Characterization of Growth-Inhibitory Activities Associated with an A-Macroglobulin of Mice1

[CANCER RESEARCH 42, 1788-1797, May 1982] 0008-5472/82/0042-OOOOS02.00 Characterization of Growth-inhibitory Activities Associated with an a-Macroglobulin of Mice1

Peter H. Koo

Program in Microbiology and Immunology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272

ABSTRACT treated animals (13). A humoral recognition factor which me diates recognition of tumor cells by macrophages has been An a-macroglobulin (AMG) of similar size and proteinase- reported as an opsonic a2M (1) and was demonstrated to be binding activity as those of human a2-macroglobulin was puri depleted in the plasma of patients with malignant disease (35). fied to homogeneity from mouse plasma. Even after additional Humoral recognition factor was reported to promote chemo- purification steps, AMG still retains a growth-inhibitory activity taxis and phagocytosis of macrophages and is capable of and a more complex subunit structure than does human Â«2- inhibiting growth of primary and metastatic tumors in animals macroglobulin. AMG can inhibit the DMA synthesis of all types (10, 11, 38). The rat (2) and mouse (21) homologs of human of murine tumor cells tested in vitro. This activity is cytostatic, a2M also have been reported to inhibit growth of murine tumor dose dependent, and unaffected by the serum concentration cells in vivo and in vitro, respectively. In addition to its protein in culture. Because this inhibitory activity is resistant to heat, ase-binding property, a2M of humans and animals has also pH 3, and methylamine, it is apparently unrelated to the pro- been demonstrated to combine in vitro with a number of mac- teinase-binding activity which is labile to all these treatments. romolecules not known to be proteinases. These include hu Furthermore, in contrast to the proteinase-binding activity, the man macrophage activation factor (29), human lymphotoxin inhibitory activity can be partially removed from AMG by acid and chemotactic factors (34), L-asparaginase (41), encephalit- dialysis. Gel filtration of the dialysate yields two fractions (M, ogenic basic protein (30), histone H4 (43), cationic aspartate 12,000 and 1,000 to 5,000) which potently inhibit murine aminotransferase (6), and nerve growth factor (36). Therefore, tumor cells but stimulate both the B- and T-lymphocyte reactiv human a2M might modulate the activities of cells also via the ities to mitogens in vitro. The growth-inhibitory activities in macromolecules that it carries and delivers to target cells. The these fractions are resistant to digestions by chymotrypsin, purpose of this investigation is to evaluate critically the purity RNase, and DNase. We conclude from this study that AMG of a mouse AMG which possesses both growth-inhibitory and does not inhibit tumor growth by virtue of its proteinase-binding trypsin-binding activities and to determine whether or not the activity; it may inhibit tumor cells via the small biomediators it proteinase-binding activity or any of the AMG-carried macro- carries. molecules might partake in the inhibition of cell growth. In this report, we demonstrate that homogeneous AMG does not INTRODUCTION inhibit DNA synthesis of tumor cells by virtue of its proteinase- binding property. Potent small-molecular-weight tumor-inhibi Many proteinase inhibitors with different modes of action and tory factors have been extracted from AMG and have been inhibition have been demonstrated to reduce selectively the in shown not to depress mitogen-induced lymphocyte reactivities. vitro growth of transformed cells when compared to nontrans- formed cells (8, 39). Carcinogen-induced and phorbol ester- MATERIALS AND METHODS promoted tumorigenesis in mouse skin (16, 46), chemically induced (24) and radiation-induced (5, 18) transformation of Animals and Cell Lines. Eight- to 20-week-old female C3HeB/Fe, C3H10T1/2 cells, and in vitro invasion of malignant hamster C57BL/6, and DBA/2 mice were purchased from The Jackson Labo cells (26) also were reported to be inhibited by a number of ratory, Bar Harbor, Maine. The tumors used were as follows: a spon proteinase inhibitors. In all these reports, the actual mechanism taneous embryonal ovarian carcinoma originated in a female C3H by which the proteinase inhibitors impair the growth and/or mouse at The Jackson Laboratory (12); P815-X2, a methylcholanthrene-induced mastocytoma of DBA/2 origin; EL-4, a methylcholan- activities of cells is still not known. It has been postulated that threne-induced thymoma of C57BL/6 origin; L51 78Y, a methylcholan- these inhibitors might block proteinase-like activities respon threne-induced lymphoma of DBA/2 origin. These were all kindly sible for unrestrained growth of transformed and neoplastic provided by Dr. Christopher Henney, formerly of The Johns Hopkins cells and invasiveness of malignant tumors. University. L1210, a methylcholanthrene-induced lymphoma of DBA/ Human a2M2 is known primarily as a major serum proteinase 2 origin, was obtained from A. D. Little, Inc., Cambridge, Mass.; and inhibitor of very wide specificity (42). A rat homolog of similar L1210/MR, a spontaneously regressing leukemic line, was derived structure and function has also been isolated from turpentine- from our laboratory (22). Tumor cell lines were maintained in ascites form in syngeneic animals, except for L5178Y which was maintained ' This work was supported in part by USPHS Research Grant 1 R01 CA 24337 in liquid culture medium. from the National Cancer Institute. 2 The abbreviations used are: o2M. o-2 macroglobulin; AMG, murine a-mac Purification of AMG. Whole blood of C3HeB/FeJ mice was obtained roglobulin; PBS. phosphate-buffered saline (0.01 M sodium phosphate buffer (pH either by cardiac puncture or via sinus plexus and was collected into 7.2) containing 0.15 M NaCI]; SOS, sodium dodecyl sulfate; TSME, N"-tosyl-L- plastic tubes in 1/9 volume 3.8% sodium citrate containing soybean arginine methyl ester; RPMI, Roswell Park Memorial Institute; dThd, thymidine; trypsin inhibitor (Worthington Biochemical Corp., Freehold, N. J.) to MOT, mouse ovarian tumor; PHA, phytohemagglutinin; LPS, lipopolysaccharide of cell wall from Escherichia col:; HBSS, Hanks' balanced salt solution without achieve a final concentration of about 0.2 mg/ml in whole blood. To every 33 ml of plasma, 0.8 g BaCI2 and 1.65 g BaSO< were added. Received October 15, 1981; accepted January 26. 1982. After having been stirred for 30 min at 0Â°, the precipitates were

1788 CANCER RESEARCH VOL. 42

removed by centrifugation at 3000 x g. The supernatant was treated tics, Los Angeles, Calif.) in a humidified atmosphere of 5% CO? in air. at 0Â°with polyethylene glycol 4000 (M, 3000 to 3700; J. T. Baker The viability of cells was assessed by the trypan blue exclusion method. Chemical Co., Phillipsburg, N. J.) to 4% saturation. The precipitates Cells were harvested by filtration (glass microfiber papers; Brandel, were then removed by centrifugation, and the supernatant was again Rockville, Md.) and washed successively with PBS, 5% trichloroacetic brought to 12% saturation with polyethylene glycol. The precipitates acid, and methanol. The radioactivity incorporated was counted in thus obtained were collected by centrifugation and dissolved in 10 to Aquasol scintillation fluid (New England Nuclear, Boston, Mass.). The 20 ml PBS. The sample was applied to a column (5 x 84 cm) of control proteins used in the assays were heat-inactivated normal mouse Sepharose CL6B (Pharmacia Fine Chemicals, Piscataway, N. J.) con serum of C3HeB/FeJ, human a2M (17), heat-activated mouse albumin jugated with Cibacron Blue F-3GA dye (45) in PBS. The first major (Miles Laboratories, Inc., Elkhart, Ind.), heat-inactivated human albumin protein eluted from the column was pooled and concentrated. (Miles Laboratories), and normal human serum. The percentage of To determine the apparent molecular weight of AMG and to further suppression of tumor cell growth was calculated as follows: purify an AMG sample, AMG (100 to 200 mg/10 ml) obtained from the Blue-Sepharose column was applied to a Sepharose CL6B column (3.5 % of suppression x 89 cm) in PBS. The column has previously been calibrated with cpm incorporated by tumor in the test sample \ molecular weight standards which included homogeneous human Â«.M radiolabeled with 125I(17), human apoferritin, human y-globulin, and cpm incorporated by tumor in the control solution/ ovalbumin (Worthington). x 100% To further remove possible impurities in the preparation, a pooled AMG sample from Sepharose CL6B column was diluted 2-fold with Growth-inhibitory titers were determined by plotting the percentage of deionized water and then applied to a Whatman DE52 column (2 x 30 suppression of tumor cell growth against the reciprocal of the appro cm) which was equilibrated previously in 5 mw sodium phosphate priate dilution of the test samples used in treating the culture. One unit buffer, pH 7.2, in 75 mw NaCI. The column was first eluted by the of growth-inhibitory activity is that amount of test sample required to equilibration buffer and then by a linear gradient composed of 300 ml reduce tumor cell growth by 50%. each of the 0.02 and 0.30 M sodium phosphate buffers, pH 6.2. Effect of AMG on the Viability and the DMA Synthesis of Tumor Fractions (5.5 ml) were collected and assayed for trypsin-binding and Cells in Vitro. MOT cells (1 x 106) were incubated in cultures contain growth-inhibitory activities (see below). The purity of AMG was deter ing 2 ml of complete medium in the presence of AMG (3.8 mg/ml) or mined by SDS:polyacrylamide gel electrophoresis (see below). The RPMI Medium 1640 alone as control. After 4 hr of incubation at 37Â°, fractions containing high trypsin-binding activity were pooled, dialyzed the supernatants (respectively labeled as the "used-AMG" and "used- RPMI") were collected by centrifugation. The cell pellets were washed into PBS, and concentrated by vacuum dialysis. Electrophoresis. The purity of AMG was judged by the criteria of in RPMI Medium 1640 and then resuspended in complete medium 5% disc polyacrylamide gel electrophoresis with a stacking gel at pH containing 5% heat-inactivated human serum. A separate MOT sample 8.9 and a running gel at pH 9.5 (9). The apparent molecular weights of (5 x 10Vml) in complete medium was incubated at 0Â°for 4 hr and AMG subunits and other peptides were determined by 5 to 15% (w/v) was designated as the control cells. The viability of cells was deter SDS:polyacrylamide slab gel electrophoresis. Gels were stained with mined by the trypan blue exclusion method. The RPMI Medium 1640- Coomassie Brilliant Blue and destained as described (25). The SDS:gel and AMG-treated viable cells (1 x 10s) were again separately cultured electrophoresis molecular weight standards (myosin, yÃŸ-galactosidase, for 27 hr at 37Â°CO2 incubator in 0.21 ml complete medium containing phosphorylase B, bovine serum albumin, and ovalbumin) were obtained 5% heat-inactivated human serum and 1 /iCi [3H]dThd. The amounts of from Bio-Rad Laboratories, Richmond, Calif. radioactivity incorporated in cells were determined as above. To deter The electrophoretic mobility of AMG was determined by comparing mine whether the used-AMG supernatant has lost any of its suppressive with the serum proteins on cellulose acetate membranes (Sepraphore activity, the viable control cells (1 x 105) kept at 0Â° above were III; Gelman Instruments Co., Ann Arbor, Mich.) electrophoresed at 350 incubated, respectively, in 0.21 ml each of fresh AMG (3.8 mg/ml), V for 45 min in Tris:barbital:sodium barbital buffer at pH 8.8. The RPMI Medium 1640, the used-AMG supernatant, and the used-RPMI membranes were fixed and stained in 5% Ponceau S in 7.5% aqueous supernatant in the same culture medium as described above. All trichloroacetic acid and destained in 5% acetic acid. cultures in triplicates were incubated for 27 hr, and the cells were Immunoelectrophoresis was performed in 1% agarose on a micro- harvested, washed, and counted for radioactivity as above. slide (7x5 cm) in a barbital buffer (ionic strength, 0.05), pH 8.6. Mitogen-induced Lymphocyte Transformation Assays. Spleen or Electrophoresis was carried out for 90 min at about 5Â°at a current of lymph node cells (5 x 105) from C57BL/6 mice were cultured in flat- 34 ma and 100 V. Immunodiffusion was allowed to proceed at 5Â°in a bottomed Microtest II plates in the presence or absence of PHA (0.45 humidified box for 1 to 2 days. fig/ml; Wellcome Research Laboratory, Beckenham, England) or LPS Trypsin-binding Assay. AMG was assayed for its functional capacity (8.7 fig/ml; Difco Laboratories, Detroit, Mich.) in 0.21 ml complete of preserving the proteinase activity from inhibition by soybean trypsin medium supplemented with 5% heat-inactivated human serum. Test inhibitor (31 ). Briefly, trypsin (0.30 nm) was incubated at 25Â°with AMG samples were added at the start of cultures and then were incubated (5 to 100 jig) in a buffer medium (0.30 ml) containing 100 mw CaCI2 in a humidified atmosphere of 5% CO2 in air for 48 hr. [3H]dThd (1 juCi/ and 50 mM Tris-CI, pH 8.1. Soybean trypsin inhibitor (10 fig in 0.10 ml) 10 Â¡i\)was added for the last 18 hr of incubation. The cells were was then added, and the mixture was again incubated for 5 min at 25Â°. harvested and washed, and radioactivity was counted as described At the end of incubation, 0.60 ml 1.66 mw TSME in 50 DIM Tris-CI above for MOT cells. buffer, pH 8.1, was added. Hydrolysis of TSME was determined by Heat Inactivation. AMG (10 mg/ml PBS) was distributed into sep change in absorbance at 247 nm. In the absence of AMG, there was arate test tubes, and the tubes were incubated in a 56 or 100Â° water negligible hydrolysis of TSME. bath. The tubes were withdrawn at various time intervals and immedi Growth-inhibition Assay. Murine neoplastic cells (5 x 10" to 1 x ately placed into an ice bath. Aliquots (100 Â¿il)were taken for deter 105) were cultured in 0.20 to 0.22 ml RPMI Medium 1640 containing mining growth-inhibitory and trypsin-binding activities of AMG. penicillin (50 units/ml), streptomycin (50 jig/ml), and 0.2 mw L-gluta- Methylamine Inactivation. AMG (7 to 8 mg/ml) in 0.01 M sodium mine (all obtained from Grand Island Biological Co., Grand Island, phosphate buffer, pH 7.2, containing 0.15 M CaCI2 was incubated in N. Y.), which will be referred to as complete medium. [3H]dThd (1 fiCi, the presence or absence of 0.05 M methylamine for 1 hr at the ambient 73.5 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, Calif), and test sam temperatures and for 3 hr at 0Â°. After incubation, the samples were ples were added at the start of cultures which were incubated at 37Â° dialyzed separately in 1000 volumes of PBS for 20 hr at 4Â°.The PBS- in flat-bottomed microtiter plates (No. 3040 MicroTest II; Falcon Plas dialyzed AMG samples and the dialysates were then sterilized by

MAY 1982 1789

Millipore filtration. (M, 150,000), ovalbumin (M, 45,000), myoglobin (M, 17,000), cyto- Extraction of Small-Molecular-Weight Growth-inhibitory Factors chrome c (M, 12,500), and bacitracin (M, 1,450) (Worthington). from AMG. AMG (20 to 30 mg/ml) was dialyzed by a seamless, W- Proteinase Digestion. A control solution and the pooled column ethylmaleimide-treated cellulose dialysis tubing (VWR Scientific Inc., fractions containing growth-inhibitory factors purified by superfine Chicago, III.; average pore radius, 24 A; r/ie x '/4 inch) into 100 ml 0.2 Sephadex G-75 column were dialyzed into HBSS (with Ca2* and Mg2*; M acetic acid (pH 3.0) for 24 to 48 hr at 4Â°. The dialysate was Grand Island Biological Co.) by Spectrapor-6 dialysis membrane (M, concentrated either by lyophilization, and the residues were redis- cutoff, 2000; VWR Scientific Inc.). The dialyzed solutions were then solved in deionized water or by Amicon ultrafiltration with an UM-2 incubated with chymotrypsin (final concentration, 500 /ig/ml; Sigma Diaflow membrane (Amicon Corporation, Lexington, Mass.). A similar Chemical Co., St. Louis, Mo.) in PBS for 1 hr at 37Â° volume (100 ml) of 0.2 M acetic acid containing 1 to 2 ml PBS was Nuclease Digestion. Sample or HBSS control was incubated in 1% similarly concentrated and used as a buffer control. AMG inside the (w/w) of RNase (bovine pancreatic type III-A; Sigma) or DNase I (bovine dialysis bag was again dialyzed into PBS at 4Â°before it was centrifuged pancratic; Worthington) for 1 hr at 37Â° to remove copious protein precipitates in the solution. Throughout the Antisera. Anti-AMG serum was prepared in New Zealand White same period, a separate AMG sample was dialyzed in PBS at 4Â°and rabbits (27) immunized with homogeneous AMG obtained from Blue- marked as "untreated" AMG. The concentrated dialysate, dialyzed Sepharose CL6B column chromatography. Anti-AMG sera have also AMG, untreated AMG, and the buffer control were sterilized by Millipore been raised in the chickens. Each chicken received i.m. 2 to 3 mg filters (0.22 um). Aliquots (100 ul) were added to cultures containing 1 AMG in an emulsified Freund's complete adjuvant:AMG mixture (1:1, x 105 MOT cells and 1 jiCi [3H]dThd in 0.21 ml complete medium. The v,v). The chickens were similarly boosted 25 days later before they cultures were incubated for 18 hr before the cells were harvested and were bled about 1 month afterwards. The rabbit and chicken antisera washed, and the radioactivity was counted as described. Trypsin- were stored at â€”70Â°.Rabbit anti-human a2M serum and anti-normal binding activities of these samples were also determined in parallel. mouse serum were obtained from Miles Laboratories. Purification and Localization of Small-Molecular-Weight Growth- Other Methods. Protein determination was carried out according to inhibitory Factors by Superfine Sephadex G-75 Column Chromatog- the procedures of Lowry ef al. (28) Dialysis of small-molecular-weight raphy. A concentrated dialysate of AMG (about 0.6 mg proteins in 0.5 materials (M, < 20,000) was carried out by Spectrapor-6 dialysis to 1.5 ml) was applied to a superfine Sephadex G-75 column (1.1 x 57 membrane (M, cutoff, 2,000; VWR Scientific Inc.). cm) preequilibrated in 0.2 M NaCI. Fractions (1.2 ml) were collected at a flow rate of about 5 ml/hr, and the absorbance was measured at 215 nm. The samples in the selected fractions were sterilized by Millipore RESULTS (0.22 /am) filtration and then assayed for growth-inhibitory activity. For tumor inhibition assays, 0.15-ml aliquots of the selected effluent frac Physicochemical and Immunochemical Properties of AMG. tions (1.2 ml) were added to cultures containing 5 x 104 L1210/MR A convenient and reproducible large-scale procedure has been cells in 0.07 ml complete medium containing 20% heat-inactivated designed here to derive homogeneous and biologically active human serum and 2 ftCi [3H]dThd. Cultures were incubated for 18 hr AMG from mouse plasma. In order to maintain biological activity before they were harvested, and the radioactivity was counted. To test and to prevent degradation of AMG by serum proteinases, care the effects of these samples on the lymphocyte responses in vitro, was taken during the processing of blood not to activate plasma identical doses (100 to 150 n\) from the same or neighboring fractions proteases or the blood-clotting system. To achieve these ends, were assayed for their effects on the PHA- or LPS-induced lymphocyte blood was collected into plastic containers containing sodium transformation reactions. Tumor cells or spleen cells were harvested citrate and soybean trypsin inhibitor. Several clotting factors and washed, and radioactivity was counted as above. The molecular weight standards used for column calibration were human -/-globulin were removed by barium chloride and barium sulfate precipi tation, and much of the fibrinogen was removed by 4% poly ethylene glycol. In order to prevent possible fragmentation of

28 AMG at higher temperatures, all the preparations were per formed between 0 and 10Â°.Blue-Sepharose column chroma tography was used to obtain large quantities of AMG. The first major protein eluted from a large Blue-Sepharose CL6B column

_ CD (Chart 1) was determined to be a proteinase-binding protein by >1 a trypsin-binding assay as described. The purity of the protein Ãz ase-binding protein ranged from 90 to 100% as judged by 5% disc polyacrylamide gel electrophoresis along the elution pro UJ O 0.3 g1? r! file (Fig. ÃŒA).Thetrypsin-binding specific activity of the protein is usually between 0.4 and 0.9 AA247/min/mg, and the final m -3 oc yield is about 1.0 to 1.4 mg per ml plasma. O 08 02 O When analyzed by SDSrgel under reducing condition, the mouse protein contained subunits of various molecular weights (M, 185,000, 163,000, 155,000, and 35,000) (Fig. 18), with -f-fâ€”yâ€” the M, 163,000 and M, 35,000 components occurring in higher 120 140 160 180 200 220 240 260 280 concentrations. This is in complete contrast to the reported FRACTION NO. subunit composition of human a2M which has only one subunit Chart 1. Fractionation of normal mouse plasma by a Blue-Sepharose CL6B size (Mr 185,000). Therefore, it is necessary to determine column. Normal mouse plasma (100 ml) was fractionated by polyethylene glycol as described in "Materials and Methods" before the redissolved precipitates in whether the mouse protein may be contaminated by other 15 ml PBS were applied to a Blue-Sepharose column (84.4 x 5 cm). The proteins or may generate aggregation and breakdown products absorbance of fractions (5.5 ml) eluted was determined spectrophotometrically from various subunit components. at 280 nm, and trypsm-binding activity was also determined as described. The results show that only the first A280 absorption peak contained high trypsin- To remove any impurities which might still be associated with binding activity. the proteinase-binding protein, it was further purified by both

1790 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Growth Inhibition by an AMG gel filtration and ion-exchange column chromatography. Gel filtration of this proteinase-binding protein by Sepharose CL6B yielded a single symmetrical absoprtion peak, eluted from the no column slightly ahead of human a2M (Fig. 2). Therefore, the purified protein appears to be homogeneous in size and has 100 either the same or a slightly larger apparent native molecular GJ weight (M, 800,000) than that of a2M (M, 725,000). Further 90 JE H fractionation of this protein by a DE52 column yielded only one a A280absorption peak (Fig. 2), and the samples collected along 80* the absorption profile all contained a subunit composition iden tical to that obtained from the Blue-Sepharose column in Fig. 70 18. Besides occurring as a single protein on the cellulose acetate membrane at pH 8.8, this proteinase-binding protein also exhibits an a-electrophoretic mobility that is slightly faster 60 than human t*2M (data not shown). In addition, the DE52 purified protein contained only one cross-reacting component SO

when reacted with a rabbit antiserum to AMG or to normal o mouse serum (Fig. 3). Therefore, it appears very unlikely that 40 -o the purified mouse protein contained any significant amounts x of contaminants. Owing to its large size and a-electrophoretic 30 - mobility, this protein is termed as AMG. AMG did not cross-react immunochemically with human a2M 20 as judged by Ouchterlony double immunodiffusion against the anti-AMG sera obtained from 3 different rabbits and 2 different 10 chickens as well as against a rabbit anti-human Â«2Mserum. Therefore, these 2 proteins have very different primary struc tures as is in part suggested by their differences in the SOS 1 3 5 7 9 11 13 subunit compositions. FINAL PROTEIN CONCENTRATION PER Tumor-inhibitory Property of AMG. To localize tumor-inhib , CULTURE, mg/ml itory activities in the fractions of Blue-Sepharose column (Chart Chart 2. Inhibition of DNA synthesis of MOT cells by AMG. heat-inactivated normal mouse serum (NMS), human aÂ¡M,heat-inactivated mouse albumin (mouse 1), an aliquot (100 Â¿il)ofeach fraction was added to MOT cells ALB), human albumin (human ALB) or heat-inactivated human serum (WHS). The (1 x 10s) in 0.10 ml complete medium containing 1 /iCi [3H] cultures (in triplicate) were incubated for 18 hr before they were harvested and counted for radioactivity as described in "Materials and Methods." The results dThd. The mixtures were incubated for 18 hr before the cells show that AMG was by far the most suppressive to MOT cells than were any of were harvested and radioactivity was counted. Two major the control proteins tested. Normal mouse serum and human ,< M were slightly tumor-inhibitory activities were localized. One activity was suppressive only at much higher concentrations. TdR, dThd. found to cofractionate with AMG, while the other actvity was localized approximately between Fractions 240 and 280 (Chart pressed by at least 42 to 48% even after 27 hr of incubation. 1). It is not known whether these activities are in any way Therefore, AMG does not appear to be cytotoxic but can exert related. a longer-term cytostatic effect on these cells. Table 3 also AMG was found to be a far more potent inhibitor than were shows that the used-AMG sample which has been incubated any of the control proteins tested, and its inhibitory effect was previously with MOT cells did not lose its inhibitory potency. In dose dependent (Chart 2). The specific activity of AMG purified other words, the MOT dose used did not significantly absorb from Blue-Sepharose and DE52 columns ranged from 2.5 to 5 out the growth-inhibitory activity of AMG. The control super units/mg AMG. The recovery of growth-inhibitory activity is natant (used-RPMI), on the other hand, appeared to slightly between 3 and 7 units/ml plasma. AMG also inhibited the enhance the growth of MOT cells. growth of all types of murine ascites tumor cells tested (Table Effect of Heat on Growth-inhibitory and Proteinase-binding 1). The AMG dose required to achieve 50% of tumor inhibition Activities of AMG. To determine the relationships between varied with AMG preparations, but usually this dose of AMG is growth-inhibitory and proteinase-binding activities, AMG was between 0.5 and 2 mg/ml. Furthermore, growth-inhibitory ac incubated at 56 or 100Â°as described in "Materials and Meth ods." Table 4 shows that proteinase-binding activity was very tivity of AMG was unaffected by an increasing concentration of human serum in the culture (Table 2). On the contrary, at heat-labile whereas growth-inhibitory activity was rather heat- higher human serum concentrations, the growth-inhibitory ef stable. fect of AMG became more evident because the added serum Effect of Methylamine on Growth-inhibitory and Protein stimulated the growth of tumor cells in the absence of AMG. ase-binding Activities of AMG. Methylamine has been de Effect of AMG on the Viability of MOT Cells in Vitro. Since scribed to neutralize proteinase-binding activity of human a2M AMG is inhibitory to tumor cell growth, experiments were (44). Since trypsin-binding activity of AMG can also be inacti carried out to determine if it is cytotoxic or cytostatic to tumor vated by methylamine,3 we have investigated the effect of cells. The results in Table 3 show that the viability of the methylamine on growth-inhibitory activity. AMG (6 to 7 mg/ml) washed AMG-treated MOT cells was not much different from was incubated with or without 50 nriMmetÃlylamine as described that of the control groups (RPMI Medium 1640-treated cells at 0 or 37Â°), but the growth of the AMG-treated cells was sup- 1N. W. Hudson, J. M. Kehoe, and P. H. Koo, submitted for publication.

MAY 1982 1791

Table 1 Table 3 Eflect of AMG on DNA synthesis and viability of various murine tumor cell lines Effect of AMG on viability and DNA synthesis of tumor cells in culture in vitro In Experiment 1, tumor cells (1 x 10s) were incubated with AMG or PBS viabilitySample123456CulturesMOT % of of sup pression control in 0.22 ml complete medium for 18 hr in the presence or absence of 1 fiCi [3H]dThd. In Experiment 2, similar procedures as above were carried out, oflivecells84.284.886.2%ofdeadcells15.815.213.8%ofMOTgrowth30-6.4425254-4.8 except that a different AMG preparation was used, and the cell viability was not 0Â°MOTin RPMI at determined. All the cultures were carried out in triplicate. RPMIforafter incubation with AMG 37Â°MOT4 hr at concentra of inhibi AMGforafter incubation with tion in cul tion of [3H}- 37Â°MOT4 hr at Experi ture (mg/ dThd incor of viable "fresh"AMG6MOTfrom Sample 1 + mentline1 Cell ml)4.21.004.21.004.01.04.01.04.01.04.01.0%poration59200655703522302347312717%cells55 "used-AMG"CMOTfrom Sample 1 + 5-X2EL42 P81 Â±4a56 Â±571 "used-RPMI"Cfrom Sample 1 + Â±361 supernatant%

Â±558 " Percentage of suppression of MOT cell growth was calculated with respect Â±766 to the control Sample 1. All triplicate cultures were incubated for 27 hr before Â±4 the cells were harvested. The fresh AMG is an AMG preparation which has never been incubated with MOTL1210L1210/MRL5178YFinal MOT cells. 0 The used-AMG and used-RPMI supernatants were obtained from Samples 3 and 2. respectively, after 4-hr incubation as described in "Materials and Methods."

Table 4 Effect of heat treatment on trypsin-binding and growth-inhibitory activities of AMG of sup Time of heat treatment incorpo pression of activity (AA/ (hr)a0 0 Average Â±S.D. of triplicate determinations. rated(cpm)19,347 MOT growth63 min/100/ig)0.047

0.5 (56Â°) Table 2 19,537 63 0.003 4 (56Â°) 21,972 58 0.001 Effect of serum concentration on tumor suppression by AMG 0.5(100Â°)PBS 25,90352,449% 510Trypsin-binding0.0010 (cpm)HBSS incorporated Ofsup-MOTgrowth334595072 controlpHJdThd manserumin AMG was incubated in PBS at 56 or 100Â°. At the time intervals indicated, cultures051015[3H]dThdcontrols20,806 aliquots of AMG samples were taken and immediately cooled to 0Â°.The trypsin- 2,032Â°87,734Â± binding and growth-inhibitory assays were carried out as described in "Materials 1,94335,849Â± and Methods." 18.72974,216Â± 6,32937,207Â± b Percentage of suppression of MOT growth was calculated with respect to 7,775128,720Â± 5,03835,958Â± Â±20,043AMG"13,665 Â±5,769% the PBS control. The results are expressed as averages of triplicate determina tions. Percentage of suppression was calculated with respect to the HBSS controls for the same human serum concentrations. 6 AMG (1.0 mg in 100 /il HBSS) was added at the start of the culture to each Table 5 of the triplicate cultures (0.21 ml) containing 1 x 105 MOT cells, 2 /iCi [3H]dThd, Effect of methylamine on growth-inhibitory and trypsin-binding activities of AMG and heat-inactivated human serum at the indicated concentrations in complete inhibition by AMG and medium. The cultures were incubated for 18 hr as described before cells were Methylamine AMG%ofsuppres harvested, washed, and counted for radioactivity incorporated. 0 Mean Â±S.D. binding activity sion of (AA/ in "Materials and Methods" and then was assayed for both [3H]dThd incorporated MOT min/ 100 growth"49.794(cpm) proteinase-binding and growth-inhibitory activities. The results SamplesAMG M9>0.035 Â± 3,874b 59 in Table 5 demonstrate that methylamine-treated AMG lost >87% of the original trypsin-binding activity while growth-in Methylamine AMG 46.709 Â± 1,386 62 0.005 PBS (AMG dialysate) 121,440 Â±15,868 0 hibitory activity remained intact. PBS (methylamine AMG dialy 122,201 Â±27,343 0Trypsin- Effects of Enzymatic Digestions on Growth-Inhibitory Ac sate)Tumor Homogeneous AMG (7.4 mg/ml, 0.9 ml) was incubated with methylamine tivity. To investigate the chemical nature of the growth-inhibi as described in "Materials and Methods." Percentage of suppression of MOT tory factors associated with AMG, the pooled partially purified growth is expressed as an average of triplicate determinations and was calculated growth-inhibitory factors (M, 12,000 and 1,000 to 5,000) were with respect to the respective PBS dialysate controls. incubated at 37Â°in the presence or absence of chymotrypsin, 0 Mean Â±S.D. RNase, or DNase as described in "Materials and Methods." The results as shown in Table 6 demonstrate that the growth- by acid dialysis, AMG (94 mg in 3 to 4 ml PBS, containing inhibitory factors are resistant to chymotrypsin and nuclease about 400 units of inhibitory activity) was dialyzed into 0.2 M digestions. acetic acid at 4Â°for 24 hr. The dialysate thus obtained was Effect of Acid Dialysis on Growth-inhibitory and Protein then lyophilized to dryness, and the residues (about 0.6 mg ase-binding Activities. To determine whether or not these proteins) were redissolved in 2 ml deionized water. The di activities are labile to acid and whether any of the tumor- alyzed AMG, the AMG dialysate, and the dialysate control were prepared and assayed as described in "Materials and Meth- inhibitory factors may be dissociable from the AMG molecules

1792 CANCER RESEARCH VOL. 42

ods." The acid-dialyzed AMG lost about 84% of the total no effect. The specific inhibitory activity of the AMG dialysate growth-inhibitory activity and all of its proteinase-binding activ samples ranged from about 170 to 400 units/mg protein. ity. The dialysate solution contained 152 units or 38% of the Effect of AMG Dialysates on Lymphocyte Transformation total growth-inhibitory activity and none of the proteinase-bind Reactions. An untreated AMG, acid-dialyzed AMG, and the ing activity. The results in Chart 3A show that acid-dialyzed concentrated dialysate, prepared as described above, were AMG lost much of its growth-inhibitory activity. However, di- tested for their effects on the PHA-induced lymphocyte trans alyzed AMG when assayed at higher concentration (>3 mg/ formation reaction. Table 7 shows that the same AMG sample ml) was almost as suppressive as was untreated AMG. The as described in Chart 3A, which suppressed MOT growth, had dialysate solution (Chart 38), on the other hand, was potently very low immunosuppressive activity. Also, the concentrated inhibitory to the DMA synthesis of MOT cells at very low protein AMG dialysate which contained potent growth-inhibitory activ concentrations, whereas the lyophilized acetic acid control had ity stimulated the PHA-induced lymphocyte transformation re action instead. Table 6 Other Physicochemical Properties of the AMG Dialysate Effect of chymotrypsin and nucleases on the activities of growth-inhibitory Materials. Homogenous AMG (30 mg in 1.5 ml PBS) or PBS factors control were dialyzed in 0.2 M acetic acid successively for 24 of sup and 48 hr. The 24- and 48-hr dialysates were separately pressionofMOTgrowth"01152S356541525415352 incor lyophilized to dryness, and the residues were then redissolved Source"HBSS porated(cpm)80,02371,25938,70337.73635.21136,91279,47338,11537,16780,78137,51938,104%in 1.5 ml deionized water and 1.1 ml PBS, respectively. Both controlsChymotrypsin the first dialysate (A28o 0.85) and the second dialysate (A28o controlSG-750 1SG-75 Fraction 0.46) have a similar A28o:A260ratio of about 1.04 and absorb 2SG-75Fraction maximally at both 222 to 226 and 270 to 272 nm wavelengths. chymotrypsinSG-75Fraction 1 and To determine the apparent molecular weights of protein com chymotrypsinRNaseFraction 2 and ponents occurring in the dialysates, one-fifth amounts of each controlSG-75 RNaseSG-75Fraction 1 and of the 2 dialysate samples were applied to a reducing 12% RNaseDNaseFraction 2 and SDS:slab gel. Fig. 4 shows that both of the acid dialysates controlSG-75 DNaseSG-75Fraction 1 and contained similar components with the relative molecular Fraction 2 and DNase[3H]dThd masses largely restricted within about 10,000 to 20,000 dal- The SG-75 Fraction 1 (M, about 12.000), and SG-75 Fraction 2 (M, 1.000 tons. Probably owing to the porosity of the gel used, other to 5,000) which contained potent growth-inhibitory activities were the pooled smaller components were barely detectable. The protein com Fractions 25 to 29 and 35 to 37. respectively (Chart 3). The results are presented as averages of triplicate samples. ponents at the top of Gels 2 to 4 were impurities in the buffers. Percentage of inhibition of DNA synthesis by MOT cells was calculated with Fractionation of Growth-inhibitory Activities by Superfine respect to the average of triplicate samples of the HBSS control. Sephadex G-75 Column Chromatography. The acid dialysate c SG-75. superfine Sephadex G-75 column. prepared as described above from 95 mg AMG after 48-hr dialysis was concentrated and then dialyzed into PBS by an

Table 7 Effect of acid-dialyzed AMG and the dialysate on the PHA-induced lymphocyte reactivity The PHA-induced lymphocyte transformation reaction was carried out accord ing to the procedure described in "Materials and Methods." All the cultures were carried out in triplicate. transformationSampleAMG,PHA-induced lymphocyte

[AMG], mg/ml suppres sion(orstimula 100 B tion)oflympho incorporated(cpm)75,297 N-AMG tration(mg/ml)0.250.06250.01560.00.250.06250.01560.00.270.0680.0170.0040.0[3H]dThdcytereactivity14350128(43)0(83)(52)(23)(16)0Trypsin-bindingactivity(AWmin/100N)0.0670.0050.006 !Â¡ DIALYSATE 60 Â±4,673s64,198 untreatedDialyzed Â±14,30062,997 Q-H Â±60566,259 Â±6,79674,210Â± C/3 20 AMGAMG 5,41760.838 0.05 0.10 0.15 Â±2.90994,818 Â±85466.259 [PROTEIN], mg/ml 6,796213.407Â± Chart 3. Effect of acid dialysis of AMG on DNA synthesis of MOT cells. Details appear under "Materials and Methods." A, dialyzed AMG. when compared to dialysateFinalproteinconcen14,939100,Â± the untreated AMG, had lost much of its growth-inhibitory activity. However, it 875 Â±66281,256 Â±10,76376,703 was still suppressive at higher AMG concentrations. B, The acid dialysate Â±7,81866,259 contained potent inhibitors which suppressed about 93% of MOT growth at 0.15 Â± 6,796%of mg/ml protein concentration. Again the inhibitory activity is dose dependent. The " Mean Â±S.D. lyophilized acetic acid control resuspended in PBS as above was inactive.

MAY 1982 1793

Amicon UM-2 Diaflow membrane. Column chromatography of contained material(s) which were markedly (500 to 800%) the AMG dialysate (A280 1.48, in 1.5 ml HBSS) by Sephadex stimulatory to lymphocyte transformation reactions. G-75 resolved at least 4 A215absorption peaks. However, only 2 well-separated peaks corresponding to apparent molecular DISCUSSION weights of about 12,000 and 1,000 to 5,000, respectively, contained growth-inhibitory activities (Chart 4X\). The void vol A proteinase-binding protein of size and nature similar to ume fraction occurred at Tube 14. The same inhibitors at the those of human a2M has been isolated in mice. The isolated same concentrations used did not suppress the PHA- and LPS- mouse protein has a slightly faster a-electrophoretic mobility induced lymphocyte transformation reactions, but instead they than does human a2M at pH 8.8, so it is designated AMG. The stimulated [3H]dThd incorporation by these cells (Chart 4B). In purified AMG has been demonstrated previously to possess addition, the fourth A215 peak material (M, > 1,000) eluted not only a trypsin-binding activity but also a growth-inhibitory activity (21). In this study, we have attempted to answer the following questions. Does AMG inhibit DNA synthesis of tumor cells by virtue of its proteinase-binding property? If not, what is then the likely mechanism of tumor inhibition by AMG? Our present data demonstrate that AMG used in this inves tigation is homogeneous as judged by the criteria of native polyacrylamide gel electrophoresis, immunoelectrophoresis, and gel filtration. However, in reducing SDS: gel, even the most purified AMG preparation consists of 2 major (M, 163,000 and 35,000) and one minor (M, 185,000) subunit; occasionally an additional minor component (M, 155,000) was also observed. The M, 163,000 and M, 35,000 components have been re cently shown to be covalently bound subunits of AMG.4 The M, 185,000 and M, 155,000 proteins share identical primary structures with that of the M, 163,000 protein; thus they may be the variant subunit and a spontaneous degradation product of AMG, respectively.3-4 Since under the nonreducing condi tions in SDS:gel only one protein component of about M, 400,000 is detected,4 all the above-described components must be covalently linked to AMG via disulfide bonds and are 20 30 40 50 60 not copurified impurities. The purified AMG has been reproducibly demonstrated to FRACTION NO. possess not only a trypsin-binding activity but also a growth- inhibitory activity. AMG exerts a dose-dependent inhibition on the ability of murine tumor cells to incorporate dThd (Chart 2). When compared with other protein controls, AMG is by far the most inhibitory of all the serum proteins tested. Mouse serum is only slightly inhibitory, probably because it also contains AMG-associated inhibitory activity. It is not known why human a2M is less inhibitory than AMG, nor is there any evidence at present to suggest that AMG may be homologous to the human recognition factor (1 ). Our present data also demonstrate that AMG is capable of inhibiting DNA synthesis of all types of murine tumors tested (Table 1) but not proliferative response of lymphocytes to PHA mitogen (Table 7). This finding may be potentially important in cancer therapy, especially if AMG does not adversely affect other normal tissues. Growth-inhibitory activity of AMG is not affected by the presence of human serum. This suggests that AMG does not suppress tumor cell growth by neutralizing or depleting growth-promoting substances in culture media, nor can any serum components neutralize growth-inhibitory activity 10 20 30 40 50 of AMG. Once tumor cells are exposed to AMG, the apparently FRACTION NO. viable cells do not resume dThd incorporation even after pro longed incubation in fresh culture media in the absence of Chart 4. Fractionation of AMG acid dialysate by superfine Sepharose G-75 AMG. Therefore, AMG appears to exert a somewhat longer- column chromatography. A. at least 2 species of tumor-inhibitory factors (M, about 12,000 and 1,000 to 5,000) were localized. B, the same aliquots (0.15 ml) term and perhaps direct cytostatic effect on tumor cells. The as above from the same fractions when added to C3HeB/Fej splenocytes (5 x question of whether or not AMG-treated cells still maintain 105 in 0.21 ml complete medium containing 5% heat-inactivated human serum) stimulated both the PHA- and LPS-induced lymphocyte transformation reactions instead. TdR. dThd. ' N. W. Hudson, and P. H. Koo. Biochim. Biophys Acta, in press.

1794 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Growth Inhibition by an AMG protein synthesis and other cellular functions is currently being lymphotoxins (M, 12,000 to 15,000) which can promote tumor investigated. regression of a human basal cell carcinoma and a mouse Although a2M is well known for its proteinase-binding activity, lymphoma (33). Whether or not the M, 12,000 mouse growth- several lines of evidence presented here clearly demonstrate inhibitory factor described here is a homolog of these human that growth-inhibitory activity of AMG is independent of pro lymphotoxins is not known. Mouse lymphotoxins, like AMG- teinase-binding activity. Growth-inhibitory activity is stable to associated growth-inhibitory activities, are also stable to acid heat (56Â°, 1 hr; or 100Â°, 30 min), methylamine, and pH 3 (pH 2) and heat (100Â° for 15 min) (20), but all mouse lympho treatment, whereas trypsin-binding activity is very labile to toxins reported to date have molecular weights between 90,- these treatments. Therefore, we conclude that the growth- 000 and 150,000. inhibitory and trypsin-binding sites either occur in separate These AMG-associated growth-inhibitory factors share cer macromolecules or reside in separate sites of the same AMG tain properties with several murine growth-inhibitory factors molecule. known to exist. The AMG-associated factors, like interferons Human a2M is known to be able to combine with a number a and ÃŸandmacrophage migration-inhibitory factors, are stable of biologically active macromolecules (42). In this study, we to acid (pH 2 to 3) and heat (56Â°,30 min). AMG, like interfÃ©rons were able to dissociate from AMG a bulk of the total growth- (3, 14), can also exert long-term cytostatic effects on tumor inhibitory activity by acid dialysis. The recovered dialysate is cells. Mouse interferons have been reported to inhibit a variety potently inhibitory to DMA synthesis of the tumor cells (Chart of murine cancer cells in vitro (7, 15) and in vivo (14), but both 3B) but exerted a dose-dependent enhancement of lymphocyte suppressing and enhancing effects of interferon preparations reactivity (Table 7). This suggests that the growth-inhibitory on the immune system have been described (40). However, the activity may selectively inhibit neoplastic cells while not ad AMG-associated tumor-inhibitory factors have apparent molec versely affecting lymphocyte proliferation. After acid dialysis, ular weights much smaller than any of the smallest murine a small amount of growth-inhibitory activity appears to still interferons (M, 20,000) (19) and macrophage migration-inhib remain with dialyzed AMG and exert a dose-dependent, and itory factors (M, 48,000 to 67,000) (23) known to exist. perhaps synergistic, suppressive effect on tumor cell growth In conclusion, this investigation demonstrates that homoge (Chart 3A). About 46% of the total growth-inhibitory activity neous AMG does not inhibit the growth of tumor cells by virtue originally present in AMG was lost after dialysis, probably of its proteinase-binding activity. One mechanism of growth through the lyophilization and concentration procedures used. inhibition by AMG is via small-molecular-weight inhibitors that Fractionation of the acid dialysate material by a superfine it carries. This investigation also presents the first direct evi Sephadex G-75 column yields 2 growth-inhibitory fractions dence that suggests AMGs as natural carriers for several small- with molecular weights of about 12,000 and 1,000 to 5,000, molecular-weight bioactive macromolecules in vivo. For this respectively (Chart 4). These components are potently inhibi reason, AMG may be a convenient, if not a unique, source for tory to tumor cells but not to lymphocytes; instead, they appear obtaining certain bioactive molecules for investigation. The to stimulate lymphocyte reactivities to mitogens. In addition, a findings of an apparently selective effect of growth-inhibitory potent immunostimulatory activity is also detected in the frac factors on murine cancer cells while not adversely affecting tions separate from growth-inhibitory activities. This stimulatory normal lymphocytes suggests potentially important therapeutic factor affects the responses of both T- and B-lymphocytes. In application but needs further investigation. conclusion, the present data suggest that AMG may carry several growth-inhibitory and immunostimulatory factors larger than Mr 1000. REFERENCES The chemical nature of these growth-inhibitory factors has 1. Allen, C . Saba, T. M., and Molnar, J. Isolation, purification and characteri not yet been established. They resist nuclease digestions; zation of opsonic protein. J. Reticuloendothel. Soc., 13: 410-423, 1973. therefore, they are not nucleic acids. The active centers of 2. Adrenne, M. V., and Chaplain, R. A. The inhibitory effect of o-2-macroglob- these molecules may reside in chymotrypsin-resistant pep- ulin on tumor growth. Experientia (Basel), 29: 1271-1272. 1973. 3. Balkwill, F. R., Watling, D., and Taylor-Papadimitriou, J. Inhibition by lym- tides, carbohydrates, or lipids. phoblastoid interferon of growth of cells derived from the human breast. Int. The endotoxic LPS from Gram-negative bacteria possess J. Cancer, 22. 258-265, 1978. 4. Berendt, M. J., North, R. J., and Kirstein, D. P. The immunological basis of many diverse biological properties, including induction of tumor endotoxin-induced tumor regression. Requirement for T-cell-mediated im necrosis (32) and tumor regression (4). However, it is unlikely munity. J. Exp. Med., Â»48.1550-1559, 1978. that growth-inhibitory activities associated with AMG are due 5. Borek, C., Miller, R., Pain, C., and Troll, W. Conditions for inhibiting and enhancing effects of the protease inhibitory antipain on x-ray induced to endotoxin effect. Not only was AMG used in the study neoplastic transformation in hamster and mouse cells. Proc. Nati. Acad. Sci. homogeneous as judged by various criteria of purity, but also U. S. A., 76. 1800-1803, 1979. a major proportion of the growth-inhibitory activity resides in 6. Boyde, T. R. C., and Pryme, I. F. Alpha2-macroglobulin binding of trypsin, chymotrypsin, papain, and cationic aspartate aminotransferase. Clin. Chim. small-molecular-weight components tightly associated with Acta, 2i: 9-14, 1968. AMG. LPS prepared from bacteria by phenol:water extraction 7. Brouty-Boye, D., and Tovey, M. G. Inhibition by interferon of thymidine is a polydisperse mixture of aggregated molecules (M, 500,- uptake in chemostat cultures of L1210 cells. Intervirology. 9. 243-244. 1978. 000) (37). Smaller subunits of molecular weights of 14 to 8. Chou, I-N., Black, P. H., and Roblin, R. Effects of protease inhibitors on 15,000 have been dissociated from LPS only by deoxycholate growth of 3T3 and SV3T3 cells. In: B. Clarkson and R. Baserga (eds.). Vol. 1, pp. 339-350. Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory. and acid hydrolysis (3) but were biologically inactive. 1974. Human a2M and albumin have been reported to carry a 9. Davis, B. J. Disc electrophoresisâ€”II. Method and application to human human lymphotoxin obtained from human lymphoblast cell line serum proteins. Ann. N. Y. Acad. Sci., 121: 404-427, 1964. RPMI 1788 in culture (34). The acid-extractable fractions of 10. DiLuzio, N. R. Macrophages, recognition factors and neoplasia. In: J. W. Rebuck, C. W. Berard, and M. R. Abell (eds.). The Reticuloendothelial supernatant preparations of the lymphoblast culture contain System, Vol. 16, pp. 49-64. Baltimore: The Williams & Wilkins Co., 1975.

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11. DiLuzio, N. R.. McNamee, R., Olcay, !.. Kitahama, A., and Miller, H. Inhibition 1970. of tumor growth by recognition factors. Proc. Soc. Exp. Biol. Med., 745: 31. Mosher, D. F., and Wing, D. A. Synthesis and secretion of a2-macroglobulin 311-315, 1974. by cultured human fibroblasts. J. Exp. Med., 743: 462-467, 1976. 12. Fekete, E., and Ferrigno, M. A. Studies on a transplantable teratoma of the 32. Oettgen. H. F., Hoffman, M. K., Clarkson, B. D., and Old, L. J. Effects of mouse. Cancer Res., 12: 438-440, 1952. endotoxin and endotoxin-induced mediators on cancer and on the immune 13. Gordon, H. The a-macroglobulins of rat serum. Biochem. J., 159: 643-650, system. In: M. Sela (ed.), The Role of Non-Specific Immunity in the Preven 1976. tion and Treatment of Cancer, pp. 121-141. Amsterdam: North-Holland 14. Gresser, I., and Bourali, C. Antitumor effects of interferon preparations in Publishing Co., 1979. mice. J. Nati. Cancer Inst., 45. 365-376, 1970. 33. Papermaster, B. W. Purification and characterization of lymphokine fractions 15. Gresser, I., Brouty-Boye, D., Thomas, M. J., and Macieira-Coelho, A. Inter associated with tumor immunotherapeutic activity. In: L. W. Mozes, J. feron and cell division. I. Inhibition of the multiplication of mouse leukemia Schultz, W. A. Scott, and R. Werner (ed.), Cellular Responses to Molecular L1210 cells in vitro by interferon preparations. Proc. Nati. Acad. Sci. Modulators, pp. 271-285. New York: Academic Press, Inc., 1981. U. S. A., 66: 1052-1057, 1970. 34. Papermaster. B. W., Gilliland, C. D., Smith, M.. Buchok, S.. McEntire. J. E., 16. Hozumi, M., Ogawa. M., Sugimura, T., Takeuchi, T., and Umezawa, H. Butler, R. C., Specter, S., and Friedman, H. Tumor regression induced in Inhibition of tumorigenesis in mouse skin by leupeptin, a protease inhibitor mice by partially purified lymphokine fractions. Discussion paper. Ann. N. Y. from Actinomycetes. Cancer Res., 32: 1725-1728. 1972. Acad. Sci., 332: 451-459. 1979. 17. Hudson, N. W., Koo, P. H., and Kehoe. J. M. Structural comparison of a 35. Pisano, J. C.. Jackson, J. P., DiLuzio, N. R., and Ichinose, H. Dimensions of murine a2-antiproteinase with human a2-macroglobulin. Fed. Proc., 39: humoral recognition factor depletion in carcinomatous patients. Cancer 1688, 1980. Res., 32: 11-15, 1972. 18. Kennedy, A. R., and Little, J. B. Effects of protease inhibitors on radiation 36. Ronne, H., Anundi, H., Rask, L., and Peterson, P. A. Nerve growth factor transformation in vitro. Cancer Res., 41: 2103-2108, 1981. binds to serum alpha-2-macroglobulin. Biochem. Biophys. Res. Commun.. 19. Knight, E., Jr. Purification and characterization of interferons. In: I. Gresser 87:330-336, 1979. (ed.). Interferon 1980. Vol. 2. pp. 1-12. New York: Academic Press, Inc., 37. Rudbach, J. A. Immunogenicity of lipopolysaccharides. In: R. F. Beers, Jr., 1980. and E. G. Bassett (eds.). The Role of Immunological Factors in Infectious, 20. Kolb, W. P., and Granger, G. A. Lymphocyte in vitro cytotoxicity: character Allergic and Autoimmune Processes, pp. 29-41. New York: Raven Press, ization of mouse interferon. Cell Immunol., 7: 122-132, 1970. 1976. 21. Koo, P. H. Tumor suppression by a-macroglobulins of mice. Fed. Proc., 37: 38. Saba, T. M., and Cho, E. Alteration of tumor growth by a purified alpha-2- 1337, 1978. glycoprotein. J. Reticuloendothel. Soc.. 22: 583-595, 1977. 22. Koo, P. H. Immunostimulatory factors specifically associated with a spon 39. Schnebli, H. P. The effects of protease inhibitors on cells in vitro. In: E. taneously regressing subline of murine leukemia L1210. J. Immunol., 727: Reich, D. B. Rifkin, and E. Shaw (eds.). Proteases and Biological Control, 373-379, 1981. Vol. 2, pp. 785-794. Cold Spring Harbor. N. Y.: Cold Spring Harbor Labo 23. KÃ¼hner,A. L., and David, J. R. Partial characterization of murine migration ratory, 1975. inhibitory factor (MIF). J. Immunol., 116: 140-145, 1976. 40. Sonnenfeld, G. Modulation of immunity by interferon. Lymphokine Rep., 7: 24. Kuroki, T., and Drevon, C. Inhibition of chemical transformation in C3H/ 113-132, 1980. 10TV4 cells by protease inhibitors. Cancer Res., 39: 2755-2761, 1979. 41. Soru, E. Interaction of o2-macroglobulin with L-asparaginase. Mol. Cell. 25. Laemmli, U. K. Cleavage of structural proteins during the assembly of the Biochem., 23: 185-192, 1979. head of bacteriophage T4. Nature (Lond.) 227: 680-685. 1970. 42. Starkey, P. M., and Barrett, A. J. a2-Macroglobulin, a physiological regulator 26. Latner, A. L., Longstaff, E., and Pradhan, K. Inhibition of malignant cell of proteinase activity. In: A. J. Barrett (ed.), Proteinases in Mammalian Cells invasion in vitro by a proteinase inhibitor. Br. J. Cancer, 27:460-464,1973. and Tissues, Vol. 2. pp. 663-696. Amsterdam: North-Holland Publishing 27. Liu, S. H., Koo, P. H., and Cebra, J. J. Effect of carrier priming on the Co., 1977. distribution of anti-hapten antibodies between lgG1 and lgG2 Â¡sotypesin the 43. Stoller, B. D., and Rezuke, W. Separation of anti-histone antibodies from hyperimmune guinea pig. J. Immunol., 113: 677-687, 1974. nonimmune histone-precipitating serum proteins, predominantly a2-macro- 28. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein globulin. Arch. Biochem. Biophys., 790: 398-404, 1978. measurement with the Folin phenol reagent. J. Biol. Chem., 793: 265-275, 44. Swenson, R. P., and Howard. J. B. Structural characterization of human a2- 1951. macroglobulin subunits. J. Biol. Chem., 254: 4452-4456, 1979. 29. McDaniel. M. C.. Laudico, R., and Papermaster, B. W. Association of 45. Travis, J., Bowen, J., Tewksbury, D.. Johnson, D., and Pannell, R. Isolation macrophage-activation factor from a human cultured lymphoid cell line with of albumin from whole human plasma and fractionation of albumin-depleted albumin and a2-macroglobulin. Clin. Immunol. Immunopathol., 5: 91-104, plasma. Biochem. J., 757: 301-306, 1976. 1976. 46. Troll, W., Klassen, A., and Janoff, A. Tumorigenesis in mouse skin: inhibition 30. McPherson, T. A., Marchalonis, J. J.. and Lennon, V. Binding of encephalit- by synthetic inhibitors of proteases. Science (Wash. D. C.), 769: 1211- ogenic basic protein by serum a-globulins. Immunology. 79: 929-933, 1213, 1970.

abcdefghijklm Sabcdefgh ijklmS

Fig. 1. Disc polyacrylamide gel electrophoresis of the fractions obtained from Blue-Sepharose column chromatography. Fractions a to m were Tubes 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, and 190 of Chart 1, respectively. Protein samples (20 /ig) in indicated fractions were applied to a 5% polyacrylamide slab gel (At or were reduced and run in 10% SDStpolyacrylamide slab gel (B) as described in "Materials and Methods." S, molecular weight standards. Gel A shows that the first eluted A2Sopeak was composed of a homogeneous protein in Fractions c to ft. Gel B shows that the same fractions when reduced in SDS yielded several subunit components. The 4 identifiable protein components in these column fractions in Gel B have apparent molecular weights of about 185,000. 163,000, 155,000, and 35,000.

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abc d e A S

Fig. 2. Fractionation of proteinase-binding protein by gel filtration and ion-exchange column chromatography. Left, proteinase-binding protein (about 190 mg in 10 ml) from Pool a of Fig. 1 was applied to a Sepharose CU6B column (3.5 x 89 cm). A single symmetrical A28oabsorp tion peak was obtained near the fractions where the '"l- labeled human a2M (28 x 106 cpm/5 /ig) was also local ized (arrow). Right, elution profile of the pooled sample ''A " (about 100 mg) on a Whatman DE52 column (2 x 30 cm) eluted by a linear gradient (L.G.). Only one A2eopeak was obtained. Protein samples (20 jig) in the selected tubes were again assayed for their subunit compositions by 10% SDS:gel electrophoresis. The results (see inset: L.G a to e are Tubes 53, 56, 59, 65, and 70, respectively) I show that the DE52 purified proteins along the elution profile all have the same subunit composition as do the samples in Fig. \B. Gel samples A and S are Pool A of Sepharose CL6B and molecular weight standards, re spectively.

20 40 60 80 20 40 60 80 100 FRACTION NO. FRACTION NO.

123456

Fig. 3. Immunoelectrophoresis of AMG samples. Wells 1 and 5 contained AMG (5 /ig in 5 /il) from DE52 column. Wells 2, 3, and 4 were, respectively, normal mouse serum, pooled AMG samples from a Blue-Sepharose column, and - pooled AMG from a Sepharose CL6B column. Troughs a and c contained a rabbit anti-AMG serum (0.1 ml), and Troughs b and d contained a rabbit anti-normal mouse serum. The results show that DE52-purified AMG contained only one cross-reacting material. .

Fig. 4. SDS:polyacrylamide gel electrophoresis of the AMG dialysates. An aliquot (0.1 ml) of the 24-hr (Gel 3) and the 48-hr (Gel 4) AMG dialysates and the 48-hr PBS control dialysate (Gel 2) was applied to a reducing 12% SDS:disc polyacrylamide gel. After electrophoresis, the slab gel was stained with Coomas- sie Brilliant Blue. Gels ) and 6 contained protein standards: bovine serum albumin (M, 66,500); chymotrypsinogen (M, 25,000); and cytochrome c (Mr 12,500). Gel 5 contained protein standards: ovalbumin (M, 45.000); and myoglobin (M, 17,000).

MAY 1982 1797

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Characterization of Growth-inhibitory Activities Associated with an α-Macroglobulin of Mice

Peter H. Koo

Cancer Res 1982;42:1788-1797.

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