □, S; ■, G2/M. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 3 Drug-sensitivity studies. Cell proliferation on treatment with (A) ICL-forming or (B) other agents at the indicated concentrations as compared with untreated cells. The IC50 ratios, comparing parental and FA-deficient cells, are indicated. Error bars represent SEM of 4 experiments. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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Figure 4 IR-sensitivity studies. (A) Analysis of 50 metaphases of each clone for chromosomal aberrations 30 minutes after treatment with IR at 2 Gy. Error bars represent SEM of 2 experiments. (B) Colony formation assays on IR. The relative survival of parental cells is presented in comparison with heterozygote (left graph: ■, parental cells; , FANCG+/−; , FANCC+/−/−), FANCC−/−/− (middle graph: ■, parental cells; ○, FANCC−/−/− [1]; ◊, FANCC−/− [2]), or FANCG−/− clones (right graph: ■, parental cells; ◊, FANCG−/− [1]; ▵, FANCG−/− [2]; ○, FANCG−/− [3]). Error bars represent SEM of 2–7 experiments. Gastroenterology , DOI: ( /j.gastro ) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions
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