- Email: [email protected]
Variations in antigen expression during the cell cycle of a human B-lymphoblastoid cell line
Leukemia Researt'h Vol. 9. No. 1. pp. 147-156, 1985. Printed in Great Britain.
0145-2126/8553.00 ÷ 0.00 ~ 1985 Pergamon Press Ltd.
THE CELL CYCLE OF A HUMAN B-LYMPHOBLASTOID CELL LINE
VARIATIONS
IN ANTIGEN
EXPRESSION
DURING
ASTRID H. GESCHE and LEONIE K. ASHMAN Department of Microbiology and Immunology, The University of Adelaide, Adelaide, South Australia 5000, Australia
(Received 29 May 1984. Accepted 28 June 1984) Abstract--Expression of six surface markers during the cell cycle of a human B-lymphoblastoid cell line has been examined. Cultures were synchronised by a double thymidine block, then monitored through a complete cell cycle following release. Surface marker expression was studied by manual immunofluorescence and flow cytometry. Surface topography of the cells during the cycle was examined by scanning electron microscopy. Some variation in the expression of all markers was observed during the cell cycle. Four antigens (surface membrane immunoglobulin, common ALL antigen, an immature cell marker identified by monoclonal antibody I I DI, and an ubiquitous leucocyte antigen identified by monoclonal antibody 6BI) showed maximal expression in S and/or G2 phase and minimal expression during mitosis. Thgse results could be due to changes in cell size. However, the individual patterns of expression of these four antigens during the phases of the cell cycle implied that changes in antigen density also occurred, at least in some cases. In contrast, expression of HLA-A, B, C and la antigens did not parallel cell size and was lowest during the S and G2 phases, increasing to a peak in early GI phase.
Key words: Cell surface antigens, cell cycle, human B-lymphoblastoid cell line, cell surface topography, immunofluorescence.
INTRODUCTION ANALYSIS of the expression of cell surface 'antigens' during the cell cycle may give clues to the biological function of these molecules. For example, expression of molecules with hormone receptor activity or transport functions might fluctuate in relation to the different metabolic activities at different stages of the cell cycle. Alternatively, surface 'antigens' may be cell interaction molecules involved in cellular proliferation. For example, it has been suggested that increased Ia antigen expression by murine B-cells in early GI phase is related to the requirement for I region-restricted T-cell 'help' at this stage during activation [9]. Chemicals such as DMSO and TPA which induce a maturation-like process in leukaemic cells and cell lines may also arrest cells at a certain stage of the cell cycle [13]. Maturation-induction by these chemicals provides a valuable approach to the study of leukaemic and normal haemopoietic differentiation (e.g. [3, 10]). We are using this approach as part of a study of patterns of antigen expression during blood cell differentiation. However, interpretation of the results of such studies requires a knowledge of the effects of cell cycle phases on antigen expression. In this paper, we report variations in the amounts of six surface antigens present on cells of a human B-lymphoblastoid line in different phases of the cell cycle. In the accompanying paper [1] we describe the effects of 'differentiation' inducers, DMSO and TPA, on the expression of these antigens.
Abbreviations: DMSO, dimethylsuifoxide; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; CALLA, common acute lymphoblastic leukaemia antigen; MHC, major histocompatibility complex. Correspondence to: Dr L. K. Ashman, Department of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, South Australia, 5001, Australia. 147
148
ASTRID H. GESCHE and LEONIE K. ASHMAN
MATERIALS AND METHODS Cell line The B-lymphoblastoid cell line used in these experiments was supplied to us as BALM-I [7], however it differs from published data [7, 8] in that the light chain isotype is ~. rather than ×, and it expresses the common acute lymphoblastic leukaemia antigen (CALLA) identified by monoclonal antibody J5 [11]. As reported for BALM-I [7], it is hyperdiploid. The HLA type is A27, B7, Bw4, Bw6, DR2, DR4. The line was maintained in RPMI 1640 medium (Flow Laboratories, lrvine, Scotland) supplemented with 10070 heat-inactivated foetal bovine serum (Flow), 15 mM HEPES, 100U/ml penicillin and 100 ~g/ml streptomycin sulphate, in a humidified 507o CO2 in air atmosphere at 37"C. Routine screening of the cell line for mycoplasma contamination gave negative results throughout this study.
Culture synchronisation Cultures were synchronised by a double thymidine block [16]. Preliminary experiments indicated that the optimum concentration of thymidine for synchronisation of cultures of this cell line was 2 mM. Cells at a density of 2 × 10'/ml were treated with 2 mM thymidine (Sigma, St Louis, Mo., U.S.A.) for 16 h, then the thymidine was removed by extensive washing with medium and the cells cultured for a further 8 h. Thymidine, 2 raM, was again added and the culture continued for a further 16 h. The thymidine was then washed away and cultures were set up in fresh flasks and medium at a density of 2-3 x l0 s ceUs/ml. Cultures werg monitored for cell number, DNA synthesis, and mitotic index at hourly intervals beginning immediately after removal of the second thymidine block. Cell number was determined using a Coulter Counter, Model F (Coulter Electronics, Hialea, Florida, U.S.A.). DNA synthesis was measured by the incorporation of 'H-thymidine as follows. Aliquots (0.15 ml) from the cultures were incubated in triplicate with 6 I.tCi +Hthymidine (25 Ci/mmol; Amersham International, Amersham, U.K.) in 96-well round-bottomed plates (Cat. No. 76-013-05; Linbro, McLean, Virginia, U.S.A) for 30 min at 37 ° in a humidified atmosphere of 50"/0CO2 in air. Cells were harvested onto glass fibre filters using a Titertek cell harvester, and radioactivity was determined using ACSII scintillation fluid (Amersham) in a Beckman model LS7500 13counter. The mitotic index was determined by preparation of cell smears using a cytocentrifuge (Shandon Southern Cytospin), staining with Giemsa, and microscopic examination. Approximately 500 cells per slide were scored.
Monoclonal antibodies la antigens were detected using FMC 14 [19] which was kindly supplied by Dr. H. Zola, Flinders Medical Centre, Adelaide, Australia. This antibody detects a monomorphic determinant common to HLA-DR, DC and SB (H. Zola and I. Beckman, personal communication). Monoclonal antibody 7B6 (prepared in this laboratory) was used to monitor HLA-A,B,C antigen expression. CALLA was detected using antibody .15 (Coulter Immunology, Hialeah, Florida, U.S.A.). Monoclonal antibodies I IDI and 6BI were prepared in this laboratory using human lenkaemie blasts as the immunogen. 11D 1 detects an antigen present on most human lymphoid and myeloid cell lines; it is not present on any mature blood cells, but is expressed on mitogen-activated T-cell blasts, and may therefore be a marker of proliferating cells. The molecular characteristics of the antigen have not been determined. The antigen detected by 6BI is present on all cell lines tested (including K562 and Daudi which lack HLA-A,B,C antigens) and on peripheral blood leukocytes from normal donors. It precipitates several bands in the range 60-120 kilodaltons from 3+S-methionine-labelled K562 lysates.
Analysis of cell surface antigen expression Surface membrane immunoglobulin expression was studied by direct immunofluorescence using fluoresceinlabelled polyclonal goat antiserum to human IgG + IgM + IgA (Behring, Marburg, West Germany) at a dilution of 1/25. All other antigens were detected by indirect immunofluorescence using murine monoclonal antibodies and fluorescein-labelled affinity-purified goat anti-mouse IgG + IgM prepared in this laboratory by Mr S. J. Gadd. Antibodies 7B6, I IDI and 6BI were used at a concentration of 5 gg/ml, J5 was reconstituted according to the manufacturer's instructions and diluted 1/10, and FMC 14 culture supernatant was used undiluted. These concentrations were shown to be non-limiting. Similarly, the fluorescein-labelled antibodies were used at non-limiting concentrations. For the indirect immunofluorescence studies, cells (2-5 x l0 s) in 50 ~tl of phosphate buffered saline pH 7.4 containing 10070 heat-inactivated normal rabbit serum, 0.1 07o bovine serum albumin and 0.1e/0 sodium azide, were incubated on ice for 60 rain with 50 I.tl of the appropriate monoclonal antibody solution. Cells were washed three times with 1 ml of buffer (as above but without rabbit serum), then incubated for a further 60 min on ice with fluorescein-labelled goat anti-mouse antibody. After a further three washes, cells were either suspended in 50070 glycerol in PBS and placed on slides for microscopic examination or fixed with 1070paraformaldehyde [5] for subsequent analysis by flow cytometry. A similar procedure was employed for direct immunofluorescence. Manual fluorescence microscopy was carried out using an Olympus microscope with epi-illumination (model BH2/BH-RFL-W). Scoring was carried out at a magnification of 400×, and 200-300 cells were examined per specimen. Analysis by flow cytometry was performed on a FACS IV (Becton Dickinson, Sunnyvale, California, U.S.A) at The Flinders Medical Centre, Adelaide.
&'anning Electron Microscopy Cells for examination were allowed to adhere to coverslips coated with poly-L-lysine (Sigma) [14] then fixed in I% glutaraldehyde at 4 ° for 24 h. The fixed cells were treated with 1070 (v/v) Osmium tetroxide in 0.15 M Cacodylate buffer, pH 7.2, for 90 min at 20°C, washed, and dehydrated slowly in a graded series of concentrations of acetone at room temperature. The specimens were critical point dried, mounted, and coated first with
Antigen expression during cell cycle
149
carbon, and then with gold-palladium. Coating was carried out by the staff of the Electron-Optical Centre, The University of Adelaide. The specimens were examined using an ETEC Scanning Electron Microscope (ETEC, U.S.A.) at an accelerating voltage of 20kV. RESULTS
Culture synehronisation Preliminary studies in which four independent cultures were monitored twice daily for nine days gave a population doubling time of 16 h. The cells grew exponentially from I × 10'/ml to 2 x 10+/ml over the first three days of culture. The first thymidine treatment, through inhibition of synthesis of deoxycytidine triphosphate [17], blocks DNA synthesis and arrests the cells at the end of the GI phase and throughout the S phase, resulting in partial synchrony. A second application of thymidine, appropriately timed, is necessary to produce effective synchrony. By allowing an interval greater than the length of S phase but less than the sum of the other phases to elapse between release o f the first thymidine block and initiation of the second block, most of the cells can be arrested at the end of G I [16]. Based on the doubling time of 16 h and assuming that the S phase would occupy less than half of that time, the thymidine blocks were applied for 16 h each, with a 'released' period of 8 h between them. A thymidine concentration of 2mM was found to give the best results. The effectiveness of this treatment in achieving synchronisation is illustrated in Fig. I which shows cell density,
•
9
J
O le a
"- s 8 o
&
'
0L
i' I
I
I
I
I
J
0
5
10
15
20
25
TIME
IN CULTURE
(HOURS)
FIG. 1. Synchrony of cultures after release from the second thymidine block. Cell density (A), DNA synthesis indicated by 3H-thymidineuptake (B) and mitotic index (C) are shown with respect to time after removal of excess thymidine. Each point represents the mean value from four independent cultures.
|50
ASTRID H. GESCHEand LEONIE K. ASHMAN
DNA synthesis, and mitotic index as a function of time after release of the second block. The duration of the phases of the cell cycle can be determined from these three parameters [16]. The length of the cell cycle, determined from the time between the starting points of the two peaks of DNA synthesis, was 15.6 h. This was made up of: 3.3 h in the S phase; 5.6 h in the G2 phase; 2 h in mitosis and 4.7 h in the GI phase. The similarity between the doubling time and the cell cycle duration implies that most cells in the culture were in cycle. Cultures were samples for scanning electron microscopy and antibody binding studies at 2.3, 6.3, 9.7, 12.0 and 14.8 h (respectively S, G2, M, early GI and late G1) after release from the second thymidine block. In all experiments, the abovementioned three parameters were monitored to ensure accuracy of the cell cycle phase.
Cell morphology during the cell cycle Cell surface morphology was examined by scanning electron microscopy during the S, G2, M and GI phases of the cell cycle (Fig. 2). The cell population remained heterogeneous in cell size, as well as in cell shape, throughout the S, G2, and G I phases (Fig. 2 a,b,d). In all cases, cells were covered with microvilli and blebs, both of which varied in size and quantity; some of these cells showed 'ruffles' (short, pleated folds extending above the cell surface) (Fig. 2a); others exhibited large, thin veils at their free margins (Fig. 2d). Most cells appeared to be attached to the substrate by filopodia of various length some of which showed extensive arborization (Fig. 2b). By contrast, during mitosis (Fig. 2c), a high percentage of cells was found to be rounded up. These cells appeared to have an increased number of microvilli, which were more evenly spread across the surface, and a decrease in the number of blebs, compared with the other three phases of the cell cycle. At this stage folds and veils had disappeared and thin filopodia of various lengths, showing frequent arborization at their tips, attached the cells to the substrate. In addition, forward scatter characteristics of the cells were examined by flow cytometry. While this parameter does not give an accurate measurement of cell size, particularly where the cells are asymmetric, the results in general reflected those obtained by scanning electron microscopy. The population appeared heterogeneous at all stages of the cell cycle, but with an increased proportion of smaller cells at mitosis (Fig. 3).
Surface marker studies Expression of cell surface antigens during the cell cycle was studied by both flow cytometry and manual immunofluorescence. Flow cytometry measures the integrated intensity of fluorescence over the whole cell [6] and is therefore strongly influenced by changes in cell size and surface area. Manual immunofluorescence, on the other hand, depends on localization of the fluorochrome and contrast with the background, and should be much less influenced by changes in cell size. However, the intensity of fluorescence cannot be accurately quantitated by the manual technique, and cells were scored as positive or negative based on an arbitrary cut-off point. The technique was less sensitive than flow cytometry. As few as 20°7o of cells were scored as positive in some assays, whereas almost all cells were scored as positive in all cases by flow cytometry. Five independent cultures were synchronised, sampled at the times indicated above, and assayed independently for expression of surface antigens by manual immunofluorescence. Aliquots of all five cultures were pooled, labelled in duplicate, and examined by flow cytometry. In addition, measurements were made on five independent unsynchronised cultures. The results of these experiments are summarized in Fig. 4. The manual immunofluorescence data for each antibody were analysed using the nonparametric Friedman two-way analysis of variance [18]. Where differences were observed (P <(0.05), results for the individual phases were compared using the Newman-Keuls multiple range test by rank sums [18]. By these tests, significant differences (P < 0.05) in antigen expression during the cycle were observed for Ia (S vs early G1, S vs late GI, G2 vs
l~c. 2. Cell morphology during the cell cycle. Synchronised cultures were sampled in S phase (a), G2 phase (b), M phase (c), and GI phase (d) and examined by scanning electron microscopy. Photographs were taken at 1000 x magnification.
151
Anligen expression during cell cycle
153 EARLY
i
G1
S
>" 0
G2
LATE
G 1
Z w w n-
FORWARD
SCATTER
FiG. 3. Light scattering characteristics of cells in different phases of
cycle.
Forward scatter
characteristics of cells were examined by flow cytometry at the same time as antigen binding studies were carried out. The figure shows profiles typical of those observed throughout the study.
ioo
I,~
HLA A,B,C
*
Ioo
* U
<
~I,
CALLA
o
SMla
~oo
11111tt i.° 6B1 t100 o
U
U
u. ~oo 11D1
o
o
A S G2 M GleGIL
A S G2 M GleGIL
FiG. 4. Analysis of surface antigen expression during the cell cycle. Expression of 6 surface antigens, as indicated, were analysed by manual immunofluorescence (open histograms) and flow cytometry (hatched histograms) in asynchronous cultures (A) and in cultures in S, G2, M, early and late GI phases of the cell cycle. Manual immunofluorescence data is shown as the mean percentage of positive cells __- S.E. from five independent cultures. Flow cytometry data, shown as peak fluorescence intensity, is the average result from two separate samples which usually differed by less than 10%. The peak fluorescence channel for negative controls was 4-6. The linear gain mode, setting 2 was used throughout. 95-100% of cells were fluorescent in all cases.
ASTRID H.
154
t
G
GESCHE and
LEONtE K. ASHMAN
~
G1late
0 Z gl o
~e u_
.........
~....
A
,1. . . . FLUORESCENCE INTENSITY-> F,G. 5. Surface membrane immunoglobulin expression during the cell cycle. The binding of fluorescein-labelled anti-lg to cells from an asynchronous culture (A), and from synchronised cultures in S, G2, M, early and late GI was analysed by flow cytometry. Peak fluorescence channels (gain 2, linear mode) were, respectively 86, 68, 96, 56, 80, 93. The percentage of cells positive was between 95 and 99 in all cases. Peak fluorescence channel for the negative control
was 4.
early GI), HLA-A,B,C (S vs late GI) and the antigen identified by monoclonal antibody 6B1 (S vs M, M vs late Gl). No statistically significant differences were observed in the percentage of cells scored as positive by manual immunofluorescence for surface immunoglobulin, CALLA or the antigen identified by monoclonal antibody 11Dl. In the case of surface immunoglobulin and CALLA, almost all cells were scored as positive in all cases, which would tend to mask any differences in intensity by this method of scoring. Results for CALLA are not directly comparable with those obtained for the other antigens studied by indirect immunofluorescence. These results were obtained with a new lamp in the microscope, which obviously increased the efficiency. Thus, most cells were scored as positive for CALLA which is not strongly expressed on this cell line compared with Ia (cf. FACS results). Results of FACS analysis in general confirmed those obtained by manual immunofluorescence. Thus, HLA-A,B,C and la antigen expression was highest in the Gl and lowest in the S phase. Similarly, the antigen defined by 6B1 was present in greater amount during GI and S, decreasing through G2 to a minimum in mitosis. While no statistically significant differences were observed between cell cycle phases by manual immunofluorescence in the case of CALLA, SMlg and the antigen defined by l l D l , variation in expression of these antigens was evident from FACS analysis. CALLA and the l l D l-antigen, like the 6B l-antigen, were expressed maximally in the S phase and least in M. SMIg expression was greatest in G2 and least in M. As an example, fluorescence histograms obtained using anti-SMlg are sl)own in Fig. 5.
Antigen expression during cell cycle
155
Variation during the cell cycle in Fc receptors for murine immunoglobulin (cf. [4]) was not a contributing factor in these experiments. 'Irrelevant' monoclonal antibodies of any class do not bind to this cell line (unpublished observations). In addition, 10o70 heatinactivated normal rabbit serum which blocks human Fc receptors [4] was included in all incubations.
DISCUSSION Variations in the expression of all antigens examined during the cell cycle were observed by flow cytometry and/or manual immunofluorescence. The pattern of antigen expression detected with monoclonal antibodies FMCl4 (anti-la) and 7B6 (anti-HLA A,B,C) could not be explained in terms of cell size, since they bound to the least extent in the S and G2 phases when cells are generally expected to be largest, and showed maximal binding in Gl. In addition, significant differences in binding of these antibodies were observed by manual immunofluorescence which is less influenced by changes in cell size and shape than FACS analysis. In contrast, the changes observed (by flow cytometry only) in binding of monoclonal antibodies J5 (anti CALLA) and l IDI during the cell cycle might have reflected the changes in cell size, since the highest fluorescence intensities were observed in S and G2, with a minimum in mitosis. The situation is less clear in relation to antibody 6Bl. Although the FACS results were similar to those obtained with J5 and I ! D1, in this case the manual immunofluorescence data showed significant differences in antibody binding between the phases of the cell cycle. This suggests that factors other than cell size may be involved. Similarly, the results obtained for SMIg expression cannot be simply explained on the basis of cell size despite the fact that fluorescence intensity was greatest in G2 and least in M, since the intensity was greater in the Gl than in the S phase. The question of variation in antigen density is an extremely complex one. Estimation of surface area from measurements of cell diameter or Coulter volume and using 'golf-ball' assumptions [9, 15] can be seen to be misleading if cell asymmetry and surface topography is examined by scanning electron microscopy. The best available solution to the problem at present would seem to be the use of the immuno-gold technique in conjunction with electron microscopy (e.g. [12]). The data presented in this paper do not allow us to draw firm conclusions as to variations in antigen density during the cell cycle. However, the diversity of patterns observed with the different antibodies means that at least some of the corresponding antigens must be expressed at different densities during the cell cycle. Our findings that Ia antigens and HLA-A,B,C antigens are present in greatest amounts on cells in the Gl phase of the cell cycle are at variance with those of Sarkar et al. [15]. Using another human B-cell line, W I-L2, synchronized by density-dependent arrest in G 1, these workers observed maximal expression of Ia antigen (HLA-DR) at the G2-M stage, but little change in HLA-A,B,C expression during the ceil cycle. However, other workers using murine cells have obtained results similar to those reported here. In early work using YAC lymphoma cells synchronised in various ways and polyclonal alloantiserum, Cikes and co-workers [2] showed that H-2 antigen expression was maximal in G l. More recently, Monroe and Cambier [9] demonstrated an increase in Ia antigen expression on the surface of mitogen-stimulated murine B cells during the G o to GI transition, followed by a marked decrease through the S, G2 and M phases. The increased amounts of HLA-A,B,C and la antigens on cells in the GI phase of the cell cycle must be taken into account when interpreting effects of 'differentiation' inducers such as DMSO and TPA on the expression of these antigens. The arrest of cells in the G1 phase of the cell cycle brought about by these agents [l, 13] could account for some of the changes in HLA-A,B,C and Ia expression which are reported in the accompanying paper [l].
156
ASTRID H. GESCHE and LEOnlE K. ASHMAN
Acknowledgements--This work was supported by a grant from the Anti-Cancer Foundation of the Universities of South Australia. L.K.A. is Senior Research Fellow of the Rotary Peter Nelson Leukaemia Research Fund. We are grateful to Anne Sheldon of the Tissue Typing Laboratory, Red Cross Blood Centre, Adelaide for HLA typing the cell line, and to Joe Webster of the Department of Immunology, Flinders Medical Centre, Adelaide for operating the FACS IV. Sean Flaherty of the Department of Anatomy and Histology, The University of Adelaide, kindly assisted with the scanning electron microscopy and Bohdan Stankewytsch-Janusch of the Department of Physiology, The University of Adelaide, advised on the statistical analysis of the data.
REFERENCES i. ASHMAN L. K. & GESCHE A. (1985) Surface antigen expression by a human B-lymphoblastoid cell line treated with 'differentiation' inducers, dimethylsulfoxide and tetradecanoylphorhol acetate. Leukemia Res. 9, 157. 2. CIKES M., FRlaERG S. & KLEIN G. (1972) Quantitative studies of antigen expression in cultured murine lymphoma cells II. Ceil-surface antigens in synchronised cultures. J. natn. Cancer Inst. 49, 1607. 3. DELLAD., NEWMAN R., GREAVESM. F., GOLDSTEIN G. & KUNG P. (1981) Induction of differentiation in T-ALL cell lines with phorbol ester (TPA). In Leukaemia Markers (KNAPP W.. Ed.). p. 293. Academic Press, London. 4. GADD S. J. & ASHMAN L. g. (1983) Binding of mouse monoclonal antibodies to human leukaemic cells via the Fc receptor: a possible source of "false positive" reactions in specificity screening. Clin. Exp. Immunol. 54, 811. 5. LANIER L. L. & WARNER N. L. 0981) Paraformaldehyde fixation of haemopoietic cells for quantitative flow cytometry (FACS) analysis. J. lmmunol. Meth. 47, 25. 6. MILLER R. G., LALANDE M. E., McCUTCHEOn M. J., STEWART S. S. & PRICE G. B. (1981) Usage of the flowcytometer-cell sorter. J. Immunol. Meth. 47, 13. 7. MINOWADA J., OSHIMURA M., TSUBOTA T., HIGay D. J. & SANDaERG A. A. (1977) Cytogenetic and lmmunoglobulin markers of human leukaemic B-cell lines. Cancer Res. 37, 3096. 8. MINOWADAJ., JANOSSYG., GREAVESM. F., TSUBOTAT., SRIVASTAVAB. 1. S., MORIKAWAS. & TATSUMIE. (1978) Expression of an antigen associated with acute lymphoblastic leukaemia in human leukaemialymphoma lines. J. hath. Cancer lnst. 60, 1269. 9. MONROE J. G. & CAMmER J. C. (1983) Level of mla expression on mitogen-stimulated murine B lymphocytes is dependent on position in cell cycle. J. lmmunol. 130, 626. 10. PERUSSIAB., LEBMAND., IP S. M., ROVErA G. & TRINCHtERI G. (1981) Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukaemia cells (HL60) treated with chemical inducers. Blood 58, 836. 11. Rrrz J., PESANDO J. M., NOTIS-McCoNARTY J., LAZARUSH. & SCHLOSSMANS. F. (1980) A monoclonal antibody to human acute lymphoblastic leukaemia antigen. Nature 283, 583. 12. ROTH J. & BINDER M. (1978) Colloidal gold, ferritin and peroxidase as markers for electron microscopic double labelling lectin techniques. J. Histochem. Cytochem. 26, 163. 13. ROVERA G., OLASHAW N. & MEO D, 0980) Terminal differentiation in human promyelocytic leukaemic cells in the absence of DNA synthesis. Nature 284, 69. 14. SANDERSS. K., ALEXANDERE. L. & BRAYLANR. C. (1975) A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy. J. Cell Biol. 67, 476. ]5. SARKARS., GLASSYM. C., FERRONE S. & JONES O. W. (1980) Cell cycle and the differential expression of HLA-A,B and HLA-DR antigens on human B lymphoid cells. Proc. ham. Acad. Sci. 77, 7297. 16. VOLPEP. & EREMENDO-VoLPET. (1970) A method for measuring the length of each phase of the cell cycle in spinner's cultures. Exp. Cell Res. 60, 456. 17. XEROS N. (1962) Deoxyriboside control and synchronisation of mitosis. Nature 194, 682. 18. ZAR J. H. (1974)Biostatistical Analysis. Prentice-Hall, New Jersey. 19. ZOLA H., MCNAMARA P. J., MOORE H . A . , SMART I. J., BROOKSD. A., BECKMAN 1. G. R. & BRADLEYJ. (1983) Maturation of human B lymphocytes - - studies with a panel of monoclonal antibodies against membrane antigens. Clin. exp. Immunol. 52, 655.
0145-2126/8553.00 ÷ 0.00 ~ 1985 Pergamon Press Ltd.
THE CELL CYCLE OF A HUMAN B-LYMPHOBLASTOID CELL LINE
VARIATIONS
IN ANTIGEN
EXPRESSION
DURING
ASTRID H. GESCHE and LEONIE K. ASHMAN Department of Microbiology and Immunology, The University of Adelaide, Adelaide, South Australia 5000, Australia
(Received 29 May 1984. Accepted 28 June 1984) Abstract--Expression of six surface markers during the cell cycle of a human B-lymphoblastoid cell line has been examined. Cultures were synchronised by a double thymidine block, then monitored through a complete cell cycle following release. Surface marker expression was studied by manual immunofluorescence and flow cytometry. Surface topography of the cells during the cycle was examined by scanning electron microscopy. Some variation in the expression of all markers was observed during the cell cycle. Four antigens (surface membrane immunoglobulin, common ALL antigen, an immature cell marker identified by monoclonal antibody I I DI, and an ubiquitous leucocyte antigen identified by monoclonal antibody 6BI) showed maximal expression in S and/or G2 phase and minimal expression during mitosis. Thgse results could be due to changes in cell size. However, the individual patterns of expression of these four antigens during the phases of the cell cycle implied that changes in antigen density also occurred, at least in some cases. In contrast, expression of HLA-A, B, C and la antigens did not parallel cell size and was lowest during the S and G2 phases, increasing to a peak in early GI phase.
Key words: Cell surface antigens, cell cycle, human B-lymphoblastoid cell line, cell surface topography, immunofluorescence.
INTRODUCTION ANALYSIS of the expression of cell surface 'antigens' during the cell cycle may give clues to the biological function of these molecules. For example, expression of molecules with hormone receptor activity or transport functions might fluctuate in relation to the different metabolic activities at different stages of the cell cycle. Alternatively, surface 'antigens' may be cell interaction molecules involved in cellular proliferation. For example, it has been suggested that increased Ia antigen expression by murine B-cells in early GI phase is related to the requirement for I region-restricted T-cell 'help' at this stage during activation [9]. Chemicals such as DMSO and TPA which induce a maturation-like process in leukaemic cells and cell lines may also arrest cells at a certain stage of the cell cycle [13]. Maturation-induction by these chemicals provides a valuable approach to the study of leukaemic and normal haemopoietic differentiation (e.g. [3, 10]). We are using this approach as part of a study of patterns of antigen expression during blood cell differentiation. However, interpretation of the results of such studies requires a knowledge of the effects of cell cycle phases on antigen expression. In this paper, we report variations in the amounts of six surface antigens present on cells of a human B-lymphoblastoid line in different phases of the cell cycle. In the accompanying paper [1] we describe the effects of 'differentiation' inducers, DMSO and TPA, on the expression of these antigens.
Abbreviations: DMSO, dimethylsuifoxide; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; CALLA, common acute lymphoblastic leukaemia antigen; MHC, major histocompatibility complex. Correspondence to: Dr L. K. Ashman, Department of Microbiology and Immunology, The University of Adelaide, GPO Box 498, Adelaide, South Australia, 5001, Australia. 147
148
ASTRID H. GESCHE and LEONIE K. ASHMAN
MATERIALS AND METHODS Cell line The B-lymphoblastoid cell line used in these experiments was supplied to us as BALM-I [7], however it differs from published data [7, 8] in that the light chain isotype is ~. rather than ×, and it expresses the common acute lymphoblastic leukaemia antigen (CALLA) identified by monoclonal antibody J5 [11]. As reported for BALM-I [7], it is hyperdiploid. The HLA type is A27, B7, Bw4, Bw6, DR2, DR4. The line was maintained in RPMI 1640 medium (Flow Laboratories, lrvine, Scotland) supplemented with 10070 heat-inactivated foetal bovine serum (Flow), 15 mM HEPES, 100U/ml penicillin and 100 ~g/ml streptomycin sulphate, in a humidified 507o CO2 in air atmosphere at 37"C. Routine screening of the cell line for mycoplasma contamination gave negative results throughout this study.
Culture synchronisation Cultures were synchronised by a double thymidine block [16]. Preliminary experiments indicated that the optimum concentration of thymidine for synchronisation of cultures of this cell line was 2 mM. Cells at a density of 2 × 10'/ml were treated with 2 mM thymidine (Sigma, St Louis, Mo., U.S.A.) for 16 h, then the thymidine was removed by extensive washing with medium and the cells cultured for a further 8 h. Thymidine, 2 raM, was again added and the culture continued for a further 16 h. The thymidine was then washed away and cultures were set up in fresh flasks and medium at a density of 2-3 x l0 s ceUs/ml. Cultures werg monitored for cell number, DNA synthesis, and mitotic index at hourly intervals beginning immediately after removal of the second thymidine block. Cell number was determined using a Coulter Counter, Model F (Coulter Electronics, Hialea, Florida, U.S.A.). DNA synthesis was measured by the incorporation of 'H-thymidine as follows. Aliquots (0.15 ml) from the cultures were incubated in triplicate with 6 I.tCi +Hthymidine (25 Ci/mmol; Amersham International, Amersham, U.K.) in 96-well round-bottomed plates (Cat. No. 76-013-05; Linbro, McLean, Virginia, U.S.A) for 30 min at 37 ° in a humidified atmosphere of 50"/0CO2 in air. Cells were harvested onto glass fibre filters using a Titertek cell harvester, and radioactivity was determined using ACSII scintillation fluid (Amersham) in a Beckman model LS7500 13counter. The mitotic index was determined by preparation of cell smears using a cytocentrifuge (Shandon Southern Cytospin), staining with Giemsa, and microscopic examination. Approximately 500 cells per slide were scored.
Monoclonal antibodies la antigens were detected using FMC 14 [19] which was kindly supplied by Dr. H. Zola, Flinders Medical Centre, Adelaide, Australia. This antibody detects a monomorphic determinant common to HLA-DR, DC and SB (H. Zola and I. Beckman, personal communication). Monoclonal antibody 7B6 (prepared in this laboratory) was used to monitor HLA-A,B,C antigen expression. CALLA was detected using antibody .15 (Coulter Immunology, Hialeah, Florida, U.S.A.). Monoclonal antibodies I IDI and 6BI were prepared in this laboratory using human lenkaemie blasts as the immunogen. 11D 1 detects an antigen present on most human lymphoid and myeloid cell lines; it is not present on any mature blood cells, but is expressed on mitogen-activated T-cell blasts, and may therefore be a marker of proliferating cells. The molecular characteristics of the antigen have not been determined. The antigen detected by 6BI is present on all cell lines tested (including K562 and Daudi which lack HLA-A,B,C antigens) and on peripheral blood leukocytes from normal donors. It precipitates several bands in the range 60-120 kilodaltons from 3+S-methionine-labelled K562 lysates.
Analysis of cell surface antigen expression Surface membrane immunoglobulin expression was studied by direct immunofluorescence using fluoresceinlabelled polyclonal goat antiserum to human IgG + IgM + IgA (Behring, Marburg, West Germany) at a dilution of 1/25. All other antigens were detected by indirect immunofluorescence using murine monoclonal antibodies and fluorescein-labelled affinity-purified goat anti-mouse IgG + IgM prepared in this laboratory by Mr S. J. Gadd. Antibodies 7B6, I IDI and 6BI were used at a concentration of 5 gg/ml, J5 was reconstituted according to the manufacturer's instructions and diluted 1/10, and FMC 14 culture supernatant was used undiluted. These concentrations were shown to be non-limiting. Similarly, the fluorescein-labelled antibodies were used at non-limiting concentrations. For the indirect immunofluorescence studies, cells (2-5 x l0 s) in 50 ~tl of phosphate buffered saline pH 7.4 containing 10070 heat-inactivated normal rabbit serum, 0.1 07o bovine serum albumin and 0.1e/0 sodium azide, were incubated on ice for 60 rain with 50 I.tl of the appropriate monoclonal antibody solution. Cells were washed three times with 1 ml of buffer (as above but without rabbit serum), then incubated for a further 60 min on ice with fluorescein-labelled goat anti-mouse antibody. After a further three washes, cells were either suspended in 50070 glycerol in PBS and placed on slides for microscopic examination or fixed with 1070paraformaldehyde [5] for subsequent analysis by flow cytometry. A similar procedure was employed for direct immunofluorescence. Manual fluorescence microscopy was carried out using an Olympus microscope with epi-illumination (model BH2/BH-RFL-W). Scoring was carried out at a magnification of 400×, and 200-300 cells were examined per specimen. Analysis by flow cytometry was performed on a FACS IV (Becton Dickinson, Sunnyvale, California, U.S.A) at The Flinders Medical Centre, Adelaide.
&'anning Electron Microscopy Cells for examination were allowed to adhere to coverslips coated with poly-L-lysine (Sigma) [14] then fixed in I% glutaraldehyde at 4 ° for 24 h. The fixed cells were treated with 1070 (v/v) Osmium tetroxide in 0.15 M Cacodylate buffer, pH 7.2, for 90 min at 20°C, washed, and dehydrated slowly in a graded series of concentrations of acetone at room temperature. The specimens were critical point dried, mounted, and coated first with
Antigen expression during cell cycle
149
carbon, and then with gold-palladium. Coating was carried out by the staff of the Electron-Optical Centre, The University of Adelaide. The specimens were examined using an ETEC Scanning Electron Microscope (ETEC, U.S.A.) at an accelerating voltage of 20kV. RESULTS
Culture synehronisation Preliminary studies in which four independent cultures were monitored twice daily for nine days gave a population doubling time of 16 h. The cells grew exponentially from I × 10'/ml to 2 x 10+/ml over the first three days of culture. The first thymidine treatment, through inhibition of synthesis of deoxycytidine triphosphate [17], blocks DNA synthesis and arrests the cells at the end of the GI phase and throughout the S phase, resulting in partial synchrony. A second application of thymidine, appropriately timed, is necessary to produce effective synchrony. By allowing an interval greater than the length of S phase but less than the sum of the other phases to elapse between release o f the first thymidine block and initiation of the second block, most of the cells can be arrested at the end of G I [16]. Based on the doubling time of 16 h and assuming that the S phase would occupy less than half of that time, the thymidine blocks were applied for 16 h each, with a 'released' period of 8 h between them. A thymidine concentration of 2mM was found to give the best results. The effectiveness of this treatment in achieving synchronisation is illustrated in Fig. I which shows cell density,
•
9
J
O le a
"- s 8 o
&
'
0L
i' I
I
I
I
I
J
0
5
10
15
20
25
TIME
IN CULTURE
(HOURS)
FIG. 1. Synchrony of cultures after release from the second thymidine block. Cell density (A), DNA synthesis indicated by 3H-thymidineuptake (B) and mitotic index (C) are shown with respect to time after removal of excess thymidine. Each point represents the mean value from four independent cultures.
|50
ASTRID H. GESCHEand LEONIE K. ASHMAN
DNA synthesis, and mitotic index as a function of time after release of the second block. The duration of the phases of the cell cycle can be determined from these three parameters [16]. The length of the cell cycle, determined from the time between the starting points of the two peaks of DNA synthesis, was 15.6 h. This was made up of: 3.3 h in the S phase; 5.6 h in the G2 phase; 2 h in mitosis and 4.7 h in the GI phase. The similarity between the doubling time and the cell cycle duration implies that most cells in the culture were in cycle. Cultures were samples for scanning electron microscopy and antibody binding studies at 2.3, 6.3, 9.7, 12.0 and 14.8 h (respectively S, G2, M, early GI and late G1) after release from the second thymidine block. In all experiments, the abovementioned three parameters were monitored to ensure accuracy of the cell cycle phase.
Cell morphology during the cell cycle Cell surface morphology was examined by scanning electron microscopy during the S, G2, M and GI phases of the cell cycle (Fig. 2). The cell population remained heterogeneous in cell size, as well as in cell shape, throughout the S, G2, and G I phases (Fig. 2 a,b,d). In all cases, cells were covered with microvilli and blebs, both of which varied in size and quantity; some of these cells showed 'ruffles' (short, pleated folds extending above the cell surface) (Fig. 2a); others exhibited large, thin veils at their free margins (Fig. 2d). Most cells appeared to be attached to the substrate by filopodia of various length some of which showed extensive arborization (Fig. 2b). By contrast, during mitosis (Fig. 2c), a high percentage of cells was found to be rounded up. These cells appeared to have an increased number of microvilli, which were more evenly spread across the surface, and a decrease in the number of blebs, compared with the other three phases of the cell cycle. At this stage folds and veils had disappeared and thin filopodia of various lengths, showing frequent arborization at their tips, attached the cells to the substrate. In addition, forward scatter characteristics of the cells were examined by flow cytometry. While this parameter does not give an accurate measurement of cell size, particularly where the cells are asymmetric, the results in general reflected those obtained by scanning electron microscopy. The population appeared heterogeneous at all stages of the cell cycle, but with an increased proportion of smaller cells at mitosis (Fig. 3).
Surface marker studies Expression of cell surface antigens during the cell cycle was studied by both flow cytometry and manual immunofluorescence. Flow cytometry measures the integrated intensity of fluorescence over the whole cell [6] and is therefore strongly influenced by changes in cell size and surface area. Manual immunofluorescence, on the other hand, depends on localization of the fluorochrome and contrast with the background, and should be much less influenced by changes in cell size. However, the intensity of fluorescence cannot be accurately quantitated by the manual technique, and cells were scored as positive or negative based on an arbitrary cut-off point. The technique was less sensitive than flow cytometry. As few as 20°7o of cells were scored as positive in some assays, whereas almost all cells were scored as positive in all cases by flow cytometry. Five independent cultures were synchronised, sampled at the times indicated above, and assayed independently for expression of surface antigens by manual immunofluorescence. Aliquots of all five cultures were pooled, labelled in duplicate, and examined by flow cytometry. In addition, measurements were made on five independent unsynchronised cultures. The results of these experiments are summarized in Fig. 4. The manual immunofluorescence data for each antibody were analysed using the nonparametric Friedman two-way analysis of variance [18]. Where differences were observed (P <(0.05), results for the individual phases were compared using the Newman-Keuls multiple range test by rank sums [18]. By these tests, significant differences (P < 0.05) in antigen expression during the cycle were observed for Ia (S vs early G1, S vs late GI, G2 vs
l~c. 2. Cell morphology during the cell cycle. Synchronised cultures were sampled in S phase (a), G2 phase (b), M phase (c), and GI phase (d) and examined by scanning electron microscopy. Photographs were taken at 1000 x magnification.
151
Anligen expression during cell cycle
153 EARLY
i
G1
S
>" 0
G2
LATE
G 1
Z w w n-
FORWARD
SCATTER
FiG. 3. Light scattering characteristics of cells in different phases of
cycle.
Forward scatter
characteristics of cells were examined by flow cytometry at the same time as antigen binding studies were carried out. The figure shows profiles typical of those observed throughout the study.
ioo
I,~
HLA A,B,C
*
Ioo
* U
<
~I,
CALLA
o
SMla
~oo
11111tt i.° 6B1 t100 o
U
U
u. ~oo 11D1
o
o
A S G2 M GleGIL
A S G2 M GleGIL
FiG. 4. Analysis of surface antigen expression during the cell cycle. Expression of 6 surface antigens, as indicated, were analysed by manual immunofluorescence (open histograms) and flow cytometry (hatched histograms) in asynchronous cultures (A) and in cultures in S, G2, M, early and late GI phases of the cell cycle. Manual immunofluorescence data is shown as the mean percentage of positive cells __- S.E. from five independent cultures. Flow cytometry data, shown as peak fluorescence intensity, is the average result from two separate samples which usually differed by less than 10%. The peak fluorescence channel for negative controls was 4-6. The linear gain mode, setting 2 was used throughout. 95-100% of cells were fluorescent in all cases.
ASTRID H.
154
t
G
GESCHE and
LEONtE K. ASHMAN
~
G1late
0 Z gl o
~e u_
.........
~....
A
,1. . . . FLUORESCENCE INTENSITY-> F,G. 5. Surface membrane immunoglobulin expression during the cell cycle. The binding of fluorescein-labelled anti-lg to cells from an asynchronous culture (A), and from synchronised cultures in S, G2, M, early and late GI was analysed by flow cytometry. Peak fluorescence channels (gain 2, linear mode) were, respectively 86, 68, 96, 56, 80, 93. The percentage of cells positive was between 95 and 99 in all cases. Peak fluorescence channel for the negative control
was 4.
early GI), HLA-A,B,C (S vs late GI) and the antigen identified by monoclonal antibody 6B1 (S vs M, M vs late Gl). No statistically significant differences were observed in the percentage of cells scored as positive by manual immunofluorescence for surface immunoglobulin, CALLA or the antigen identified by monoclonal antibody 11Dl. In the case of surface immunoglobulin and CALLA, almost all cells were scored as positive in all cases, which would tend to mask any differences in intensity by this method of scoring. Results for CALLA are not directly comparable with those obtained for the other antigens studied by indirect immunofluorescence. These results were obtained with a new lamp in the microscope, which obviously increased the efficiency. Thus, most cells were scored as positive for CALLA which is not strongly expressed on this cell line compared with Ia (cf. FACS results). Results of FACS analysis in general confirmed those obtained by manual immunofluorescence. Thus, HLA-A,B,C and la antigen expression was highest in the Gl and lowest in the S phase. Similarly, the antigen defined by 6B1 was present in greater amount during GI and S, decreasing through G2 to a minimum in mitosis. While no statistically significant differences were observed between cell cycle phases by manual immunofluorescence in the case of CALLA, SMlg and the antigen defined by l l D l , variation in expression of these antigens was evident from FACS analysis. CALLA and the l l D l-antigen, like the 6B l-antigen, were expressed maximally in the S phase and least in M. SMIg expression was greatest in G2 and least in M. As an example, fluorescence histograms obtained using anti-SMlg are sl)own in Fig. 5.
Antigen expression during cell cycle
155
Variation during the cell cycle in Fc receptors for murine immunoglobulin (cf. [4]) was not a contributing factor in these experiments. 'Irrelevant' monoclonal antibodies of any class do not bind to this cell line (unpublished observations). In addition, 10o70 heatinactivated normal rabbit serum which blocks human Fc receptors [4] was included in all incubations.
DISCUSSION Variations in the expression of all antigens examined during the cell cycle were observed by flow cytometry and/or manual immunofluorescence. The pattern of antigen expression detected with monoclonal antibodies FMCl4 (anti-la) and 7B6 (anti-HLA A,B,C) could not be explained in terms of cell size, since they bound to the least extent in the S and G2 phases when cells are generally expected to be largest, and showed maximal binding in Gl. In addition, significant differences in binding of these antibodies were observed by manual immunofluorescence which is less influenced by changes in cell size and shape than FACS analysis. In contrast, the changes observed (by flow cytometry only) in binding of monoclonal antibodies J5 (anti CALLA) and l IDI during the cell cycle might have reflected the changes in cell size, since the highest fluorescence intensities were observed in S and G2, with a minimum in mitosis. The situation is less clear in relation to antibody 6Bl. Although the FACS results were similar to those obtained with J5 and I ! D1, in this case the manual immunofluorescence data showed significant differences in antibody binding between the phases of the cell cycle. This suggests that factors other than cell size may be involved. Similarly, the results obtained for SMIg expression cannot be simply explained on the basis of cell size despite the fact that fluorescence intensity was greatest in G2 and least in M, since the intensity was greater in the Gl than in the S phase. The question of variation in antigen density is an extremely complex one. Estimation of surface area from measurements of cell diameter or Coulter volume and using 'golf-ball' assumptions [9, 15] can be seen to be misleading if cell asymmetry and surface topography is examined by scanning electron microscopy. The best available solution to the problem at present would seem to be the use of the immuno-gold technique in conjunction with electron microscopy (e.g. [12]). The data presented in this paper do not allow us to draw firm conclusions as to variations in antigen density during the cell cycle. However, the diversity of patterns observed with the different antibodies means that at least some of the corresponding antigens must be expressed at different densities during the cell cycle. Our findings that Ia antigens and HLA-A,B,C antigens are present in greatest amounts on cells in the Gl phase of the cell cycle are at variance with those of Sarkar et al. [15]. Using another human B-cell line, W I-L2, synchronized by density-dependent arrest in G 1, these workers observed maximal expression of Ia antigen (HLA-DR) at the G2-M stage, but little change in HLA-A,B,C expression during the ceil cycle. However, other workers using murine cells have obtained results similar to those reported here. In early work using YAC lymphoma cells synchronised in various ways and polyclonal alloantiserum, Cikes and co-workers [2] showed that H-2 antigen expression was maximal in G l. More recently, Monroe and Cambier [9] demonstrated an increase in Ia antigen expression on the surface of mitogen-stimulated murine B cells during the G o to GI transition, followed by a marked decrease through the S, G2 and M phases. The increased amounts of HLA-A,B,C and la antigens on cells in the GI phase of the cell cycle must be taken into account when interpreting effects of 'differentiation' inducers such as DMSO and TPA on the expression of these antigens. The arrest of cells in the G1 phase of the cell cycle brought about by these agents [l, 13] could account for some of the changes in HLA-A,B,C and Ia expression which are reported in the accompanying paper [l].
156
ASTRID H. GESCHE and LEOnlE K. ASHMAN
Acknowledgements--This work was supported by a grant from the Anti-Cancer Foundation of the Universities of South Australia. L.K.A. is Senior Research Fellow of the Rotary Peter Nelson Leukaemia Research Fund. We are grateful to Anne Sheldon of the Tissue Typing Laboratory, Red Cross Blood Centre, Adelaide for HLA typing the cell line, and to Joe Webster of the Department of Immunology, Flinders Medical Centre, Adelaide for operating the FACS IV. Sean Flaherty of the Department of Anatomy and Histology, The University of Adelaide, kindly assisted with the scanning electron microscopy and Bohdan Stankewytsch-Janusch of the Department of Physiology, The University of Adelaide, advised on the statistical analysis of the data.
REFERENCES i. ASHMAN L. K. & GESCHE A. (1985) Surface antigen expression by a human B-lymphoblastoid cell line treated with 'differentiation' inducers, dimethylsulfoxide and tetradecanoylphorhol acetate. Leukemia Res. 9, 157. 2. CIKES M., FRlaERG S. & KLEIN G. (1972) Quantitative studies of antigen expression in cultured murine lymphoma cells II. Ceil-surface antigens in synchronised cultures. J. natn. Cancer Inst. 49, 1607. 3. DELLAD., NEWMAN R., GREAVESM. F., GOLDSTEIN G. & KUNG P. (1981) Induction of differentiation in T-ALL cell lines with phorbol ester (TPA). In Leukaemia Markers (KNAPP W.. Ed.). p. 293. Academic Press, London. 4. GADD S. J. & ASHMAN L. g. (1983) Binding of mouse monoclonal antibodies to human leukaemic cells via the Fc receptor: a possible source of "false positive" reactions in specificity screening. Clin. Exp. Immunol. 54, 811. 5. LANIER L. L. & WARNER N. L. 0981) Paraformaldehyde fixation of haemopoietic cells for quantitative flow cytometry (FACS) analysis. J. lmmunol. Meth. 47, 25. 6. MILLER R. G., LALANDE M. E., McCUTCHEOn M. J., STEWART S. S. & PRICE G. B. (1981) Usage of the flowcytometer-cell sorter. J. Immunol. Meth. 47, 13. 7. MINOWADA J., OSHIMURA M., TSUBOTA T., HIGay D. J. & SANDaERG A. A. (1977) Cytogenetic and lmmunoglobulin markers of human leukaemic B-cell lines. Cancer Res. 37, 3096. 8. MINOWADAJ., JANOSSYG., GREAVESM. F., TSUBOTAT., SRIVASTAVAB. 1. S., MORIKAWAS. & TATSUMIE. (1978) Expression of an antigen associated with acute lymphoblastic leukaemia in human leukaemialymphoma lines. J. hath. Cancer lnst. 60, 1269. 9. MONROE J. G. & CAMmER J. C. (1983) Level of mla expression on mitogen-stimulated murine B lymphocytes is dependent on position in cell cycle. J. lmmunol. 130, 626. 10. PERUSSIAB., LEBMAND., IP S. M., ROVErA G. & TRINCHtERI G. (1981) Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukaemia cells (HL60) treated with chemical inducers. Blood 58, 836. 11. Rrrz J., PESANDO J. M., NOTIS-McCoNARTY J., LAZARUSH. & SCHLOSSMANS. F. (1980) A monoclonal antibody to human acute lymphoblastic leukaemia antigen. Nature 283, 583. 12. ROTH J. & BINDER M. (1978) Colloidal gold, ferritin and peroxidase as markers for electron microscopic double labelling lectin techniques. J. Histochem. Cytochem. 26, 163. 13. ROVERA G., OLASHAW N. & MEO D, 0980) Terminal differentiation in human promyelocytic leukaemic cells in the absence of DNA synthesis. Nature 284, 69. 14. SANDERSS. K., ALEXANDERE. L. & BRAYLANR. C. (1975) A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy. J. Cell Biol. 67, 476. ]5. SARKARS., GLASSYM. C., FERRONE S. & JONES O. W. (1980) Cell cycle and the differential expression of HLA-A,B and HLA-DR antigens on human B lymphoid cells. Proc. ham. Acad. Sci. 77, 7297. 16. VOLPEP. & EREMENDO-VoLPET. (1970) A method for measuring the length of each phase of the cell cycle in spinner's cultures. Exp. Cell Res. 60, 456. 17. XEROS N. (1962) Deoxyriboside control and synchronisation of mitosis. Nature 194, 682. 18. ZAR J. H. (1974)Biostatistical Analysis. Prentice-Hall, New Jersey. 19. ZOLA H., MCNAMARA P. J., MOORE H . A . , SMART I. J., BROOKSD. A., BECKMAN 1. G. R. & BRADLEYJ. (1983) Maturation of human B lymphocytes - - studies with a panel of monoclonal antibodies against membrane antigens. Clin. exp. Immunol. 52, 655.