American Journal of Pathology, Vol. 140, No. 3, March 1992 Copyright X) American Association of Pathologist
Neutralizing Antibodies Against Transforming Growth Factor p Potentiate the Proliferation of Ki-1 Positive Lymphoma Cells Further Evidence for Negative Autocrine Regulation by Transforming Growth Factor p
S. R. Newcom,* K. K. Tagra,* and M. E. Kadint From the Division of Hematology/Oncology, Department of Medicine,* Emory University School of Medicine, Atlanta, Georgia, and the Department of Pathology, Beth Israel Hospital, and the Harvard University School of Medicine,t Boston, Massachusetts
Activated lymphocytes and malignant lymphoma cells derived from them (Ki-1 positive lymphoma cells) share similar mechanisms ofproliferation. To further examine the inhibitory role of endogenous transforming growthfactor ,B (TGFO) in Ki-1 positive lymphoma cells, the authors studied anti-TGFO antibodies and measured their effect on proliferation A monoclonal antibody (TIA5) prepared against a unique antigenic epitope of high molecular weight Hodgkin's TGFI3 and a polyclonal rabbit antibody prepared against highly purified 25,000 D porcine platelet TGFO1 were used Both antibodies are shown here to inhibit the biological activity of Hodgkin's TGFI and to crossreact with their respective antigens in immunoblotting DNA synthesis by Ki-I lymphoma cells was increased 138-fold by anti-TGFI3 antibody and 262-fold by anti-Hodgkin's TGF. Exogenous TGF I suppression was completely reversed by anti-TGFI31 antibody and IL-2-inducedproliferation was markedly potentiated (41 fold). L-428 Reed-Sternberg cells secrete physiologically active TGFO but havefewer than 500 TGF receptor sitesper cell; no significant proliferative response was measured for either anti-TGFi3 or anti-Hodgkin's TGFf These results show the suppressive effect of exogenous TGFI31 on indolent Ki-I lymphoma cells and suggest that the endogenous secretion of high molecular weight physiologically active TGFOI is important in
maintaining the indolent nature of this low-grade Ki-1 positive lymphoma (Am J Pathol 1992, 140:
Transforming growth factor ,B (TGF,) is a member of a family of multifunctional polypeptides that induce anchorage-independent growth of nontransformed fibroblasts13 and inhibit the proliferation of many cells including epithelial cells4.5 and lymphocytes.67 It has recently been established that there are at least three TGFfi mammalian proteins, each encoded by different genes.8 A previous study has reported that Ki-1 positive human lymphoma cells and activated human lymphocytes share similar mechanisms of proliferation.9 After activation of lymphocytes, Interleukin-2 (IL-2) is secreted and its membrane receptor (R) is formed within 4-8 hours.6 Although mRNA for TGFI1 can be identified within 2 hours of lymphocyte activation, secretion of TGFI does not occur for 24 hours and reaches a maximum at 72-96 hours after stimulation.6 The effect of TGF,B is inhibitory and the clonal expansion of activated lymphocytes is terminated. A low-grade malignant activated lymphocyte cell line expresses 18,000 high affinity IL-2 R per cell, undergoes proliferation in response to IL-2, has 4200 TGFo1 R per cell, and is partially inhibited by exogenous TGF,B1.9 These Ki-1 positive lymphoma cells secrete 1 fM physiologically active TGF,B/cell/day. To further investigate the Supported in part by grants CA-50739 and RR-MO1-01032 from the National Institutes of Health, Bethesda, Maryland, grant CD-485 from the American Cancer Society, Atlanta, Georgia, and grant 2630 from the Council for Tobacco Research. Accepted for publication October 22, 1991. Address reprint requests to Dr. Samuel R. Newcom, Department of Medicine, Emory University, Woodruff Research Extension Building, 46 Armstrong St., Atlanta, GA 30303.
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possible role of this endogenous, physiologically active, autocrine TGF, in suppression of malignant Ki-1 lymphoma cell growth, we used a monoclonal antibody prepared against a unique epitope of high molecular weight Hodgkin's TGF,B (TiA5)10 and a polyclonal rabbit antibody prepared against highly purified 25,000 D porcine platelet TGFP1. We tested these antibodies to determine if inhibition of endogenous TGFP could be accomplished and if this inhibition would affect the proliferation of these Ki-1 positive malignant lymphoma cells.
Materials and Methods Cell Cultures Ki-1 positive lymphoma cells (Mac-1) are a continuously proliferating human lymphoma cell culture established from the malignant cells in the blood of a patient with cutaneous T-cell lymphoma.9 This patient had been treated 1 0 years previously for stage IIA mixed cellularity Hodgkin's disease. Ki-1 lymphoma cells were demonstrated to be mycoplasma free and to express CD-2 (T11), CD-25 (IL-2R), T-9, and CD-30 (Ki-1) antigens. CD-8 was expressed weakly. CCL-64 cells were mink lung epithelial cells obtained from the American Type Culture Collection (ATCC) (Rockville, MD). L-428 ReedSternberg cells are from nodular sclerosing Hodgkin's disease and have been extensively characterized.11,12 All cells were maintained in complete medium containing Iscove's modified Dulbecco's medium (IMDM) supplemented with fetal calf serum (10%), insulin (5 jig/ml), selenium (5 ng/ml), transferrin (5 ,ug/ml), 2 mM 1-glutamine, and penicillin-streptomycin (1 %).
rum is purified by Staph A chromatography and has been shown to neutralize both TGFp1 and TGF,2.13 This antibody does not crossreact with acidic or basic fibroblast
growth factor, platelet-derived growth factor, or epidermal growth factor. A non-neutralizing anti-TGFpi antibody, which is highly specific for TGF,1 but does crossreact with TGF,B2, is used for immunoblot detection (R & D Systems, Inc., Minneapolis, MN).13 Anti-Hodgkin's TGF3 (TiA5) is a monoclonal IgGi murine antibody prepared against high molecular weight TGFP.10 This monoclonal antibody partially neutralizes Hodgkin's TGF,B (70%) from the L-428 Reed-Sternberg cell line but not TGFp1 from human or porcine platelets.10 Immunoblot and enzyme-linked immunoadsorbent assay (ELISA) indicate that T1A5 crossreacts with a unique epitope on Hodgkin's TGFO distinct from TGFP1.10
TGF3 Bioassay The method for detecting inhibition of epithelial cell DNA synthesis has been previously described14 and is a modification of the original method published by Tucker et al.4 Briefly, mink lung epithelial cells (CCL-64) are plated into the microtiter wells of a flat-bottomed 96-well plate (1i4 cells/well in 1 % fetal calf serum). Triplicate test wells were used for each test condition; after 24 hours, the cells were at a stable rate of DNA synthesis [==3000 counts per minute (CPM)]. A 12-point titration curve of TGFI1 was constructed and the result was used to measure the efficacy of anti-TGF,li. Inhibitory concentrations of TGFoi were incubated with varying concentrations of the neutralizing antibody. 3H-thymidine (0.5 ,uCi, New England Nuclear, Boston, MA) was added to each well for 16 hours and the cells were collected on glass filters, dried, and counted in a scintillation counter with Hydrofluor scintillation fluid (National Diagnostics, Manville, NJ).
Growth Factors Human platelet TGFB1 was purchased from R and D Systems, Inc. (Minneapolis, MN). This preparation has been demonstrated to be homogeneous by analysis on SDS-polyacrylamide gels and to be identical to other human TGF,1 s in bioassay, receptor binding, and aminoacid sequence.13 Recombinant IL-2 was purchased from Cetus Corp. (Emeryville, CA). One unit of IL-2 is defined as the amount of IL-2 required to induce half-maximal stimulation of an IL-2-dependent T-cell line (CTLL-2).
Anti-TGFP Antibodies Anti-TGFIl is a polyclonal rabbit antibody (R & D Systems, Inc., Minneapolis, MN) prepared by injection of highly purified TGFp1 from porcine platelets. The antise-
Ki-1 Lymphoma and L-428 Reed-Sternberg Cell Growth Measurements To measure DNA synthesis, lymphoma cells were prepared as a single-cell suspension in unsupplemented IMDM. Cells were cultured for 48 hours in flat-bottomed microtiter dishes in the presence of control media, TGFP1, IL-2, anti-TGF,B1, or anti-Hodgkin's TGF, (Ti A5). 3H-thymidine (0.5 ,uCi) was added for 16 hours, and the cells were collected on glass filters, dried, and counted in a scintillation counter.
Immunoblot Detection of TGFI Using a modification of the Laemmli method15 TGF,B from Ki-1 lymphoma cells or TGFp1 were equilibrated in 2%
Anti-TGFP in Ki-1 Lymphoma 711 AJP March 1992, Vol. 140, No. 3
SDS electrophoresis buffer and electrophoresed into 4-30% polyacrylamide gradient gels. Biological activity was tested by slicing gels horizontally and removing the growth factor by electroelution. Immunoblotting was performed using nitrocellulose paper and an 18-hour transfer with cooling (80 v). Reaction of anti-TGF,1 and antiHodgkin's TGF, was detected using a biotinylated affinity-purified antibody specific for the appropriate species. Biotin was detected using alkaline phosphataseconjugated avidin. Control blots were performed with second antibody, with the avidin conjugate, and with nitroblue tetrazolium to detect nonspecific staining.
Neutralizing Activity of Anti-TGFI1 Antibodies Polyclonal anti-TGF31 has been previously shown to neutralize TGF,B1 in the AKR-2B semi-solid agar assay.13 To further assess and quantitate the anti-TGF31 activity of this antibody, a CCL-64 inhibition assay was performed and a titration curve constructed. Maximum inhibition of CCL-64 cell DNA synthesis is achieved at a concentration of 2.5 ng/ml TGFp1 (1000 pM) and the inhibition is nearly complete at 98.7%. The titration curve from one experiment is shown in Figure 1. The monoclonal antibody directed at a unique epitope on the Hodgkin's TGFI (Ti A5) did not neutralize TGFp1 in this assay. Polyclonal anti-TGFp1 completely blocked TGF,1 activity at 10 pLg/ml. The titration curve for anti-TGFp1 is shown in Figure 1 (Bottom).
Epithelial Cell Inhibition (%) 100 90
Role of Autocrine TGFP in Ki-1 Lymphoma Cell Growth Regulation Ki-1 lymphoma cells are responsive to IL-2 but, after passage 2, have not required exogenous IL-2 for survival.9 These cells are serum-dependent, however, and enter G. after 48-72 hours in serum-free medium. To determine the role of TGF,1 in maintaining Go in serum-free culture, we exposed resting Ki-1 lymphoma cells to various concentrations of Staph A-purified polyclonal antiTGFP1. There was a marked increase in DNA synthesis suggesting that this growth factor is important in regulating the proliferation of these cells. The data are presented in Figure 2 and show a 1 38-fold increase in DNA synthesis after complete neutralization of endogenous TGFp1. In contrast, L-428 Reed-Sternberg cells do not require IL-2 or serum factors for survival and do not enter Go in serum-free medium. Anti-TGFI1 and control IgG had no effect on L-428 Reed-Sternberg cell proliferation (Figure 3A, C).
Anti - TGFB 1 Antibody (Dilution) Figure 1. Anti-TGC1 aakt* of rabbitpo4lo a1 antbody. Top: To c n>rm doe biologial aaci ofhigb,y purfIed human platelet TGF1, epit*elialcell (CCL-64) DNA swdx i*niion ua nmeue over a range of concentratons of TGF1 (20-5000 pM). Near complete (99%) hbiin was obtaned after 24bous of posure to 1000-5000 pM TGF51. Bottom: To test die inhibition of the biologial am&hy of TGF1 usingpo4coa anti-TG1 abody, an epe 7ev was performied using CC-64 epitbelial cells. Maxuintwn (99%) inhibition of d*e DNA exsis of dese cells was acheved wit 1000 pM TGF1 (25 ng/ml) This actiy was complete retsed using pocdonal antiTGF1 (10 pg/ml).
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30 28 26 24 22 20 18 16 14 12 10 8
24 22 20 18 16 14 12
0 0 a. 4
Anti-TGFf1 (R & D)
C. ? x 0
30 28 2624 22 20 18 16 14 12 10 8 6 4 2 0
302826 24 2220 18 16 14 12 10 8642-
Non-immune Rabbit IgG
Non-immune Murine IgG
Role of Autocrine TGFI1 in IL-2-induced Ki-1 Lymphoma Cell Proliferation Ki-1 lymphoma cells have 18,000 high affinity IL-2 receptors/cell (Kd = 4.05pM).9 It has been shown previously that exogenous TGFI1 inhibits IL-2-induced DNA synthesis in these cells by approximately 40-45%9 and colony formation in methycellulose by 60-80%.16 Ki-1 Iymphoma cells secrete approximately 1 fM physiologically active TGF,31/cell/day.9 To determine if this autocrine TGF,1 was inhibiting IL-2-induced proliferation, we studied the role of anti-TGFpi1 in the proliferation induced by recombinant IL-2. In Figure 4, the DNA synthesis of Ki-1 lymphoma cells after exposure to IL-2 (100 U/ml) is shown. There is a fivefold increase in DNA synthesis. In this experiment, exogenous TGF,1 (2 ng/ml) decreased IL-2-induced DNA synthesis by 24%. However, antiTGFp1 increased the DNA synthesis to a maximum of 21,200 + 1900 counts per minute or a 42-fold increase above the serum-free rate and a 1 9-fold increase above
Figure 2. The role of endogenous TGF in Ki-1 lymphoma cell growth. A: Ki-1 lymphoma cells were incubated in increasing concentrations of rabbit polyclonal neutralizing anti-TGFf1. After 48 hours, the highest concentration (10 pg/ml) increased the DNA synthesis by 138fold (from 226 + 10 CPM to 31,097 + 4548 CPM). B: T1AS, monoclonal anti-Hodgkin's TGF3 induced a 262-fold increase in DNA synthesis (59,250 517 CPM). Non-immune rabbit IgG (C) and murine IgG (D) did not produce significant changes in DNA synthesis.
the IL-2-induced DNA synthesis rate. A second experiment comparing Ki-1 lymphoma cells and L-428 ReedSternberg cells is shown in Table 1. At increasing concentrations of IL-2, TGF,1 inhibited IL-2-dependent Ki-1 lymphoma cell DNA synthesis by 24-50%. L-428 ReedSternberg cells were unresponsive to IL-2 and not significantly inhibited by exogenous TGF,1 (1-11%). AntiTGFo1 completely neutralized the activity of exogenous TGFI1, and apparently, endogenous TGFP1, resulting in Ki-1 lymphoma DNA synthesis increase of 942-8146% above baseline (10-82-fold). In contrast, no significant increase was noted in L-428 Reed-Sternberg cell DNA synthesis (19-58%; less than onefold). Anti-TGFpi1 was able to induce DNA synthesis rates in the Ki-1 lymphoma cells that approached those of the highly malignant L-428 Reed-Sternberg cells. These findings suggest that, after malignant transformation, autocrine TGFP remains an important, although imperfect, suppressor of Ki-1 lymphoma cell proliferation. L-428 Reed-Sternberg cells have lost suppression by exogenous TGF1 and
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A. 3028 26
28. 26. 22. 20.
16. 14 .
2. 0. 1.25
5 Anti-TGFpI (R & D) 2.5
30 28 26 24 22 20 18 1614 -
.1. Figure 3. The role of endogenous TGF in L-428 Reed-Stemnberg cell growth. A: L-428 cells were incubated in increasing concentrations of rabbit polyclonal neutralizing anti-TGFp3lfor 48 hours. B: L-428 cells were incubated in increasing concentrations of T1A5 murine monoclonal antiHodgkin's TGFj and controls (C, D) for 48 hours. There was no significant change in DNA synthesis from that measured in serum-free medium (99,794 ± 7454).
have little ized.
268 24222018. 16~ 1412-
when endogenous TGFP is neutral-
The Close Relationship of the Function of Autocrine TGF, from Ki-1 Lymphoma Cells to Hodgkin's TGF,3 Hodgkin's TGF, is a macromolecule-containing TGF,1 that is secreted in a physiologically active form by both fresh Hodgkin's cells17 18 and a long-term Hodgkin's cell line (L-428).14 Monoclonal antibodies have been prepared that recognize unique epitopes on the Hodgkin's TGF, molecule that are distinct from TGFP1.1° To determine if neutralization of one of these epitopes would neutralize the inhibitory activity of autocrine TGF, from Ki-1 lymphoma cells, we used the murine monoclonal antibody T1A5. A marked increase in DNA synthesis was noted in Ki-1 lymphoma cells (Figure 2B) but not L-428 Reed-Sternberg cells (Figure 3B). Nonimmune murine IgG did not affect DNA synthesis.
Non-immune Rabbit IgG
Non-immune Murine IgG
The Close Relationship of the Structure of Autocrine TGFP from Ki-1 Lymphoma Cells to Hodgkin's TGF, Transforming growth factor P from Ki-1 lymphoma cells was collected in serum-free IMDM and concentrated 100 times. After dialysis and exchange into 2% SDS electrophoresis buffer, the Ki-1 lymphoma TGF, was electrophoresed into 4-30% polyacrylamide gradient gels and transferred to nitrocellulose paper. Figure 5 shows the immunoblot obtained using T1A5 anti-Hodgkin's TGF, and anti-TGF,1. Similar to Hodgkin's TGFP, Ki-1 lymphoma TGF, contains the unique T1A5 epitope that is distinct from TGFi1 (Lanes 1 and 2). Similar to Hodgkin's TGF,, Ki-1 lymphoma TGFP contains the TGFpl epitope (Lanes 4 and 5). This high molecular weight form of Hodgkin's TGFO is physiologically active when electroeluted from the gel slice at this high molecular weight.14 Similar to Hodgkin's TGFP, Ki-1 lymphoma TGFP contains the homodimer of TGF,1 that, after cleavage of disulfide bonds by 2-mercaptoethanol and boiling, is fully
714 Newcom, Tagra, and Kadin AJP March 1992, Vol. 140, No. 3
[~~//B/// rz717 IL-2
S ._ _
IL-2 + Polyclonal Anti - TGFB1 Antibody reduced to the biologically inactive monomer (12,500 D) (Lanes 4 and 5) similar to TGF,B1 from platelets3 (Lane 6).
Discussion Ki-1 positive lymphomas express the Ki-1 activation antigen19'20 which was originally identified on the L-428 Reed-Sternberg cell line21 and was later shown to be transiently expressed on normal lymphocytes following activation.22'23 Some non-Hodgkin's lymphomas have also been shown to express this lymphocyte activation antigen and therefore may be histogenetically related to both the malignant Reed-Sternberg cell and nonmalignant activated lymphocytes.19'20'24'25 The present study of the proliferative events in a Ki-1 positive lymphoma may offer insight into the derangements occur-
Figure 4. The inhibitoty effect of exogenous Interleukin-2 (100 TGF1 (2 ng/ml) U/ml)-induced DNA synthesis by Ki-1 lymphoma cells was confirmed here. To test the secete by role of on
__ly_pLg/ml) mphoma pLg/ml-20 was used anti-TGFf1 to inhibit TGF1 biological IL-2 activity. In this experiment IL-2 was shown to neat AB induce DNA synthesis which was patially inhibited (24%) by exogenous TGFP(1. This inhibition was reversed and IL-2 was markedly potentiated by anti-TGFp1 (42-fold). ~
ring in the transformation of normal lymphocytes to malignant lymphoma cells. When normal T lymphocytes are activated, IL-2R are formed and IL-2 is secreted.6 These events are accompanied by DNA synthesis, proliferation, and expansion of the lymphocyte population. Although mRNA for TGF,B can be identified within 2 hours of activation, secretion of TGF, does not occur for 24 hours and reaches a maximum at 72-96 hours after stimulation. The effect of TGF,B on IL-2-stimulated human T lymphocytes has been analyzed and shown to be inhibitory, perhaps by decreasing the number of IL-2R.6 TGF,3 suppresses DNA synthesis 60-80% in T lymphocytes6 and 65% in B lymphocytes.7 Therefore, TGF,B appears important in limiting the expansion of nonmalignant activated lymphocytes. The failure of the inhibitory response to occur at any step could result in the clonal expansion of the dysfunctional
Table 1. Comparison of the Role of Anti-TGFI3 in Ki-1 Lymphoma Cell and L-428 Reed-Stemnberg Cell Proliferation IL-2 Supp* TGF,1 Anti-TGFo1 (2 ng/ml) % Increaset (%) (100 ±g/ml) (U/ml) (CPM)
Mac-1 Ki-1 lymphoma cells 3 465 ± 330 10 2,854 ± 287 30 3,673 ± 1198 100 3,106 ± 609 L-428 Reed-Sternberg cells 3 61,595 ± 9812 10 61,557 ± 4733 30 63,512 ± 4217 100 65,954 ± 7054
230 2,176 1,955 2,369
± ± ± ±
59,474 ± 61,037 ± 56,379 ± 61,991 ±
76 382 206 403
18,966 ± 5508 40,251 ± 4999 51,793 7464 24,696 1720
6749 4626 6795 1654
3 1 11 6
71,249 72,569 88,827 74,527 ±
6569 1826 3957 8293
8,146 1,749 2,549 942 20 19 58 20
* Supp = the percentage suppression of IL-2-dependent DNA synthesis produced by TGF,p1. t Percentage increase of anti-TGFp1 DNA synthesis compared with maximally suppressed DNA synthesis. Cell cultures were incubated in the test conditions for 48 hours before the addition of 3H-thymidine as described. Serum-free CPM for Ki-1 lymphoma cells were 257 ± 178. Serum-free CPM for L-428 Reed-Sternberg cells were 51,940 ± 3381.
in Ki-1 Lymphoma 715 AJP March 1992, Vol. 140, No. 3
Figure 5. Polyacrylamide gel electrophoresis was performed using samples equilibrated in 2% SDS buffer. After electrophoresis all proteins were blotted onto nitrocellulose paper (0.45 L) using 80 vfor 24 hours. Top: Lanes 1 and 2 were were labeled with TlA5 monoclonal anti-Hodgkin's TGFf3. Lane 1 (human platelet TGF1) does not crossreact with antiHodgkin's TGF1. Lane 2 (Ki-1 Lymphoma TG1) crossreacts with anti-Hodgkin's TGF and has a similar high molecular weight double band (-300,000 daltons). Bottom: Lanes 4-6 were labeled with anti-TGFMI1. Lanes 4 and 5 (fully reduced Ki-1 Lymphoma TGFO) demonstrate the appropriate 12,500 dalton TGFI1 monomer. Lane 6 (human platelet TGF41) confirs the cross reactivity of the antibody with its homodimer antigen (TGFf1). 7bis study confirms the molecular identity of Ki-1 lympboma high molecular weight TGF with Hodgkin's TGFf3.0"14
cell. Although endogenous TGF,B appears much more important, we have shown previously that exogenous TGFp1 suppressed IL-2--induced DNA synthesis in Ki-1 lymphoma cells (passage 25) 40-45%9 or approximately 50% of the suppression obtained in activated lymphocytes. This result correlated with a 50% reduction in IL-2R number. In the present study, Ki-1 lymphoma cells (pas-
sage 45) showed a similar 24-50% suppression with exogenous TGF,B1. Although these results must be interpreted cautiously in view of the unmeasured endogenous effects of TGF,1, others have shown that progressive loss of exogenous TGFI suppression correlated with progressive accumulation of malignant features in human colon carcinoma,26 H-ras transformed he-
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patocytes and epithelial cells,27 metastatic H-ras transformed murine fibroblasts,28 and neoplastic rat tracheal epithelial cells.',' One of us (M.E.K.) has shown that Ki-1 positive lymphoma cell lines established at a later time in the clinical course of the Mac-1 patient had lost sensitivity to suppression by TGFI3i16 suggesting that in vivo passage of these lymphoma cells has produced a progressive loss of sensitivity to exogenous TGFI suppression of proliferation. TGFoi from platelets is normally bound to alpha-2macroglobulin and stored as an inactive high molecular weight form.31 32 Recent work suggests that a binding protein containing repeat segments similar to epidermal growth factor is also present.3 The events resulting in activation are incompletely understood. Activation has been shown experimentally by cellular environmental acidification,34 cellular coculture of endothelial cells with pericytes,35 displacement of TGFi from the alpha-2macroglobulin by heparin,36 enzymatic treatment of the macromolecule by plasmin,37 and enzymatic removal of carbohydrates from the complex by endoglycosidase F.38 The marked secretion of physiologically active TGFI by leukemia cells in the adult T-cell leukemiaAymphoma syndrome appears to be related to direct transactivation of the TGFI3 gene by the HTLV-1 tax protein.39 The TGFP secreted by fresh and cultured Reed-Sternberg cells14,17 is secreted in a high molecular weight, activated form. Activated TGFP is identified in lymph-node sections of nodular sclerosing Hodgkin's disease.i' Inhibition of proteases does not block activation of Hodgkin's TGFp, 14 coculture with other cells is unnecessary,12 and unique epitopes have been identified on this high molecular weight Hodgkin's TGFP from the L-428 ReedSternberg cell line.10 We have shown in this study that the TGFP secreted by a Ki-1 positive lymphoma cell line (Mac-1) is physiologically active, contains the TGFpl antigen, has a high molecular weight, and contains a unique epitope (TlA5), all features identical to Hodgkin'sderived TGF3. To further test the hypothesis that the progressive loss of TGFO responsiveness in Ki-1 lymphoma cells is associated with increasing proliferative activity, we used a potent neutralizing antibody against TGFpl to inhibit the endogenous high molecular weight TGFoi produced by these cells. There was a marked (138-fold) increase in DNA synthesis. Interaction of the T1A5 monoclonal antibody, demonstrated to crossreact with the TGFP secreted by these lymphoma cells in immunoblotting, was also markedly inhibitory (a 262-fold increase in DNA synthesis) as previously reported when Ti A5 was directed at Hodgkin's TGFI3.10 This result suggests that the T1A5 epitope is important in mediating the biological activity of Hodgkin's-related TGF~. The mechanism by which TGFfi normally inhibits Ki-i
lymphoma-cell proliferation is uncertain. Some studies, including ours, have shown that IL-2R are decreased.69 Other nonlymphocyte cell culture systems, however, have found other mechanisms that are not necessarily exclusive but may be part of the inhibitory cascade. These observations of TGF,3 actions include induction of c-sis mRNA,41 inhibition of platelet-derived growth-factor (PDGF) binding,42 inhibition of PDGF-R subunit synthesis,4 regulation of cell adhesion receptors,44 inhibition of transin/stromelysin gene expression,45 enhanced junB sinoof proto-oncogene expression, 4647 and suppression c-myc proto-oncogene expression.48 By indirect evidence suppression of c-myc by TGFo1l appears to be mediated by the retinoblastoma gene product.49,50 We have shown in this study that Ki-1 positive lymphoma cells secrete a high molecular weight TGF,B biologically active at physiologic pH and apparently identical to that secreted by L-428 Reed-Sternberg cells.14 This high molecular weight TGFI expresses the T1A5 epitope shared with Hodgkin's TGF,B. Inhibition of the TGF,B secreted by Ki-1 positive lymphoma cells markedly potentiates DNA synthesis and provides additional evidence that TGF,B is a potent autocrine inhibitor of cell proliferation in some indolent Ki-1 positive lymphoma cells. Loss of this autocrine inhibition, as shown here for L-428 Reed-Sternberg cells, is associated with features of a high-grade malignancy.
References 1. Roberts AB, Frolik CA, Anzano MA, Spom MB: Transforming growth factors from neoplastic and non-neoplastic tissues. Fed Proc 1983, 42:2621-2626 2. Moses HL, Branum EL, Proper JA, Robinson RA: Transforming growth factor production by chemically transformed
cells. Cancer Res 1981, 41:2842-2848 3. Spom MB, Roberts AB: Transforming growth factor-p: Multiple actions and potential clinical applications. JAMA 1989, 262:938-941 4. Tucker RF, Shipley GD, Moses HL, Holley RW: Growth inhibitor from BSC-1 cells closely related to platelet type 1 transforming growth factor. Science 1984, 226:705-707 5. McPherson JM, Sawamura SJ, Ogawa Y, Dinely K, Carrillo P, Piez KA; The growth inhibitor of African green monkey (BSC-1) cells is transforming growth factors beta 1 and beta 2. Biochemistry 1989, 28:3442-3447 6. Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Sporn MB, Fauci AS: Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986, 163:1037-1050 7. Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, Spom MB, Fauci AS: Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes. J Immunol 1986, 137:3855-3860
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8. Graycar JL, Miller DA, Arrick BA, Lyons RM, Moses HL, Derynck R: Human transforming growth factor-33: recombinant expression, purification, and biological activities in comparison with transforming growth factors-pl and ,2. Mol Endocrinol 1989, 3:1977-1989 9. Newcom SR, Kadin ME, Ansari AA: Production of transforming growth factor-beta activity by Ki-1 positive lymphoma cells and analysis of it role in the regulation of Ki-1 positive lymphoma growth. Am J Pathol 1988, 131:569-577 10. Newcom SR, Muth LH, Parker ET: Production of monoclonal antibodies that detect Hodgkin's high molecular weight transforming growth factor-P. Blood 1990, 75:2434-2437 11. Diehl V, Kirchner HH, Burichter H, Stein H, Fonatsch C, Gerdes J, Schaadt M, Hett W, Ziegler B, Ziegler A, Heintz F, Sueno K: Characteristics of Hodgkin's disease derived cell line. Cancer Treat Rep 1982, 66:615-632 12. Newcom SR, Kadin ME, Phillips C: L-428 Reed-Sternberg cells and mononuclear Hodgkin's cells arise from a single cloned mononuclear cell. Int J Cell Cloning 1988,6:417-431 13. Lucas RC: Transforming growth factor, beta. Technical Bulletin, Minneapolis, MN, R & D Systems, 1985 14. Newcom SR, Kadin ME, Ansari AA, Diehl V: The L-428 nodular sclerosing Hodgkin's cell secretes a unique TGF-p active at physiologic pH. J Clin Invest 1988, 82:1915-1921 15. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970, 227:680-685 16. Kadin ME, Cavaille-Coll MW, Morton CC: Tumor progression in Ki-1 positive cutaneous T-cell lymphomas is related to escape from inhibition by transforming growth factor, beta. Blood 1990, 76:345a 17. Newcom SR, O'Rourke L: Potentiation of fibroblast growth by nodular sclerosing Hodgkin's disease cell cultures. Blood 1982, 60:228-237 18. Newcom SR: The Hodgkin's cell in nodular sclerosis does not release Interleukin-1. J Lab Clin Med 1985,105:170-177 19. Agnarsson BA, Kadin ME: Ki-1 positive large cell lymphoma. A morphologic and immunologic study of 19 cases. Am J Surg Pathol 1988,12:264-274 20. Kadin ME, Sako D, Berliner N, Franklin W, Woda B, Borowitiz M, Ireland K, Schweid A, Herzog P, Lange B, Dorfman R: Childhood Ki-1 lymphoma presenting with skin lesions and peripheral lymphadenopathy. Blood 1986, 68:1042-1049 21. Schwab U, Stein H, Gerdes J, Lemke H, Kirchner H, Schaadt M, Diehl V: Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin's disease and a subset of normal lymphoid cells. Nature 1982, 299:65-67 22. Andreesen R, Osterhol J, Lohr GW, Bross KJ: A Hodgkin cell specific antigen is expressed on a subset of auto and allactivated T (helper) lymphoblasts. Blood 1984, 63:12991302 23. Stein H, Mason DY, Gerdes J, O'Connor N, Wainscoat J, Pallesen G, Gatter K, Falini B, Delsol G, Lemke H, Schwarting R, Lennert K: The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic
malignancies are derived from activated lymphoid cells. Blood 1985, 66:848-858 Kadin ME: Common activated helper-T-cell origin for lymphomatoid papulosis, mycosis fungoides, and some types of Hodgkin's disease. Lancet 1985, 2:864-865 Coiffier B, Berger F, Bryon P-A, Magaud J-P: T-cell lymphomas: immunologic, histologic, clinical, and therapeutic analysis of 63 cases. J Clin Oncol 1988, 6:1584-1589 Hoosein NM, McKnight MK, Levine AE, Mulder KM, Childress KE, Brattain DE, Brattain MG: Differential sensitivity of subclasses of human colon carcinoma cell lines to the growth inhibitory effects of transforming growth factor-beta 1. Exp Cell Res 1989,181:442-453 Houck KA, Michalopoulos GK, Strom SC: Introduction of a Ha-ras oncogene into rat liver epithelial cells and parenchymal hepatocytes confers resistance to the growth inhibitory effects of TGF-beta. Oncogene 1989, 1:19-25 Schwarz LC, Gingras MC, Goldberg G, Greenberg AH, Wright JA: Loss of growth factor dependence and conversion of transforming growth factor-beta 1 inhibition to stimulation in metastatic H-ras-transformed murine fibroblasts. Cancer Res 1988, 48:6999-7003 Terzaghi-Howe M: Changes in response to, and production of, transforming growth factor beta during neoplastic progression in cultured rat tracheal epithelial cells. Carcinogenesis 1989, 6:973-980 Hubbs AF, Hahn FF, Thomassen DG: Increased resistance to transforming growth factor beta accompanies neoplastic progression of rat tracheal epithelial cells. Carcinogenesis 1989,10:1599-1605 Wakefield LM, Smith DM, Flanders KC, Sporn MB: Latent transforming growth factor-1 from human platelets: a high molecular weight complex containing precursor sequences. J Biol Chem 1988, 263:7646-7654 Danielpour D, Sporn MB: Differential inhibition of TGF,B1 and ,B2 activity by alpha 2-macroglobulin. J Biol Chem 1990, 265:6973-6977 Kanzaki T, Olofsson A, Moren A, Wernstedt C, Hellman U, Miyazono K, Claesson-Welsh L, Heldin CH: TGF-pl binding protein: a component of the large latent complex of TGFp1 with multiple repeat sequences. Cell 1990, 61:1051-1061 Jullien P, Berg TM, Lawrence DA: Acidic cellular environments: activation of latent TGF-beta and sensitization of cellular responses to TGF-beta and EGF. Int J Cancer 1989, 43:886-891 Antonelli-Orlidge A, Saunders KB, Smith SR, D'Amore PA: An activated form of TGFP is produced by coculture of endothelial cells and pericytes. Proc Natl Acad Sci USA 1989, 86:4544-4548 McCaffrey TA, Falcone DJ, Brayton CT, Agarwal LA, Welt FGP, Weksler BB: Transforming growth factor-beta activity is potentiated by heparin via dissociation of the transforming growth factor-beta/alpha 2-macroglobulin inactive complex. J Cell Biol 1989, 109:441-448 Lyons RM, Gentry LE, Purchio AF, Moses HL: Mechanism of activation of latent recombinant TGFp1 by plasmin. J Cell Biol 1990, 110:1361-1367
Newcom, Tagra, and Kadin
AJP March 1992, Vol. 140, No. 3
38. Miazono K, Heldin CH: Role for carbohydrate structure in TGFI1 latency. Nature 1989, 338:158-160 39. Kim SJ, Kehrl JH, Burton J, Tendler CL, Jeang KT, Danielpour D, Thevenin C, Kim KY, Sporn MB, Roberts AB: Transactivation of the TGF,1 gene by human T lymphotropic virus type 1 tax: a potential mechanism for the increased production of TGFI1 in adult T cell leukemia. J Exp Med 1990, 172:121-129 40. Kadin ME, Agnarsson BA, Ellingsworth LR, Newcom SR: Immunohistochemical evidence of a role for transforming growth factor-beta in the pathogenesis of nodular sclerosing Hodgkin's disease. Am J Path 1990 136:1209-1214 mononuclear cell. Int J Cell Cloning 1988, 6:417-431 41. Press RB, Misra A, Gillaspy G, Samols D, Goldthwait DA: Control of the expression of c-sis m-RNA in human glioblastoma cells by phorbol ester and transforming growth factor beta 1. Cancer Res 1989, 49:2914-2920 42. Bryckaert MC, Lindroth M, Lonn A, Tobelem G, Wateson A: Transforming growth factor (TGFbeta) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding. Exp Cell Res 1988, 179:311-321 43. Gronwald RG, Seifert RA, Bowen-Pope DF: Differential regulation of expression of two platelet-derived growth factor receptor subunits by transforming growth factor-beta. J Biol Chem 1989, 264:8120-8125 44. Heino J, Ignotz RA, Hemler ME, Crouse C, Massague J: Regulation of cell adhesion receptors by transforming
growth factor-beta. Concomitant regulation of integrins that share a common beta 1 subunit. J Biol Chem 1989, 264:380-388 Kerr LD, Miller DB, Matrisian LM: TGFp1 inhibition of transin/ stromelysin gene expression is mediated through a Fos binding sequence. Cell 1990, 61:267-278 Li L, Hu JS, Olson EN: Different members of the jun protooncogene family exhibit distinct patterns of expression in response to type beta TGF. J Biol Chem 1990, 265:15561562 Pertovaara L, Sistonen L, Bos TJ, Vogt PK, Keski-Oja J, Alitalo K: Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation. Mol Cell Biol 1989, 9:1255-1262 Pientenpol JA, Holt JT, Stein RW, Moses HL: TGFo1 suppression of c-myc gene transcription: role in inhibition of keratinocyte proliferation. Proc Natl Acad Sci USA 1990, 87:3758-3762 Laiho M, DeCaprio JA, Ludlow JW, Livingston DM, Massague J: Growth inhibition by TGF,B linked to suppression of retinoblastoma protein phosphorylation. Cell 1990, 62:175185 Pietenpol JM, Stein RW, Moran E, Yaciuk P, Schlegel R, Lyons RM, Pittelkow MR, Munger K, Howley PM, Moses HL: TGFp1 inhibition of c-myc transcription and growth in keratinocytes is abrogated by viral transforming proteins with pRB binding domains. Cell 1990, 61:777-785