Damaged mitochondria in Fanconi anemia - an isolated event or a general phenomenon?

Fanconi anemia (FA) is known as an inherited bone marrow failure syndrome associated with cancer predisposition and susceptibility to a number of DNA damaging stimuli, along with a number of clinical features such as upper limb malformations, increased diabetes incidence and typical anomalies in skin pigmentation. The proteins encoded by FA-defective genes (FANC proteins) display well-established roles in DNA damage and repair pathways. Moreover, some independent studies have revealed that mitochondrial dysfunction (MDF) is also involved in FA phenotype. Unconfined to FA, we have shown that other syndromes featuring DNA damage and repair (such as ataxia-telangiectasia, AT, and Werner syndrome, WS) display MDF-related phenotypes, along with oxidative stress (OS) that, altogether, may play major roles in these diseases. Experimental and clinical studies are warranted in the prospect of future therapies to be focused on compounds scavenging reactive oxygen species (ROS) as well as protecting mitochondrial functions.

Another line of studies, dating back to 1980's, has provided consistent evidence for a role of OS in FA phenotype, such as excess oxygen sensitivity [25][26][27], in vitro and in vivo accumulation of oxidative DNA damage [28,29], and other anomalies of redox endpoints [30].Most notably, direct implications of FANC proteins in redox pathways have been reported.The FANCC protein was found to be associated with redox-related activities, namely NADPH cytochrome P450 reductase [31,32] and GST [32].The FANCG protein interacts with a P450 protein, cytochrome P450 2E1 (CYP2E1) [34], an activity also known to be involved in redox biotransformation of xenobiotics including, e.g., MMC [35,36].The FANCA and FANCG proteins were found to respond to redox state in terms of physical structure related to their ability to form disulphide bonds in the FA protein complex.Thus, FANCA, FANCC and FANCG were found to interact with redox state, also accounting for excess MMC sensitivity [31][32][33][34][35][36][37].A set of independent studies showed implications of BRCA1 (FANCD2) with OS.Dziaman et al. reported excess oxidative DNA damage in breast and ovary cancer patients with defective BRCA1 vs. cancer-free BRCA1 carriers and vs. control donors [38].Another study by Li et al. showed functional interaction of FANCD2 and the forkhead transcription factor forkhead box O 3a (FOXO3a), which colocalized with FANCD2 foci in response to OS; the authors suggested that interacting FANCD2/FOXO3a contribute to cellular antioxidant defense [39,40].
Consistent with the links of FA phenotype -and of FA proteins -with OS, and given the well-established relationships between redox pathways and MDF, a set of independent studies revealed that mitochondria are actually involved in FA phenotype, from the observation that FANG localizes to mitochondria [2].Major mitochondrial functions were found significantly altered in FA cells of genetic subtypes A, C, D2 and G, namely ATP production, mitochondrial membrane potential (∆Ψ), mitochondrial ultrastructure, defective mitochondrial peroxiredoxin-3, and oxygen consumption [1-3]; these malfunctions were not found in corrected FA cells.Another study, conducted on transcripts from bone marrow cells from FA patients vs. healthy donors, found that genes involved in mitochondrial bioenergetic pathways, i.e.Krebs cycle and electron transport chain were significantly down-regulated, approximately by 1.5to 2-fold [4].These findings, both arising from freshly drawn bone marrow cells and from lymphoblastoid cells or fibroblasts, point to an in vivo occurrence of MDF in FA patients, unconfined to FA cell cultures [1][2][3][4].
A possible scenario may be suggested for FAassociated MDF and OS: normal cell conditions undermine that mitochondria actively synthesize ATP (State 3) and the rate of electron transport is accelerated upon transferring ADP, phosphate and protons across the inner membrane.In that state almost 90% of oxygen is consumed by the respiratory chain and is reduced to water.One may assume that oxidative damage is accumulated in FA cells thus resulting in MDF and affecting both ATP production and cellular respiration.This state moves the majority of FA mitochondria toward semi-resting state (State 4), where ATP production is defective and the rate of oxygen consumption is low.All these events may result in mitochondrial abnormalities [1].Our recent data, from six FA patients as reported in Appendix I, showed down-regulation of several mitochondrial genes in cells from FA patients, confirming an involvement of MDF in FA phenotype (Fig. 1).Among those genes, nicotinamide nucleotide transhydrogenase (NNT) may play a role in detoxifying ROS as it was found that NNT knockdown resulted in impaired redox potential and increased ROS levels [41].NNT may control ROS level and cellular redox state by replenishment of GSH antioxidant systems and mitochondrial repair enzymes (thioredoxin, glutaredoxin, peroxiredoxins and phospholipid hydroperoxidase) and contribute to maintainence of the mitochondrial membrane potential through generation of a proton gradient [42,43].
An involvement of OS and MDF in FA phenotype, far from being unique, is recognized for other disorders, including mitochondrial and other genetic diseases, as well as an extensive number of diseases pertaining to a broad range of medical disciplines, and involving mitochondrial damage to cells of, e.g., brain, heart, liver, blood, kidney, lung, and eye, as reviewed recently [5,6,44,45].Table 1 shows a selection of cancer-prone and/or progeric genetic diseases, suggesting that they share clinical and Fanconi anemia patients from Andhra Mahila Sabha Hospital, Chennai, or from individuals with no symptoms of FA, was amplified using Express Art mRNA amplification kit micro version (Artus GmbH, Germany), labeled with Cy3 Post-Labeling Reactive Dye Pack (GE Healthcare UK limited, UK), fragmented and purified using Express Art Amino allyl mRNA amplification kit and YM10 columns (Millipore, USA).10.0 mg of the labeled amplified RNA was used for hybridization with the Human 40K (A+B) OciChip array.Hybridization was performed using automated hybstation HS 4800.Hybridized chips were scanned using Affymetrix 428TM array scanner at three different PMT gains.Differentially expressed genes were filtered and the results represent the most downregulated mitochondrial genes.A threshold log fold change (LFC) of 3.0 was fixed to attain FDR of less than 0.05.biochemical features both involving defective DNA repair (DDR), and revealing a direct evidence of MDF/ OS, including altered mitochondrial functions and/ or ultrastructure, higher ROS levels and imbalance of cellular bioenergetics pathways.Interestingly, many of the mitochondrial-related diseases (MRD) show involvement of DDR pathways (either at mtDNA or at nuclear DNA level).Altogether, this allows us to suggest a simplified scheme (Fig. 2), where ROS accumulated in DDR may equally affect and damage mitochondria and -at the same time -defects in mitochondria may provoke accumulation of ROS followed by OS and DNA damage.In other terms, in spite of different origins, these two classes of diseases may contribute to common -or analogous -phenotypes.
Cancer predisposition in DDR diseases is a wellestablished fact and most of the DDR evolve various malignancies.Mitochondrial dysfunction has been also associated with a wide range of solid tumors, proposed to be central to the aging process, and found to be a common factor in the toxicity of a variety of xenobiotics [101].An irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, thus mutations in mtDNA represent an early event during tumorigenesis.Due to the lack of introns, histones and limited repair mechanisms, mtDNA is more susceptible to mutations, including ROSdependent ones.Mutations in mtDNA can contribute to the development of breast [102] and colorectal cancers [103], leukemia [104] and hepatocellular carcinoma [105].There are many reasons to believe that ROS, acting both as mutagens and cellular mitogens, may play a role in tumor progression, thus suggesting a possible new avenue for the development of a treatment to suppress metastasis.In this regard, natural antioxidants should be considered for mitochondria-oriented FA therapy (mitochondrial nutrients, such as α-lipoic acid and coenzyme Q10) [6].Interestingly, several compounds used in the treatment of FA patients, whose mechanisms of action in FA are largely unknown (ouabain, curcumin, androgen analogs) were also used in the treatment of MRD, e.g.heart disease (ouabain), or AD (curcumin) [106][107][108].In MRD, these agents are known to inhibit Na(+)/K(+)-ATPase (ouabain), influence mitochondrial oxidation of cholesterol (oxandrolone, oxymetholone), prevent membrane permeability transition in mitochondria (thus reducing ROS by increasing glutathione) [106][107][108][109][110][111][112].Therefore, it is highly suggestive that the effects of the above drugs in FA are linked to mitochondrial-related ROS.In addition to inactivating ROS by antioxidants, another strategy is to use artificial uncoupling agents that decrease proton gradient and then ROS production [113].Unfortunately, therapeutic window(s) between efficacy and toxicity of such agents is too narrow.In order "to widen" the window between antioxidant and prooxidant concentrations, novel conjugates of plastoquinone and penetrating cations have been recently suggested [114].Clinical studies focusing on novel ROS-scavenging compounds as well as agents preventing mitochondria from accumulation of ROS are warranted in the prospect of future therapy.

Figure 1 :
Figure 1: Downregulation of mitochondrial genes in FA patients.Total RNA isolated from peripheral blood of 6

Figure 2 :
Figure 2: Scheme illustrating possible involvement of ROS into phenotypes of DDR and MDR diseases.