Leukemia Research 24 (1999) 129–137
`
`www.elsevier.com/locate/leukres
`
`Efficacy of N-acetylcysteine and all-trans retinoic acid in restoring
`in vitro effective hemopoiesis in myelodysplastic syndromes
`
`Agostino Cortelezzi a,*, Chiara Cattaneo a, Barbara Sarina a, Silvia Cristiani a,
`Mauro Pomati a, Ilaria Silvestris a, Marina Motta a, Adalberto Ibatici a,
`Gianluca Gornati b, Aldo Della Volpe c, Anna Teresa Maiolo a
`a Ser6izio Autonomo di Ematologia Diagnostica, Ospedale Maggiore Policlinico IRCCS, Via F. Sforza 35, 20122 Milan, Italy
`b Centro Trasfusionale, Istituti Clinici di Perfezionamento, Milan, Italy
`c Centro Trapianti di Midollo, Ospedale Maggiore Policlinico IRCCS, Milan, Italy
`
`Received 26 April 1999; accepted 20 August 1999
`
`Abstract
`
`We evaluated the in vitro effect on clonogenic potential (CFU-GM) and apoptosis in myelodysplastic syndromes (MDS)
`progenitors of an anti-oxidant (N-acetylcysteine, NAC) and/or a differentiating (all-trans retinoic acid, ATRA) agent. NAC
`significantly reduced apoptosis, both NAC and ATRA induced an increase in CFU-GM, but NAC seemed to be particularly
`effective in the high risk (HR) MDS. NAC+ATRA conferred a significant advantage in terms of CFU-GM with respect to NAC
`and ATRA alone. Tumor Necrosis Factor-a (TNF-a) levels decreased after incubation with NAC in the MDS samples. This study
`shows that ineffective hemopoiesis in MDS could benefit from both NAC and ATRA, suggesting that anti-oxidant treatment may
`play a role in guaranteeing MDS cell survival, predisposing them towards differentiation. © 2000 Published by Elsevier Science
`Ltd. All rights reserved.
`
`Keywords: Myelodysplastic syndromes; N-acetylcysteine; All-trans retinoic acid; Apoptosis; Clonogenic activity
`
`1. Introduction
`
`Increased bone marrow apoptosis and ineffective
`hemopoiesis are the two main characteristics of the
`myelodysplastic syndromes (MDS) [1–6]. The former,
`which is at least partially responsible for premature
`intramedullary cell death, is particularly pronounced in
`low-risk MDS (such as refractory anemia, RA and
`refractory anemia with ring sideroblasts, RARS),
`whereas the latter leads to the well known maturation
`arrest of bone marrow (BM) progenitors, but both
`
`Abbre6iations: ATRA, all-trans-retinoic acid; BM, bone marrow;
`BMMNC, bone marrow mononuclear cell; HR, high risk; LR, low
`risk; MDS, myelodysplastic syndromes; NAC, N-acetylcysteine; RA,
`refractory anemia; RAEB, refractory anemia with excess of blasts;
`RAEB-t, refractory anemia with excess of blasts in transformation;
`RARS, refractory anemia with ring sideroblasts; TNF-a, tumor ne-
`crosis factor-a.
`* Corresponding author. Tel.: +39-02-5503-3429/3345; fax: +39-
`02-5503-3380.
`E-mail address: cortelez@polic.cilea.it (A. Cortelezzi)
`
`determine the often severe and life-threatening cytope-
`nia(s) typical of MDS.
`It is well documented that MDS patients have in-
`creased serum Tumor Necrosis Factor-a (TNF-a) levels
`[7–11], which may be responsible for pro-apoptotic
`oxidative damage by inducing the generation of free
`radicals [12–16]. In line with this hypothesis, Peddie et
`al.
`[17] have demonstrated the presence of oxidative
`damage and a reduction in intracellular glutathione
`(GSH) in MDS CD34+ cells, and a number of in vitro
`studies have shown the ability of the antioxidant thiol
`N-acetylcysteine (NAC) to reduce the apoptosis in-
`duced by free radicals in neuronal and leukemic cell
`lines by increasing the supply of intracellular GSH
`[18–20]. NAC also acts as an antiapoptotic agent by
`inhibiting TNF-a release from lymphocytes and acces-
`sory cells [21].
`MDS bone marrow (BM) progenitors can be induced
`to differentiate and mature in vitro by using various
`differentiating agents, such as vitamin D3 and E, Ara-
`
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`
`C, butyrates and amifostine and this has provided the
`rationale for several clinical trials [22].
`ATRA is a natural metabolite of retinol, whose
`differentiating activity has also been demonstrated in
`non-APL blast cells [23]. However, when used in vivo,
`the ability of ATRA alone to restore defective hemo-
`poiesis in MDS has not always been demonstrated
`[24–30].
`On the basis of these observations we incubated
`MDS progenitor cells in vitro first with NAC alone and
`after 24 h with ATRA alone or in combination in order
`to explore their activity in terms of enhanced clono-
`genic potential
`(CFU-GM), reduced apoptosis and
`stimulated differentiation. The rationale of the sequence
`(first NAC then ATRA) was based on the hypothesis of
`first reducing the degree of apoptosis by means of an
`antioxidant, and then stimulating differentiation via
`ATRA. The possible role of TNF-a in inducing free
`radicals was evaluated by determining TNF-a levels in
`bone marrow plasma and in supernatants of liquid
`cultures.
`
`2. Patients
`
`The BM cells were obtained from 25 patients with de
`novo MDS (21 males and 4 females, with a median age
`of 68 years: range 34–86) and seven normal bone
`marrow donors, after they had given their informed
`consent. On the basis of the FAB criteria, the patients
`were classified as being affected by RA (n=6), RARS
`(n=7), RAEB (n=10) and RAEB-t (n=2). For the
`purpose of the study, we classified RA+RARS as low
`risk (LR) MDS and RAEB+RAEB-t as high risk
`(HR) MDS.
`
`3. Methods
`
`3.1. Cell preparation
`
`The BM samples were collected in preservative-free
`heparin. The mononuclear cells (MNCs) were separated
`by means of gradient centrifugation using Ficoll
`Lymphoprep (Nicomed Pharma Oslo, Norway), evalu-
`ated for apoptosis and then suspended in IMDM
`(Gibco Europe, Paisley, UK) with 10% FCS (Hyclone,
`Logan, Utah, USA ) at a concentration of 1×106/ml
`and incubated at 37°C in 5% CO29NAC 0.5 mM for
`24 h. After this time, some of the cells9NAC (Sigma,
`Saint Louis, MO, USA) were evaluated for CFU-GM
`and apoptosis and others were cultured9ATRA 5 mM
`(kindly provided by Roche) for a further 24 h. After a
`total culture time of 48 h, the cells incubated with
`ATRA, NAC, NAC+ATRA as well as the control
`cells were again tested for CFU-GM and apoptosis.
`
`Due to the scanty cell recovery from MDS BM, the
`samples incubated for 48 h with NAC alone were
`evaluated in only 12/25 cases. The viability after liquid
`incubation was always \90% evaluated by the Trypan
`blue exclusion test.
`
`3.2. Apoptosis
`
`Apoptosis was evaluated by means of a TdT/dUTP
`assay using the commercially available ‘In Situ Cell
`Death
`Fluorescein Detection Kit’
`(Boehringer
`Mannheim, Germany). Briefly, 1–2×106 cells were
`fixed in cold ethanol 70% in PBS, and stored at 4°C
`until use. Before staining, the cells were washed in PBS,
`permeabilized with 100 ml TritonX-100 0.1% in sodium
`citrate 0.1% for 2 min on ice, and again washed twice in
`PBS. After incubation for 1 h at37°C with dUTP
`FITC, with and without TdT, samples were again
`washed in PBS, resuspended in 500 ml of PBS and made
`ready for cytofluorimetric analysis. A total of 1×104
`events were analyzed.
`
`3.3. CFU-GM
`
`A volume of 1×105 cells/ml in IMDM were cultured
`in 1 ml of a mixture containing 20% FCS, 0.3% agar,
`GM-CSF 200 U/ml, IL-3 100 U/ml and SCF 8 U/ml
`(Genzyme Cambridge, MA, USA) and the plates were
`incubated in humidified air with 5% CO2 at 37°C for 14
`days. The aggregates containing \50 cells were scored
`as colonies, whereas those containing B50 cells were
`scored as clusters. All of the cultures were set up in
`quadruplicate. In order to exclude interference due to
`the absence of reducing equivalents in the agar assay,
`clonogenic tests using methylcellulose (HF 4434, Stem
`Cell Technologies Vancouver, Canada) were also made
`in a subset of patients.
`
`3.4. TNF-h le6els in BM plasma and liquid culture
`
`The plasma samples obtained by spinning the BM
`samples at 3000 rpm were stored at −20°C until used,
`and then investigated for their TNF-a content by
`means of a commercially available immunoenzymatic-
`assay kit (Medgenix Diagnostic, Fleurus, Belgium). The
`TNF-a levels in the supernatants of 24 h liquid cultures
`were also evaluated. The minimum detectable dose of
`TNF-a was 3.0 pg/ml.
`
`3.5. Statistical analysis
`
`The Wilcoxon matched pairs signed ranks test was
`used to evaluate within-group differences, and the
`Mann–Whitney test for differences between groups. A
`P value of B0.05 was considered to be statistically
`significant.
`
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`A. Cortelezzi et al. /Leukemia Research 24 (2000) 129–137
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`131
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`4. Results
`
`4.1. E6aluation of apoptosis in fresh bone marrow
`mononuclear cells (BMMNCs)
`
`The apoptosis evaluated in fresh MDS BMMNCs
`was higher than that observed in the normal samples
`(1.6790.16 SE versus 1.4990.27, n.s.). Apoptosis was
`even higher in the LR group (LR: 2.0290.22, PB0.05
`versus normals).
`
`4.2. E6aluation of apoptosis after 24 h of incubation
`with NAC
`
`In the normal samples, apoptosis was significantly
`(PB0.05) less after NAC incubation in comparison
`with controls (3.0390.74 versus 4.190.84). It was also
`less after NAC stimulation in the MDS samples as a
`whole (2.1290.52 versus 3.4490.66) (PB0.01), as
`well as in the two subgroups (LR: 2.0690.68 versus
`3.6790.93; HR: 2.1890.83 versus 3.1690.96) (PB
`0.05 and PB0.01, respectively) (Fig. 1).
`
`4.3. Clonogenic assay (CFU-GM) after 24 h of
`incubation with NAC
`
`The number of CFU-GM was significantly higher in
`the NAC treated normal than in the controls (195.69
`49.72 versus 153.8945.98) (PB0.05). A significant
`increase in the number of CFU-GM was also observed
`in the MDS patients after NAC incubation (MDS:
`73.39912.88
`57.3911.24, PB0.01; LR:
`versus
`78.69914.59 versus 61.85913.79, PB0.01; HR:
`66.5923.54 versus 51.4919.34, PB0.05) (Fig. 2).
`
`4.4. E6aluation of apoptosis after 48 h of incubation
`with NAC9ATRA
`
`ATRA alone did not modify the percentage of apop-
`tosis in either the normals or MDS samples, but incu-
`bation with NAC+ATRA led to a significant decrease
`in the apoptotic rate in both the normal (2.3690.61
`versus 3.7490.97, PB0.05) and MDS samples (2.069
`0.44 versus 3.7890.7, PB0.01). The same was ob-
`served in both the LR (PB0.05) and HR (P=0.06)
`subgroups (LR: 1.6290.34 versus 3.390.69; HR:
`2.7190.96 versus 4.3691.23) (Fig. 3). Apoptosis was
`also less in the 12 MDS samples evaluated after 48 h
`NAC stimulation than in controls (1.5290.85 versus
`3.1691.08, PB0.05) (Fig. 3a).
`
`4.5. Clonogenic assay (CFU-GM) after 48 h of
`incubation with NAC9ATRA
`
`Both ATRA and NAC+ATRA significantly in-
`creased clonogenic activity in both the normal (PB
`0.05) and MDS samples (PB0.01) (normal: CTRL
`172.2936.06, ATRA 329.8962.56, NAC+ATRA
`361965.29; MDS: CTRL 59.67915.28, ATRA
`74.76914.95, NAC+ATRA 87.59919.56).
`However although incubation with ATRA alone in-
`creased clonogenic activity in both MDS subgroups,
`this was statistically significant only in the LR patients
`(PB0.01); the increase after incubation with NAC+
`ATRA was statistically significant
`in both the LR
`(PB0.01) and HR patients (PB0.05) (mean values
`LR: CTRL 78.58921.83, ATRA 100.67921.64,
`NAC+ATRA 110.08929.06; HR: CTRL 34.449
`18.7, ATRA 40.22913.59, NAC+ATRA 55.119
`
`Fig. 1. Effect of NAC on apoptosis after 24 h of liquid culture. * PB0.05, ** PB0.01 versus controls.
`
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`Fig. 2. Effect of NAC on clonogenic activity (CFU-GM) after 24 h of liquid culture. * PB0.05, ** PB0.01 versus controls.
`
`20.09) (Fig. 4). The NAC+ATRA combination con-
`ferred a significant (PB0.05) advantage in terms of
`clonogenic potentials with respect to ATRA in the
`MDS samples as a whole.
`After 48 h incubation with NAC alone, the increase
`in the clonogenic activity of the MDS samples was
`(61.5912.63 versus 42.759
`statistically significant
`10.48, PB0.05) in comparison with the control sam-
`ples, but significantly (PB0.05) less than that observed
`after incubation with NAC+ATRA (69913.61) (Fig.
`4a).
`Analysis of the colony/cluster ratio after 24 and 48 h
`always revealed a moderate and non-significant increase
`in the MDS samples as a whole (24 h: CTRL 29.429
`11.48, NAC 35.99912.92; 48 h: CTRL 24.8698.38,
`ATRA 37.35912.89, NAC 32.83916.33, NAC+
`ATRA 37.27910.91) as well as in the LR (24 h: CTRL
`45.93920.41, NAC 56.06920.46;
`48 h: CTRL
`37.13913.84, ATRA 54.98921.04, NAC 52.039
`29.12, NAC+ATRA 49.49916.17) and HR patients
`(24 h: CTRL 11.0894.98, NAC 13.6996.66; 48 h:
`CTRL 11.0596.55, ATRA 17.52911.46, NAC
`13.6398.81, NAC+ATRA 22913.02). We never ob-
`served a reduction in the colony/cluster ratio of any
`patient sample or under any culture conditions.
`The data obtained using methylcellulose assay were
`comparable to those derived from agar assay (data not
`shown).
`
`4.6. TNF-h le6els in BM plasma and liquid culture
`
`BM plasma TNF-a levels were significantly higher
`(PB0.01) in the MDS patients (36.9493.58, range
`15.53–70.81 pg/ml) than in the normal subjects (8.389
`
`0.44, range 3.06–13.77 pg/ml). No difference was found
`between the LR and HR subgroups, although the
`highest TNF-a (70.81 pg/ml) value was observed in a
`LR patient (Fig. 5). However, the patients with TNF-a
`levels of \50 pg/ml also responded to NAC in terms
`of CFU-GM and apoptosis.
`The TNF-a levels in the supernatants of 24 h BM
`MDS liquid cultures showed a significant (PB0.05)
`decrease after incubation with NAC (16.9996.22) in
`comparison with the control samples (26.7295.78)
`(Fig. 6).
`
`5. Discussion
`
`The apparently bizarre behaviour of MDS hemato-
`poiesis, which is expressed in the form of peripheral
`cytopenia associated with normo-hypercellular bone
`marrow, is the consequence of increased apoptosis and
`the maturation arrest of MDS hemopoietic progenitors.
`It is also known that high levels of TNF-a (a pro-apop-
`totic agent) are found in the serum of MDS patients,
`and so the documented oxidative damage to MDS
`CD34+ cells could also be the result of TNF-a in-
`duced free radical production.
`On the basis of these observations, we evaluated the
`effect of an antioxidant
`(and anti-apoptotic) agent
`(NAC) and a differentiating agent (ATRA), alone and
`in association, on clonogenic activity and apoptosis in
`MDS BM mononuclear cells.
`Our study showed the in vitro ability of NAC to
`enhance hemopoiesis in MDS, even in the presence of a
`large percentage of blasts. After 24 and 48 h of incuba-
`tion with NAC, we observed an increase in the number
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`133
`
`of CFU-GM in the MDS samples as a whole, as well as
`in the LR and HR groups. A consensual decrease in the
`apoptotic rate was seen after 24 and 48 h NAC stimula-
`tion. These data indirectly suggest that apoptosis plays
`a role even in the MDS HR subset. Moreover, ATRA
`did not have any adverse effects on erythroid colonies,
`evaluated in methylcellulose assay (data not shown).
`NAC alone, in fact, slightly enhanced the number of
`BFU-E colonies whereas
`the association ATRA-
`NAC neither increased nor decreased the number of
`colonies.
`As a result of the physiological senescence of cul-
`tured cells, the apoptosis evaluated in the control sam-
`ples (i.e. after incubation with medium alone) was
`higher than that observed in the fresh samples. More-
`over, the level of apoptosis was particularly high in the
`MDS HR subgroup. Since our observations are derived
`
`from a mixed population of all BMMNCs, we cannot
`be sure whether or not this phenomenon is due to
`CD34+ cells. However this singular behaviour may be
`explained by the poor self-renewal of MDS blasts,
`which are less capable of proliferation than leukemic
`blasts, as has previously been pointed out by Aul et al.
`[31]. A propensity towards apoptosis therefore seems to
`coexist with enhanced proliferation in HR MDS cells,
`and this may be responsible for the relative indolence of
`RAEB and RAEB-t.
`The efficacy of NAC may be due to an increase in
`intracellular glutathione (GSH) levels in MDS CD34+
`cells, which in turn provides protection against the
`apoptosis induced by free radicals. Furthermore, NAC
`the release of TNF-a from the
`may also prevent
`lymphocytes still present in BM liquid cultures, a hy-
`pothesis that is apparently supported by the decreased
`
`Fig. 3. Effect of NAC and/or ATRA on apoptosis after 48 h of liquid culture. * PB0.05, ** PB0.01, ° P=0.06 versus controls.
`
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`Fig. 4. Effect of NAC and/or ATRA on clonogenic activity (CFU-GM) after 48 h of liquid culture. * PB0.05, ** PB0.01 versus controls.
`
`release of TNF-a in the supernatant of the liquid
`cultures. NAC was also effective in the patients with
`the highest TNF-a levels.
`On the contrary, and as expected, ATRA did not
`modify the apoptotic rate of MDS or normal BM cells,
`and a significantly enhanced clonogenic activity was
`only observed in the MDS samples as a whole and in
`the LR sub-group, thus suggesting that only patients
`with a low degree of BM blasts retain differentiative
`ability. The fact that, in all of the MDS samples, the
`
`NAC+ATRA combination seemed to confer an ad-
`vantage over the use of either substances alone in terms
`of clonogenic activity suggests that anti-oxidant treat-
`ment may play a key role in guaranteeing MDS cell
`survival and predisposing them towards differentiation.
`There was no evidence that NAC or ATRA add any
`leukemogenic effect on MDS BM cells, as the number
`of clusters remained the same after stimulation with
`either substance, whereas the number of colonies al-
`ways increased.
`
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`135
`
`Fig. 5. Bone marrow plasma TNF-a levels. * PB0.05, ** PB0.01 versus controls.
`
`It has recently been reported that combined therapy
`with ciprofloxacin, pentoxifylline and dexamethasone is
`efficacious in reducing TNF-a levels and improving
`hemopoiesis in MDS patients [9]. Moreover, List et al.
`[32] have demonstrated that the aminothiol amyfostine
`is capable of improving the in vitro clonogenic activity
`
`of MDS hemopoietic precursors. The in vivo results seem
`to be encouraging [33], although Bowen et al.
`[34]
`observed a poor response rate to amyfostine therapy. Our
`own in vitro results strongly suggest that a substance with
`a very low toxicity profile such as NAC could be included
`in the non-cytotoxic therapy of MDS.
`
`Fig. 6. TNF-a levels after 24 h cultures. * PB0.05 versus controls.
`
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`
`Acknowledgements
`
`A Cortelezzi provided the concept, design, drafting
`of the paper, critical revision and gave final approval. C
`Cattaneo provided statistical expertise and contributed
`to the concept design, drafting of the paper, critical
`revision. B Sarina provided statistical help, assisted in
`drafting the paper and gave final approval. S Cristiani
`assisted with data interpretation, provided statistical
`expertise, administrative support and helped with data
`assembly and gave final approval. M Pomati helped
`with analysis of the data. I Silvestris collected and
`helped with the data analysis. M Motta provided study
`materials and helped to assemble the data. A Ibatici
`provided study materials. AT Maiola provided funding
`for the project.
`
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