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1116
mune neutropenia due to anti-SH. 5th European Symposium on Platelet
and Granulocyte Immunobiology, S’Agaro´, Girona, Spain, May 9-12,
1998, p 52 (abstr)
6. Hessner MJ, Curtis BR, Endean DJ, Aster RH: Determination of
neutrophil antigen NA gene frequencies in five different ethnic groups
CORRESPONDENCE
by the polymerase chain reaction with sequence-specific primers
(PCR-SSP). Transfusion 36:895, 1996
7. Lin M, Chen CC, Wang CL, Lee HL: Frequencies of neutrophilspecific antigens among Chinese in Taiwan. Vox Sang 66:247, 1994
(letter)
Mitomycin C–Induced DNA Damage in Fanconi Anemia: Cross-Linking or Redox-Mediated Effects?
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 (Fac2/2). 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-28deoxyguanosine (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
al17 provided 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.19,20 That 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 chemilumines-
cence (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 Fac2/2 mice
to different oxygen levels, with or without MMC administration. As
additional endpoints worth being evaluated in Fac2/2 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.
Giovanni Pagano
Italian National Cancer Institute
G. Pascale Foundation
Naples, Italy
Adriana Zatterale
Cytogenetics Unit, Elena D’Aosta Hospital
Naples, Italy
Ludmila G, Korkina
Russian Institute of Pediatric Hematology
Moscow, Russia
REFERENCES
1. Carreau M, Gan OI, Liu L, Doedens M, McKerlie C, Dick JE,
Buchwald M: Bone marrow failure in the Fanconi anemia group C
mouse model after DNA damage. Blood 91:2737, 1998
2. Joenje H, Youssoufian H, Kruyt FAE, dos Santos C, Wevrick R,
Buchwald M: Expression of the Fanconi anemia gene FAC in human
cell lines: Lack of effect of oxygen tension. Blood Cells Mol Dis
21:182, 1995
3. Saito H, Hammond AT, Moses RE: The effect of low oxygen
tension on the in vitro replicative life span of human diploid fibroblast
cells and their transformed derivatives. Exp Cell Res 217:272, 1995
4. Nakayama K, Nakayama KI, Negishi I, Kuida K, Sawa H, Loh
DY: Targeted disruption of Bcl-2ab in mice: Occurrence of gray hair,
polycystic kidney disease, and lymphocytopenia. Proc Natl Acad Sci
USA 91:3700, 1994
5. Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ: Bcl-2-deficient
mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys,
and hypopigmented hair. Cell 75:229, 1993
6. Motoyama N, Wang F, Roth KA, Sawa H, Nakayama KI,
Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S, Loh DY: Massive
cell death of immature hematopoietic cells and neurons in Bcl-xdeficient mice. Science 267:1506, 1995
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
CORRESPONDENCE
1117
7. Takeuchi T, Morimoto K: Increased formation of 8-hydroxydeoxyguanosine, an oxidative DNA damage, in lymphoblasts from Fanconi’s
anemia patients due to possible catalase deficiency. Carcinogenesis
14:1115, 1993
8. Degan P, Bonassi S, De Caterina M, Korkina LG, Pinto L,
Scopacasa F, Zatterale A, Calzone R, Pagano G: In vivo accumulation
of 8-hydroxy-28-deoxyguanosine in DNA correlates with release of
reactive oxygen species in Fanconi’s anaemia families. Carcinogenesis
16:735, 1995
9. Pagano G, Korkina LG, Brunk UT, Chessa L, Degan P, Del
Principe D, Kelly FJ, Malorni W, Pallardo´ F, Pasquier C, Scovassi I,
Zatterale A, Franceschi C: Congenital disorders sharing oxidative stress
and cancer proneness as phenotypic hallmarks: Prospects for joint
research in pharmacology. Med Hyp 51:253, 1998
10. Nordenson I: Effect of superoxide dismutase and catalase on
spontaneously occuring chromosome breaks in patients with Fanconi’s
anemia. Hereditas 86:147, 1977
11. Raj AS, Heddle JA: The effect of superoxide dismutase, catalase
and L-cysteine on spontaneous and on mitomycin C induced chromosomal breakage in Fanconi’s anemia and normal fibroblasts as measured
by the micronucleus method. Mutat Res 78:59, 1980
12. Joenje H, Arwert F, Eriksson AW, de Koning H, Oostra AB:
Oxygen-dependence of chromosomal aberrations in Fanconi’s anaemia.
Nature 290:142, 1981
13. Nagasawa H, Little JB: Suppression of cytotoxic effect of
mitomycin-C by superoxide dismutase in Fanconi’s anemia and dyskeratosis congenita fibroblasts. Carcinogenesis 4:795, 1983
14. Dallapiccola B, Porfirio B, Mokini V, Alimena G, Isacchi G,
Gandini E: Effect of oxidants and antioxidants on chromosomal
breakage in Fanconi’s anemia lymphocytes. Hum Genet 69:62, 1985
15. Schindler D, Hoehn H: Fanconi anemia mutation causes cellular
susceptibility to ambient oxygen. Am J Hum Genet 43:429, 1988
16. Poot M, Gross O, Epe B, Pflaum M, Hoehn H: Cell cycle defect
in connection with oxygen and iron sensitivity in Fanconi anemia
lymphoblastoid cells. Exp Cell Res 222:262, 1996
17. Ruppitsch W, Meisslitzer C, Hirsch-Kauffmann M, Schweiger
M: Overexpression of thioredoxin in Fanconi anemia fibroblasts
prevents the cytotoxic and DNA damaging effect of mitomycin C and
diepoxybutane. FEBS Lett 422:99, 1998
18. Kuge S, Jones N: YAP1-dependent activation of TRX2 is
essential for the response of Saccharomyces cerevisiae to oxidative
stress by hydroperoxides. EMBO J 13:655, 1994
19. Gutteridge JMC, Quinlan GJ, Wilkins S: Mitomycin C-induced
deoxyribose degradation inhibited by superoxide dismutase. A reaction
involving iron, hydroxyl and semiquinone radicals. FEBS Lett 167:37,
1984
20. Pritsos CA, Sartorelli AC: Generation of reactive oxygen radicals through bioactivation of mitomycin antibiotics. Cancer Res 46:
3528, 1986
21. Clarke AA, Philpott NJ, Gordon-Smith EC, Rutherford TR: The
sensitivity of Fanconi anaemia group C cells to apoptosis induced by
mitomycin C is due to oxygen radical generation, not DNA crosslinking. Br J Haematol 96:240, 1997
22. Liebetrau W, Runger TM, Mehling BE, Poot M, Hoehn H:
Mutagenic activity of ambient oxygen and mitomycin C in Fanconi’s
anaemia cells. Mutagenesis 12:69, 1997
23. Rumyantsev AG, Samochatova EV, Afanas’ev IB, Korkina LG,
Suslova TB, Cheremisina ZP, Maschan AA, Durnev AD, Lurye BL: The
role of free oxygen radicals in the pathogenesis of Fanconi’s anemia.
Ter Arkh 61:32, 1989
24. Korkina LG, Samochatova EV, Maschan AA, Suslova TB,
Cheremisina ZP, Afanas’ev IB: Release of active oxygen radicals by
leukocytes of Fanconi’s anemia patients. J Leukoc Biol 52:357, 1992
Response
First regarding the references, we believe that one reference by
Takeuchi et al1 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 Fancc2/2 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 Fancc2/2 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.
1. FA cells were shown to be specifically sensitive to interstrand
crosslinks and not intrastrand crosslinks confirming the specificity
of the defect in crosslink repair.2
2. FA cells were shown to lack a repair complex that specifically
binds DNA crosslinks.3
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.
4. 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.4
5. FA cells have been shown to be defective in double-strand break
repair.5
Oxidative DNA damage is repaired by the BER pathway, which may
share steps with the crosslink repair pathway. Thus, increased sensitiv-
ity 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 Fancc2/2 mice, and we would
be more than willing to provide Dr Pagano with the Fancc 2/2 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.
Madeleine Carreau
Manuel Buchwald
Program in Genetics and Genomic Biology
Research Institute
Hospital for Sick Children
Toronto, Ontario, Canada
REFERENCES
1. Takeuchi T, Morimoto K: Increased formation of 8-hydroxydeoxyguanosine, an oxidative DNA damage, in lymphoblasts from Fanconi’s
anemia patients due to possible catalase deficiency. Carcinogenesis
14:1115, 1993
2. Fujiwara Y, Nakamura M, Yokoo S: A new anticancer platinum
compound, (2)-(R)-2-aminomethyl-pyrrolidine(1,1-cyclobutanedicar-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1118
boxylato) platinum(II): DNA interstrand crosslinking, repair and lethal
effects in normal human, Fanconi’s anaemia and xeroderma pigmentosum cells. Br J Cancer 67:1285, 1993
3. Hang B, Yeung AT, Lambert MW: A damage-recognition protein
which binds to DNA containing interstrand cross-links is absent or
defective in Fanconi anemia, complementation group A cells. Nuclic
Acids Res 21:4187, 1993
CORRESPONDENCE
4. Yamashita T, Kupfer G, Naf D, Suliman A, Joenje H, Asano S,
D’Andrea AD: The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation. Proc Natl Acad Sci USA
95:13085, 1998
5. Escarceller M, Buchwald M, Singleton B, Jeggo P, Jackson S,
Moustacchi E, Papadopoulo D: Fanconi anemia C gene product plays a
role in the fidelity of blunt DNA end-joining. J Mol Biol 279:375, 1998
Is the Oral Methionine Loading Test Insensitive to the Remethylation Pathway of Homocysteine?
To the Editor:
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in
the remethylation pathway of homocysteine. The study by Girelli et al1
showed that the C677T mutation of the gene encoding for MTHFR is
common in Italy, is not associated with coronary atherosclerotic disease,
and influences the plasma levels of total homocysteine (tHcy) only in
subjects who have low serum concentrations of folic acid. These
findings are important confirmations of previous reports.2-6 In addition
to the above issues, Girelli and his coworkers addressed the problem of
the influence of the MTHFR genotype on the increases of plasma tHcy
levels after an oral methionine loading. The methionine loading test,
which was originally developed to detect heterozygosity for the
deficiency of cystathionine-b-synthase, a key enzyme in the transsulfuration pathway of homocysteine,7 is useful not only to detect
subjects with defects of the trans-sulfuration pathway, but also to
identify subjects at risk for thrombosis.8,9 It has been suggested that
abnormal post-methionine loading (PML) increases in the plasma
levelsof tHcy reflect abnormalities of the trans-sulfuration pathway of
homocysteine metabolism and are insensitive to the remethylation
pathway, in contrast with fasting plasma levels of tHcy, which are
sensitive to the remethylation pathway.10,11 If this hypothesis were true,
the T/T mutation at nucleotide 677 of MTHFR, which is associated with
an enzyme activity of about 50% of normal, should not influence the
results of the methionine loading test. Girelli et al commented that their
results agree with this hypothesis, because they showed that homozygosity for the C677Tmutation of MTHFR is not associated with abnormally
high PML tHcy increments.1 However, they found higher PML tHcy
increments in individuals carrying the mutant T/T genotype (25.5
µmol/L) than in those with normal (C/C) or heterozygous (C/T)
genotypes (23.6 µmol/L for both), although the difference was not
statistically significant (Table 1). In addition, they found that individuals
with low serum levels of folate, which plays an important role in the
remethylation pathway, tended to have higher PML tHcy increments
than those with high folate levels, independently of their genotype (24.9
v 22.9 for C/C; 25.1 v 23.5 for C/T; 28.8 v 21.4 for T/T). In our opinion,
these data do not allow a definite conclusion regarding the influence of
defects of the remethylation pathway on the methionine loading test. At
least four additional reports with an experimental design similar to that
of Girelli et al have been published thus far.2,3,5,6 All of them clearly
showed that the fasting tHcy levels in plasma are significantly higher in
the T/T genotype of MTHFR than in the other genotypes; however, the
results of the methionine loading test were less consistent. Table 1
shows that all the studies, perhaps with the exception of that by Jacques
et al,6 showed that the PML increases of tHcy in plasma were higher in
the T/T genotype of MTHFR than in the normal C/C genotype, although
the statistical significance was reached in only two.2,5 Since the
publication of our study in 1997,2 we screened more individuals for
MTHFR genotype and the methionine loading test. Our most recent
analysis of the data obtained in 399 healthy controls and 250 patients
with previous episodes of arterial or venous thrombosis confirmed our
previous findings that the PML increases of tHcy levels in plasma are
significantly higher in individuals with the T/T genotype (18.2 6 7.5,
Table 1. Increases of Plasma tHcy After an Oral Methionine Loading
as a Function of MTHFR Genotypes
PML tHcy Increase (µmol/L)
MTHFR Genotype at nt 677
Study
Jacques et al,
19966 †
Cattaneo et al,
19972 †
Legnani et al,
19973 †
Verhoef et al,
19975 ‡
Girelli et al,
19981 ‡
No. of
Subjects
C/C
C/T
T/T
365
17.9
18.5
18.2
NS
231
14.4
14.8
18.6
.006
63
17.0
19.0
23.1
NS
318
23.6
26.4
27.7
,.05
415
23.6
23.6
25.5
NS
P*
Values of PML tHcy increase are arithmetic means (†) or geometric
means (‡). The dose of oral methionine was 3.8 g/m2 body surface
area in the study by Cattaneo et al, and 100 mg/kg body weight in the
other studies.
Abbreviation: NS, not significant.
*Genotype T/T v genotype C/C.
arithmetic mean 6 SD) than in those with the C/C or the C/T genotypes
of MTHFR (14.1 6 5.6, P , .001). The differences remained highly
statistically significant (P , .001) also after adjustment for the serum
levels of folate and cobalamin, and the plasma levels of vitamin B6,
which is the essential cofactor in the trans-sulfuration pathway of
homocysteine.5 Among the 649 subjects studied, 142 had the T/T
genotype of MTHFR (80 controls and 62 cases); 14 of them (9.9%) had
high fasting tHcy levels, 15 (10.6%) had both high fasting levels and
high PML increases of tHcy, and 6 (4.2%) had high PML increases of
tHcy. Therefore, the methionine loading test allowed the identification
of 6 additional subjects with the MTHFR variant who had normal
fasting levels of tHcy.
In conclusion, the available experimental evidence indicates that the
fasting levels of tHcy in plasma are very sensitive to an abnormality of
the remethylation pathway of homocysteine, such as that associated
with the T/T genotype of MTHFR; however, they do not allow the
conclusion that the PML increases of tHcy levels in plasma only reflect
abnormalities of the trans-sulfuration pathway of homocysteine, because they also tend to be abnormal in individuals with the T/T
genotype of MTHFR and in those with low serum levels of folic acid.
Marco Cattaneo
Rossana Lombardi
Anna Lecchi
Maddalena L. Zighetti
A. Bianchi Bonomi Hemophilia and Thrombosis Center
Department of Internal Medicine
IRCCS Ospedale Maggiore
University of Milano
Milano, Italy
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1999 93: 1116-1118
Mitomycin C−Induced DNA Damage in Fanconi Anemia: Cross-Linking or
Redox-Mediated Effects?
Giovanni Pagano, Adriana Zatterale and Ludmila G, Korkina
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