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Origin of Microcells in the Human Sarcoma Cell Line HT-1080

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Origin of Microcells in the Human Sarcoma Cell Line HT-1080 73 Origin of microcells in the human sarcoma cell line HT-1080 Indulis Bui¸kis, L¯ıga Harju and numbers of microcells in tumour tissue markedly in- Talivaldis¯ Freivalds ∗ crease after chemotherapy, irradiation or immunother- Latvian Institute of Experimental and Clinical apy. Medicine, R¯ıga, LV-1004, Latvia Various ideas have been proposed for the origin of the microcells. Microcells in tumour tissue have been Received 17 July 1998 attributed to host immunocompetetive cell (macro- Revised 5 January 1999 phage progenitors, lymphocytes, etc.) and migration Accepted 17 February 1999 from blood capillaries into tumour tissue [21,24,48]. Other authors have considered microcells to origi- The aim of this study was to investigate the development nate from large damaged tumour cells undergoing of microcells in the human sarcoma cell line HT-1080 af- programmed cell death (apoptosis) [20,41]. Apop- ter interference with thiophosphamidum. We found that dam- totic events are manifested by tumour cell shrink- aged interphase macrocells located at the projection of the age, nucleus condensation and fragmentation, inter- nucleolus may form one or several microcells. The micronu- nucleosomal DNA cleavage and formation of apop- clei of the microcells intensively incorporate the thymidine 0 tic bodies (microcells) with low DNA content [6,16, analogue 5-bromo-2 -deoxyuridine and strongly express ar- 28,52]. Evidence also suggests that hypodiploid mi- gyrophilic nucleolar organiser region proteins. At an early crocells develop from large tumour cells by multipo- phase of the development, the micronuclei contain frag- mented DNA, but in subsequent phases, the micronuclei ac- lar mitosis [14] or by segregation of chromosomes in cumulate polymeric DNA, simultaneously with an increase metaphase – blocked cytogenesis [29]. in their size. After desintegration of the damaged macrocell, In previous investigations, we observed microcell the microcells appear in the intercellular space. The micro- development from perinuclear bodies of interphase nu- cells can enter mitosis and they strongly express the lung re- clei in damaged macrocells of the Jungarian hamster sistance protein. Electron microscopic observations suggest fibroblastoma cell line 4/21 [9,10]. This path of mi- that coiled bodies are involved in the development of the mi- crocell formation morphologically differed from cell crocells. Since the observed path of microcell formation dif- death via apoptosis. fers from apoptotic cell fragmentation into apoptotic bod- The aim of this study was to investigate the devel- ies, we propose a new term for this microcell development: sporosis. We suggest that self-renewal of the tumour stem opment of microcells in the established human sar- cells is likely based on sporosis. coma cell line HT-1080 after application of the cyto- Keywords: Microcells, sporosis, immortality toxic drug – thiophosphamidum. 1. Introduction 2. Materials and methods Microcells (small cells) are a natural component of 2.1. Cell culture tumour tissues. Microcells are distingnished by their roundish or oval form, scanty cytoplasm, and homo- The human sarcoma cell line HT-1080 [37] was geneously and intensively stained nuclei. The relative maintained at 37◦Cin25cm2 Karell flasks in Eagle medium supplemented with 10% bovine serum, glu- *Corresponding author: Talivaldis¯ Freivalds, Latvian Institute of Experimental and Clinical Medicine, O. Vatsiesha street 4, Riga, LV- tamine and antibiotics (in air). The cells were passaged 1004, Latvia. Fax: +371 7612038. at 4 to 7 day intervals. Analytical Cellular Pathology 18 (1999) 73–85 ISSN 0921-8912 / $8.00 1999, IOS Press. All rights reserved 74 I. Bui¸kis et al. / Origin of microcells 2.2. Induction of microcells with 0.1 M phosphate buffered saline (PBS), pH 7.2. The cells were fixed in 70% ethanol (in glycine buffer, Development of microcells was induced with thio- 50 mmol, pH 2.0) for 20 min at −20◦C. phosphamidum. Cells grown in maintenance medium Some of the cell samples after labelling with BrdU (2–3 × 105 cells/ml) were placed in culture flasks. The and washing with PBS were incubated in fresh main- cells were grown on coverslips at 37◦C for 3–4 days. tenance medium at 37◦C for 24, 48 or 72 h and then Then the medium was replaced with fresh medium fixed. containing thiophosphamidum in a concentration of Double-stranded DNA was denatured with 2 N HCl 10 or 20 µg/ml. The cells were exposed to thiophos- at room temperature for 20 min. The cells were neu- phamidum at 37◦C for 24 h. The selective medium was tralised with 0.1 M sodium borate buffer, pH 8.6. The than removed, the cells were washed 2× with Hank’s cover slips were washed with PBS and the cells were balanced salt solution and fresh maintenance medium covered with anti-BrdU monoclonal antibody work- was added. ing solution in PBS (1 : 100, pH 7.2) for 30 min at The cell samples for investigation were taken imme- 37◦C. The cells were washed with PBS and than incu- diately after 24 h incubation with thiophosphamidum, bated with anti-mouse-IG-AP, Fab-fragment solution and at 24, 48 and 72 h intervals after resupply with a (in PBS, 1 : 100, pH 7.2) for 30 min at 37◦C. The cover fresh medium. slips were washed with PBS and cells were stained with colour-substrate solution in Tris-HCl-buffer (con- 2.3. Light microscopy taining NBT and X-phosphate, and 5 mM levamisole, pH 9.2) at room temperature (RT) for 30 min. The cells The cells on coverslips were fixed in Carnoy’s mix- and nuclei were counterstained correspondingly with ture (acetic acid : chloroform: 96% ethanol – 1 : 3 : 6) methyl green-pyronine or by the Feulgen procedure. at 4◦C for 10 min, washed in 70% ethanol, air dried and Anti-BrdU mouse monoclonal antibody and anti- stained with methyl green-pyronine and azure-eosin. mouse-IG-AP, Fab-fragments, were obtained from DNA was stained by Feulgen procedure. Boehringer Mannheim, Germany. 2.4. AgNOR staining 2.6. In situ detection of DNA breaks The cells were fixed in Carnoy’s mixture. One step Two methods were used for in situ detection of AgNOR staining was conducted following the method DNA breaks: (1) terminal deoxynucleotidyl trans- described in Ploton et al. [34]. Staining mixtures were ferase (TdT)-mediated DIG-11-dUTP nick end la- prepared ex tempore from one part of 2% gelatine so- belling (TUNEL) method of Gavrieli et al. [19] and lution in 1% formic acid and two parts of 50% silver (2) holoenzyme of DNA polymerase-I (DNA Pol. I)- nitrate solution. The cells were stained at 70◦C for 5– mediated DIG-11-dUTP incorporation into DNA nicks 10 min. The slides were than washed in water and sub- (nick translation) method of Oberhammer et al. [31]. sequently in 5% sodium hyposulfite solution. Nuclear TdT and DNA Pol. I were purchased from Boehringer, chromatin was counterstained by the Feulgen proce- Mannheim. dure. The cells on cover slips were fixed at 4◦C for 15 min in 4% paraformaldehyde solution, prepared in 0.1 M 2.5. Bromo-20-deoxyuridine (BrdU) labelling and Tris/HCl buffered saline (TBS), pH 7.4, then washed in detection distilled water and air dried. The cells were incubated in 0.1 M TBS, pH 7.4, containing 0.025% Triton X- BrdU is a thymidine analogue that can be incorpo- 100 (Serva) for 30 min at RT, then washed with TBS, rated into cellular DNA of cell cycle S-phase cells. pH 7.4. The cells were deproteinated with proteinase K Immunocytochemical assay was used for detection of (10 µg/ml, 0.1 M TBS, pH 7.5) for 20 min at RT. BrdU incorporated into cellular DNA. Samples were pre-treated with the respective reaction At the time of cell sampling, the culture medium buffer for 15 min at RT (TUNEL: 30 mM Tris/HCl, from flasks was aspirated and BrdU-labelling medium pH 7.4, 1% bovine serum albumin (BSA), 140 mM (final concentration of BrdU – 10 µmol) was added. sodium cacodylate, 1 mM CoCl2; nick translation: 50 ◦ The cells were incubated with BrdU at 37 Cfor mM Tris/HCl, pH 7.4, 1% BSA, 20 mM MgCl2), then 60 min. The cover slips with the cells were washed incubated for 1 h at 37◦Cin25µl of the respective I. Bui¸kis et al. / Origin of microcells 75 reaction buffer containing 1 µl d-NTPs (DIG DNA la- 2.8. Electron microscopy belling mixture, Boehringer Mannheim) and the rel- evant enzyme (TUNEL: 7.5 U TdT; nick translation: Cell suspensions prepared by mechanical detach- 6 U DNA Pol. I). This reaction was stopped with ment of the monolayer were fixed with cold 2.5% glu- 0.1 M Tris/HCl buffer, pH 7.4, containing 0.3 M NaCl taraldehyde solution in 0.1 M sodium cacodylate buffer and 30 mM sodium citrate (for TUNEL) or 50 mM pH 7.0 for 20 min and postfixed with 1% osmic acid Tris/HCl buffer, pH 7.4, containing 20 mM EDTA (for solution for 1 h. The cell pellet was embedded in Epon. nick translation). Ultra thin sections were contrasted by uranylacetate After extensive washing with 0.1 M TBS, pH 7.4, and lead citrate, and examined using a JEM-100 C the incorporation of digoxigenin-labelled dUTP into electron microscope. DNA was detected using monoclonal anti-digoxigenin Fab-fragments (1 : 250, TBS, pH 7.4, 1 h at 37◦C) linked directly to alkaline phosphatase (Boehringer 3. Results Mannheim), followed by colour development with NBT/X-phosphate in Tris/HCl buffer containing 5 mM 3.1. General morphology levamisole, pH 9.2, for 30 min at RT.
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