`@1998 Cancer Research Campagn
`
`Autonomous proliferation and bcl-2 expression involving
`haematopoietic cells in patients with myelodysplastic
`syndrome
`
`C Bincolettol, STO Saad2, E Soares da Silva2 and MLS Queiroz3
`Departments of 'Physiology, 2Clinical MedkineHemocentre and 3PhafrmacoogHemocentre. State University of Campinas, Faculty of Medical Sciences,
`Unicamp, Brazil
`
`Summary In this work, we investigated the autonomous proliferation, bcl-2 expression and number of apoptotic cells in the bone marrow of
`patients with confirmed diagnosis of myelodysplastic syndromes (MDS). Normal bone marrow cells obtained from donors of the Clinical
`Hospital of this university were used as a control. The autonomous proliferation, evaluated by lonal culture without exogenous growth factor,
`and the number of apoptotic cells in bone marrow kept for 10 days in liquid cultures at 3TC and 5% carbon dioxide, were significantly greater
`in MDS patients than in control subjects (P = 0.001, Wilcoxon). However, bcl-2 expression, measured by immunocytochemistry, was
`significantly lower in MDS patients than in normal individuals (P = 0.002, Wilcoxon). These results suggest that the high proliferation activity
`in MDS patients may be counteracted by the high level of medullar cell death, which might be related to the lower bcl-2 expression.
`
`Keywords: apoptosis; autonomous cell proliferation; bc/-2; myelodysplastic syndrome
`
`Myelodysplastic syndromes (MDS) consist of a group of acquired
`haemopoietic disorders with evidence of trilineage dysplasia and an
`incidence of 30% of eventual transformation into acute myeloid
`leukaemia (AML) (Ganser and Hoelzer. 1992: Loffler et al. 1992:
`Willemze et al. 1993). An apparent paradox in MDS is that patients
`with these disorders have peripheral cytopenias. despite frequently
`having normo- or hypercellular bone marrow (Raza et al. 1995).
`These contradictory findings may be explained by an excessive
`intramedullary cell death in the face of normal or even enhanced
`rates of proliferation. Recently. some studies have suggested that
`the increased programmed cell death. or apoptosis. may cancel the
`normal or high proliferation activity in MDS patients (Raza et al.
`1995). One reason for this increased number of apoptotic cells
`may be transcriptionally deregulated bcl-2 expression. bcl-2 was
`initially detected at a translocation breakpoint in B-cell follicular
`lymphomas and was subsequently shown to have a role in
`preventing apoptosis (Korsmeyer. 1992: Vaux. 1993). The protein
`is found in various fetal tissues destined for long-term survival and
`in adult tissues in which apoptosis has an important homeostatic
`role (Hockenbery et al. 1991). bcl-2 is expressed in the myeloid
`lineage at the myeloblastic and promyelocytic stages of differentia-
`tion. diminishing with the maturation of cells into granulocytes
`(Delia et al. 1992). Furthermore. the early cell death observed in
`bone marrow cells of MDS patients suggests abnormalities in the
`cell cycle control pathways. which can also affect cellular prolifer-
`ation. For example. suppression of the apoptotic-enhancing effect
`of deregulated c-mvc by either bcl-2 or mutated p53 allows expres-
`sion of an unopposed proliferate signal (Vaux et al. 1988: Green et
`
`Received 23 July 1997
`Revised 16 February 1998
`Accepted 3 March 1998
`Correspondence to: MLS Queiroz. Department of Pharmacology. FCM,
`Unicamp, PO Box 6111. CEP 13084-100, Campnas. S.P., Brazil
`
`al. 1994). Moreover. the association between proliferation activity.
`bc1-2 expression and apoptosis has not been previously determined.
`In the present work. we examined bcl-2 expression. autonomous
`colony
`(without exogenous growth factors) and
`formation
`apoptosis in bone marrow cells from MDS patients with the aim of
`identifying the process involved in the ineffective haemopoiesis
`observed in this clonal disorder.
`
`MATERIAL AND METHODS
`Cases
`bc1-2 expression was evaluated in
`all
`the patients
`(n = 15).
`Autonomous colony formation (CFU-C) and apoptosis were studied
`in 12 and 11 patients respectively. The MDS patients were defied
`according to the FAB cooperative group (Bennett et al. 1982). Bone
`marrow cells from 19 blood donors of the Clinical Hospital in this
`universitv were used as control subjects (n = 11 for apoptosis. n = 15
`for bcl-2 expression and n = 19 for autonomous proliferation). All
`subjects gave informed consent and the study was approved by the
`ethics committee of this hospital. A maximum of 3 ml of bone
`marrow was allowed to be used for this study.
`
`Bone marrow cell separation
`Mononuclear cells were separated from 3 ml of heparinized bone
`marrow by 30 mm
`centrifutation at 400g in Ficoll-Hypaque
`(density 1.077g ml-l: Pharmacia Fine Chemicals. Uppsala. Sweden).
`The cells from the interface were washed three times with RPMI-
`1640 (Sigma. St Louis. MO. USA) and counted for autonomous
`colony formation culture. bc1-2 expression and apoptosis.
`
`Assay for autonomous colony formation (CFU)
`Assay with mononuclear cell suspensions was performed in 2 ml
`of agar cultures in 35-mm Petri dishes using 5 x l0W cells ml. The
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`Figure 3 Percentage of apoptotic cells from bone marrow of MDS patients
`and control subjects, obtained after 10 days in liquid cultures (P= 0.001,
`Wicoxon)
`
`bc-2 expression (immunocytochemistry)
`Cytocentrifuge preparations of mononuclear cells were fixed in
`acetone at 40C and washed in Ths-buffered saline containing
`Tween (TBSlTween 20, 50 mm Tns-HCI, 0.9% sodium chloride,
`0.05% Tween 20, pH 7.6). Slides were incubated in a 1:40 dilution
`of monoclonal mouse antibody to human bcl-2 oncoprotein in
`phosphate-buffered saline (PBS) containing 2% bovine serum
`albumin for 1 h, a 1:150 dilution of goat biotinylated anti-mouse
`immunoglobulin in PBS for 45 min and with alkaline phosphatase-
`conjugated streptavidin (Dako) for 45 min. Between incubations,
`slides were washed thoroughly with TBSiTween. Alkaline phos-
`phatase activity was detected using a substrate of 0.2% naphthol
`AS-MX phosphate. 2% dimethylformamide, 0.24% levamisole and
`0.1% Fast Red TR salt in 0.01 M Ths-HCL, pH 8.1. Mononuclear
`cells were counterstained using haematoxylin. All chemicals were
`from Sigma (Maung et al, 1994). The percentage of positive bcl-2
`mononuclear cells present in MDS patients and in control subjects
`was calculated after counting at least 300 cells.
`
`Apoptotc cells
`Viability of mononuclear cells isolated from heparinized bone
`marrow was determined by the trypan blue dye exclusion test.
`The cells were resuspended in 20% serum/RPMI-1640 medium
`(Gibco-USA) supplemented with 2 mm 1-' glutamnine, 100 U ml-'
`penicillin and 100 gg mll streptomycin. The cells were seeded at
`a density of 5 x 101 cells ml-l and incubated in an atmosphere of
`95% air/5% carbon dioxide at 370C for 10 days. Cytocentrifuge
`preparations from MDS bone marrow cells kept for 10 days in
`culture were performed. The slides were then stained with haema-
`toxylin and the apoptotic cells were determined under high power
`
`Patents
`
`Control nAexts
`
`Figure 1
`Autonorous colony proliferaton in Fe absence of exogenous
`growth fators of bone marrow cels from MDS patients and control subjects
`(P= 0.001, Wllcoxon)
`
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`Percentage of mononuclear bcl-2+ cell expression from bone
`Figwe 2
`marrow of MDS patents and control subjects (P = 0.002, Wicoxon)
`
`medium used was Iscove's modified Dulbecco medium (Sigma)
`containing 20% fetal calf serum (Sigma) and 0.6% agar. Colony
`formation was studied without addition of any exogenous growth
`factors. The plates were incubated at 370C in 5% carbon dioxide in
`air at 100% humidity. Colonies were counted after 14 days at 35 x
`magnification using a dissection microscope (Metcalf, 1984).
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`Brtihs Joukmal of Cancer (1998) 78(5), 621-624
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`(40x objective) in accordance with Koshida et al (1997) as
`follows: overall shrinkage and homogeneously dark basophilic
`nuclei: presence of nuclear fragments (apoptotic bodies); sharply
`delineated cell borders surrounded by empty space: homogeneous
`eosinophilic cytoplasm. The percentage of apoptotic cells was
`calculated after counting at least 300 cells.
`
`Statisticals analysis
`Statistical comparison of the results from MDS patients and
`control subjects was performed using the Wilcoxon test. A result
`of P < 0.05 was considered statistically significant.
`
`RESULTS
`The growth and differentiation of early bone marrow progenitor
`cells (CFU/5 x 10' cells ml-') in the absence of any exogenous
`growth factors,
`patients with a confirmed diagnosis of
`in
`myelodysplastic syndrome (MDS) were significantly higher than
`in control subjects (P = 0.001. Wilcoxon, Figure 1). Out of 12
`patients. only two presented values similar to that of control
`subjects. The percentage of positive mononuclear bc1-2 cells was
`reduced in MDS patients in relation to normal individuals (P =
`0.002. Wllcoxon, Figure 2). However, the percentage of apoptotic
`cells was significantly increased in MDS bone marrow cells in
`relation to the percentage in control subjects (P = 0.001, Wilcoxon,
`Figure 3). We did not observe a correlation between bcl-2 expres-
`sion. autonomous proliferation and FAB classification. It was not
`possible to perform the correlation between bcl-2 expression
`and apoptosis as almost all cells were in an advanced stage of
`apoptosis by the tenth day of culture (Figure 3).
`
`DISCUSSION
`Myelodysplastic syndromes (MDS) are clonal disorders of pluri-
`potent haematopoietic stem cells, generally of unknown aetiology.
`occurring predominantly in the elderly, characterized by ineffective
`haematopoiesis leading to blood cytopenias despite of the presence
`of a hypercellular or normocellular bone marrow (Fenaux. 1996).
`Recently. some studies have suggested that an important factor
`involved in the peripheral cytopenias in MDS patients is an
`increase in programmed cell death (apoptosis). In this regard, a
`high range of apoptosis was observed in this study when the MDS
`cells were cultivated in liquid cultures and evaluated morphologi-
`cally. These results corroborate the findings reported by Raza et al
`(1995). who observed more than 75% of apoptosis in stromal bone
`marrow cells of the MDS patients using the in situ end-labelling
`technique (ISEL). These findings are complementary as the ISEL
`technique allows the study of apoptosis at the very early stages after
`initial changes in DNA levels, whereas our morphological
`approach reveals the late stages of apoptosis. Therefore. based on
`these results and other reports (Clark and Lampert. 1990: Yoshida,
`1993: Raza et al. 1995: Yoshida et al. 1995: Bogdanovic et al.
`1997). we suggest that apoptosis is a mechanism responsible. at
`least in part. for the ineffective haematopoiesis in MDS.
`Alterations in the bc1-2 expression are involved in the regulation
`of apoptosis (Gajewaki and Thompson. 1996: Kroemer. 1997). as
`well as in the sensitivity of cells to a variety of cytotoxic drugs
`(Kamesaki et al. 1993). In this regard. we observed a low bcl-2
`expression in mononuclear MDS cells, suggesting that this proto-
`oncogene may be involved in the high rate of cell death observed
`
`bcl-2 expression in MDS 623
`
`in this study. These findings suggest impairment in the pathways
`involved in proliferation. differentiation and cell death. In this
`field. we observed autonomous colony formation in the absence of
`any exogenous haemopoietic growth factors in MDS patients.
`These results support the hypothesis. in the literature. that early
`cell death cancels high or normal proliferation activity (Raza et al.
`1995). Autonomous proliferation activity seems to be related to
`the autocrine production of some growth factors in acute myeloid
`leukaemia (AML) (Young and Griffin. 1986: Bradbury et al. 1994:
`Bradbury and Russell: 1995. Russel et al. 1995: Hu et al. 1996).
`However. Shetty et al (1996). reported a relative absence or unde-
`tectable levels of granulocyte-macrophage colony-stimulating
`factor (GM-CSF) in MDS patients. which denotes another mecha-
`nism involved in the progression of this disease. In this regard.
`Soligo et al (1996). observed an overexpression of GM-CSF and c-
`kit receptors in MDS patients. suggesting an increased sensitivity
`of bone marrow progenitors. leading to an autonomous colony
`formation without exogenous growth factors. On the other hand.
`patients with MDS have normal or elevated levels of erythropoi-
`etin (Epo) (Jacobs et al. 1989) and activation of StatS by Epo is
`impaired in these patients (Hoefsloot et al. 1997). Moreover. alter-
`ations in genes that control the proliferation activity and cell death
`can be involved in the autonomous colony formation observed in
`our study. such as the c-mvc oncogene (Nowak. 1992. Rajapaksa et
`al. 1996) or a high p21 ras expression (Silva et al. 1997) Another
`factor that could help to explain our results is the recent finding in
`our laboratory (unpublished data) showing a high p53 expression
`in MDS patients. The p53 overexpression might be associated with
`bcl-2 mRNA and protein reduction. probably because the 5'
`untranslated region of the bcl-2 gene contains a p53-negative
`responsive element, through which p53 may directly or indirectly
`transcriptionally down-regulate the expression of bcl-2 (Haldar et
`al. 1994: Miyashita et al. 1994: Lepelley et al. 1995). Furthermore.
`p53 stimulates the expression of bar. a gene that encodes a domi-
`nant inhibitor of the bcl-2 protein (Miyashita et al. 1994).
`Although these results suggest a participation of bcl-2 expres-
`sion in the high rate of cell death in MDS patients. further investi-
`gations are necessary to clarify
`the molecular mechanisms
`involved in the progress of this disease. It has been demonstrated
`that the cell-surface receptor FAS/APO-I (CD95) is able to trigger
`apoptosis in a variety of cell types (Karawajew et al. 1997).
`However. in relation to the FAS expression on MDS cells. Munker
`et al (1996). described in 17 MDS patients that the average value
`of soluble CD95 was not statistically different from normal control
`subjects and no correlation was found with the FAB type.
`In conclusion. the MDS cells with a higher baseline level of
`growth stimulation may contribute to additional mutations and
`progression of MDS to AML. Finally. autonomous proliferation
`activity might be a good marker for myelodysplastic syndrome
`diagnosis.
`
`ACKNOWLEDGEMENTS
`Fundaqdo de Amparo a Pesquisa do Estado de Sdo Paulo e
`Conselho Nacional de Desenvolvimento e Pesquisa (CNPq).
`
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`Britih Jounal of Cancer (1998) 78(5), 621-624
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`0 Cancer Research Campaign 1998
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