Flow cytometric characterization of the response of Fanconi's anemia cells to mitomycin C treatment

Abstract

DNA flow histogram analysis, using 33342 Hoechst as a stain, has been used to detect the effect of the potentially bifunctional alkylating agent, mitomycin C (MMC) on dermal fibroblasts from patients with Fanconi's anemia (FA), a hereditary human disease characterized by pancytopenia, hypersensitivity to DNA-crosslinking agents, congenital abnormalities, and a predisposition for neoplasia. At 24 or 48 hr after a 2-hr exposure to 0.05 or 0.10 pg/ml MMC, 3HdT incorporation was reduced to a greater extent in FA cells than in normal cells. Cells sorted from the last half of S phase showed a slightly greater inhibition of 3HdT incorporation than did those sorted from the first half of S.Fanconi's anemia cells exhibited a marked accumulation in the Gz + M peak of flow histograms following exposure to MMC. Twenty-four hr after treatment with 0.05 pg/ ml MMC, the G2 + M fraction of FA cells (eight lines) increased to more than 0.5 from a control value of approximately 0.2. Both normals (six lines) and heterozygotes (eight lines) showed, on the average, much less of a Gz + M increment than did FA cells, even after exposure to 0.1 pg/ml MMC. Examination of cells sorted from the Gz + M peak revealed that MMC-treated FA cells were blocked prior to mitosis.To determine whether the response of FA cells was specific for a bifunctional alkylating agent, cells were also treated with ethylmethanesulfonate, a monofunctional agent. Twenty-four hours after exposure to 0.25 or 0.5 mg/ml ethylmethanesulfonate, FA and normal cells showed similar, small increases in the G2 + M peak. The results suggest the utility of flow cytometry in the diagnostic evaluation of fibroblasts from patients suspected of having Fanconi's anemia.Key terms: Fanconi's anemia, DNA crosslinking agent, mitomycin C, flow cytometry, 33342 Hoechst, Gz block, Cell cycle analysis, DNA repair Fanconi's anemia (FA), a syndrome of pancytopenia and a variable constellation of congenital anomalies (12,34,44), has been shown to be associated with chromosomal instability (8, 42, 47) and a predisposition for cancer (5,16, 43). Cells from patients with FA show increased sensitivity to agents capable of producing DNA crosslinks. This sensitivity is evident as decreased survival (51), increased chromosome breakage (3, 29,40) and a reduction in both the rate of progression through I This research was supported by grants CD-36F from the American Cancer Society and GM21121 from the National Institute of General Medical Sciences.Presented at the VIII Conference on Analytical Cytology and Cytometry, Wentworth-by-the-Sea, New Hampshire, May 19-25, 1981. the cell cycle (41) and in the proportion of mitotic cells (17, 51), following exposure to such agents.A reduction in the ability of cells from patients with Fanconi's anemia to remove DNA crosslinks, caused e.g. by mitomycin C (MMC), was observed in some studies (15) but not in others (13, 25). There are also isolated reports that cells from patients with FA exhibit other defects, such as a reduced ability to remove damage induc...

FANCG Is Phosphorylated at Serines 383 and 387 during Mitosis

“…24,2004 FANCG IS PHOSPHORYLATED AT SERINES 383 AND 387 8577 (13). After deletion of poor quality spectra and conversion to .dta file format, SEQUEST was used to search against a FANCG database.…”

“…Indeed, such sensitivity results in chromosomal breakage, a phenotype utilized in a clinical test for FA. The reaction to cross-linking may also be manifest by the exhibition of G 2 delay, which has been shown by some to be an S-phase defect in FA cells (12,24,28). Nonetheless, no biochemical mechanism has been elucidated to explain these findings.…”

High-Throughput Screening Identifies Novel Agents Eliciting Hypersensitivity in Fanconi Pathway–Deficient Cancer Cells

“…Thus, the increased sensitivity of FA pathway-deficient cells to 80136342 seemed to be mechanistically distinct from the hypersensitivity to ICL agents or irradiation and could not be attributed to a cellular impairment in ICL or DSB repair. On the other hand, 80136342 mimicked the hypersensitivity phenotype of ICL agents with regard to cell cycle dysregulation (10,(30)(31)(32). FANCC À/À/À cells and, to a lesser extent, FANCG À/À cells had a more accentuated G 2 (but not M) phase arrest at low concentrations of 80136342 than FA pathwayproficient cells, suggesting that the commonly observed G 2 cell cycle abnormalities of FA pathway-deficient cells may not exclusively reflect the cellular response to DNA damage but might occur upon non-genotoxic influences as well.…”

Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

“…Another phenotype associated with FA cells is increased G 2 accumulation after MMC treatment (26,49,50). This phenotype is reversed in mutant cells after provision of the specific complementing cDNA.…”