Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 131-135, January 1975
Mitogenic Activity of Blood Components. I. Thrombin and Prothrombin (chick embryo fibroblasts/growth factors in plasma and serum/wound healing)
LAN BO CHEN AND JOHN M. BUCHANAN Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139
Contributed by John M. Buchanan, October 15, 1974 ABSTRACT When added to a culture medium of resting confluent chick embryo fibroblasts in the absence of serum, thrombin (EC 3.4.21.5) is able to stimulate DNA synthesis 12 hr later and to cause a substantial increase in cell number over a period of 4 days. As compared to thrombin, prothrombin exhibits low mitogenic activity. However, in the presence of purified Factor Xa (EC 3.4.21.6) and Factor V, prothrombin is converted to thrombin by "thromboplastin activity" supplied by the fibroblasts. Prothrombin, either purified or as a constituent of plasma or serum, may thus be considered to be a reservoir of mitogenic activity in tissue culture unless antithrombin is present in the culture medium in amounts sufficient to neutralize the thrombin formed. By use of a specific inhibitor of proteases, and by separation of prothrombin by absorption on BaSO4, we estimate that the potential mitogenic activity of prothrombin is approximately 3050% of the total activity that can be obtained by treatment of fibrinogen-free plasma with thromboplastin. In addition to its mitogenic activity, thrombin can also stimulate the migration of cells. These experiments with thrombin illustrate that well-characterized proteases of blood can act as potent mitogens and suggest that they may play a role in the process of wound healing.
Many lines of evidence suggest that the control of cell growth either in vivo or in culture is mediated by agents in the extracellular fluid. Even though there are several agents capable of stimulating growth of a variety of cell types, such as serum (1-3), plasma (4), hormones (5-7), antigens (9), plant lectins (10, 11), and proteases (12, 13), very little progress has been made in any instance on the elucidation of the mechanism of cell growth, in particular the initial interaction of mitogens with cell surface components. In some ways the proteases offer a unique opportunity for the study of changes on the cell surface during mitogenic stimulation since (1) many of the enzymes have been obtained in a chemically pure state and (2) the specificities of their proteolytic activities have been carefully documented. During the process of blood clotting a number of proteolytic enzymes are produced and quite probably contribute to the mitogenic activity of this fluid. However, the total mitogenic activity of coagulating plasma is not realized in serum, since neutralizing or inactivating proteins are present in plasma in abundant amounts (14). Thrombin (EC 3.4.21.5) is both a product and catalyst of the blood clotting system and occupies a central position in reaction sequences. As part of a general program to separate and identify actual or potential mitogenic components of serum (or plasma), we have examined highly purified thromAbbreviations: PBS, phosphate-buffered saline; PhMeSO2F,
phenyl methyl sulfonyl fluoride. 131
bin and prothrombin to determine whether they have mitogenic activity when added to resting chick embryo fibroblasts. MATERIALS AND METHODS
Cell Culture and Growth Measurements. Secondary cultures of chick embryo fibroblasts were prepared by a standard procedure (15). Confluent resting cultures were prepared by seeding 7 to 8 X 105 cells per 35-mm culture plate in 2 ml of Dulbecco modified Eagle's medium supplemented with 0.5% (v/v) calf serum. On day 2 the medium was changed. On day 4 the medium was replaced with 1.5 or 2 ml of Dulbecco modified Eagle's medium containing the fractionated materials to be tested for their mitogenic activity. Twelve hours later 10 IAl of [3H]thymidine (0.2 mCi/ml in 50% ethanol, 6.7 Ci/mmol, New England Nuclear Co.) were added, and the incubation was continued for 1 hr. The acid-insoluble fraction was collected and the radioactive DNA was measured. When cell growth was measured by counting the number of cells as a function of time, cell cultures were prepared as described above except that the number of cells seeded was reduced to 3 to 4 X 105 per 35-mm culture plate. After the medium was replaced with Dulbecco modified Eagle's medium containing the materials to be tested, the cultures were incubated at 370 for 4 days and the cell count measured each day by a Coulter counter. Treatment of Fibrinogen-Free Plasma with Phenyl Methyl Sulfonyl Fluoride (PhMeSO2F). Fibrinogen-free plasma was prepared by dialysis of oxalated plasma (16) against water and by further heat treatment for 30 min at 560. One milliliter of a standard solution of brain thromboplastin was mixed with 9 ml of a solution containing 10% fibrinogen-free bovine plasma in Dulbecco modified Eagle's medium. After incubation at 370 for 2 hr, the precipitate was removed. Nine milliliters of supernatant fluid were then mixed with 1 ml of a solution of 10 mg of PhMeSO2F dissolved in 2-propanol. After 3 hr the solution was dialyzed exhaustively first against phosphate-buffered saline (PBS) containing 5% 2-propanol and then against PBS. Finally, the solution was dialyzed against Dulbecco modified Eagle's medium overnight at 4°. A similar treatment was carried out with a control sample of fibrinogenfree bovine plasma in which the Ph-AeSO2F solution was replaced with an equal volume of 2-propanol. Fractionation of Oxalated Bovine Plasma by BaSO4 Treatment. Plasma was treated with BaSO4 according to a procedure described by Rosenberg and Waugh (16) modified from a previous method of Goldstein et al. (17). In this procedure 56 g of BaSO4 (Baker) were mixed with 1600 ml of plasma.
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FiG. 1. Mitogenic activity as a function of concentration of thrombin (0-10 ,.g/ml, 0), calf serum (0-5 mg/ml, A), and bovine prothrombin (0-10 ,ug/ml, 0). Resting chick embryo fibroblasts were incubated for 12 hr with Dulbecco modified Eagle's medium containing proteins to be tested. The cells in 1.5 ml of medium were then incubated for 1 hr with 10 ml of [3H]thymidine (0.2 mCi/ml; 6.7 Ci/mmol) and the DNA was collected. The background radioactivity of cells incubated with serum-free medium is 2400 cpm, and has been subtracted from the values reported in the figure.
The unabsorbed supernatant material (Fraction I) was removed by centrifugation and the crude prothrombin fraction was eluted from the washed BaSO4 cake with 5% sodium citrate (Fraction II). In order to test for mitogenic activity the fibrinogen in Fraction I was removed by dialysis. Fraction II was dialyzed against PBS. Aliquots of all the dialyzed fractions were then treated with Ca2+ and thromboplastin. Estimation of Total Mitogenic Activity of Fractions I and II. In many of the experiments reported in this paper concerned primarily with the elution pattern of specific proteins during chromatography on Sephadex G-200, the exact proportionality of mitogenic activity to protein concentration was not important. However, when quantitative estimation of mitogenic activity was desired (for example, Fraction I and II), we have taken care to use concentrations of solutions in which the mitogenic response as measured by the incorporation of [3H]thymidine into DNA was proportional to the concentration of solution under test. A unit of activity has been defined by Pierson and Temin (2). Column Chromatography on Sephadex G-200. The column of Sephadex G-200 (3.3 X 50 cm) was equilibrated at 40 with PBS supplemented with penicillin (0.1 mg/ml) and streptomycin (0.1 mg/ml) to prevent bacterial growth. The flow rate was 10-15 ml/hr. The eluate was collected in fractions of 2.5 ml. Preparation of Thrombin, Prothrombin, Factor Xa (EC 3.4.21.6) and Factor V. Highly purified bovine thrombin was prepared as described by Baughman and Waugh (18), and bovine prothrombin by an unpublished procedure of C. WI. Becker and D. F. Waugh. Topical thrombin was purchased from Parke-Davis Co. The purity of the samples was tested by electrophoresis on polyacrylamide gel columns containing 0.1% sodium dodecyl sulfate (19). The specific activity of
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FIG. 2. Comparison of capacity of serum, thrombin, and prothrombin to stimulate cell division. Cells were seeded at a density of 4 X 105 per 35-mm plate. They were incubated in Dulbecco modified Eagle's medium containing the following additions. A. none, 0; B. Factor V, X; C. prothrombin, 0; D. Factor Xa plus Factor V, 0; E. Factor Xa, *; F. thrombin, *; G. prothrombin, Factor X., and Factor V, *; H. serum, O. When added, the materials were present in the following concentrations: thrombin, 2.5 Mg/ml; prothrombin, 4 gg/ml; Factor X., 0.02 Mg/ml; Factor V, 0.01 Mug/ml; and serum, 5 mg/ml..
thrombin is about 2000 NIH units/mg. Factor Xa, which was prepared by the method of Fujikawa et al. (20, 21), was kindly supplied by Dr. Richard Leary and Dr. Earl W. Davie. Factor V was a partially purified preparation isolated by a modification of the method of Esnouf and Jobin (22). Highly purified thrombin and prothrombin were labeled with 125I according to the method of Marchalonis (23). Measurement of Cell Migration. Resting cultures with a density of 4.4 X 105 cells per 35-mm plate were wounded with a razor blade as described by Burk (24). After wounding, the debris was aspirated, the cells were washed twice, and fresh medium containing thrombin (2.5 ,g/ml) or serum (5 mg/ml) was added. After 16 hr, the cultures were photographed under the microscope. The number of cells migrating into the wound was counted from photographs. RESULTS The Mitogenic Activity of Thrombin. In Fig. 1 are shown the results of an experiment in which purified bovine thrombin, bovine prothrombin, and calf serum were compared for their capacity to stimulate DNA synthesis 12 hr after addition to resting cultures of chick embryo fibroblasts. Serum was added within a range of 0-5 mg/ml of medium, and thrombin and prothrombin within a range of 0-10,ug/ml. As seen, the mitogenic response obtained with serum and thrombin were very similar within the ranges of concentrations used. On the other hand, prothrombin exhibits a very low mitogenic activity. This experiment was then repeated measuring the increase in actual cell count over a period of 4 days but with fixed concentrations of the mitogenic agents, 5 mg/ml for serum, 2.5 ,Mg/ml for thrombin, and an equivalent amount of prothrombin (4,ug/ml) (Fig. 2). The increase in cell count for thrombin and calf serum was in the expected proportion based on the response to these reagents at the indicated concentrations in the DNA synthesis assay. Again only a relatively weak response was obtained when prothrombin was added alone to the culture medium.
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TABLE 1. Estimation of mitogenic activity in BaSO4-treated fractions Protein concenFrac- Volume tration tion (ml) (mg/ml) I II
1600 80
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Mitogenic activity (units/mg) 1.47 125
Total activity % of total (units recovered X 10-4) activity 68.4 18 8.4 31.4
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Prothrombin as a Source of Thrombin Mitogenic Activity in Cell Culture. These experiments utilizing two kinds of assays to measure cell proliferation both indicated that thrombin is a potent mitogenic agent and that prothrombin is not. Possibly the low response of prothrombin may be attributed to its slow conversion to thrombin. However, in the presence of Factors Xa and V and with a source of thromboplastin, one might expect to observe a conversion of prothrombin to thrombin. This conversion would occur if the residual prothrombin of serum (approximately 10 gg/ml) reacts with Factor X0 and V of serum and if "thromboplastin activity" is provided by the cells themselves (25). In a model system we have measured the mitogenic activity arising from the incubation of a mixture of purified prothrombin, purified Factor Xa, and Factor V with confluent chick embryo fibroblasts (Fig. 2). The increase in cell count over a period of 4 days was compared with that resulting from incubations of each component alone or in incomplete combinations and with the mitogenic activity of thrombin added at a level of 2.5 Mg/ml or serum present in a concentration of 5 mg/ml. A major part of the increase in mitogenic activity of the plate containing prothrombin, Factor Xa, and Factor V over that observed in the control vessel is the result of conversion of prothrombin to thrombin and reflects indirectly the thromboplastin activity of cells that are undergoing division. These experiments illustrate that prothrombin, although not a mitogen itself, can be a source of mitogenic activity for cells in culture. Treatment of Oxalated Bovine Plasma with PhMeSO2F. Fig. 3 shows that after thromboplastin treatment Ph.MeSO2F can inhibit mitogenic activity as much as 50% when fibrinogen-free plasma is tested at a concentration of 2.5% in Dulbecco modified Eagle's medium. This result suggests that thromboplastin-treated plasma contains a mitogenic protease that can be inhibited by PhMeSO2F, a known potent protease inhibitor. The inhibitory effect of PhMeSO2F on thrombin has been reported (14). Several of the proenzymes of the blood clotting system are converted into active form either by the action of thromboplastin or by a secondary reaction of thrombin formed in the primary reaction. Since some of these are serine proteases and may exhibit mitogenic activity, it is possible that they may account for a part of the inhibition of mitogenic activity of plasma by Ph.6eSO2F. However, from a quantitative point of view (26) prothrombin is by far a major component of the proenzymes of the blood clotting system, and, therefore, after conversion to thrombin probably accounts for a large part of the mitogenic activity lost upon reaction of PhMeSO2F with thromboplastin-treated plasma. Estimation of Mitogenic Activity Attributed to Prothrombin as Determined with Fractions of Plasma Treated with BaSO4. In order to bring further evidence to bear on the conclusion that the activity inhibited by PhIVIeSO2F is, in fact, mainly
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(0) inhibitor.
thrombin, we have obtained two fractions of plasma prepared by absorption of proteins onto BaSO4 and elution under conditions specified in Materials and Methods. Fraction I includes the portion of proteins not absorbed, and Fraction II contains practically all of the prothrombin and Factor X of plasma along with some Factor VII, Factor V, and Factor IX. Table 1 summarizes the results of the analysis of Fractions I and II after treatment with thromboplastin. Fraction I contained about 68% of the total recovered mitogenic activity of plasma. The specific activity of this fraction in terms of mitogenic activity per mg of protein was approximately equal to that commonly found in serum and did not depend on treatment with thromboplastin. Fraction II, not treated with thromboplastin, shows only low mitogenic activity when assay was performed by the incorporation of [3H]thymidine into DNA 12 hr after the initiation of stimulation. Only measurement of those cells stimulated during the early period of the incubation will be included in the first wave of DNA synthesis. However, when Fraction II, which contains also Factor X and Factor V, was treated with an excess of thromboplastin, it was found to exhibit substantial amounts of mitogenic activity. Fraction II contained about 30% of the total recovered activity but has a specific activity about 125 times greater than that of calf serum. The wash fractions contained very little mitogenic ac-
tivity. Chromatography of Treated and Untreated Fraction II on Sephadex G-200. When Fraction II untreated with thromboplastin was applied on a column of Sephadex G-200 (3.3 X 50 cm), three major peaks of protein were observed, as indicated
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FIG. 5. Elution pattern of 125I-labeled prothrombin and thrombin mixed with untreated Fraction II. Fraction II (4.5 ml) not treated with thromboplastin was mixed either with (A) 125I-labeled prothrombin or (B) 125I-labeled thrombin and added to a Sephadex G-200 column (3.3 X 50 cm). The conditions of chromatography are the same as those described in Fig. 4. The symbols used are: *, absorbance at 280 nm, and 0, radioactivity of 12-I-labeled proteins. The arrows represent the volumes at which materials with absorption at 280 nm or with mitogenic activity were eluted from the column.
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(A) before and (B) after treatment with thromboplastin and (C) chromatography of a commercial sample of thrombin. To a column of Sephadex G-200 (3.3 X 50 cm) were added: A and B, 4.5 ml of Fraction II at a concentration of 8.4 mg/ml; or C, 2 ml of commercial thrombin (50 mg/ml). The eluate was collected in fractions of 2.5 ml. The samples (0.5 ml) were tested for absorbance at 280 nm (-) and for mitogenic activity (0). The values of all experiments have been corrected for the background radioactivity of the control samples incubated in the absence of serum (approximately 2000 cpm). In the plates containing 2% serum the radioactivity of [3H] thymidine incorporated into DNA was 37,500, 25,000, and 62,000 cpm for the experiments reported in Fig. 4A, B, and C, respectively. The peaks of materials eluting from the column with absorption at 280 nm are indicated by arrows at the appropriate volumes of effluent.
by the arrows in Fig. 4A. A low level of mitogenic activity was found in peak 3. However, when 4.5 ml of Fraction II from the BaSO4 fractionation procedure was first incubated with 1 ml of brain thromboplastin at 370 for 2 hr, and the supernatant formed after centrifugation at 12,000 X g was then applied to Sephadex G-200, 'peak 3 as measured by absorbancy at 280 nm was completely missing, and a new peak, peak 4, appeared (Fig. 4B). More interesting, not only was all of the activity of Fraction II located in peak 4, but also the specific activity of the mitogenicity of peak 4 was very high as compared with that of peak 3 of the untreated preparation. Since the activity observed in peak 4 emerged only after Fraction II had been treated with thromboplastin, and since Fraction II contained abundant amounts of prothrombin, it is very likely that peak 3 contained the relatively nonmitogenic prothrombin (Fig. 4A) and peak 4, (Fig. 4B) the mitogenic thrombin. Further support for the belief that peak 3 is prothrombin
and that peak 4 is thrombin comes from the results of experiments reported in Fig. 5A and B, respectively. Highly purified samples of electrophoretically pure thrombin and prothrombin were labeled with 125J and mixed with untreated Fraction II. As may be seen in Fig. 5A, the labeled prothrombin migrates at the position previously designated for prothrombin in Fig. 4A. 1251-labeled thrombin (Fig. 5B) migrates at the position expected for thrombin from Fig. 4B. The peak of mitogenic activity shown for treated Fraction II contains two shoulders on either side of the main central peak (Fig. 4B). The shoulders may represent an artifact of the assay procedure, since at the center of the peak the concentration of thrombin in individual samples was probably too high to fall in the proportional range of the assay.
Chromatography of a Commercial Sample of Thrombin. When Parke-Davis thrombin was fractionated on Sephadex G-200, three major protein peaks were observed (Fig. 4C). The peak of protein located in the lower-molecular-weight range of the column effluent contained abundant thrombin and stimulated DNA synthesis of resting cells to a significant level. However, some mitogenic activity was also observed in the high-molecular-weight peak. Since some purified '251-labeled thrombin also migrates in this high-molecular-weight fraction when mixed with Fraction II (Fig. 5B), it is likely that the mitogenic activity observed in the high-molecular-weight peak may result from the binding of thrombin to high-molecularweight proteins. Migration of Cells Stimulated by Serum or by Thrombin. A wounded culture was washed twice with 3 ml of Dulbecco modified Eagle's medium containing no serum and then incubated with 1.5 ml of medium alone or with medium containing either 2.5 ,g/ml of thrombin or 5 mg/ml of calf serum.
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The number of cells migrating into a wound in 16 hr/3-mm length was determined from photographs. For the plates containing medium alone, thrombin, or serum, the number was 0, 27, and 37, respectively. DISCUSSION The experiments reported in this communication have established that thrombin is a potent mitogenic agent. Its activity has been established both by measurement of the DNA synthesis during the first wave of cell division at 12 hr and by actual cell count over a period of 4 days. A model system con-
sisting of purified prothrombin, purified Factor Xa, Factor V, and chick fibroblasts was capable of yielding mitogenic activity as evidenced by a substantial increase in cell number over a 4-day period. These experiments support the speculation that prothrombin of plasma (about 100 ug/ml) and the residual prothrombin of serum (approximately 10 Ag/ml) may contribute to the mitogenic activity of these fluids by conversion to thrombin by the reaction of Factors Xa and V present in the fluids and the thromboplastin activity supplied by the chick cells. The actual contribution of prothrombin as a source of mitogenic activity, however, is difficult to estimate under physiological circumstances, since thrombin is readily neutralized by antithrombin of plasma and serum (14). We would propose, however, that in the microenvironment of a clotting area that the activated proteases might initiate the process of wound healing and that thrombin might contribute to this for a period either before neutralization is complete or as a result of the sequestering of active thrombin, for example, by fibrin (27). * The experiments with the protease inhibitor PhMeSO2F together with the studies with fractions of plasma obtained by absorption on BaSO4 were carried out to obtain a rough estimate of the potential contribution of prothrombin to the total mitogenic activity of this fluid. After treatment of fibrinogen-free plasma with thromboplastin, we observed that 50% of the mitogenic activity was lost upon reaction with
PhMeSO2F. Then after treatment of plasma with BaSO4, the absorbed fraction (Fraction II) contained approximately 30% of the total mitogenic activity of the plasma fractions. We have shown that a peak of protein corresponding to prothrombin is present in an elution pattern of untreated Fraction II when applied to a column of Sephadex G-200. Upon treatment of Fraction II with thromboplastin, this peak of protein is no longer found upon chromatography, but a new peak of protein appears at a further point in the elution pattern. This new peak of protein coincides with the location of thrombin in the elution profile. The mitogenic activity also corresponds to the location of thrombin with shoulders on either side of the central peak. Although other components in Fraction II may account for some of the mitogenic activity appearing in peak 4, it is likely that most of this activity may be ascribed to thrombin. The disappearance of the prothrombin-containing peak 3 and appearance of thrombin-containing peak 4 after thromboplastin treatment of plasma is further circumstantial evi-
dence for this conclusion. The treatment of chick fibroblasts with thrombin results in an enhancement of their capacity to migrate. This agrees *
W. J. Landis and D. F. Waugh, unpublished results.
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with the general observation that most of the mitogens for fibroblasts also stimulate the migration of such cell types. We wish to thank Dr. David F. Waugh, Dr. Carter M. Becker, and Miss P. M. McIlvaine for their generous gifts of highly purified thrombin and prothrombin and Fractions I and II mentioned in this communication. We also wish to express our appreciation to Dr. P. W. Robbins and Mrs. Lin-Huey Chen for supplying chick embryo fibroblasts. We also wish to acknowledge the capable assistance of Mr. Norman Hochella. This work was supported by grants-in-aid from the National Science Foundation (BMS 17669) and the National Cancer Institute (CA 02015), National Institutes of Health. L.B.C. is a predoctoral fellow of the Johnson and Johnson Co. 1. Todaro, G., Matsuya, Y., Bloom, S., Robbins, A. & Green, H. (1967) "Growth regulating substances for animal cells in culture," WistarInst. Symp. Monogr. no. 7, 87-101. 2. Pierson, R. W. & Temin H. M. (1972) J. Cell. Physiol. 79, 319-329. 3. Holley, R. W. & Kiernan, J. A. (1968) Proc. Nat. Acad. Sci. USA 60, 300-304. 4. Balk, S. D., Whitfield, J. F., Youdale, T. & Braun, A. C. (1973) Proc. Nat. Acad. Sci. USA 70, 675-679. 5. Gospodarowicz, D. (1974) Nature 249, 123-127. 6. Temin, H. M. (1967) J. Cell. Physiol. 69, 377-384. 7. Dulak, N. C. & Temin, H. M. (1973) J. Cell. Physiol. 81, 161-170. 8. Holley, R. W. & Kiernan, J. A. (1974) Proc. Nat. Acad. Sci. USA 71, 2908-2911. 9. Edelman, G. M. (1974) "Control of proliferation in animal cells," in Cold Spring Harbor Conferences on Cell Proliferation, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Vol. 1, pp. 357-377. 10. Fahey, J. L. (1974) "Control of proliferation in animal cells," in Cold Spring Harbor Conferences on Cell Proliferation, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Vol. 1, pp. 379392. 11. Vaheri, A., Ruoslahti, E. & Hovie, T. (1974) "Control of proliferation in animal cells," in Cold Spring Harbor Conferences on Cell Proliferation, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Vol. 1, pp. 305-312. 12. Burger, M. M. (1970) Nature 227, 171-171. 13. Sefton, B. M. & Rubin, H. (1970) Nature 227, 843-845. 14. Magnusson, S. (1971) in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), Vol. III, pp. 277-321. 15. Rein, A. & Rubin, H. (1968) Exp. Cell Res. 49, 666-678. 16. Rosenberg, R. D. & Waugh, D. F. (1970) J. Biol. Chem. 245, 5049-5056. 17. Goldstein, R., LeBolloc'h, A., Alexander, B. & Zonderman, E. (1959) J. Biol. Chem. 234, 2857-2866. 18. Baughman, D. J. & Waugh, D. F. (1967) J. Biol. Chem. 242, 5252-5259. 19. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 20. Fujikawa, K., Legaz, M. E. & Davie, E. W. (1972) Biochemistry 11, 4882-4891. 21. Fujikawa, K., Legaz, M. E. & Davie, E. W. (1972) Biochemistry 11, 4892-4899. 22. Esnouf, M. P. & Jobin, F. (1967) Biochem. J. 102, 660665. 23. Marchalonis, J. J. (1969) Biochem. J. 113, 299-305. 24. Burk, R. R. (1973) Proc. Nat. Acad. Sci. USA 70, 369372. 25. Zacharski, L. R. & McIntyre, 0. R. (1973) Blood 41, 679685. 26. Esnouf, M. P. & MacFarlane; R. G. (1968) in Advances in Enzymology and Related Areas of Molecular Biology, ed. Nord, F. F. (Interscience Publishers, New York), Vol. 30, pp. 255-315. 27. Waugh, D. F. & Livingstone, B. J. (1951) J. Phys. Colloid
Chem. 55, 1206-1218.
Mitogenic Activity of Blood Components. I. Thrombin and Prothrombin (chick embryo fibroblasts/growth factors in plasma and serum/wound healing)
LAN BO CHEN AND JOHN M. BUCHANAN Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139
Contributed by John M. Buchanan, October 15, 1974 ABSTRACT When added to a culture medium of resting confluent chick embryo fibroblasts in the absence of serum, thrombin (EC 3.4.21.5) is able to stimulate DNA synthesis 12 hr later and to cause a substantial increase in cell number over a period of 4 days. As compared to thrombin, prothrombin exhibits low mitogenic activity. However, in the presence of purified Factor Xa (EC 3.4.21.6) and Factor V, prothrombin is converted to thrombin by "thromboplastin activity" supplied by the fibroblasts. Prothrombin, either purified or as a constituent of plasma or serum, may thus be considered to be a reservoir of mitogenic activity in tissue culture unless antithrombin is present in the culture medium in amounts sufficient to neutralize the thrombin formed. By use of a specific inhibitor of proteases, and by separation of prothrombin by absorption on BaSO4, we estimate that the potential mitogenic activity of prothrombin is approximately 3050% of the total activity that can be obtained by treatment of fibrinogen-free plasma with thromboplastin. In addition to its mitogenic activity, thrombin can also stimulate the migration of cells. These experiments with thrombin illustrate that well-characterized proteases of blood can act as potent mitogens and suggest that they may play a role in the process of wound healing.
Many lines of evidence suggest that the control of cell growth either in vivo or in culture is mediated by agents in the extracellular fluid. Even though there are several agents capable of stimulating growth of a variety of cell types, such as serum (1-3), plasma (4), hormones (5-7), antigens (9), plant lectins (10, 11), and proteases (12, 13), very little progress has been made in any instance on the elucidation of the mechanism of cell growth, in particular the initial interaction of mitogens with cell surface components. In some ways the proteases offer a unique opportunity for the study of changes on the cell surface during mitogenic stimulation since (1) many of the enzymes have been obtained in a chemically pure state and (2) the specificities of their proteolytic activities have been carefully documented. During the process of blood clotting a number of proteolytic enzymes are produced and quite probably contribute to the mitogenic activity of this fluid. However, the total mitogenic activity of coagulating plasma is not realized in serum, since neutralizing or inactivating proteins are present in plasma in abundant amounts (14). Thrombin (EC 3.4.21.5) is both a product and catalyst of the blood clotting system and occupies a central position in reaction sequences. As part of a general program to separate and identify actual or potential mitogenic components of serum (or plasma), we have examined highly purified thromAbbreviations: PBS, phosphate-buffered saline; PhMeSO2F,
phenyl methyl sulfonyl fluoride. 131
bin and prothrombin to determine whether they have mitogenic activity when added to resting chick embryo fibroblasts. MATERIALS AND METHODS
Cell Culture and Growth Measurements. Secondary cultures of chick embryo fibroblasts were prepared by a standard procedure (15). Confluent resting cultures were prepared by seeding 7 to 8 X 105 cells per 35-mm culture plate in 2 ml of Dulbecco modified Eagle's medium supplemented with 0.5% (v/v) calf serum. On day 2 the medium was changed. On day 4 the medium was replaced with 1.5 or 2 ml of Dulbecco modified Eagle's medium containing the fractionated materials to be tested for their mitogenic activity. Twelve hours later 10 IAl of [3H]thymidine (0.2 mCi/ml in 50% ethanol, 6.7 Ci/mmol, New England Nuclear Co.) were added, and the incubation was continued for 1 hr. The acid-insoluble fraction was collected and the radioactive DNA was measured. When cell growth was measured by counting the number of cells as a function of time, cell cultures were prepared as described above except that the number of cells seeded was reduced to 3 to 4 X 105 per 35-mm culture plate. After the medium was replaced with Dulbecco modified Eagle's medium containing the materials to be tested, the cultures were incubated at 370 for 4 days and the cell count measured each day by a Coulter counter. Treatment of Fibrinogen-Free Plasma with Phenyl Methyl Sulfonyl Fluoride (PhMeSO2F). Fibrinogen-free plasma was prepared by dialysis of oxalated plasma (16) against water and by further heat treatment for 30 min at 560. One milliliter of a standard solution of brain thromboplastin was mixed with 9 ml of a solution containing 10% fibrinogen-free bovine plasma in Dulbecco modified Eagle's medium. After incubation at 370 for 2 hr, the precipitate was removed. Nine milliliters of supernatant fluid were then mixed with 1 ml of a solution of 10 mg of PhMeSO2F dissolved in 2-propanol. After 3 hr the solution was dialyzed exhaustively first against phosphate-buffered saline (PBS) containing 5% 2-propanol and then against PBS. Finally, the solution was dialyzed against Dulbecco modified Eagle's medium overnight at 4°. A similar treatment was carried out with a control sample of fibrinogenfree bovine plasma in which the Ph-AeSO2F solution was replaced with an equal volume of 2-propanol. Fractionation of Oxalated Bovine Plasma by BaSO4 Treatment. Plasma was treated with BaSO4 according to a procedure described by Rosenberg and Waugh (16) modified from a previous method of Goldstein et al. (17). In this procedure 56 g of BaSO4 (Baker) were mixed with 1600 ml of plasma.
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FiG. 1. Mitogenic activity as a function of concentration of thrombin (0-10 ,.g/ml, 0), calf serum (0-5 mg/ml, A), and bovine prothrombin (0-10 ,ug/ml, 0). Resting chick embryo fibroblasts were incubated for 12 hr with Dulbecco modified Eagle's medium containing proteins to be tested. The cells in 1.5 ml of medium were then incubated for 1 hr with 10 ml of [3H]thymidine (0.2 mCi/ml; 6.7 Ci/mmol) and the DNA was collected. The background radioactivity of cells incubated with serum-free medium is 2400 cpm, and has been subtracted from the values reported in the figure.
The unabsorbed supernatant material (Fraction I) was removed by centrifugation and the crude prothrombin fraction was eluted from the washed BaSO4 cake with 5% sodium citrate (Fraction II). In order to test for mitogenic activity the fibrinogen in Fraction I was removed by dialysis. Fraction II was dialyzed against PBS. Aliquots of all the dialyzed fractions were then treated with Ca2+ and thromboplastin. Estimation of Total Mitogenic Activity of Fractions I and II. In many of the experiments reported in this paper concerned primarily with the elution pattern of specific proteins during chromatography on Sephadex G-200, the exact proportionality of mitogenic activity to protein concentration was not important. However, when quantitative estimation of mitogenic activity was desired (for example, Fraction I and II), we have taken care to use concentrations of solutions in which the mitogenic response as measured by the incorporation of [3H]thymidine into DNA was proportional to the concentration of solution under test. A unit of activity has been defined by Pierson and Temin (2). Column Chromatography on Sephadex G-200. The column of Sephadex G-200 (3.3 X 50 cm) was equilibrated at 40 with PBS supplemented with penicillin (0.1 mg/ml) and streptomycin (0.1 mg/ml) to prevent bacterial growth. The flow rate was 10-15 ml/hr. The eluate was collected in fractions of 2.5 ml. Preparation of Thrombin, Prothrombin, Factor Xa (EC 3.4.21.6) and Factor V. Highly purified bovine thrombin was prepared as described by Baughman and Waugh (18), and bovine prothrombin by an unpublished procedure of C. WI. Becker and D. F. Waugh. Topical thrombin was purchased from Parke-Davis Co. The purity of the samples was tested by electrophoresis on polyacrylamide gel columns containing 0.1% sodium dodecyl sulfate (19). The specific activity of
I
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FIG. 2. Comparison of capacity of serum, thrombin, and prothrombin to stimulate cell division. Cells were seeded at a density of 4 X 105 per 35-mm plate. They were incubated in Dulbecco modified Eagle's medium containing the following additions. A. none, 0; B. Factor V, X; C. prothrombin, 0; D. Factor Xa plus Factor V, 0; E. Factor Xa, *; F. thrombin, *; G. prothrombin, Factor X., and Factor V, *; H. serum, O. When added, the materials were present in the following concentrations: thrombin, 2.5 Mg/ml; prothrombin, 4 gg/ml; Factor X., 0.02 Mg/ml; Factor V, 0.01 Mug/ml; and serum, 5 mg/ml..
thrombin is about 2000 NIH units/mg. Factor Xa, which was prepared by the method of Fujikawa et al. (20, 21), was kindly supplied by Dr. Richard Leary and Dr. Earl W. Davie. Factor V was a partially purified preparation isolated by a modification of the method of Esnouf and Jobin (22). Highly purified thrombin and prothrombin were labeled with 125I according to the method of Marchalonis (23). Measurement of Cell Migration. Resting cultures with a density of 4.4 X 105 cells per 35-mm plate were wounded with a razor blade as described by Burk (24). After wounding, the debris was aspirated, the cells were washed twice, and fresh medium containing thrombin (2.5 ,g/ml) or serum (5 mg/ml) was added. After 16 hr, the cultures were photographed under the microscope. The number of cells migrating into the wound was counted from photographs. RESULTS The Mitogenic Activity of Thrombin. In Fig. 1 are shown the results of an experiment in which purified bovine thrombin, bovine prothrombin, and calf serum were compared for their capacity to stimulate DNA synthesis 12 hr after addition to resting cultures of chick embryo fibroblasts. Serum was added within a range of 0-5 mg/ml of medium, and thrombin and prothrombin within a range of 0-10,ug/ml. As seen, the mitogenic response obtained with serum and thrombin were very similar within the ranges of concentrations used. On the other hand, prothrombin exhibits a very low mitogenic activity. This experiment was then repeated measuring the increase in actual cell count over a period of 4 days but with fixed concentrations of the mitogenic agents, 5 mg/ml for serum, 2.5 ,Mg/ml for thrombin, and an equivalent amount of prothrombin (4,ug/ml) (Fig. 2). The increase in cell count for thrombin and calf serum was in the expected proportion based on the response to these reagents at the indicated concentrations in the DNA synthesis assay. Again only a relatively weak response was obtained when prothrombin was added alone to the culture medium.
Proc. Nat. Acad. Sci. USA 72
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133
TABLE 1. Estimation of mitogenic activity in BaSO4-treated fractions Protein concenFrac- Volume tration tion (ml) (mg/ml) I II
1600 80
78 8.4
Mitogenic activity (units/mg) 1.47 125
Total activity % of total (units recovered X 10-4) activity 68.4 18 8.4 31.4
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Prothrombin as a Source of Thrombin Mitogenic Activity in Cell Culture. These experiments utilizing two kinds of assays to measure cell proliferation both indicated that thrombin is a potent mitogenic agent and that prothrombin is not. Possibly the low response of prothrombin may be attributed to its slow conversion to thrombin. However, in the presence of Factors Xa and V and with a source of thromboplastin, one might expect to observe a conversion of prothrombin to thrombin. This conversion would occur if the residual prothrombin of serum (approximately 10 gg/ml) reacts with Factor X0 and V of serum and if "thromboplastin activity" is provided by the cells themselves (25). In a model system we have measured the mitogenic activity arising from the incubation of a mixture of purified prothrombin, purified Factor Xa, and Factor V with confluent chick embryo fibroblasts (Fig. 2). The increase in cell count over a period of 4 days was compared with that resulting from incubations of each component alone or in incomplete combinations and with the mitogenic activity of thrombin added at a level of 2.5 Mg/ml or serum present in a concentration of 5 mg/ml. A major part of the increase in mitogenic activity of the plate containing prothrombin, Factor Xa, and Factor V over that observed in the control vessel is the result of conversion of prothrombin to thrombin and reflects indirectly the thromboplastin activity of cells that are undergoing division. These experiments illustrate that prothrombin, although not a mitogen itself, can be a source of mitogenic activity for cells in culture. Treatment of Oxalated Bovine Plasma with PhMeSO2F. Fig. 3 shows that after thromboplastin treatment Ph.MeSO2F can inhibit mitogenic activity as much as 50% when fibrinogen-free plasma is tested at a concentration of 2.5% in Dulbecco modified Eagle's medium. This result suggests that thromboplastin-treated plasma contains a mitogenic protease that can be inhibited by PhMeSO2F, a known potent protease inhibitor. The inhibitory effect of PhMeSO2F on thrombin has been reported (14). Several of the proenzymes of the blood clotting system are converted into active form either by the action of thromboplastin or by a secondary reaction of thrombin formed in the primary reaction. Since some of these are serine proteases and may exhibit mitogenic activity, it is possible that they may account for a part of the inhibition of mitogenic activity of plasma by Ph.6eSO2F. However, from a quantitative point of view (26) prothrombin is by far a major component of the proenzymes of the blood clotting system, and, therefore, after conversion to thrombin probably accounts for a large part of the mitogenic activity lost upon reaction of PhMeSO2F with thromboplastin-treated plasma. Estimation of Mitogenic Activity Attributed to Prothrombin as Determined with Fractions of Plasma Treated with BaSO4. In order to bring further evidence to bear on the conclusion that the activity inhibited by PhIVIeSO2F is, in fact, mainly
0 0 0
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(0) inhibitor.
thrombin, we have obtained two fractions of plasma prepared by absorption of proteins onto BaSO4 and elution under conditions specified in Materials and Methods. Fraction I includes the portion of proteins not absorbed, and Fraction II contains practically all of the prothrombin and Factor X of plasma along with some Factor VII, Factor V, and Factor IX. Table 1 summarizes the results of the analysis of Fractions I and II after treatment with thromboplastin. Fraction I contained about 68% of the total recovered mitogenic activity of plasma. The specific activity of this fraction in terms of mitogenic activity per mg of protein was approximately equal to that commonly found in serum and did not depend on treatment with thromboplastin. Fraction II, not treated with thromboplastin, shows only low mitogenic activity when assay was performed by the incorporation of [3H]thymidine into DNA 12 hr after the initiation of stimulation. Only measurement of those cells stimulated during the early period of the incubation will be included in the first wave of DNA synthesis. However, when Fraction II, which contains also Factor X and Factor V, was treated with an excess of thromboplastin, it was found to exhibit substantial amounts of mitogenic activity. Fraction II contained about 30% of the total recovered activity but has a specific activity about 125 times greater than that of calf serum. The wash fractions contained very little mitogenic ac-
tivity. Chromatography of Treated and Untreated Fraction II on Sephadex G-200. When Fraction II untreated with thromboplastin was applied on a column of Sephadex G-200 (3.3 X 50 cm), three major peaks of protein were observed, as indicated
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Proc. Nat. Acad. Sci. USA 72
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FIG. 5. Elution pattern of 125I-labeled prothrombin and thrombin mixed with untreated Fraction II. Fraction II (4.5 ml) not treated with thromboplastin was mixed either with (A) 125I-labeled prothrombin or (B) 125I-labeled thrombin and added to a Sephadex G-200 column (3.3 X 50 cm). The conditions of chromatography are the same as those described in Fig. 4. The symbols used are: *, absorbance at 280 nm, and 0, radioactivity of 12-I-labeled proteins. The arrows represent the volumes at which materials with absorption at 280 nm or with mitogenic activity were eluted from the column.
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(A) before and (B) after treatment with thromboplastin and (C) chromatography of a commercial sample of thrombin. To a column of Sephadex G-200 (3.3 X 50 cm) were added: A and B, 4.5 ml of Fraction II at a concentration of 8.4 mg/ml; or C, 2 ml of commercial thrombin (50 mg/ml). The eluate was collected in fractions of 2.5 ml. The samples (0.5 ml) were tested for absorbance at 280 nm (-) and for mitogenic activity (0). The values of all experiments have been corrected for the background radioactivity of the control samples incubated in the absence of serum (approximately 2000 cpm). In the plates containing 2% serum the radioactivity of [3H] thymidine incorporated into DNA was 37,500, 25,000, and 62,000 cpm for the experiments reported in Fig. 4A, B, and C, respectively. The peaks of materials eluting from the column with absorption at 280 nm are indicated by arrows at the appropriate volumes of effluent.
by the arrows in Fig. 4A. A low level of mitogenic activity was found in peak 3. However, when 4.5 ml of Fraction II from the BaSO4 fractionation procedure was first incubated with 1 ml of brain thromboplastin at 370 for 2 hr, and the supernatant formed after centrifugation at 12,000 X g was then applied to Sephadex G-200, 'peak 3 as measured by absorbancy at 280 nm was completely missing, and a new peak, peak 4, appeared (Fig. 4B). More interesting, not only was all of the activity of Fraction II located in peak 4, but also the specific activity of the mitogenicity of peak 4 was very high as compared with that of peak 3 of the untreated preparation. Since the activity observed in peak 4 emerged only after Fraction II had been treated with thromboplastin, and since Fraction II contained abundant amounts of prothrombin, it is very likely that peak 3 contained the relatively nonmitogenic prothrombin (Fig. 4A) and peak 4, (Fig. 4B) the mitogenic thrombin. Further support for the belief that peak 3 is prothrombin
and that peak 4 is thrombin comes from the results of experiments reported in Fig. 5A and B, respectively. Highly purified samples of electrophoretically pure thrombin and prothrombin were labeled with 125J and mixed with untreated Fraction II. As may be seen in Fig. 5A, the labeled prothrombin migrates at the position previously designated for prothrombin in Fig. 4A. 1251-labeled thrombin (Fig. 5B) migrates at the position expected for thrombin from Fig. 4B. The peak of mitogenic activity shown for treated Fraction II contains two shoulders on either side of the main central peak (Fig. 4B). The shoulders may represent an artifact of the assay procedure, since at the center of the peak the concentration of thrombin in individual samples was probably too high to fall in the proportional range of the assay.
Chromatography of a Commercial Sample of Thrombin. When Parke-Davis thrombin was fractionated on Sephadex G-200, three major protein peaks were observed (Fig. 4C). The peak of protein located in the lower-molecular-weight range of the column effluent contained abundant thrombin and stimulated DNA synthesis of resting cells to a significant level. However, some mitogenic activity was also observed in the high-molecular-weight peak. Since some purified '251-labeled thrombin also migrates in this high-molecular-weight fraction when mixed with Fraction II (Fig. 5B), it is likely that the mitogenic activity observed in the high-molecular-weight peak may result from the binding of thrombin to high-molecularweight proteins. Migration of Cells Stimulated by Serum or by Thrombin. A wounded culture was washed twice with 3 ml of Dulbecco modified Eagle's medium containing no serum and then incubated with 1.5 ml of medium alone or with medium containing either 2.5 ,g/ml of thrombin or 5 mg/ml of calf serum.
Proc. Nat. Acad. Sci. USA 72
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The number of cells migrating into a wound in 16 hr/3-mm length was determined from photographs. For the plates containing medium alone, thrombin, or serum, the number was 0, 27, and 37, respectively. DISCUSSION The experiments reported in this communication have established that thrombin is a potent mitogenic agent. Its activity has been established both by measurement of the DNA synthesis during the first wave of cell division at 12 hr and by actual cell count over a period of 4 days. A model system con-
sisting of purified prothrombin, purified Factor Xa, Factor V, and chick fibroblasts was capable of yielding mitogenic activity as evidenced by a substantial increase in cell number over a 4-day period. These experiments support the speculation that prothrombin of plasma (about 100 ug/ml) and the residual prothrombin of serum (approximately 10 Ag/ml) may contribute to the mitogenic activity of these fluids by conversion to thrombin by the reaction of Factors Xa and V present in the fluids and the thromboplastin activity supplied by the chick cells. The actual contribution of prothrombin as a source of mitogenic activity, however, is difficult to estimate under physiological circumstances, since thrombin is readily neutralized by antithrombin of plasma and serum (14). We would propose, however, that in the microenvironment of a clotting area that the activated proteases might initiate the process of wound healing and that thrombin might contribute to this for a period either before neutralization is complete or as a result of the sequestering of active thrombin, for example, by fibrin (27). * The experiments with the protease inhibitor PhMeSO2F together with the studies with fractions of plasma obtained by absorption on BaSO4 were carried out to obtain a rough estimate of the potential contribution of prothrombin to the total mitogenic activity of this fluid. After treatment of fibrinogen-free plasma with thromboplastin, we observed that 50% of the mitogenic activity was lost upon reaction with
PhMeSO2F. Then after treatment of plasma with BaSO4, the absorbed fraction (Fraction II) contained approximately 30% of the total mitogenic activity of the plasma fractions. We have shown that a peak of protein corresponding to prothrombin is present in an elution pattern of untreated Fraction II when applied to a column of Sephadex G-200. Upon treatment of Fraction II with thromboplastin, this peak of protein is no longer found upon chromatography, but a new peak of protein appears at a further point in the elution pattern. This new peak of protein coincides with the location of thrombin in the elution profile. The mitogenic activity also corresponds to the location of thrombin with shoulders on either side of the central peak. Although other components in Fraction II may account for some of the mitogenic activity appearing in peak 4, it is likely that most of this activity may be ascribed to thrombin. The disappearance of the prothrombin-containing peak 3 and appearance of thrombin-containing peak 4 after thromboplastin treatment of plasma is further circumstantial evi-
dence for this conclusion. The treatment of chick fibroblasts with thrombin results in an enhancement of their capacity to migrate. This agrees *
W. J. Landis and D. F. Waugh, unpublished results.
Mitogenic Activity of Blood Components I
135
with the general observation that most of the mitogens for fibroblasts also stimulate the migration of such cell types. We wish to thank Dr. David F. Waugh, Dr. Carter M. Becker, and Miss P. M. McIlvaine for their generous gifts of highly purified thrombin and prothrombin and Fractions I and II mentioned in this communication. We also wish to express our appreciation to Dr. P. W. Robbins and Mrs. Lin-Huey Chen for supplying chick embryo fibroblasts. We also wish to acknowledge the capable assistance of Mr. Norman Hochella. This work was supported by grants-in-aid from the National Science Foundation (BMS 17669) and the National Cancer Institute (CA 02015), National Institutes of Health. L.B.C. is a predoctoral fellow of the Johnson and Johnson Co. 1. Todaro, G., Matsuya, Y., Bloom, S., Robbins, A. & Green, H. (1967) "Growth regulating substances for animal cells in culture," WistarInst. Symp. Monogr. no. 7, 87-101. 2. Pierson, R. W. & Temin H. M. (1972) J. Cell. Physiol. 79, 319-329. 3. Holley, R. W. & Kiernan, J. A. (1968) Proc. Nat. Acad. Sci. USA 60, 300-304. 4. Balk, S. D., Whitfield, J. F., Youdale, T. & Braun, A. C. (1973) Proc. Nat. Acad. Sci. USA 70, 675-679. 5. Gospodarowicz, D. (1974) Nature 249, 123-127. 6. Temin, H. M. (1967) J. Cell. Physiol. 69, 377-384. 7. Dulak, N. C. & Temin, H. M. (1973) J. Cell. Physiol. 81, 161-170. 8. Holley, R. W. & Kiernan, J. A. (1974) Proc. Nat. Acad. Sci. USA 71, 2908-2911. 9. Edelman, G. M. (1974) "Control of proliferation in animal cells," in Cold Spring Harbor Conferences on Cell Proliferation, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Vol. 1, pp. 357-377. 10. Fahey, J. L. (1974) "Control of proliferation in animal cells," in Cold Spring Harbor Conferences on Cell Proliferation, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Vol. 1, pp. 379392. 11. Vaheri, A., Ruoslahti, E. & Hovie, T. (1974) "Control of proliferation in animal cells," in Cold Spring Harbor Conferences on Cell Proliferation, eds. Clarkson, B. & Baserga, R. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Vol. 1, pp. 305-312. 12. Burger, M. M. (1970) Nature 227, 171-171. 13. Sefton, B. M. & Rubin, H. (1970) Nature 227, 843-845. 14. Magnusson, S. (1971) in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), Vol. III, pp. 277-321. 15. Rein, A. & Rubin, H. (1968) Exp. Cell Res. 49, 666-678. 16. Rosenberg, R. D. & Waugh, D. F. (1970) J. Biol. Chem. 245, 5049-5056. 17. Goldstein, R., LeBolloc'h, A., Alexander, B. & Zonderman, E. (1959) J. Biol. Chem. 234, 2857-2866. 18. Baughman, D. J. & Waugh, D. F. (1967) J. Biol. Chem. 242, 5252-5259. 19. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 20. Fujikawa, K., Legaz, M. E. & Davie, E. W. (1972) Biochemistry 11, 4882-4891. 21. Fujikawa, K., Legaz, M. E. & Davie, E. W. (1972) Biochemistry 11, 4892-4899. 22. Esnouf, M. P. & Jobin, F. (1967) Biochem. J. 102, 660665. 23. Marchalonis, J. J. (1969) Biochem. J. 113, 299-305. 24. Burk, R. R. (1973) Proc. Nat. Acad. Sci. USA 70, 369372. 25. Zacharski, L. R. & McIntyre, 0. R. (1973) Blood 41, 679685. 26. Esnouf, M. P. & MacFarlane; R. G. (1968) in Advances in Enzymology and Related Areas of Molecular Biology, ed. Nord, F. F. (Interscience Publishers, New York), Vol. 30, pp. 255-315. 27. Waugh, D. F. & Livingstone, B. J. (1951) J. Phys. Colloid
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