Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane

Abstract

Adult T cell leukemia derived factor (ADF)/thioredoxin (Trx) is known to be an important intracellular antioxidant involved in a number of redox reactions such as ribonucleotide reductase (RNR) as well as of tyrosinase. Since RNR is a key enzyme of nucleotide metabolism and DNA synthesis, a reduced Trx level would result in reduced enzymatic activity and cause DNA damage. Furthermore, Trx is considered to be an effective regulator of redox sensitive gene expression. The role of Trx in nucleotide metabolism and gene expression may be an explanation for increased chromosomal instability as well as hypersensitivity towards oxygen, ROI and ROI generating agents. The activity of tyrosinase, the key enzyme of melanin biosynthesis, is influenced by the thioredoxin level and by superoxide radicals. Low thioredoxin levels and high superoxide concentrations activate tyrosinase causing hyperpigmentation of the skin. In addition to the observed high superoxide concentration in Fanconi anemia (FA) patients, a low thioredoxin level might be responsible for the hyperpigmentation (cafe è-aulait spots) in this disease. We observed that overexpression of the thioredoxin cDNA in FA fibroblasts completely abolished the DNA damaging effects of mitomycin C and diepoxybutane and inhibited the constitutive activity of the nuclear factor U UB (NF-U UB) in SV40 transformed FA fibroblasts. However, spontaneous chromosomal breakage was not affected.z 1998 Federation of European Biochemical Societies.

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia

“…Mutant cells exhibit spontaneous chromosomal breakage 38 and increased sensitivity to the development of cytogenetic anomalies on in vitro treatment with cross-linking agents 39 such as MMC 40 and DEB. 41 Additional functions for FA proteins have also been described, including modulation of cytokine-mediated signaling, [42][43][44][45][46][47][48][49][50] responsiveness to oxidative stress, [51][52][53][54][55][56][57] and chromatin remodeling. 58 Moreover, at least 2 members of the FA genes (FANCC and FANCD2) are multifunctional.…”

Oxidative stress-related mechanisms are associated with xenobiotics exerting excess toxicity to Fanconi anemia cells.

“…Well-established mechanistic information has shown that MMC-associated toxicity is dependent on oxygen levels (Clarke et al 1997;Korkina et al 2000) and is removed by low-molecularweight antioxidants (Raj and Heddle 1980) and by thioredoxin (Trx) overexpression (Ruppitsch et al 1998). Recent reports demonstrate that a) MMC exposure results in an increase in oxidative DNA damage (Pagano et al 2001); b) FA cells fail to accumulate higher MMC adduct levels in DNA than control cells (Rutherford et al 1999;Will et al 1998); and c) the ratio of MMC biadducts to monoadducts (reflective of a DNA repair defect) shows no difference in FA cells compared with normal cells (Rutherford et al 1999).…”

“…Diepoxybutane (DEB) toxicity may be indirectly associated with redox mechanisms because its epoxide structure implies susceptibility to GSH scavenging (Bartók and Láng 1980). Similar to MMC, DEB-associated toxicity correlates with oxygen levels and oxidative DNA damage (Pagano et al 2001), whereas DEB-induced cytotoxicity and DNA damage are decreased by Trx overexpression (Ruppitsch et al 1998). DEB toxicity is modulated by GSH, and in turn, DEB results in GSH depletion Spanò et al 1998;Vlachodimitropoulos et al 1997).…”

“…A consistent body of evidence has been accumulated since then, both related to phenotypic redox abnormalities and related to the involvement of FA proteins in redox pathways (Ahmad et al 2002;Bogliolo et al 2002;Pagano et al 1998;Pagano and Korkina 2000;Pagano and Youssoufian 2003), as summarized in Table 4. FA cells are characterized by a) excess oxidative DNA damage ] in hydrogen peroxide-exposed FA cells (Takeuchi and Morimoto 1993), and in freshly drawn white blood cells from FA patients (Degan et al 1995); b) increased oxygen sensitivity (Joenje et al 1981) and sensitivity to free iron (Poot et al 1996); c) correction of chromosomal abnormalities by either antioxidant enzymes or by low-molecularweight antioxidants (Dallapiccola et al 1985;Nordenson 1977;Raj and Heddle 1980); d) excess plasma levels of clastogenic factor (Emerit et al 1995) and of tumor necrosis factor-α (TNF-α; Dufour et al 2003;Schulz and Shahidi 1993); e) involvement of redoxactive cytokines, such as TNF-α and interferon-γ (Dufour et al 2003;Pearl-Yafe et al 2003), and of inducible nitric oxide synthase (Hadjur and Jirik 2003); and d) an involvement of Trx, which corrects MMC and DEB sensitivity in FA cells (Ruppitsch et al 1998) and occurs in lower-than-normal levels in FA fibroblasts (Kontou et al 2002). Together, a well-established body of evidence on FA cellular phenotype, along with the roles for some FA proteins in redox pathways (Bogliolo et al 2002;Pagano and Youssoufian 2003), combine to support a central role for oxidative stress both in FA cellular phenotype and in FA protein functions.…”

Control of Trx1 redox state modulates protection against methyl methanesulfonate-induced DNA damage via stabilization of p21

“…Trx1 interacts directly with various intracellularsignalling molecules, as well as with transcription factors including AP-1 and nuclear factor kB (7,8), thereby affecting cell growth and cell survival. A recent study showed that overexpression of Trx in Fanconi's anaemia fibroblasts prevented the cytotoxic and DNA-damaging effects of mitomycin C and diepoxybutane (9). The cytosolic isoforms of Trx (Trxh1) were shown to be responsible for sensitivity to DNAdamaging agents, including MMS, in the green alga Chlamydomonas reinhardtii (10).…”