Pediatr Blood Cancer

REVIEW Fanconi Anemia (FA) and Crosslinker Sensitivity: Re-Appraising the Origins of FA Definition Giovanni Pagano,

MSc,

1

* Marco d’Ischia,

The commonly accepted definition of Fanconi anemia (FA) relying on DNA repair deficiency is submitted to a critical review starting from the early reports pointing to mitomycin C bioactivation and to the toxicity mechanisms of diepoxybutane and a group of nitrogen mustards causing DNA crosslinks in FA cells. A critical analysis of the literature prompts revisiting the FA phenotype and

Key words:

PhD,

2

and Federico V. Pallardo ,

MD, PhD

3

crosslinker sensitivity in terms of an oxidative stress (OS) background, redox-related anomalies of FA (FANC) proteins, and mitochondrial dysfunction. This re-appraisal of FA basic defect might lead to innovative approaches both in elucidating FA phenotypes and in clinical management. Pediatr Blood Cancer # 2015 Wiley Periodicals, Inc.

bioactivation; crosslinkers; diepoxybutane; FANC proteins; Fanconi anemia; glutathione; melphalan; mitomycin C; oxidative stress

INTRODUCTION Fanconi anemia (FA) is generally defined as a genetic disease characterized by bone marrow failure, excess risk of malignancies, and by excess sensitivity to crosslinkers, that is, agents causing DNA interstrand crosslinks (ICL). FA’s crosslinker sensitivity is commonly attributed to deficiency in DNA repair pathways since early reports dating back to mid-1970s [1–8], in analogy with other genetic diseases categorized for DNA repair defects [9,10]. A mechanistic alternative to this theory is provided by the body of literature pointing to toxicity mechanisms of crosslinkers, such as mitomycin C (MMC), diepoxybutane (DEB) used in FA diagnosis, and of a nitrogen mustard, cyclophosphamide (CP) used in pretransplant conditioning. Some of these xenobiotics either require biotransformation leading to formation of alkylating and toxic derivatives (MMC, CP), or their toxicities involve redox-related effectors such as glutathione (GSH) for DEB. The present review critically discusses the work that found metabolic errors in FA phenotype and the redox functions of FA (FANC) proteins. This re-appraisal may lead to an unprecedented impetus in elucidating FA’s cellular and clinical phenotype, along with long-awaited efforts in improving FA’s clinical management.

CROSSLINKER-ASSOCIATED TOXICITY MECHANISMS Early research on new drugs for antineoplastic chemotherapy prompted investigations on toxicity mechanisms of candidate antineoplastic drugs, including biotransformation leading to directly acting derivatives/metabolites [11]. This was especially the case for various xenobiotics resulting in DNA alkylation and in particular, some agents causing ICL, which were termed crosslinkers because they produce disarrangement of the DNA chains. A different focus originally arose for the other major xenobiotic used for FA diagnostic purposes, DEB, which was discovered as a directly acting carcinogenic and mutagenic metabolite of butadiene, utilized in the production of synthetic rubber [12].

of cell extracts activating MMC to its directly acting derivatives. Subsequent studies found that MMC bioactivation is associated with redox mechanisms involving NADPH cytochrome P-450 reductase producing reactive oxygen species (ROS) with formation of semiquinone radicals [15]; moreover, MMC toxicity was found to strictly depend on hypoxia [16]. MMC bioactivation was found to result in lipid peroxidation that was inhibited by antioxidant activities (superoxide dismutase [SOD] and catalase [CAT]), by glutathione (GSH), and ascorbic acid (AA) [17–22].

Diepoxybutane Diepoxybutane has long been known as a directly acting mutagen following 1,3-butadiene biotransformation [23]. Subsequent investigations elucidated the role of GSH and GSH-related activities (GSH S-transferase, GST) in coping with DEB-induced toxicity. As shown in Table II, Pelin et al. [24] reported that GST activity was involved in a major detoxification pathway for DEB, and Landi et al. [25] found that DEB-induced sister chromatid exchange (SCE) was dependent on the expression of GST, while GSH depletion was found to enhance DEB toxicity [26,27]. Other studies reported on redox modulation of DEB toxicity, such as expression of two important antioxidant enzymes, thioredoxin (Trx), and GSH peroxidase (GSH-Px) in FA cells, along with DEBinduced ROS formation, and inhibition of CYP2E1 activity [28–32]. Altogether, the available database on DEB-induced toxicity points to combined redox-dependent mechanisms, including ROS formation and dependence on GSH-related detoxification, 1

Istituto Nazionale Tumori Fondazione G. Pascale—Cancer Research Center at Mercogliano (CROM), Mercogliano (AV), Italy; 2Department of Chemical Sciences, University of Naples “Federico II,” Naples, Italy; 3University of Valencia—INCLIVA, CIBERER (Centro de Investigaci on Biome´dica en Red de Enfermedades Raras), Valencia, Spain Conflict of interest: Nothing to declare.



Mitomycin C As shown in Table I, early reports by Iyer and Szybalski since 1963 [13,14] demonstrated that MMC toxicity requires the addition

2015 Wiley Periodicals, Inc. DOI 10.1002/pbc.25452 Published online in Wiley Online Library (wileyonlinelibrary.com).

C

Correspondence to: Giovanni Pagano, Istituto Nazionale Tumori Fondazione G. Pascale—Cancer Research Center at Mercogliano (CROM)—IRCCS, Mercogliano (AV), 83013, Italy. E-mail: [email protected] Received 3 November 2014; Accepted 12 January 2015

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TABLE I. Historical Summary of Early Reports of MMC Bio-Reductive Activation (1960’s–1980’s) Authors (years) Iyer & Szybalski (1963–1964) Handa & Sato (1975) Bachur et al. (1979)

Kennedy et al. (1980) Teicher et al. (1981) Tomasz & Lipman (1981) Rockwell et al. (1982) Trush et al. (1982) Gutteridge et al. (1984)

Main findings

References

MMC in vitro effect on DNA requires a cell extract addition; formation of links between complementary DNA strands by metabolically activated MMC Quinone-containing anticancer chemicals form semiquinone radicals NADPH cytochrome P-450 reductase catalyzes the single-electron reduction of quinone antibiotics (including MMC); MMC is activated to free radical intermediates which transfer their single electron to O2 to form
O2 MMC toxicity is enhanced in hypoxic cells and requires NADPH-generating system ROS formation during MMC bioactivation

[13,14] [15] [16]

[17] [18–20]

MMC biotransformation generates lipid peroxidation-associated chemiluminescence; lipid peroxidation was inhibited by SOD, GSH, ascorbic acid, and CAT MMC stimulates deoxyribose degradation with peroxidation products under hypoxia. This damage is inhibited by scavengers of the
OH radical, Fe chelators, CAT and SOD.

[21] [22]

MMC, mitomycin C; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase.

by testifying a role for cell redox systems in modulating DEB toxicity. Thus, one may consider the excess DEB sensitivity of FA cells as related to abnormal redox status, and specifically, to anomalies in GST expression.

Melphalan and Cyclophosphamide The alkylating action of several nitrogen mustards has long been related to GSH depletion that is involved in detoxification and repair of cellular injury, along with induction of oxidative damage and with increased hypoxia sensitivity [33–37]. Melphalan (MEL) toxicity was tested in early reports as a crosslinker in FA cells [8] (see below). As shown in Table III, subsequent studies found that MEL toxicity is enhanced by GSH depletion and hypoxia [38–39] by increasing DNA fragmentation, peroxide accumulation, and endogenous formation of H2O2 [40–43]. Altogether, the direct MEL-associated crosslinking activity can be related to the multifold relationships of MEL toxicity with GSH levels, GSH-related activities, and with MEL-induced prooxidant state. Analogous to MEL, as shown in Table III, CP toxicity was found to relate with GSH depletion, and CP biotransformation resulted in ROS-forming active metabolites, along with oxidative DNA

damage and showing protection by antioxidants [44–51]. A specific relationship of CP toxicity with FA phenotype was reported by Joenje and Oostra [52], who found a clastogenic effect of nonactivated CP in FA lymphocyte cultures, suggesting a spontaneous—and excess—propensity of FA cells in CP bioactivation. This finding provided direct evidence for a metabolic anomaly of FA cells in CP bioactivation, while this subject was not further investigated and still deserves ad hoc studies.

OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION IN FA PHENOTYPE As shown in Table IV, a series of independent investigations related FA phenotype with OS since the pioneering report by Ingrid Emerit in 1977 [53], who found SOD-induced decrease of FA cell chromosomal instability, which was confirmed in MMC-treated FA cells. Excess oxygen and iron sensitivity was reported in FA cells, both involving cell viability and G2 cell growth phase arrest [54–58]. Excess oxidative DNA damage was observed in FA lymphoblasts, which was enhanced in MMC-exposed cells, and was partly attributed by Takeuchi and Morimoto to decreased CAT

TABLE II. Historical Summary of Published Evidence for DEB Toxicity Mechanisms Authors (years) Pelin et al. (1996) Landi et al. (1996) Vlachodimitropoulos et al. (1997) Spano` et al. (1998) Ruppitsch et al. (1998) Erexson & Tindall (2000) Davies et al. (2005) Yadavilli et al. (2007) Hartman et al. (2014)

Main findings

References

GSTT1 activity as a major detoxification pathway for DEB SCE induction by DEB is enhanced in GSTT1-null homozygotes Increased DEB sensitivity in lymphocytes from GSTT-deficient donors Glutathione depletion enhances DEB toxicity Overexpression of thioredoxin in FA cells abolished MMC- and DEB-induced DNA damage GSH-Px is involved in the detoxification of DEB-induced DNA damage; DEB causes ROS formation DEB sensitivity was significantly increased in GSTT1-null FA patients; GSTM1 influenced time to BMF in FA-C patients DEB induced apoptosis and ROS formation; trans-membrane potential in DEBinduced apoptosis DEB inhibits CYP2E1 activity

[24] [25] [26] [27] [28]

DEB, diepoxybutane; GST, GSH S-transferase; SCE, sister chromatid exchange; CYP, cytochrome P 450. Pediatr Blood Cancer DOI 10.1002/pbc

[29] [30] [31] [32]

Crosslinking Agents and Fanconi Anemia

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TABLE III. Historical Summary of Published Evidence for Toxicity Mechanisms of Melphalan and Cyclophosphamide Agents Melphalan (MEL)

Authors (years) Roizin-Towle (1985) Lunel-Orsini et al. (1995) Vahrmeijer et al. (1999)

de Wilt et al. (1999) Troyano et al. (2001) Matsura et al. (2004) Cyclophosphamide (CP)

Crook et al. (1986) LeBlanc & Waxman (1990) Stankiewicz & Skrzydlewska (2003)

Murata et al. (2004) Selvakumar et al. (2006) Sadir et al. (2007) Kasapovic´ et al. (2010) Liu et al. (2012)

Main findings

References

Increased toxicity by GSH depletion and hypoxia GSH depletion increased MEL toxicity GSH depletion caused a fourfold increase in DNA fragmentation and a sevenfold increase in the fraction of apoptotic cells Hypoxia enhanced anti-tumor activity of MEL GSH depletion enhanced peroxide accumulation and apoptosis H2O2 formation, phosphatidylserine oxidation, cyt C release in cytosol CP metabolite acrolein was effective in GSH depletion CP and acrolein modulate hepatic P-450 expression CP is biotransformed into ROS-forming active metabolites; decreased SOD, GPx, GR, and CAT; decreased GSH, Vit C and E 4-hydroperoxyCP caused oxidative DNA damage (8-oxodG) Protection by a-lipoic acid of CYP-induced DNA damage Protection by antioxidants (melatonin) of CYP-induced OS CP causes oxidative DNA damage and decreases antioxidant activities (Cu,ZnSOD, GPx, GR) and GSH levels CP metabolite acrolein impairs the cytoskeleton and induces OS

[38] [39] [40]

[41] [42] [43] [44] [45] [46]

[47] [48] [49] [50] [51]

GPx, glutathione peroxidase; GR, glutathione reductase; 8-oxodG, 8-Oxo-20 -deoxyguanosine.

activity [59], while Trx overexpression counteracted MMC- and DEB-induced toxicity in FA fibroblasts [28]. Unconfined to in vitro testing, a series of independent studies conducted on blood cells and biological fluids from patients with FA found an in vivo prooxidant state assessed on a set of redox endpoints, including oxidative DNA damage, GSH imbalance (expressed as GSH:GSSG ratio), WBC chemiluminescence, methylglyoxal levels, and SOD expression [60–63]. The formation and toxicity of aldehydes have been focused in recent years as a hallmark in FA phenotype. Aldehydes are established biochemical correlates of OS in a broad variety of physiological and pathological conditions (reviewed in [64,65]). Polyunsaturated lipids and sugars are typical sources of OS-related aldehydes, such as 4-hydroxynonenal, acrolein, MDA, and glyoxal/ methylglyoxal, whose formation is an index of, and responsible for,

extensive cell damage. Besides being a footprint of ROS generation, aldehydes can mediate a number of toxicity pathways which may ultimately lead to DNA cross-linking [65]. Several aldehydes have been implicated in formation of covalent adducts and crosslinks targeted to dG [66]. A series of reports have focused on a deficiency of aldehyde dehydrogenase 2 (ALDH2) in FANCD2 cells and fancd2 ( / ) mice [67–69], relating ALDH2 deficiency to inability to counteract acetaldehyde toxicity. The relevance of these data has found support by the report of Hira et al. [70], who found that a ALDH2 variant is associated with accelerated progression of bone marrow failure in Japanese patients with FA. However, whether an ALDH2 defect does actually play a significant and specific role in acetaldehyde detoxification awaits verification, in view of the mentioned excess in vivo levels of other aldehydes in patients with FA [62,63]. Moreover, ALDH2 displays low specificity in aldehyde

TABLE IV. Early Studies Assessing OS-Related Mechanisms in FA Cells Authors (years) Nordenson (1977) Joenje et al. (1981) Joenje & Oostra (1983) Nagasawa & Little (1983)

Saito et al. (1993) Takeuchi & Morimoto (1993) Poot et al. (1996) Ruppitsch et al. (1998)

Main findings

References

Chromosomal instability of FA cells is decreased by SOD The rate of chromosomal breaks in FA cells is decreased by low-level oxygen The influence of low-level oxygen in enhanced in MMC-treated FA cells MMC cytotoxicity is suppressed by SOD in FA cells; “Hypersensitivity to MMC cytoxicity in FA cells may involve among other factors a deficiency in the inactivation of certain free radical intermediates” “The sensitivity to MMC in different FA lines varied from normal level to extreme hypersensitivity, whereas all the FA lines showed similar hypersensitivity to oxygen” FA-A cells have increased susceptibility to oxidative DNA damage; this increased susceptibility is possibly due to decreased CAT activity A G2 phase defect is dependent upon the oxygen concentration; exposure to excess iron was very toxic to FA cells at 20%, but less toxic at 5% oxygen Overexpression of thioredoxin cDNA in FA fibroblasts abolished the DNA damaging effects of MMC and DEB

[53] [54] [55] [56]

Pediatr Blood Cancer DOI 10.1002/pbc

[57] [58] [59] [28]

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clearance by counteracting 4-hydroxynonenal toxicity [71]. The relative impact of aldehyde mediated toxicity versus other OSrelated pathways of cell damage remains little understood whereby excess aldehyde formation (or a detoxification defect) stands at present as only one of the many possible contributory factors produced in a pro-oxidant state. This is the case, for example, for oxidative DNA damage (8-OHdG) that occurs in FA phenotype [58,61–63], and displays a cascade of adverse effects including mutagenesis [72,73]. Thus, a possible attempt to improve FA’s clinical course by means of aldehyde detoxifying agents might not compensate the basic—and multi-faceted—pro-oxidant state featured in FA phenotype. A few studies tested some different antioxidants in FA knock-out mice reporting protective effects of some antioxidants (tempol and resveratrol, but not N-acetylcysteine) [74–76]. These findings might suggest an appropriate design of further animal studies, and possibly, of clinical trials. Closely related to redox impairment, mitochondrial abnormalities have been reported in FA cells of four genetic subtypes (A, C, D2, and G), pointing to multi-faceted links between mitochondrial dysfunction in FA cellular phenotype and the available database on the roles for OS in FA cells [77–79]. Alterations in mitochondrial morphology were reported by Kumari et al. [78], including damaged mitochondrial membranes, abnormal shapes, and possible events of mitophagy consistent with decreased mitochondrial transmembrane potential (DCm) and overproduction of ROS in FA cells. We have recently evaluated transcriptomal analyses on RNA obtained from bone marrow cells from patients with FA versus healthy volunteers, and identified significant changes in the expression of genes involved in bioenergetic pathways and in antioxidant activities [80]. Altogether, an in vivo, as yet unexplored, involvement of MDF in patients with FA may be viewed as a realistic working hypothesis based on the current database and prompting further investigations in this subject.

REDOX-RELATED ROLES OF FANC PROTEINS Some FANC proteins have been associated with several redoxrelated activities and/or interactors, as shown in Table V. Independent studies by Kruyt et al. and by Futaki et al. found that FANCC and FANCG proteins bind to and regulate the activity of cytochrome-P450 [81,82], microsomal membrane proteins involved in electron transfer, and long recognized as effectors in MMC bioactivation [83]. A direct involvement of BRCA1 and BRCA2 (FANCD1) proteins was reported in the repair of 8oxoguanine damage, thus, implying their direct role in redox pathways [84], as oxidation of guanine is a well-known parameter of OS and key factor in cancer proneness. Other investigations found functional interactions of FANCC and FANCG proteins with NADPH cytochrome P450 reductase and by redox regulation of GSTP1 [85]. Park et al. found that FANCA and FANCG proteins were sensitive to redox conditions since their monomers became multimers following H2O2 treatment [86]. The links of the FANCD2 protein with OS were reported in independent studies that found interactions with ATM (Ataxia–Telangiectasia mutated protein) and FOXO3a (forkhead box O3) exerting coordinated actions with FANCD2 consistent with the established functions of both ATM and FOXO3a in response to oxidative DNA damage [87– 89]. An extensive database points to the multiple links of the FANCJ (FANCJ/BACH1/BRIP1) protein with OS. This protein displays functional interaction with BRCA1 in breast and ovary cancer suppression [90,91] and in turn, BRCA1 is known to be involved in OS- related mechanisms [92]. Moreover, FANCJ/BACH1/BRIP1 competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants [93]. FANCJ/BACH1/BRIP1 features a helicase activity and an ATPase activity, a twofold activity that is recognized for other RECQ helicases that are associated with ATPase activity [94]. FANCJ/BACH1/BRIP1 regulates heme

TABLE V. Redox-Related Functions of FANC Proteins Consistent With a Prooxidant State in FA Cells Authors (years) Kruyt et al. (1998) Futaki et al. (2002) Le Page et al. (2000) Cumming et al. (2001) Park et al. (2004) Li et al. (2010) Castillo et al. (2011) Du et al. (2012) Cantor and Andreassen (2006) Levran et al. (2005) Dhakshinamoorthy et al. (2005) Mukhopadhyay et al. (2006) Kitamuro et al. (2003) Okada et al. (2010) Suhasini et al. (2009)

Main findings

References

FANCC protein binds to NADPH CYP-P450 reductase, involved in electron transfer FANCG interacts with cytochrome P450 2E1 (CYP2E1), which is associated with the production of ROS and the bioactivation of carcinogens BRCA1 and BRCA2 proteins are involved in the repair of 8-oxoguanine damage FANCC protein interacts glutathione S-transferase P1-1, which catalyzes the detoxification of xenobiotics and by-products of oxidative stress FANCA and FANCG respond to oxidative damage by forming complexes Functional interaction of the FANCD2 protein and the forkhead box O 3a (FOXO3a) FANCD2 colocalized with FOXO3a in response to oxidative stress; FANCD2 as interactor with Ataxia telangiectasia mutated protein (ATM) OS-induced FANCD2 ubiquitination interacts with FA-BRG1–promoter complex RECQ helicase associated with ATPase activity FANCJ/BACH1/BRIP1 interacts with heme oxygenase-1; BRCA 1 ARE-NAD(P)H:quinone oxidoreductase-1; sensing thymin glycol damage FANCG protein is found in mitochondria; wild-type but not mutant FANCG physically interacts with the mitochondrial peroxidase PRDX3 Bach1 as hypoxia-inducible repressor for the heme oxygenase-1 gene

[81] [82]

FANCJ/BACH1/BRIP1 senses oxidative DNA damage

[84] [85] [86] [87] [88] [89] [90] [91] [93] [77] [95] [96] [97]

FANC, proteins encoded by FA defective genes; BRCA, breast cancer-related genes and proteins; RECQ helicase, family of helicase enzymes; ARE, antioxidant response element; PRDX3, peroxiredoxin-3. Pediatr Blood Cancer DOI 10.1002/pbc

Crosslinking Agents and Fanconi Anemia oxygenase-1 [95,96], senses oxidative DNA damage, and is stimulated by replication protein A to unwind the damaged DNA substrate in a strand-specific manner [97]. Altogether, the database on interactions of several FANC proteins points to multiple molecular interactions of at least five FANC (A, C, D2, G, and J) proteins with redox-related mechanisms.

DEFECTIVE DNA REPAIR: TOWARD THE ROOTS OF A SHARED AXIOM By far, the most shared opinion, defective DNA repair with excess sensitivity to crosslinkers is associated with FA definition and provides the basis for most of the FA literature up to recent reviews [9,10]. Albeit currently assumed as an undisputed axiom, this theory dates back to a few early studies that may be analyzed under a historical viewpoint. The pioneering studies by Sasaki [1,2] tested the susceptibility to chromosome breakage of peripheral blood lymphocytes from patients with FA exposed to a number of clastogens (including MMC). The increased susceptibility to compounds resulting in DNA ICL was interpreted as an indication that the FA cells are defective in DNA repair mechanisms. No mention was provided to the toxicity mechanisms of the tested clastogens, namely related to MMC requirement of bioactivation, as available in the previous and contemporary literature [15–19]. Excision repair ability was investigated in FA cells in terms of MMC cell killing and by DNA sucrose sedimentation compared to HeLa cells and Xeroderma pigmentosum (XP) cells [3,4]. The results showed that FA DNA sedimented much faster after MMC treatment suggesting a possible impairment in FA cell’s ability to unhook crosslinks [4]. However, no reference was provided to the available literature, whether the excess MMC sensitivity of FA cells might be due to an abnormal propensity to activate MMC to its directly acting metabolites [3,4]. Latt et al. [5] found a decreased SCE induction in FA cells versus normal cells, while MMC treatment resulted in a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. The authors suggested that chromosomal breaks and rearrangements in FA cells might result from a defect in repair of DNA damage. Unlike the above studies, Auerbach and Wolman [6] in 1976 investigated the effects of DEB and methyl methane sulfonate (MMS) as directly acting mutagens by prolonged exposure of FA fibroblasts versus XP and versus control cells, and by evaluating the frequency of chromosomal aberrations after DEB or MMS removal. This study was designed by assuming the direct mutagenic action for DEB, as still currently recognized, while the GSH-related modulation of DEB toxicity (Table II) would be reported some 20 years later [24–26]. A quite singular conclusion was offered in the report by Raj and Heddle [7], who tested the induction of chromosomal breakage in FA and normal cells with or without MMC treatment and cultured with SOD, or CAT, or l-cysteine. These authors found that antioxidant treatments resulted in decreased chromosomal breaks both in FA and in normal cells, thus, they stated that the effect was not specific, by hence concluding for the occurrence of DNA repair deficiency. Opposite conclusions were provided by Nagasawa and Little [56], who found that survival of MMC-treated FA cells was increased by adding SOD, unlike MMC-treated normal cells. These authors suggested that the hypersensitivity to “MMC cytoxicity in Pediatr Blood Cancer DOI 10.1002/pbc

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FA cells may involve among other factors a deficiency in the inactivation of certain free radical intermediates [56].” A major support to the theory of DNA repair deficiency was provided by Ishida and Buchwald [8]. This report evaluated growth inhibition of FA lymphoblasts exposed to a number of mono- and bifunctional crosslinking agents including, among others, MMC and MEL, and found that FA cells are more sensitive than normal cell lines to all DNA crosslinking agents examined. Interestingly, these authors stated that “MMC requires activation by a NADPHgenerating system [14] before it will cross-link DNA while melphalan does not [98], yet FA cells have similar sensitivities to the two drugs. [...] It is probable, therefore, that the defect(s) in FA is at the level of repair of the damage in DNA [8].” About MMC biotransformation, one may comment on the quite outdated citation from Iyer and Szybalski [14], published almost 20 years before this report by disregarding the recent and contemporary literature providing evidence for redox-related MMC biotransformation [15– 19]. On the other hand, one should recognize that studies of redoxrelated toxicity mechanisms of MEL were not yet available. Thus, this basic report on FA-associated DNA repair deficiency reached a conclusion partly unaware of the up-to-date literature (MMC bioactivation), and partly surpassed by following investigations (MEL-associated redox toxicity mechanisms). Altogether, the founding publications supporting the theory of DNA repair deficiency in FA either failed to consider the available literature pointing to MMC bioactivation requirements or could not foresee the subsequent evidence for redox toxicity (GSH-related) mechanisms of DEB and MEL. That theory, though presently shared almost universally in the scientific community, cannot be regarded as an undisputable truth, as far as its origins can be critically reconstructed in a historical framework, displaying some inborn pitfalls as discussed here.

RESEARCH PROSPECTS The prevailing opinion attributing FA’s basic defect to impaired DNA repair has led to the current state of art in clinical practice, as yet confined to androgen therapy and/or to optimized transplantation protocols. A long-lasting, so far unsuccessful prospect treatment has been targeted to gene therapy. We can hope that new developments make feasible this therapy. On the other hand, the hitherto overlooked evidence for OS and MDF in FA relies on a body of independent reports encompassing redox toxicity mechanisms of FA-related crosslinkers, multifaceted evidence for OS in FA cells and patients, redox-related functions of several FANC proteins, and impaired mitochondrial function in FA cells from four genetic subtypes. Further studies designed to test this theory might contribute to basic elucidation of FA phenotype and might provide prospects for innovative clinical interventions in patients with FA.

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