Mutagenesis, Vol. 17, No. 1, 25-30,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Alternative metabolic pathways for energy supply and resistance to apoptosis in Fanconi anaemia
Laboratory of Mutagenesis, National Cancer Research Institute, IST, Lgo R. Benzi 10, 16132 Genoa and 1 Department of Oncology, Biology and Genetics, University of Genoa, Italy
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Abstract |
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Deregulation of control of the apoptotic process in Fanconi anaemia (FA) appears to be one of the main features of this disease at the cellular level. We show here that FA cells are resistant to treatments with rhodamine-1,2,3 and doxycycline, which both interfere with mitochondrial functionality by different mechanisms. In contrast, normal lymphoblastoid cells are severely affected by these treatments, which result in acute ATP depletion and a significant enhancement of the fraction of cells undergoing apoptotic cell death. FA cells are very sensitive to the action of 2-deoxy-D-glucose (2dG) and iodoacetic acid (IAA), two inhibitors of glycolytic metabolism. The ability of FA cells to sustain metabolic insults interfering with energy production and balance may be linked with the pathological manifestations of the disease, including susceptibility to acute myeloid leukemia. These findings suggest that FA genes may be involved in a pathway that mediates a protective response to stress. We suggest that a peculiar metabolic regulation in FA cells could explain both defective apoptosis and susceptibility to oxidative stress.
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Introduction |
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Fanconi anaemia (FA) is a complex chromosomal instability disorder for which at least seven genetic complementation groups (FANCA to FANCG) have been characterized (Carreau and Buchwald, 1998
). Patients belonging to complementation groups FANCA and FANCC account for ~80% of affected sufferers. While the FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG (Garcia-Higuera et al., 2001) genes have recently been cloned, the biochemical function of the corresponding proteins is still largely unknown (Kupfer et al., 1997; Mian and Moser, 1998). It has been suggested that enhanced cell death involving the sequential activation of caspases and production of reactive oxygen species leads to depletion of stem and progenitor haemopoietic cells and to the development of aplastic anaemia in FA patients (Nagata and Goldstein, 1995). However, apoptosis appears to be deregulated in FA (Ridet et al., 1997), possibly due to defective signalling through the Fas receptor (Kruyt et al., 1996; Marathi et al., 1996). A number of recent studies revealed that FA cells are resistant to apoptotic cell death induced by different oxidants (Monti et al., 1997; Ridet et al., 1997).
Apoptosis is an active suicide mechanism used under certain circumstances by cells undergoing growth impairment due to a lack of proliferative stimuli, growth factor withdrawal, cellular damage or inhibition of the energy production pathways (Weil et al., 1996). Apoptosis is particularly sensitive to the redox state of the cells and is strictly associated with alterations in mitochondrial functionality (Kannan and Jain, 2000). A reduction in mitochondrial transmembrane polarization potential mediated by oxidative stress, enhanced TNF-
, NAD+ depletion and altered cytokine expression all play a role in FA (Bagnara et al., 1993; Rosselli et al., 1994).
Mitomycin C (MMC) and other DNA crosslinking agents to which FA cells are particularly sensitive act not only at the nuclear level but also inside the mitochondria (Clarke et al., 1997). These agents induce superoxide anion production, damage mitochondrial DNA, alter the cellular redox equilibrium and interfere with the production of ATP (Pritsos et al., 1997), which lead, as a consequence, to diminished cell growth and survival, cell cycle arrest at different phases and enhanced apoptosis. The intriguing relationships between apoptotic susceptibility and alterations in redox state and energy metabolism prompted us to study the response of FA cells to a number of metabolic inhibitors known to interfere with ATP production and the consequence of these treatments on proliferation and survival of these cells
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Materials and methods |
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Cells
EUFA-274-L and HSC536 cells, referred to hereafter as cell lines FANCA and FANCC, respectively, were obtained after Epstein–Barr virus (EBV) transformation of lymphocytes from two FA patients belonging to complementation groups A and C. COR FANCA and COR FANCC are spontaneous in vitro revertants from cell lines EUFA-274-L and HSC536, respectively. These cell lines show a normal response to MMC (Marathi et al., 1996
). Two cell lines, VU-362 and HSC93, derived from normal lymphocytes immortalized by EBV were used as controls. For the purposes of simplicity, the data reported in the tables and figures refer to HSC93 cells, since the response of VU-623 cells was very similar. All these cell lines were kindly provided by Dr H.Joenje (University of Amsterdam, Amsterdam, The Netherlands). The cells were routinely maintained in RPMI-1640 medium containing 1 mM L-glutamine, 1 mM pyruvate and 100 U/ml penicillin/streptomycin, supplemented with 10% heat-inactivated fetal calf serum, and were grown at 5% CO2 at 37°C. Cell viability was determined using the trypan blue exclusion assay. In the experiments performed to test the ability of the cell lines to use alternative precursors for the production of energy, cells were grown in media of different compositions, while concomitantly growing in the presence of doxycycline. The media employed were: RPMI, RPMI supplemented with 25 mM glucose, Dulbecco's modified Eagle's medium (DMEM), DMEM supplemented with 1 mM sodium pyruvate and DMEM supplemented with 1 mM sodium pyruvate plus 25 mM glucose.
Treatments
Chemicals used in this study were from Sigma Chemical Co. (St Louis, MO) unless otherwise specified. In the case of chronic treatments, the drug used was diluted to the assay concentration directly in complete growth medium. Rhodamine-1,2,3 (Fluka Chemie AG, Buchs, Switzerland) (2.5 µM) was prepared from a 1 mg/ml stock solution in 100% ethanol. Doxycycline (15 µM) was from a 6 mg/ml stock solution in 70% ethanol. Culture medium was replaced every 48 h with fresh medium already containing the appropriate drug at the final concentration. 2-Deoxy-D-glucose (2dG) (5 mM) and iodoacetic acid (IAA) (10 mM) were prepared from 100-fold concentrated stock solutions directly in growth medium.
Mitochondrial membrane polarization (MMP) analysis
MMP was measured according to the method described (Polla et al., 1996). Aliquots of cell suspensions (1.0x106) were incubated in complete medium with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolcarbocyanine iodide, 10 µg/ml) at room temperature in the dark for 10 min. Cells were then washed twice in phosphate-buffered saline (PBS) and analysed in a Perkin Elmer LS 50 Spectrofluorimeter. Excitation at 488 nm and emission in the range 500–625 nm enabled visualization and quantification of the fraction of cells with depolarized mitochondrial membranes.
Cell cycle analysis and apoptosis
After treatment with the various agents, cells were washed with PBS, resuspended and fixed overnight at 4°C in 250 µl of PBS and 750 µl of 95% ethanol. Samples were then centrifuged at 1000 r.p.m. for 5 min at 4°C and the pellet resuspended in RNase A (1 mg/ml) in PBS and incubated for 20 min at 37°C. The samples were then washed (PBS, 0.5% Tween 20) and centrifuged twice (1000 r.p.m. for 5 min at 4°C). The cells were finally resuspended in a solution of propidium iodide (10 mg/ml) in PBS and stained for at least 2 h before analysis. For the cell cycle analysis, a FACScan (Beckton Dickinson) was used and 20 000 events were recorded for each sample. Cell cycle distribution and apoptosis were analysed with ModFit software (Beckton Dickinson).
ATP quantification
Intracellular ATP quantification was achieved using the procedure described in the Sigma ATP reaction kit 366-UV protocol. Briefly, phosphoglycerate phosphokinase and glyceraldehyde phosphate dehydrogenase catalyse ATP reduction to ADP concomitant with NADH reduction to NAD. By determining the decrease in absorbance at 340 nm, a measurement of the ATP originally present in the sample was obtained.
Statistical analysis
Data were statistically examined by one-way ANOVA and unpaired two-tailed Student's t-test using InStat software for the Macintosh.
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Results |
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Rhodamine-1,2,3 is a lipophilic cation that accumulates specifically within the mitochondrial membranes. It induces membrane depolarization, affects the efficiency of electron transport and impairs the respiratory chain and oxidative phosphorylation. While HSC93 and VU-362 (normal) cells showed a significant loss of viability (Figure 1
), only a slight decrease in survival of FANCA and FANCC cells was induced by chronic treatment with rhodamine-1,2,3. The behaviour of the two FA cell lines was very similar. Survival of COR FANCA and COR FANCC cells was intermediate between the parental FA and control cell lines.
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Continuous treatment with doxycycline inhibits synthesis of mitochondrially encoded proteins by 50% at every cell division (van der Bogert et al., 1992). Figure 2
shows survival of the different cell lines as a function of time of exposure to doxycycline. As for rhodamine-1,2,3, drug sensitivity was highest in HSC93 and VU-362 cells, lowest for FANCA and FANCC cells and intermediate in the FA revertant cell lines.
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To test the ability of the various cell lines to use alternative precursors for the production of energy, each cell line was maintained in growth media of different compositions while concomitantly being exposed to doxycycline. No compound present in the standard DMEM medium formulation is absent in RPMI, but DMEM contains higher concentrations of almost all amino acids and vitamins. As shown in Figure 3
, the richer formulation of DMEM promoted increased survival of all cell lines and, particularly, of HSC93 and VU-362 cells (compare Figure 3A and B). Survival was further increased by the addition of glucose to both media (data not shown). The addition of glucose to RPMI or of pyruvate to DMEM did not greatly change the response of the cell lines. Since no compound present in the standard DMEM medium formulation is absent in RPMI, a single factor is unlikely to account for the different effects on cellular proliferation in the presence of doxycycline.
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Inhibition of glucose utilization by the metabolic inhibitor 2dG resulted in a rapid decrease in cell viability of the two FA cell lines more rapidly than of the control or revertant cells (Figure 4A
). IAA, an anti-metabolite known to inactivate the glycolytic enzyme glyceraldehyde 3-phosphate dehydro-genase by covalent modification of a cysteine residue within its active site and thus irreversibly inhibiting glycolysis, induced a significant reduction in survival of all the cell lines (Figure 4B). This effect was less prominent in HSC93 and VU-362 cells.
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Apoptotic induction following treatment with rhodamine-1,2,3 or 2dG was followed by quantification of the sub-G1 peak in a cell cycle analysis (Table I
), as well as by quantification of the fraction of cells presenting MMP (Table II). The treatment with rhodamine-1,2,3 induced apoptotic cell death to a significant extent in HSC93 and VU-362 cells. In striking contrast to this behaviour, 2dG treatment induced significant cell death in FANCA cells (Table I). An increase was also seen in FANCC, COR FANCA and COR FANCC cells, but it was not statistically significant. MMP quantification (Table II) performed under the same conditions confirmed that normal cells are particularly sensitive to treatment with rhodamine-1,2,3, while FANCA and FANCC cells are most influenced by treatment with 2dG. COR FANCA and COR FANCC cells showed intermediate levels of depolarization.
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The impairment of mitochondrial functions induced by rhodamine 1,2,3 resulted in a block in the production of ATP (Figure 5
) Rhodamine-1,2,3-induced MMP reduced the intracellular ATP content of HSC93 and VU-362 cells to values <35% of the initial level (Figure 5A), while both FA cell lines maintained ATP at >70% of the basal level up to 72 h after treatment. A similar result was obtained after inhibition of mitochondrial protein synthesis induced by doxycycline, where a reduction in intracellular ATP level for HSC93 cells to 40% of the basal level was found within the first 48 h (Figure 5B) and a further decrease ensued thereafter. These data fit with findings relative to the MMP quantification reported in Table II
. Both the FA lines were more resistant to treatment, with the FANCA line showing a modest reduction of ATP to 74%. COR FANCA and COR FANCC cells behaved similarly to HSC93.
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The block of glucose uptake and inhibition of glycolysis achieved by treatments with 2dG and IAA led to a rapid decrease in ATP for all cell lines (Figure 6
). The behaviour of the two COR cell lines was always intermediate between control and mutant cells and these cells appeared to maintain some of the biochemical characteristics of the mutants. This finding may be associated with the fact that these clones were specifically isolated for normalization to MMC sensitivity.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Enhanced apoptosis could explain the haemopoietic defect in FA, due to loss of stem cells, as well as many of the developmental defects characteristic of the disease (Philpott et al., 1995
). However, the route to apoptosis in FA has been reported to be both quantitatively and qualitatively abnormal or abortive (Clarke et al., 1998). Upon exposure to MMC, FANCC cells undergo mitochondrial membrane depolarization and apoptosis (Guillouf et al., 1999). 2-Deoxy-D-ribose (d-Rib), an inducer of apoptosis, specifically interferes with mitochondrial redox balance and induces mitochondrial membrane depolarization. Monti et al. (1997) reported that apoptosis induced by d-Rib and TNF- was significantly lower in FA cells. These reports, as well as the knowledge that the activity of MMC inside the mitochondria is strictly modulated by the oxygen tension (Clarke et al., 1997; Pritsos et al., 1997), prompted us to speculate as to the existence of a possible functional defect in mitochondria from FA cells as the factor responsible for defective apoptosis. The action of rhodamine-1,2,3 is similar to that of d-Rib and FA cells treated with this agent were still able to grow and survived. Similar results were observed in cells treated with doxycycline (data not shown). While a difference in uptake of these drugs between FA and normal cells cannot be excluded, this does not seem to provide a satisfactory explanation when the results of the experiments with 2dG and IAA are considered. The ability of FA cells to survive when mitochondrial functionality is inhibited by rhodamine-1,2,3 and doxycycline may seem paradoxical, since the consequent ATP shortage would limit cell survival. We found, however, that FA cells are able to produce ATP under conditions where mitochondrial functionality is inhibited. While all the cells used in this study were able to maintain a certain amount of cellular homeostasis using glucose as the main metabolic fuel and are able to switch their metabolic requirements normally when allowed to do so, this condition appears to be favoured in FA cells.
The inhibition of glucose utilization on treatment of the cells with 2dG and IAA induced rapid ATP depletion that strongly affected the survival of FA cells. From the standpoint of cellular functionality, the same result is expected from inhibition of the mitochondrial or glycolitic pathways (Marton et al., 1997), provided that ATP production is maintained above a certain level (Sweet and Singh, 1995).
In conclusion, our data suggest that FA cells are able to resist apoptotic cell death induced by different treatments that interfere with cellular redox state and ATP production. The maintenance of ATP production appears to be associated mainly with enhanced glycolysis in FA, suggesting the presence of a functional abnormality of the mitochondria in these cells. An analogous behaviour has been reported in rat liver endothelial cells with functional mitochondrial impairment (Nishimura et al., 1998) and, significantly, these findings have been reported in association with the cytoskeletal abnormalities that are typical of FA cells (Kletsas et al., 1998).
Finally, it is noteworthy that the slightly different behaviour shown by FA cells belonging to the two complementation groups employed in this study suggests that the different FA genes, although coordinated within the same biochemical pathway, have different roles (D'Andrea and Grompe, 1997). This difference may also account in part for the different physiopathological manifestations of the disease in patients belonging to the various complementation groups.
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Acknowledgments |
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We are greatly indebted to Prof. Hans Joenje for supplying the cell lines. We are grateful to the Associazione Italiana per la Ricerca sulla Fanconi Anemia (AIRFA) for encouragement and support. This research was partially supported by the EC EUROS project (BMH4-CT98-3107 DG12-SSMI) and CNR Progetto Finalizzato ACRO.
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Notes |
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2 To whom correspondence should be addressed. Tel: +39 010 56 00 910; Fax: +39 010 56 00 992; Email: deganpol{at}hp380.ist.unige.it
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Received on February 13, 2001; accepted on August 10, 2001.
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