Mitomycin C–Induced DNA Damage in Fanconi Anemia: Cross-Linking or Redox-Mediated Effects?

Giovanni Pagano, Adriana Zatterale and Ludmila G, Korkina

To the Editor:

The article by Carreau et al1 reports on the in vivo effects of mitomycin C (MMC) in mice carrying the Fanconi anemia (FA) group C mutation (Fac−/−). Among the mechanistic scenarios underlying FA pathogenesis, the authors refer to a phenotypic feature of FA cells related to oxygen hypersensitivity. Unfortunately, the use of citations on this subject appears to be quite inappropriate. First, the authors attributed a “secondary” role for oxygen sensitivity in FA cells2 which, however, may have been made oxygen-resistant after the immortalization procedure. In fact, the loss of O2 sensitivity in transformed cells has been recognized as a general phenomenon, not confined to FA cell lines.3 A general statement was then made1 about the published results of studies which “have demonstrated overproduction of reactive oxygen species (ROS) and increased susceptibility to oxygen, as well as an increase in ROS-induced DNA lesions, particularly 8-hydroxy-2′-deoxyguanosine (8OHdG).” Unfortunately, the three references reported4-6 (cited as 37-39 in the report) neither dealt with FA nor with ROS-induced DNA damage. The above statement about excess ROS production and 8OHdG formation in FA was true, but rather should refer to the reports by Takeuchi and Morimoto7 and Degan et al.8 It is worthwhile to consider the subject of oxidative stress in FA based on both in vitro and ex vivo evidence, as reviewed by us recently.9

A role for oxidative stress in FA has been documented for two decades, with reports providing evidence for an improvement of either chromosomal instability or cell growth after exposure of either primary lymphocyte cultures or fibroblasts from FA patients to: (1) catalase or superoxide dismutase, (2) low-molecular-weight antioxidants, or (3) decreased oxygen levels.10-14 A G2 cell cycle delay, observed in FA cells, was counteracted by culturing cells in 5% O2,15 and a major role was suggested for free iron in inducing G2 arrest in FA cells.16 The report by Takeuchi and Morimoto7 provided evidence for excess oxidative DNA damage (8OHdG) in FAA cells challenged with H2O2 that was related, at least in part, to catalase deficiency. A recent report by Ruppitsch et al17provided elegant evidence for the loss of both MMC and diepoxybutane (DEB) sensitivity of FAA cells transfected with cDNA causing overexpression of thioredoxin, a nonenzymatic antioxidant protein.18 Hence, both exogenous and endogenous antioxidants can decrease the phenotypic defect of FA cells, both including O2 and MMC sensitivity. In turn, the action mechanisms of MMC can either be ascribed to DNA cross-linking or to redox cycling, as reported in early studies of MMC.19 20That MMC sensitivity in FA cells may be attributed to redox mechanisms rather than to DNA cross-linking has been shown by four independent reports11 13 21 22 focused on as many different endpoints (chromosomal instability, cytotoxicity, apoptosis, and mutagenesis). Together, the results of these studies showed that: (1) MMC-induced toxicity was confined to normoxic conditions which, unlike hypoxia, were associated to enhanced redox-cycling mechanisms, not to DNA cross-linking,21 22 and (2) MMC toxicity was both removed by antioxidant enzymes and by low-molecular-weight antioxidants.11 13

The observation of redox abnormalities in FA is not confined to in vitro conditions. A series of ex vivo studies provided evidence for abnormal O2 metabolism in FA patients and in their parents. Freshly drawn white blood cells from both FA homozygotes and heterozygotes produced excess ROS as detected by luminol-dependent chemiluminescence (LDCL),23 24 and displayed excess 8OHdG levels that were significantly correlated with LDCL as well as with chromosomal instability.8 Thus, both ex vivo and in vitro evidence pointed to a direct link between ROS formation, oxidative DNA damage, and chromosomal breakages in FA.

Based on the available evidence, one might suggest that the authors1 could carry out a new series of experiments by exposing Fac−/− mice to different oxygen levels, with or without MMC administration. As additional endpoints worth being evaluated in Fac−/− mice, one might suggest to include the evaluation of oxidative DNA damage as well as of ROS-detoxyfying activities. This study could provide a formidable insight both into the FAC defect and the in vivo action mechanisms of MMC.

In conclusion, the current view attributing the FA-associated defect(s) to the phenotypic sensitivity to MMC and DEB related to cross-linking mechanisms may be viewed as a fading dogma relying on the definition of FA as a DNA repair disorder. While no conclusive evidence has thus far related FA gene products to any function in DNA repair, a thriving body of evidence has associated MMC (and DEB) sensitivity to an impairment of redox balance in FA cells, both in vitro and in vivo. This evidence should no longer be disregarded in the forthcoming studies of FA.



First regarding the references, we believe that one reference by Takeuchi et al1-1 was omitted due to formatting of the paper and was overlooked on our part. The references cited as 37-39 regard the Bcl2 knockout mice and are discussed and referred to later in the paper.

Second, our paper dealt with mitomycin C (MMC) hypersensitivity of the Fancc−/− mouse model we generated. We believe that our discussion is in fact an overview of the possible in vivo effects of MMC,and we did not dismiss oxygen radical formation as a possible effect during the metabolism of MMC. Nonetheless, one still does not know if reactive oxygen species (ROS) formation is responsible for the hypersensitivity of the Fancc−/− mice treated with MMC, although we believe that the effect we observed may result from a defect in DNA repair. In fact, more information is now becoming available regarding a DNA repair defect in FA.

FA cells were shown to be specifically sensitive to interstrand crosslinks and not intrastrand crosslinks confirming the specificity of the defect in crosslink repair.1-2
FA cells were shown to lack a repair complex that specifically binds DNA crosslinks.1-3
The increased ROS-induced lesion 8OHdG, in FA patients cells also supports the idea of a lack of a repair mechanism; without repair, the lesions remain in the DNA.
FANCA and FANCC have been shown to interact in a complex and translocate to the nucleus; this implies a more direct role of FANCC in repair.1-4
FA cells have been shown to be defective in double-strand break repair.1-5

Oxidative DNA damage is repaired by the BER pathway, which may share steps with the crosslink repair pathway. Thus, increased sensitivity of FA cells to MMC caused by either oxidative damage or crosslinks, or both, support the notion of an altered repair mechanism.

We did, however, discuss the possible effect of ROS formation in the toxicity of MMC. Although MMC is known to induce a wide variety of lesions in the DNA, several papers have described the inability of FA cells to repair crosslinked DNA as the principal cause of MMC sensitivity. Again, we do not dismiss ROS formation as a possible mechanism in the toxicity of MMC in the Fancc−/− mice, and we would be more than willing to provide Dr Pagano with the Fancc −/− mice if he wishes to test his hypothesis.

Until we find the true function of the FA proteins, one can only speculate on the defects present in FA cells.


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