Med Oncol (2007) 24:419–424 DOI 10.1007/s12032-007-0031-y

ORIGINAL PAPER

The cell growth, morphology and immunocytochemistry of novel cell line established from a bone marrow of the patient with therapy-related myelodysplastic syndrome, entitled PC-MDS Gordana Bogdanovic´ Æ Dimitar Jakimov Æ Bratislav Stojiljkovic Æ Vladimir Jurisˇic´

Received: 29 March 2007 / Accepted: 1 May 2007 / Published online: 1 June 2007
Humana Press Inc. 2007

Abstract We report on cell growth, morphology, and immunocytochemistry of the first human cell line, PCMDS, derived from a bone marrow of a patient with therapy-related myelodysplastic syndrome who had no overt leukemia post-MDS phase. This cell population consisted of fast-growing mononuclear cells. Standard cytochemistry methods for detection of MPO, lipids, glycogen and ANAE gave results as follows: MPO and SBB negative while PAS and ANAE positive. Positive cytochemical staining and immunophenotype analyses indicated that PC-MDS cells have some characteristics of the early myeloid precursor cell. As the first t-MDS derived cell line it could be a new tool in evaluation of complex biology of MDS and also serves as a model for diverse in-vitro research. Keywords Human cell line
MDS
Therapy
Cell growth
Immunocytochemistry
Morphology

Introduction Cell lines represent important resource for research in a variety of fields and disciplines. The experimental use of continuous human tumor cell lines represents a valuable tool for understanding the biology, pathogenesis, and

treatment [1] options for various malignancies including myelodysplastic syndrome (MDS). MDS are heterogeneous group of clonal hematopoietic stem cell disorders, characterized by ineffective hematopoiesis, and impaired maturation of hematopoietic cells, progressive cytopenias, and an increased risk of developing acute myeloid leukemia (AML) [2]. Natural history of MDS ranges from indolent chronic course to rapid leukemic progression [3–8]. Continuous malignant hematopoietic cell lines have been established from the whole spectrum of MDS variants and from different stages of the disease. Drexler [1] recently reported on 10 cell lines derived from the various MDS subtypes and 17 cell lines from the patients with post-MDS leukemia. Among MDS subtypes-derived cell lines only one cell line was derived from the patient with therapy-related MDS/AML (t-MDS/ t-AML) [9]. We here described cell growth, morphology and immunocytochemistry characteristics of the first human cell line obtained in our laboratory. Cell line was derived from a bone marrow of a patient with t-MDS who had no overt signs or symptoms of post-MDS leukemia.

Patient and methods Case history

G. Bogdanovic´
D. Jakimov
B. Stojiljkovic Institute of Oncology Sremska Kamenica, Sremska Kamenica, Serbia V. Jurisˇic´ (&) School of Medicine, University of Kragujevac, 34000 Kragujevac, Serbia e-mail: [email protected]

A 21-year-old Caucasian man, with diagnosis of pancytopenia and secondary MDS, was admitted to daily hospital of the Institute of Oncology Sremska Kamenica, Serbia for control sternal aspiration biopsy. At that time bone marrow sample was also taken and used for cell line establishing. The patient’s disease history started in 1981, when he was 10 years old, and when an acute lymphoblastic leukemia

420

(ALL) was diagnosed. He was treated by standard polychemotherapy for children ALL from 1981 to 1983. A remission was achieved in 1982. Several years later (1985), aplastic anemia was developed. Patient was treated with blood transfusions. Secondary hemosiderosis and hepatitis virus C (HCV) were diagnosed in 1990 and 1991, respectively. In 1991, secondary MDS, FAB subtype RA, with hypoplastic bone marrow and pancytopenia was diagnosed. Cytogenetic analysis (G-banding technique) performed on bone marrow specimen showed normal male karyotype. Patient was treated with blood-derivative transfusions, iron-chelating agents, and shortly with erythropoetin without success. At the time when bone marrow aspiration was made, the patient had no signs or symptoms of acute leukemia and the result of the bone marrow analysis confirmed diagnosis of MDS, FAB subtype RA. Due to hemosiderosis, dilatative myocardiopathy was developed and overt clinical manifestations of hepatic cirrhosis appeared due to hemosiderosis, myocardiopathy, and HCV infection. Patient died in April 1998 from cardiovascular insufficiency. He had no signs of progression of hematological disease. The MDS was probably related to cytotoxic treatment he received for ALL. As myelodysplasia was developed several years after chemotherapy, secondary MDS was termed t-MDS. Establishment of the cell line Two milliliters of heparinized bone marrow aspirate, without erythrocyte separation, was seeded into tissue culture flask (Costar, 25 cm2, Badhoevedorp, The Netherlands) containing 4.5 ml RPMI 1640 medium (Sigma– Aldrich, Taufkirchen, Germany) supplemented with 10% of fetal calf serum (FCS) (Sigma–Aldrich) and antibiotics (penicillin and streptomycin) (Galenika, Belgrade, Serbia). The culture was maintained at 37C in a humid atmosphere with 5% carbon dioxide. About 48 h later small ‘‘islands’’ consisting of light and round cells were found. Medium was completely replaced by fresh one, and up to the seventh day of culture, only one-third of the medium was replaced. During the first week of culture the number of ‘‘islands’’ consisting of adherent fibroblast-like cells was increased. A monolayer was produced 15 days after seeding and then the first subculture was made using 0.5% trypsin in PBS. The subcultured cells continued to grow as adherent fibroblast-like cells for the next 2 weeks i.e., until the second subculture. Viability was 73%. At the same time, detached, round cells (viability 61%) were found in the supernatant. After the second subculture, the elongated or triangular cells were partly adhered to the flask bottom. The number of cells that floated freely as small clusters or single, round cells similar in size was rapidly increased. At that time, we tested cell growth in different media. As no

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differences in cell number or morphology were noticed, the cells were cultured in RPMI 1640 medium (Sigma– Aldrich) supplemented with 2 mM glutamine, 10% FCS (Veterinary Institute, Novi Sad, Serbia) and antibiotics. The adherent cells completely disappeared after third subculture and cells kept on growing as a suspension of freely floating cells. Viability of cells was high ranging from 96 to 98%. Cell line was tested for EBV and mycoplasma, and results were negative for both analyses. The cells were frozen at –80C, in freezing medium (RPMI 1640 80% + FCS 10% + DMSO 10%). Later on, samples of PC-MDS cell line were frozen and used from time to time for different in-vitro assays. Cell growth and morphology PC-MDS cells (105 cells/ml) were plated in triplicate into 24-well plates and harvested on 2, 3, 4, 5, 6, and 7 days after inoculation. Cell count was performed by dye exclusion test with trypan blue. The mean population doubling time was determined during the exponential growth phase. Results presented the mean of triplicate from two independent experiments. Cytospin smears of PC-MDS cells were prepared, air dried, and stained by May Grunwald–Giemsa method (MGG). Morphological analyses were performed by light microscopy (Leica DM 2500, Wetzlar, Germany). Enzyme cytochemistry Cytospin smears of PC-MDS cells were stained by standard cytochemical staining procedures for the presence of myeloperoxidase (MPO), lipids (Sudan black B—SBB), glycogen (periodic acid-Schiff—PAS), and non-specific esterase (alpha-naphthyl acetate esterase (ANAE) not inhibited with sodium fluoride. All chemicals were of analytical grade and obtained from Sigma–Aldrich. Cytochemical enzyme patterns were studied by light microscopy. Stain positivity for MPO, SBB and PAS was determined if the reactivity was present in more than 3% of the cell population but ANAE required >20% positivity [10]. Immunocytochemistry analyses Cytospin smears of the PC-MDS cells were fixed in cold methanol, air dried and stored at –20C until use. Immunocytochemistry (ICH) analyses for the expression of vimentin, leukocyte common antigen (LCA) epithelial membrane antigen (EMA), Ki-67, and p53 protein were performed using LSAB2 kit (HRP rabbit/mouse AEC, K 0677, DAKO, Glostrup, Denmark) according to manufacturer’s instruction. Prediluted primary monoclonal antibodies

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421 6th passages

Cell number (log)

were used: vimentin—N1521, mouse-antihuman MoAb, clone V9; leukocyte common antigen—LCA, N1514, mouse-antihuman MoAb, clones 2B11&PD7/26; epithelial membrane antigen-EMA, N1504, mouse-antihuman MoAb clone E29/EP1, and p53-N1581, mouse-antihuman MoAb, clone DO7. N1526, rabbit-antihuman, polyclonal antibody against Ki-67 and N-series antibodies for negative controls were also used. The ICH analysis of the MPO expression was performed using Universal APAAP kit (K 0670, DAKO) according to manufacturer’s instruction. Monoclonal antibody against MPO was mouse-antihuman MoAb, MPO-7. Negative controls were performed by replacing the primary antibody with phosphate buffer saline (PBS, pH 7.2). Antigen expression was analyzed by light microscopy. Results were given as percentage of stained cells (distribution ratio) and staining intensity. The staining intensity range was scored on a scale from – to +++ and distribution ratio as follows: <10% of stained cells as low positivity, 10–50% of stained cells as moderate positivity, and >50% of stained cells as strong positivity.

8th passages

1.5 1 0.5 0 1

24

48

72

96

120

144

Incubation time (h) Fig. 1 The cell growth curve

and large, eccentric, oval, or slightly indented nuclei that contain one or more nucleoli. Single giant cells are also present. They are 4–5 times larger then described ones and they contain several nuclei (Fig. 2a). A high mitotic index was found: 14 metaphase cells/1,000 cells. Standard cytochemistry methods for detection of MPO, lipids, glycogen, and ANAE gave results as follows: MPO (Fig. 2b) and SBB negative (Fig. 2d) and PAS (Fig. 2c) and ANAE positive (Fig. 2e). Immunocytochemistry

Results Cell growth PC-MDS cells grew in suspension as single cells. Growth medium was RPMI 1640 supplemented with 2 mM glutamine, 10% of FCS and antibiotics. When initial cell density was adjusted to 105 cells/ml, the mean saturation density was 1.58 · 106 cells/ml after 96 h. Doubling time was ~24 h and population density level was 4 (Table 1, Fig. 1). The cells can be frozen in liquid nitrogen under standard conditions. Cell viability after thawing was above 80%. Morphology and cytochemistry Morphology of PC-MDS cells stained with MGG is shown in Fig. 2a. PC-MDS cell population consists mostly of round or ovoid mononuclear cells with abundant cytoplasm

Table 1 Doubling time, saturation density and population density level Cell line

Passage Doubling time (h)

Saturation density · 106 (1.9 cm–2)

Population density level (PDL)

PC

6

23.53

1.597

4.02

PC

8

24.04

1.573

3.99

Population density level was calculated by formula: PDL=log 10(N/ N0) · 3.33, here N represents cell number at the end of culture and N0 represents initial cell number

We analyzed the presence of membrane and intracellular antigens of PC-MDS cells by immunocytochemical methods. The results showed that PC-MDS cells were positive for EMA, vimentin, LCA, and Ki-67, and negative for p53 (Fig. 3a–f). The intensity of staining was weak for EMA, LCA, and Ki-67 but moderately stained cytoplasm with antibody for vimentin. PC-MDS cells were negative for MPO by standard cytochemistry, but immunocytochemistry analysis confirmed positive result in 16% of cell population. Morphology, co-expression of epithelial and mesenchymal markers, and results of immuno- and enzyme cytochemistry indicated undifferentiated stage of PC-MDS cells that might belong to myeloid lineage.

Discussion We previously described cytogenetic characteristics [11] and apoptosis sensitivity [12] of novel human myeloid cell line, PC-MDS, obtained from a bone marrow of a patient who developed MDS several years after polychemotherapy applied for ALL patients. Here, we give new data about the cell growth, morphology and immunocytochemistry of this cell line. As MDS was developed several years after chemotherapy, we categorized it as therapy-related MDS. To date, only OHN-GM cell line was derived from the patient with therapy-related MDS/AML [9]. However, the OHNGM cell line was established from the bone marrow cells

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Fig. 2 Photography of cultured PC-MDS cells stained with MGG (a), MPO (b), PAS (c), SBB (d) and ANAE (e)

collected at leukemic phase of disease. It is worth to point out that our patient had no hematological signs of progression to leukemia at the time when bone marrow sample was taken. A year before the bone marrow cells were taken for culturing the patient had already developed hemosiderosis, a common complication of MDS. According to our best knowledge PC-MDS is the first cell line obtained from the bone marrow of the patient with t-MDS, FAB subtype RA. From the very beginning, cell growth in culture was independent of external growth factor other than those from fetal calf serum. Standard cytological staining and preliminary immunocytochemical analyses indicated undifferentiated cell population. Further cell characterization was done according to the guidelines for characterization of human malignant hematopoietic cell lines [13, 14]. Classic cytology analyses, flow cytometric immunophenotyping, and cytogenetic procedures were used for characterization of the PC-MDS cell line. PC-MDS cell population consisted of mononuclear, fast growing cells. The cells grew in suspension culture at culture conditions common to hematopoietic cell lines. Moderate positivity of cells for nuclear Ki-67 antigen was in accordance with high mitotic index of PC-MDS cell

population. Co-expression of EMA and vimentin antigens and discrete staining for LCA indicated that the cell population was undifferentiated. Theoretically, differentiation of PC-MDS cells toward mesenchymal, epithelial, or even lymphoid cells might be possible. The blasts in MDS are mainly of myeloid origin [1]. The majority of cell lines derived from the various forms of MDS so far display myelocytic, monocytic, or erythroid features but some cell lines have lymphoid characteristics [1]. To determine PC-MDS cell origin, we performed routine enzyme cytochemistry and flow cytometric immunophenotyping. MGG morphology of PC-MDS cells resemble mostly to monocytic blast cells. Therefore, we performed cytochemical staining for the presence of MPO, lipids, glycogen, and non-specific esterase. PC-MDS cells were found negative for MPO and strongly positive for ANAE. Myeloperoxidase is considered as an early appearing and highly reliable intracellular myeloid lineage marker, and the most sensitive and specific molecular marker for myeloid lineage assignment in acute myeloid leukemia (AML) [10, 15]. In case of negative MPO cytochemistry, especially in diagnosis of undifferentiated and minimally differentiated AML, it is recommended to use monoclonal antibodies against MPO [10, 16] because of

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Fig. 3 Immunocytochemistry of PC-MDS cells. Cells were stained for epithelial membrane antigen (EMA, a, 40%), vimentin (b, 75% ++), leukocyte common antigen (LCA, c, 10% +), Ki-67 (d, 50% +), p53 (e, negative) and MPO (f, 16% +)

their higher sensitivity over cytochemical stains for MPO. After ICH staining we found that 16% of PC-MDS cells were positive for MPO. Some authors explain the presence of immunologically reactive, but enzymatically inactive MPO by the presence of either abnormal mature form of MPO or pro-form of MPO [17]. Some authors [18] suggested that any myeloid blasts were capable of synthesizing MPO, but were unable to process the enzyme to the mature active form. Positivity of PC-MDS cells for MPO and ANAE assigned them to myeloid lineage and further immunophenotypic studies have provided evidence that the PC-MDS cells belong generally to the myeloid lineage. The immunologic profile of PC-MDS cells regarding main plasma membrane antigens was determined by flow cytometry [11, 12, 19, 20] and disclosed expression of the following antigens: CD45+, CD33+, CD13+, CD15+, CD30+. The immunophenotype analyses confirmed that PC-MDS cells origin from hematopoietic system and have differentiating potential toward myelo-monocytic line. Positive staining for MPO and ANAE and immunophenotype results indicated that PC-MDS cells have some characteristics of the early myeloid precursor cell.

In summary, PC-MDS cell line as the first t-MDS derived cell line with its complex cytogenetic abnormalities typical for secondary MDS, sensitivity to apoptosis, as well as with completely described morphology and immunocytochemistry could be a new tool in evaluation of complex biology of MDS [21] and could serve as a model for further different in-vitro studies. Acknowledgements This work is supported by grants from Ministry for Science and Environmental Protection of Republic of Serbia. The authors thank Dr. Ivana Milosevic and Prof. Slavica KnezevicUsaj for help in enzyme and immunocitochemistry analyses.

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