Journal of the Neurological Sciences, 1978, 38: 67-75
67
© Elsevier/North-HollandBiomedicalPress
FIBRINOLYSIS I N D U C E D BY RAT GLIOMA CELLS A Mitosis-triggered Process?
K. S. Z.~NKER1, D. STAVROU2, U. OSTERKAMP2, I. WRIEDT-LOBBE1 and G. BLOMEL1 llnstitut fiir Experimentelle Chirurgie (Prof. Dr. G. Bliimel) der Technischen Universitiit Miinchen, 2Lehrstuhl fiir Allgemeine Pathologie und Neuropathologie (Prof. Dr. E. Dahme) am Institut fiir Tierpathologie der Universitiit Miinchen, Munich (F.R.G.)
(Received 1 De~mber, 1977) (Accepted 28 March, 1978)
SUMMARY Experimentally induced brain glioma cell lines were studied in cell cultures for their fibrinolytic activity; simultaneously the mitotic index was determined. It was found that the most intense fibrinolytic activity of glioma cells coincides with mitotic waves. After 68 hr incubation of SD/GI cells on a [125I]fibrin layer, [lZSI]fibrin digestion products were examined by gel chromatography. Hydroxy-urea and actinomycin D were used as inhibitors in cell cultures, investigating their effect on the expression of fibrinolysis. These studies reveal that [l~q]fibrin digestion products are mainly low molecular weight ( < 12,000 daltons) compounds and that fibrinolysis is partially inhibited by the drugs added.
INTRODUCTION In recent years, evidence has been provided that enzymes with fibrinolytic activities are involved in the growth of tumours (Rifkin, Loeb, Moore and Reich 1974; Unkeless, Dano, Kellermann and Reich 1974; Jones, Benedict, Strickland and Reich 1975) and in normal tissue (Nijs, Brassine, Coune and Tagnon 1973). Christman and Acs (1974) described a serine protease, released by transformed cells, with a fibrinolytic This study was supported by the Deutsche Forschungsgemeinschaft(SFB-51/A-19) and the Cancer Foundation of the University of Munich. Correspondence to: Prof. Dr. D. Stavrou, Lehrstuhl ftir AllgemeinePathologic und Neuropathologicam Institut for Tierpathologieder Universit~itMiinchen,Veterin~irstr.13, 8000Mtinchen22, Federal Republic of Germany.
68 activity. A plasminogen activating enzyme produced by ovarian cancer cells in tissue culture was found by/kstedt and Homberg (1976) and characterized as immunologically similar, if not identical with the plasminogen activator urokinase. In an attempt to clarify the role of plasminogen activator(s) in nebplastic processes, we have investigated the regulation of the expression of plasminogen activator during the cell cycle. Evidence that the plasminogen activator(s) is (are) active only during a certain phase of the cell cycle may contribute to the understanding of its role in the survival of transformed cells in vivo and to the development of a rational approach to the therapeutic changing of its level with exogenous agents. MATERIALS AND METHODS Cells Materials under study were a rat-astrocytoma line (C6), originally described by Benda, Someda, Messer and Sweet (1971) and a SD/G1 glioma line. The SD/GI line was derived from an experimentally induced brain glioma of a Sprague-Dawley rat; induction procedure and time is detailed by Stavrou, Haglid and R6nnb/ick (1973). Sloo-protein The glial nature of the newly established SD/G1 line was confirmed by the immuno-histochemical demonstration of the gila-specific S100-protein. The immunofluorescence was carried out according to the multiple layer method of Coons (1957). Cryostat sections of 5 #m and tissue culture specimens (SD/GI : 52nd passage) were dried and then fixed in cold acetone for 30 sec. After washing for 5 min in phosphatebuffered saline PBS, (pH 7.1), the sections were covered for 30 min with a dilution l : 8 of an antiserum against S100-protein. The purification of beef S100-protein and the production of antibodies to beef brain S100-protein in rabbits were described by Haglid and Stavrou (1973). After thorough washing (3 × 5 min) in cold PBS, a swine anti-rabbit gamma-globulin (Hyland), conjugated with fluorescein isothiocyanate was applied to the sections for 30 min. Excess fluorescent label was removed by repeated washing with PBS and the sections were mounted in buffered glycerol. All control experiments, specified by Haglid, Carlsson and Stavrou (1973), were carried out. Plasminogen activator assay The plasminogen activator generated by the cells was assayed by measuring the amount of 125I-labelled fibrin digestion products (FDP) released by the proteolytic activity generated by a mixture of endogenous plasminogen and glioma cells. At zero time, tumour cells in the appropriate medium, containing 10 ~o foetal calf serum, were seeded into 25 cm 2 Falcon plastic flasks each. The flasks had been coated with [1251]fibrinogen (New England Nuclear, 0.9 mg fibrinogen/100/zCi) and fibrinogen (human research fibrinogen, Kabi), which were converted to fibrin by thrombin. Human Fibrinogen (Research Fibrinogen, Kabi) commercially available, is still contaminated by plasminogen because iris difficult to purify fibrinogen without contamination with plasminogen. This problem, however, does not influence our experiments,
69
Fig. 1. Immunofluorescenceof the glia specificSlo0-proteinin the established SD/GI cell line (52nd passage) (A). Phase micrograph of C6-astrocytomacells growing in Falcon plastic flasks on a fibrin layer, 258 x (B).
because (see Discussion) plasminogen attached to fibrin(ogen) is not the rate-limiting factor in our test system. The concentration of [125I]fibrinogen is expressed in 1251cpm/cm 2 (on the other hand, the specific activity of [125I]fibrinogen used was 0.9 mg fibrinogen/100/~Ci); so the absolute fibrinogen-concentration of the label can be calculated. It is more important to give counts/cm 2 in the tubes than the absolute [1251]fibrinogen concentration. Details of the experiments and the specification of the media used are given in the legends to Fig. 2 and Fig. 3. At 4 hr intervals thereafter, the cell culture medium was removed and aliquots of the supernatant assayed for released FDPs in a gamma-spectrometer. The amount of solubilized FDPs in control flasks without cells has been subtracted. Prior to the seeding, cell numbers were determined in a particle volume analyser (AEG-Telefunken). Each point in the graphs represents the mean value of the average of duplicate counts of medium from 3 flasks. Percentage of [lzsI]fibrin digested was calculated from released cpm to total cpm added into the flasks, corrected by the controls.
Monitoring of the mitotic index Simultaneously with the counting of the cell culture medium, the cells were removed from the flasks by vigorously shaking and spun down by low speed centrifugation (Eppendorf cell centrifuge at 3200 g per 0.25 min). The cells were washed twice in medium and stained immediately for chromosomes according to Belling (1926). The mitotic index was determined under a light microscope.
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Fig. 2. Relationship of fibrinolytic activity to mitotic index of Ce-astrocytoma cells. Falcon plastic flasks (25 cm2) were coated with [lzst]fibrinogen, converted to [125I]fibrin(7.4 × 10a cpm/cma) by thrombin. At zero time, 6 × l0 s C6 cells in 5 ml DME medium supplemented by 10% foetal calf serum, were seeded onto the fibrin.
Gelfiltration of f125I]FDPs The iodinated FDPs, produced by SD/G1 cells during 68 hr incubation on fibrincoated Falcon plastic flasks, were chromatographed on a calibrated Biogel Agarose 1.5 m column (30 cm × 1.5 cm) at room temperature. The elution rate of the column was maintained by a Vario-LKB pump with 1.3 ml/min. The column was calibrated with the globular marker protein ferritin, catalase and cytochrome c. The column was prepared and eluted with PBS (pH 7.3); fractions of 1.5 ml were collected and the 1251 counted in a gamma spectrometer.
D NA- and D NA-dependent RNA synthesis inhibitors The effect on the fibrinolytic activity of Ce ceils was tested by the application of hydroxy-urea and actinomycin D (Serva, Heidelberg) to the cultures. The inhibitors were present at 20/~g/ml and 2/~g/ml, respectively. The concentration of inhibitors was maintained in the relevant cultures for the duration of the experiment. RESULTS The immunofiuorescence picture of the glia-specific S100-protein in the established SD/GI reveals a bright and specific fluorescence in the cytoplasm of the tissue culture cells but not in the nuclei (Fig. 1A). The morphology of C6 cells, growing on a fibrin layer is seen in Fig. 1B; the fibrin is visible as wavy shadowed lines. Fibrinolysis of C6 cells could be related to the mitotic cycle of the bulk of cells even in cultures, which had not been synchronized. Figure 2 shows the fibrinolytic activity during the first two mitotic waves in a freshly seeded C6 astrocytoma culture. It may be seen that fibrinolytic activity was detectably increased during the second, but not the first, mitotic wave. This
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Fig. 3. Theoretical and experimental cell cycle-dependent fibrinolysis of SD/G1 cells. As described in legend to Fig. 2,1.4 × 10e cells in 5 ml M199 medium containing 10 %foetal calf serum, were seeded at zero time onto [185I]fibrin(1.3 × 108 cpm/cm8) in Falcon plastic flasks. At 4 hr intervals thereafter, the cell culture medium was removed and assayed for released [z2sI]FDPs (left ordinate); t~r,t~ and t~ assumed first, second and third cell cycle. Hatched colunms (right ordinate) represent cumulative 185I-countsin the following periods: from zero time to t~, tj~and to t~. Note that t~ has almost the expected double value of t~; t~ is less than the expected (hatched plus checked columns) double of t~. This is a typical experiment of a series of 4, which were reproducible when cumulative [zzSI]FDPs counts in 3 apparent cell cycles were 12-20 % of total counts in the flasks.
was a result of the chosen low sensitivity o f the assay in which 6 × 106 cells were plated on [125I]fibrin with only 7.3 x 10 a cpm/cm 2. The second mitotic wave occurred 24 hr after the first and was more than twice as large. This is compatible with the cell cycle period of the Ce-astrocytoma, determined prior by the fraction of labelled mitosis technique. Furthermore, the data suggest that all daughter cells, resulting from the first mitotic wave, divided in the second. The fibrinolytic activity of SD/G1 cells over a period of 68 hr is shown in Fig. 3. The level of plasminogen activator remained on a plateau for 14-16 hr. This was followed by a distinct increase leading to the next plateau. The elution pattern of iodinated FDPs produced by SD/G1 cells during 68 hr on fibrin-
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Fig. 4. Chromatography of [lzSI]FDPs, released by incubation with SD/G 1 cells. The starting material, [lzsi]fibrinogen' is completely excluded from the gel and emerges at the void volume (Vo)of the column. Vo~as been determined by the exclusion of Dextran blue. [lzSI]FDPs penetrate into the gel and are separated into distinct peaks, The insert shows the calibration of the column with globular marker proteins. coated Falcon flasks is shown in Fig. 4. A first peak, eluted together with the globular marker protein ferritin; a second smaller but broader peak eluted behind catalase and a third dominant sharp peak appeared after cytochrome c. The elution characteristic of the third peak suggests that it may be due to micro-molecular FDPs (Triantaphyllopoulos 1973) similar or identical to those found in the blood of cancer patients (Girmann, Pess, Schwarze and Scheurlen 1976). Treatment of C6-astrocytoma cultures w i t h
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Fig. 5. Fibrinolytic activity of C6-cells"effect of treatment with hydroxy-urea (A) and actinomycin D (B). Cells wereallowedto grow for 18 hr without inhibitors (0--(3). Thereafter, hydroxy-urea(O--t) (20 pg/ml), and actinomycin D (V--V) were added (J). sufficiently nontoxic doses of DNA-dependent RNA- or DNA-synthesis inhibitor caused a marked decrease in fibrinolytic activity as compared to untreated cultures (Figs. 5A and 5B). By adding 2 #g/ml actinomycin D to 6 × 106 C6-cells, their fibrinolytic activity was suppressed by about 55 ~o and for hydroxy-urea (20 Fg/ml) by about 25 ~ , when data were expressed as cpm released per initially seeded cells. DISCUSSION The plasminogen activator in glioma cells was assayed by measuring the amount of soluble [l~5I]fibrin digestion products released from human fibrin containing plasminogen. This endogenous plasminogen was not the rate-limiting factor in the test system since experiments with 750 # CTA urokinase (Laboratoire Choay) completely lyse the fibrin coat within 45 min, whereas tumour cells had a maximum capacity of lysing about 35 ~o of the [125I]fibrin in 68 hr. The cell cycle of freshly seeded glioma cultures show partial synchrony; the incidence of mitosis follows a skewed Gaussian distribution. Experiments showed that by careful limitation of the number of cells plated and the amount of labelled fibrin per cm ~, assays could be arranged so that lysis was detectable only at the time of the peak of mitotic waves (Fig. 2). In this way, fibrinolysis could be related to the mitotic cycle of the bulk of cells even in cultures which had not been synchronized. If it is assumed that the stepwise advance of fibrinolytic activity after 14-16 hr (Fig. 3) is related to a cell cycle of the SD/G1 cell line, the cumulated counts during consecutive cycles should increase exponentially, reflecting the increase in mitoses. Thus, if at the mitotic time t~a a
74 bulk of SD/GI cells is in mitosis and an amount m of activator is generated, then at the following mitotic time t 2, the amount 2m and at t 3, the amount 4m of activator activity, and therefore, of [lzSI]FDPs ought to be found. As the hatched columns in Fig. 3 show, consecutive m-values increased less than expected. This may have been due to (a) daughter cells loosing synchrony or (b) dying in significant numbers or (c) cells producing less activator in following mitoses. Our data agreed well with the theoretical values for t~a and tZM,but the fibrinolytic activity at t 3 was less than the expected 9.6 ~o It has been reported by Rabes, Wirsching, Tuzcek and Iselar (1976) that natural synchrony of an unsynchronized cell population diminishes with the number of mitosis, and this may account for the lower increase in fibrinolysis at t~. Nevertheless, a superficial analysis of Figs. 3 and 4 suggests an evident increase in fibrinolytic activity at the mitotic phase of C6 and SD/GI cells. On the other hand, the effect of inhibitors, either of DNA replication or DNA-dependent RNA synthesis, seems to indicate a steady state between the solubilization of [12~I]fibrin digestion products and their uptake, at least in cell cultures. After adding the inhibitors, the supporting medium has not been changed, but surprisingly, a decrease in [lZSI]FDPs in contrast to control tubes, was observed (Figs. 5A and 5B). One possible explanation for this result may be a partially inhibited de novo synthesis of plasminogen activator, whereas the [x2~I]FDP uptake mechanism is unaffected by the added drugs. After a lag phase, plasminogen activator activity and [125I]FDP uptake has been regulated at lower levels. The possibility of error caused by dying cells during the incubation period with inhibitors cannot be ruled out completely. This interpretation may explain the decreased [125I]FDP plateaus, but there still remains a discrepancy between the measured amount of [125I]FDPs before and after adding the inhibitors. Examination of the size of [125I]FDPs reveals several discrete size classes (Fig. 4). The first peak of [125I]FDPs eluted like ferritin with an apparent molecular weight of 480,000 daltons, which is much greater than the molecular weight of human fibrinogen. For this reason, the size of the larger molecular weight fractions of [~25I]FDPs must be interpreted with caution, when examined by gel filtration, because of the pronounced tendency of FDP molecules to form aggregates; it appears that the molecules do not behave like perfect spheres, which is an important prerequisite for estimating molecular weights by gel filtration. Our finding that the most intense fibrinolytic activity of glioma cells coincides with mitotic waves is in good agreement with results obtained by Roblin, Chou and Black (1975) that the greatest expression of fibrinolytic activity corresponded with the period of active cell multiplication. Ossowski, Quigley and Reich (1975) found that cell migration appeared to depend on the presence of plasminogen. Cell migration and invasive growth of cells are obviously closely linked. Thus, the observations of mainly discontinuous and therefore still controlled synthesis and/or release of plasminogen activator might be a further parameter for the cell biology of glioma cells. ACKNOWLEDGEMENTS We thank Mrs. E. Sincini for skilful technical work and Dr. V. Eisen of the Rheum. Research Dept., The Middlesex Hospital, London, for critically reviewing the paper.
75 REFERENCES Asted, B. and L. Homberg (1976) Immunological identity of urokinase and ovarian carcinoma plasminogen activator released in tissue cultures, Nature (Lond.), 261 : 595-596. Belling, J. (1926) The iron-acetocarmine method of fixing and staining chromosomes, BioL Bull., 50: 160-162. Benda, P. H., K. Someda, J. Messer and W. Sweet (1971) Morphological and immunological studies of rat glial tumors and clonal strains in cultures, J. Neurosurg., 34: 310-320. Christman, J. K. and G. Acs (1974) Purification and characterization of a cellular fibrinolytic factor associated with oncogenic transformation - - The plasminogen activator of SV 40 transformed hamster cells, Biochim. biophys. Acta, 340: 339-347. Coons, A. H. (1957) The application of fluorescent antibodies to the study of naturally occurring antibodies, Ann. N. Y. Acad. Sci., 69: 658-662. Girmann, G., H. Pess, G. Schwarze and P. G. Scheurlen (1976) Immunosuppression by micromolecular fibrinogen digestion products in cancer, Nature (Lond.), 259: 399-401. Haglid, K. G. and D. Stavrou (1973) Water soluble and pentanol extractable proteins in human normal tissue and human brain tumours with special reference to S-100 protein, J. Neurochem., 20: 15231532. Haglid, K. G., C. A. Carlsson and D. Stavrou (1973) An immunological study of human brain tumors concerning the brain specific proteins S-100 and 14.3.2., Acta neuropath. (Berl.), 24: 187-196. Jones, P., W. Benedict, S. Strickland and E. Reich (1975) Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells, Cell, 5 : 323-329. Nijs, M., C. Brassine, A. Coune and H. J. Tagnon (1973) Direct fibrinogenolytic and fibrinolytic activity in the human prostate, Europ. J. Cancer, 9: 691-698. Ossowski, L., J. P. Quigiey and E. Reich (1975) Plasminogen, a necessary factor for cell migration in vitro. In: E. Reich, D. B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Iiol. 2, Cold Spring Harbor Laboratory, New York, 1975, pp. 901-913. Rabes, H., R. Wirsching, H. Tuzcek and G. Iselear (1976) Analysis of cell cycle compartments of hepatocytes after partial hepatectomy, Cell Tissue Kinet., 9:517-532. Rifkin, D., J. Loeb, G. Moore and E. Reich (1974) Properties of plasminogen activators formed by neoplastic human cell cultures, J. exp. Med., 139: 1317-1328. Roblin, R., L. N. Chou and P. H. Black (1975) Role of fibrinolysin T activity in properties of 3T3 and SV 3T3 cells. In: E. Reich, D. B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Vol. 2, Cold Spring Harbor Laboratory, New York, 1975, pp. 869-884. Stavrou, D., K. G. Haglid and L. R~Snnb~ick (1973) The S-100 protein in rat brain and in methylnitrosourea induced tumors of the rat nervous system, Europ. Neurol., 10: 168-178. Triantaphyllopoulos, E. (1973) The micromolecular fibrinogen derivates - - Fractionation by ultrafiltration and cation exchange chromatography, Prep. Biochem., 3 : 451-472. Unkeless, J. C., K. Dano, G. M. Kellermann and E. Reich (1974) Fibrinolysis associated with oncogenic transformation-- Partial purification and characterization of the cell factor, a plasminogen activator, J. biol. Chem., 249: 4295-4305.
67
© Elsevier/North-HollandBiomedicalPress
FIBRINOLYSIS I N D U C E D BY RAT GLIOMA CELLS A Mitosis-triggered Process?
K. S. Z.~NKER1, D. STAVROU2, U. OSTERKAMP2, I. WRIEDT-LOBBE1 and G. BLOMEL1 llnstitut fiir Experimentelle Chirurgie (Prof. Dr. G. Bliimel) der Technischen Universitiit Miinchen, 2Lehrstuhl fiir Allgemeine Pathologie und Neuropathologie (Prof. Dr. E. Dahme) am Institut fiir Tierpathologie der Universitiit Miinchen, Munich (F.R.G.)
(Received 1 De~mber, 1977) (Accepted 28 March, 1978)
SUMMARY Experimentally induced brain glioma cell lines were studied in cell cultures for their fibrinolytic activity; simultaneously the mitotic index was determined. It was found that the most intense fibrinolytic activity of glioma cells coincides with mitotic waves. After 68 hr incubation of SD/GI cells on a [125I]fibrin layer, [lZSI]fibrin digestion products were examined by gel chromatography. Hydroxy-urea and actinomycin D were used as inhibitors in cell cultures, investigating their effect on the expression of fibrinolysis. These studies reveal that [l~q]fibrin digestion products are mainly low molecular weight ( < 12,000 daltons) compounds and that fibrinolysis is partially inhibited by the drugs added.
INTRODUCTION In recent years, evidence has been provided that enzymes with fibrinolytic activities are involved in the growth of tumours (Rifkin, Loeb, Moore and Reich 1974; Unkeless, Dano, Kellermann and Reich 1974; Jones, Benedict, Strickland and Reich 1975) and in normal tissue (Nijs, Brassine, Coune and Tagnon 1973). Christman and Acs (1974) described a serine protease, released by transformed cells, with a fibrinolytic This study was supported by the Deutsche Forschungsgemeinschaft(SFB-51/A-19) and the Cancer Foundation of the University of Munich. Correspondence to: Prof. Dr. D. Stavrou, Lehrstuhl ftir AllgemeinePathologic und Neuropathologicam Institut for Tierpathologieder Universit~itMiinchen,Veterin~irstr.13, 8000Mtinchen22, Federal Republic of Germany.
68 activity. A plasminogen activating enzyme produced by ovarian cancer cells in tissue culture was found by/kstedt and Homberg (1976) and characterized as immunologically similar, if not identical with the plasminogen activator urokinase. In an attempt to clarify the role of plasminogen activator(s) in nebplastic processes, we have investigated the regulation of the expression of plasminogen activator during the cell cycle. Evidence that the plasminogen activator(s) is (are) active only during a certain phase of the cell cycle may contribute to the understanding of its role in the survival of transformed cells in vivo and to the development of a rational approach to the therapeutic changing of its level with exogenous agents. MATERIALS AND METHODS Cells Materials under study were a rat-astrocytoma line (C6), originally described by Benda, Someda, Messer and Sweet (1971) and a SD/G1 glioma line. The SD/GI line was derived from an experimentally induced brain glioma of a Sprague-Dawley rat; induction procedure and time is detailed by Stavrou, Haglid and R6nnb/ick (1973). Sloo-protein The glial nature of the newly established SD/G1 line was confirmed by the immuno-histochemical demonstration of the gila-specific S100-protein. The immunofluorescence was carried out according to the multiple layer method of Coons (1957). Cryostat sections of 5 #m and tissue culture specimens (SD/GI : 52nd passage) were dried and then fixed in cold acetone for 30 sec. After washing for 5 min in phosphatebuffered saline PBS, (pH 7.1), the sections were covered for 30 min with a dilution l : 8 of an antiserum against S100-protein. The purification of beef S100-protein and the production of antibodies to beef brain S100-protein in rabbits were described by Haglid and Stavrou (1973). After thorough washing (3 × 5 min) in cold PBS, a swine anti-rabbit gamma-globulin (Hyland), conjugated with fluorescein isothiocyanate was applied to the sections for 30 min. Excess fluorescent label was removed by repeated washing with PBS and the sections were mounted in buffered glycerol. All control experiments, specified by Haglid, Carlsson and Stavrou (1973), were carried out. Plasminogen activator assay The plasminogen activator generated by the cells was assayed by measuring the amount of 125I-labelled fibrin digestion products (FDP) released by the proteolytic activity generated by a mixture of endogenous plasminogen and glioma cells. At zero time, tumour cells in the appropriate medium, containing 10 ~o foetal calf serum, were seeded into 25 cm 2 Falcon plastic flasks each. The flasks had been coated with [1251]fibrinogen (New England Nuclear, 0.9 mg fibrinogen/100/zCi) and fibrinogen (human research fibrinogen, Kabi), which were converted to fibrin by thrombin. Human Fibrinogen (Research Fibrinogen, Kabi) commercially available, is still contaminated by plasminogen because iris difficult to purify fibrinogen without contamination with plasminogen. This problem, however, does not influence our experiments,
69
Fig. 1. Immunofluorescenceof the glia specificSlo0-proteinin the established SD/GI cell line (52nd passage) (A). Phase micrograph of C6-astrocytomacells growing in Falcon plastic flasks on a fibrin layer, 258 x (B).
because (see Discussion) plasminogen attached to fibrin(ogen) is not the rate-limiting factor in our test system. The concentration of [125I]fibrinogen is expressed in 1251cpm/cm 2 (on the other hand, the specific activity of [125I]fibrinogen used was 0.9 mg fibrinogen/100/~Ci); so the absolute fibrinogen-concentration of the label can be calculated. It is more important to give counts/cm 2 in the tubes than the absolute [1251]fibrinogen concentration. Details of the experiments and the specification of the media used are given in the legends to Fig. 2 and Fig. 3. At 4 hr intervals thereafter, the cell culture medium was removed and aliquots of the supernatant assayed for released FDPs in a gamma-spectrometer. The amount of solubilized FDPs in control flasks without cells has been subtracted. Prior to the seeding, cell numbers were determined in a particle volume analyser (AEG-Telefunken). Each point in the graphs represents the mean value of the average of duplicate counts of medium from 3 flasks. Percentage of [lzsI]fibrin digested was calculated from released cpm to total cpm added into the flasks, corrected by the controls.
Monitoring of the mitotic index Simultaneously with the counting of the cell culture medium, the cells were removed from the flasks by vigorously shaking and spun down by low speed centrifugation (Eppendorf cell centrifuge at 3200 g per 0.25 min). The cells were washed twice in medium and stained immediately for chromosomes according to Belling (1926). The mitotic index was determined under a light microscope.
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Fig. 2. Relationship of fibrinolytic activity to mitotic index of Ce-astrocytoma cells. Falcon plastic flasks (25 cm2) were coated with [lzst]fibrinogen, converted to [125I]fibrin(7.4 × 10a cpm/cma) by thrombin. At zero time, 6 × l0 s C6 cells in 5 ml DME medium supplemented by 10% foetal calf serum, were seeded onto the fibrin.
Gelfiltration of f125I]FDPs The iodinated FDPs, produced by SD/G1 cells during 68 hr incubation on fibrincoated Falcon plastic flasks, were chromatographed on a calibrated Biogel Agarose 1.5 m column (30 cm × 1.5 cm) at room temperature. The elution rate of the column was maintained by a Vario-LKB pump with 1.3 ml/min. The column was calibrated with the globular marker protein ferritin, catalase and cytochrome c. The column was prepared and eluted with PBS (pH 7.3); fractions of 1.5 ml were collected and the 1251 counted in a gamma spectrometer.
D NA- and D NA-dependent RNA synthesis inhibitors The effect on the fibrinolytic activity of Ce ceils was tested by the application of hydroxy-urea and actinomycin D (Serva, Heidelberg) to the cultures. The inhibitors were present at 20/~g/ml and 2/~g/ml, respectively. The concentration of inhibitors was maintained in the relevant cultures for the duration of the experiment. RESULTS The immunofiuorescence picture of the glia-specific S100-protein in the established SD/GI reveals a bright and specific fluorescence in the cytoplasm of the tissue culture cells but not in the nuclei (Fig. 1A). The morphology of C6 cells, growing on a fibrin layer is seen in Fig. 1B; the fibrin is visible as wavy shadowed lines. Fibrinolysis of C6 cells could be related to the mitotic cycle of the bulk of cells even in cultures, which had not been synchronized. Figure 2 shows the fibrinolytic activity during the first two mitotic waves in a freshly seeded C6 astrocytoma culture. It may be seen that fibrinolytic activity was detectably increased during the second, but not the first, mitotic wave. This
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Fig. 3. Theoretical and experimental cell cycle-dependent fibrinolysis of SD/G1 cells. As described in legend to Fig. 2,1.4 × 10e cells in 5 ml M199 medium containing 10 %foetal calf serum, were seeded at zero time onto [185I]fibrin(1.3 × 108 cpm/cm8) in Falcon plastic flasks. At 4 hr intervals thereafter, the cell culture medium was removed and assayed for released [z2sI]FDPs (left ordinate); t~r,t~ and t~ assumed first, second and third cell cycle. Hatched colunms (right ordinate) represent cumulative 185I-countsin the following periods: from zero time to t~, tj~and to t~. Note that t~ has almost the expected double value of t~; t~ is less than the expected (hatched plus checked columns) double of t~. This is a typical experiment of a series of 4, which were reproducible when cumulative [zzSI]FDPs counts in 3 apparent cell cycles were 12-20 % of total counts in the flasks.
was a result of the chosen low sensitivity o f the assay in which 6 × 106 cells were plated on [125I]fibrin with only 7.3 x 10 a cpm/cm 2. The second mitotic wave occurred 24 hr after the first and was more than twice as large. This is compatible with the cell cycle period of the Ce-astrocytoma, determined prior by the fraction of labelled mitosis technique. Furthermore, the data suggest that all daughter cells, resulting from the first mitotic wave, divided in the second. The fibrinolytic activity of SD/G1 cells over a period of 68 hr is shown in Fig. 3. The level of plasminogen activator remained on a plateau for 14-16 hr. This was followed by a distinct increase leading to the next plateau. The elution pattern of iodinated FDPs produced by SD/G1 cells during 68 hr on fibrin-
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Fig. 4. Chromatography of [lzSI]FDPs, released by incubation with SD/G 1 cells. The starting material, [lzsi]fibrinogen' is completely excluded from the gel and emerges at the void volume (Vo)of the column. Vo~as been determined by the exclusion of Dextran blue. [lzSI]FDPs penetrate into the gel and are separated into distinct peaks, The insert shows the calibration of the column with globular marker proteins. coated Falcon flasks is shown in Fig. 4. A first peak, eluted together with the globular marker protein ferritin; a second smaller but broader peak eluted behind catalase and a third dominant sharp peak appeared after cytochrome c. The elution characteristic of the third peak suggests that it may be due to micro-molecular FDPs (Triantaphyllopoulos 1973) similar or identical to those found in the blood of cancer patients (Girmann, Pess, Schwarze and Scheurlen 1976). Treatment of C6-astrocytoma cultures w i t h
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Fig. 5. Fibrinolytic activity of C6-cells"effect of treatment with hydroxy-urea (A) and actinomycin D (B). Cells wereallowedto grow for 18 hr without inhibitors (0--(3). Thereafter, hydroxy-urea(O--t) (20 pg/ml), and actinomycin D (V--V) were added (J). sufficiently nontoxic doses of DNA-dependent RNA- or DNA-synthesis inhibitor caused a marked decrease in fibrinolytic activity as compared to untreated cultures (Figs. 5A and 5B). By adding 2 #g/ml actinomycin D to 6 × 106 C6-cells, their fibrinolytic activity was suppressed by about 55 ~o and for hydroxy-urea (20 Fg/ml) by about 25 ~ , when data were expressed as cpm released per initially seeded cells. DISCUSSION The plasminogen activator in glioma cells was assayed by measuring the amount of soluble [l~5I]fibrin digestion products released from human fibrin containing plasminogen. This endogenous plasminogen was not the rate-limiting factor in the test system since experiments with 750 # CTA urokinase (Laboratoire Choay) completely lyse the fibrin coat within 45 min, whereas tumour cells had a maximum capacity of lysing about 35 ~o of the [125I]fibrin in 68 hr. The cell cycle of freshly seeded glioma cultures show partial synchrony; the incidence of mitosis follows a skewed Gaussian distribution. Experiments showed that by careful limitation of the number of cells plated and the amount of labelled fibrin per cm ~, assays could be arranged so that lysis was detectable only at the time of the peak of mitotic waves (Fig. 2). In this way, fibrinolysis could be related to the mitotic cycle of the bulk of cells even in cultures which had not been synchronized. If it is assumed that the stepwise advance of fibrinolytic activity after 14-16 hr (Fig. 3) is related to a cell cycle of the SD/G1 cell line, the cumulated counts during consecutive cycles should increase exponentially, reflecting the increase in mitoses. Thus, if at the mitotic time t~a a
74 bulk of SD/GI cells is in mitosis and an amount m of activator is generated, then at the following mitotic time t 2, the amount 2m and at t 3, the amount 4m of activator activity, and therefore, of [lzSI]FDPs ought to be found. As the hatched columns in Fig. 3 show, consecutive m-values increased less than expected. This may have been due to (a) daughter cells loosing synchrony or (b) dying in significant numbers or (c) cells producing less activator in following mitoses. Our data agreed well with the theoretical values for t~a and tZM,but the fibrinolytic activity at t 3 was less than the expected 9.6 ~o It has been reported by Rabes, Wirsching, Tuzcek and Iselar (1976) that natural synchrony of an unsynchronized cell population diminishes with the number of mitosis, and this may account for the lower increase in fibrinolysis at t~. Nevertheless, a superficial analysis of Figs. 3 and 4 suggests an evident increase in fibrinolytic activity at the mitotic phase of C6 and SD/GI cells. On the other hand, the effect of inhibitors, either of DNA replication or DNA-dependent RNA synthesis, seems to indicate a steady state between the solubilization of [12~I]fibrin digestion products and their uptake, at least in cell cultures. After adding the inhibitors, the supporting medium has not been changed, but surprisingly, a decrease in [lZSI]FDPs in contrast to control tubes, was observed (Figs. 5A and 5B). One possible explanation for this result may be a partially inhibited de novo synthesis of plasminogen activator, whereas the [x2~I]FDP uptake mechanism is unaffected by the added drugs. After a lag phase, plasminogen activator activity and [125I]FDP uptake has been regulated at lower levels. The possibility of error caused by dying cells during the incubation period with inhibitors cannot be ruled out completely. This interpretation may explain the decreased [125I]FDP plateaus, but there still remains a discrepancy between the measured amount of [125I]FDPs before and after adding the inhibitors. Examination of the size of [125I]FDPs reveals several discrete size classes (Fig. 4). The first peak of [125I]FDPs eluted like ferritin with an apparent molecular weight of 480,000 daltons, which is much greater than the molecular weight of human fibrinogen. For this reason, the size of the larger molecular weight fractions of [~25I]FDPs must be interpreted with caution, when examined by gel filtration, because of the pronounced tendency of FDP molecules to form aggregates; it appears that the molecules do not behave like perfect spheres, which is an important prerequisite for estimating molecular weights by gel filtration. Our finding that the most intense fibrinolytic activity of glioma cells coincides with mitotic waves is in good agreement with results obtained by Roblin, Chou and Black (1975) that the greatest expression of fibrinolytic activity corresponded with the period of active cell multiplication. Ossowski, Quigley and Reich (1975) found that cell migration appeared to depend on the presence of plasminogen. Cell migration and invasive growth of cells are obviously closely linked. Thus, the observations of mainly discontinuous and therefore still controlled synthesis and/or release of plasminogen activator might be a further parameter for the cell biology of glioma cells. ACKNOWLEDGEMENTS We thank Mrs. E. Sincini for skilful technical work and Dr. V. Eisen of the Rheum. Research Dept., The Middlesex Hospital, London, for critically reviewing the paper.
75 REFERENCES Asted, B. and L. Homberg (1976) Immunological identity of urokinase and ovarian carcinoma plasminogen activator released in tissue cultures, Nature (Lond.), 261 : 595-596. Belling, J. (1926) The iron-acetocarmine method of fixing and staining chromosomes, BioL Bull., 50: 160-162. Benda, P. H., K. Someda, J. Messer and W. Sweet (1971) Morphological and immunological studies of rat glial tumors and clonal strains in cultures, J. Neurosurg., 34: 310-320. Christman, J. K. and G. Acs (1974) Purification and characterization of a cellular fibrinolytic factor associated with oncogenic transformation - - The plasminogen activator of SV 40 transformed hamster cells, Biochim. biophys. Acta, 340: 339-347. Coons, A. H. (1957) The application of fluorescent antibodies to the study of naturally occurring antibodies, Ann. N. Y. Acad. Sci., 69: 658-662. Girmann, G., H. Pess, G. Schwarze and P. G. Scheurlen (1976) Immunosuppression by micromolecular fibrinogen digestion products in cancer, Nature (Lond.), 259: 399-401. Haglid, K. G. and D. Stavrou (1973) Water soluble and pentanol extractable proteins in human normal tissue and human brain tumours with special reference to S-100 protein, J. Neurochem., 20: 15231532. Haglid, K. G., C. A. Carlsson and D. Stavrou (1973) An immunological study of human brain tumors concerning the brain specific proteins S-100 and 14.3.2., Acta neuropath. (Berl.), 24: 187-196. Jones, P., W. Benedict, S. Strickland and E. Reich (1975) Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells, Cell, 5 : 323-329. Nijs, M., C. Brassine, A. Coune and H. J. Tagnon (1973) Direct fibrinogenolytic and fibrinolytic activity in the human prostate, Europ. J. Cancer, 9: 691-698. Ossowski, L., J. P. Quigiey and E. Reich (1975) Plasminogen, a necessary factor for cell migration in vitro. In: E. Reich, D. B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Iiol. 2, Cold Spring Harbor Laboratory, New York, 1975, pp. 901-913. Rabes, H., R. Wirsching, H. Tuzcek and G. Iselear (1976) Analysis of cell cycle compartments of hepatocytes after partial hepatectomy, Cell Tissue Kinet., 9:517-532. Rifkin, D., J. Loeb, G. Moore and E. Reich (1974) Properties of plasminogen activators formed by neoplastic human cell cultures, J. exp. Med., 139: 1317-1328. Roblin, R., L. N. Chou and P. H. Black (1975) Role of fibrinolysin T activity in properties of 3T3 and SV 3T3 cells. In: E. Reich, D. B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Vol. 2, Cold Spring Harbor Laboratory, New York, 1975, pp. 869-884. Stavrou, D., K. G. Haglid and L. R~Snnb~ick (1973) The S-100 protein in rat brain and in methylnitrosourea induced tumors of the rat nervous system, Europ. Neurol., 10: 168-178. Triantaphyllopoulos, E. (1973) The micromolecular fibrinogen derivates - - Fractionation by ultrafiltration and cation exchange chromatography, Prep. Biochem., 3 : 451-472. Unkeless, J. C., K. Dano, G. M. Kellermann and E. Reich (1974) Fibrinolysis associated with oncogenic transformation-- Partial purification and characterization of the cell factor, a plasminogen activator, J. biol. Chem., 249: 4295-4305.
