Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia.

Abstract

Sister chromatid exchanges, which may reflect chromosome repair in response to certain types of DNA damage, provide a means of investigating the increased chromosome fragility characteristic of Fanconi's anemia. By a recently developed technique using 33258 Hoechst and 5-bromodeoxyuridine, it was observed that the baseline frequency of sister chromatid exchanges in phytohemagglutinin-stimulated lymphocytes from four males with Fanconi's anemia differed little from that of normal lymphocytes. However, addition of the bifunctional alkylating agent mitomycin C (0.01 or 0.03 ;Ig/ml) to the Fanconi's anemia cells during culture induces less than half of the increase in exchanges found in identically treated normal lymphocytes. This reduced increment in exchanges is accompanied by a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. Many of these events occur at sites of incomplete chromatid interchange. The increase in sister chromatid exchanges induced in Fanconi's anemia lymphocytes by the monofunctional alkylating agent ethylmethane sulfonate (0.25 mg/ml) was slightly less than that in normal cells. Lymphocytes from two sets of parents of the patients with Fanconi's anemia exhibited a normal response to alkylating agents, while dermal fibroblasts from two different patients with Fanconi's anemia reacted to mitomycin C with an increase in chromatid breaks, but a nearly normal increment of sister chromatid exchanges. The results suggest that chromosomal breaks and rearrangements in Fanconi's anemia lymphocytes may result from a defect in a form of repair of DNA damage. Fanconi's anemia is a hereditary disease characterized by pancytopenia, congenital malformations, and patches of increased skin pigmentation (1). Chromosomes from affected individuals exhibit structural lability (2, 3), and there is a strong predisposition for the development of neoplasia (4, 5). Because of the high frequency of chromosomal abnormalities, predominantly breaks, it has been suggested that an error in DNA repair exists in Fanconi's anemia (5).Bifunctional alkylating agents such as mitomycin C (6), as well as y-rays (7), are unusually effective in inducing breaks in chromosomes of peripheral lymphocytes from individuals with Fanconi's anemia. Similarly, mitomycin C has been reported to be highly lethal to fibroblasts cultured from patients with Fanconi's anemia (8). Monofunctional alkylating agents exhibit less selective toxicity to Fanconi's anemia cells than do their bifunctional counterparts (6,8). One recent study has suggested that Fanconi's anemia fibroblasts may also have a reduced ability to excise thymine dimers (9).The observation that alkylating agents are able to induce large numbers of sister chromatid exchanges in chromosomes from normal cells (10-12) provides a new approach for investigating Fanconi's anemia. Since MATERIALS AND METHODSSister chromatid exchanges in human lymphocyte chromosomes were detected by fluorescence microscopy after staining with the dye 33258 Hoec...

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

“…In order to characterize the specificity of the effect of MMC on Fanconi's anemia cells, additional experiments were performed using EMS. This agent, unlike MMC, is not capable of causing appreciable DNA crosslinks and is not especially toxic to cells from patients with Fanconi's anemia (20,29). In contrast to MMC, EMS, at concentrations sufficient to more than double the sister chromatid exchange frequency in normal cells, did not effect a differential accumulation of Fanconi's anemia cells in late S or Gn + M ( Table 6).…”

“…(20). Defective sister chromatid exchange induction by mitomycin C in lymphocytes, but not fibroblasts, was observed under some conditions (29,31), that included the presence of FUdR and dC, but was not observed in cells from some patients treated by slightly different protocols (6, 35). If fibroblasts were exposed to a brief pulse of mitomycin C , those from patients with Fanconi's anemia actually ex-hibited a greater increment in SCEs than did normal cells Procedures based on metaphase chromosome analysis are compromised by the paucity of metaphase cells in cultures of FA cells (51), accentuated by exposure to mitomycin (17).…”

Genetic control of chromosome breakage and rejoining in Drosophila melanogaster: spontaneous chromosome aberrations in X-linked mutants defective in DNA metabolism.

“…In Fanconi anemia, on the other hand, exchanges of both symmetrical and asymmetrical types preferentially involve nonhomologous chromosomes. In addition, Bloom syndrome, but not Fanconi anemia, exhibits a strikingly high frequency of sister-chromatid exchange (4,5). The existence of characteristic cytological phenotypes associated with specific genetic defects makes these studies promising not only as an aid to understanding the mechanisms of formation of chromosome aberrations, but also for inferring the role of wild-type alleles at these loci in DNA metabolism.…”

Deficiency of gamma-ray excision repair in skin fibroblasts from patients with Fanconi's anemia.

“…For FA, additional clinical symptoms include pancytopenia with elevated levels of fetal hemoglobin, abnormal "cafe au lait"-type skin pigmentation, and stunted growth (2). Evidence for a defect in the repair of interstrand DNA crosslinks formed by the bifunctional, DNA-damaging agents mitomycin C and psoralen plus light has been presented, but the deficiency has not yet been characterized in molecular terms (3)(4)(5)(6). Some observations suggest a deficiency of FA cells in repairing DNA damage induced by monofunctional agents such as ethylmethanesulfonate (6) and decarbamoyl-mitomycin C (5).…”

“…Evidence for a defect in the repair of interstrand DNA crosslinks formed by the bifunctional, DNA-damaging agents mitomycin C and psoralen plus light has been presented, but the deficiency has not yet been characterized in molecular terms (3)(4)(5)(6). Some observations suggest a deficiency of FA cells in repairing DNA damage induced by monofunctional agents such as ethylmethanesulfonate (6) and decarbamoyl-mitomycin C (5). It has also been reported that the exonucleolytic degradation step in the excision repair of thymine-photodimers may be deficient in skin fibroblasts from a patient with FA after exposure to relatively high doses of ultraviolet light (7).…”