[CANCER RESEARCH 52, 6768-6773, December 15, 1992]

High Molecular Weight Transforming Growth Factor/ Is Excreted in the Urine in Active Nodular Sclerosing Hodgkin's Disease Samuel R. Newcom 2 and Krishna K. Tagra Department of Medicine, Division of Hematology /Oncology, Emory University School of Medicine, Atlanta, Georgia 30327

A B S T R A C T

To measure the in vivo secretion of high molecular weight (HMW) transforming growth factor (TGF)B by Reed-Sternberg cells from pa- tients with nodular sclerosing Hodgkin's disease, we studied the urine samples from untreated patients. The urinary proteins did not promote the proliferation of NIH-3T3 cells in monolayer culture and contained similar amounts of total TGF activity when compared with normal controls. Urinary proteins from 24 different control and test urines were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Either of two primary antibodies were used for immunoblot detection: (a) affinity column purified polyclonal anti- TGFBI prepared against platelet TGF~ or (b) monoclonai anti-HMW- TGFB prepared against HMW-TGF~ secreted by cloned L-428 Reed- Sternberg cells. All patients with active nodular sclerosing Hodgkin's disease had a detectable HMW-TGF~ (-~300,000) which cross-reacted with both anti-TGFB1 and anti-HMW-TGFB. Purification demon- strated HMW-TGF~ which was active at physiological pH. Twelve control urine samples from healthy adults and 5 follow-up samples from the Hodgkin's patients after successful treatment contained no detect- able urinary HMW-TGF~. The in vivo production of HMW-TGF~ in untreated nodular sclerosing Hodgkin's disease supports the conclusion that this growth factor is secreted in large amounts by Reed-Sternberg cells or cells stimulated by Reed-Sternberg cells.

I N T R O D U C T I O N

Reed-Sternberg ceils from patients with nodular sclerosing Hodgkin ' s disease secrete H M W 3 TGF~ (1, 2). The native mo- lecular structure of TGF8 from cloned L-428 Reed-Sternberg cells has been shown to contain TGF/~ and to mediate its activity through the high-affinity T G F ~ receptor (3, 4). The unique high molecular weight configuration of this growth fac- tor (--~300,000) is stoichiometrically opt imal at physiological pH and disrupted by acidification, proteases, or boiling (3).

Because a H M W - T G F ~ is created when platelet TGF/3 is bound to a2-macroglobulin in normal serum (5, 6), the addit ion of Reed-Sternberg cell-derived antigenic TGF~ is difficult to measure accurately. Platelet-free plasma preparat ions are also unpredictably contaminated with TGF/~1 (7). Urine represents a filtered plasma product that is not normal ly contaminated by platelet-release products and contains no T G F ~ .

Prior study of urine TGFs demonstrated their presence in the urine of patients with metastatic carc inoma (8, 9). The urine samples studied were extracted with acetic acid/ethanol, chro- matographed, and tested for NRK fibroblast colony format ion and epidermal growth factor receptor competi t ion. Both pa-

Received 3/23/92; accepted 10/5/92. The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by Grants CA 50739 and CA 30565 from the National Institutes of Health, Bethesda, MD.

2 To whom requests for reprints should be addressed, at Emory University, Woodruff Research Building, 46 Armstrong St., Atlanta, GA 30303.

3 The abbreviations used are: HMW, high molecular weight; TGF, transforming growth factor; NRK, normal rat kidney; EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; FGF, fibroblast growth factor; HDU, Hodgkin's disease urine specimen; CU, control urine specimen; IMDM, Iscove's modified Dulbecco's medium; MOPP, Mustargen, Oncovin, prednisone, and procarbazine; ABVD, Adriamycin, bleomycin, vinblastine, and dacarbazine.

tients and normal controls excreted a low molecular weight T G F activity (~6000 ) (8, 9). However, cancer patients had a significantly greater l ikelihood of demonstrat ing a second high molecular weight TGF peak (--~30,000) (9). None of the pa- tients ' urine samples contained the Mr 300,000 molecule de- scribed in nodular sclerosing Hodgkin 's disease. These authors (8, 9) did not study the urine samples of any Hodgkin 's disease patients and that is the focus of the present investigation.

M A T E R I A L S AND M E T H O D S

Study Patients and Controls

Patients with untreated nodular sclerosing Hodgkin's disease were selected for study. All patient information and specimens were provided to the investigators after full written consent had been obtained from each patient. The research plan and consent form were reviewed and approved by the institutional Human Investigations Committee. The 12 controls (6 adult females and 6 adult males) were young healthy adults in the same age range as the patients (15-40 years old). All medications were stopped for 48 h prior to study. None of the female patients or controls were known to be pregnant.

Urine Specimens

Urine collections (24 or 12 h) were obtained in refrigerated plastic containers (Nalgene, 500 ml) with 500 mg of sodium azide. The urine samples were identified by a laboratory code and numbered. Filtered specimens (0.2 ~m) were either processed immediately or stored (-70~ Urine specimens were exhaustively dialyzed in 30-liter con- tainers (4~ with constant stirring, using Mr 3500 cut-off dialysis tubing (Spectrapor, Los Angeles, CA). The dialysis fluid was Ringer's lactate (pH 8) changed each 24 h three times. The retained colloids were shell-frozen, lyophilized to dryness, and stored in desiccators at 4~ Specimens yielded 3.7-34.07 mg of dry colloid/100-2400 ml. There was no difference in the amount of colloid/volume obtained from the Hodgkin's disease patients and the controls.

Bioassays

NIH-3T3 and NRK-49F cells were a kind gift (George Todaro, M.D., Bethesda, MD). CCL-64 epithelial cells were obtained from the American Type Culture Collection (Rockville, MD). Early passage al- iquots of cells were cryopreserved in liquid nitrogen for study. Thawed cells were maintained in IMDM supplemented with calf serum (10%) or fetal calf serum (10%), L-glutamine (2 raM), and penicillin-streptomycin (1%). All cell cultures were placed in 37~ incubators with 7.5% CO2 and maximum humidity. Cell viability and concentration were deter- mined each passage using trypan blue dye exclusion and hemocytome- ter cell counting. Cells were passaged to maintain a concentration of 1 x 105 to 2 x 106 cells/ml.

Adherent 3T3 Cell Proliferation (FGF-like Activity). To determine fibroblast proliferation on plastic surfaces, 3T3 cells were removed from contact-inhibited cultures using trypsin (0.25%) and replated in 35-ram test dishes (104 cells/dish). All cultures, including controls, received fresh media weekly. Test dishes received supplements (200 ng FGF every other day, 10 ug urine protein every other day, or 50% nodular sclerosing Hodgkin's lymph node serum-free conditioned me- dium weekly). The initial cell count was confirmed by trypsinizing the adherent monolayer from 2 test dishes after 18 h and counting the resulting cell suspension using an automatic cell counter (Coulter,

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URINARY TGFd IN HODGKIN'S DISEASE

Table 1 Characteristics of nodular sclerosing Hodgkin's disease patients Patient Stage Treatment Time of urine collection

HDU 1A IIB Radiotherapy Prior to treatment HDU 2A IIIB MOPP x 6 cycles Prior to treatment HDU 2B 6 mo of treatment, clinical complete remission HDU 3A IVB MOPP/ABVD • 12 Prior to treatment HDU 3B 6 mo of treatment, partial remission HDU 3C 8 mo of treatment, clinical complete remission HDU 3D 10 mo of treatment, clinical complete remission HDU 4A IIIB MOPP • 6 cycles Prior to treatment HDU 4B 6 mo of treatment, clinical complete remission HDU 4C 3 mo of treatment, clinical complete remission HDU 5A IIIB MOPP x 6 cycles Prior to treatment HDU 5B Clinical complete remission after 3 cycles of treatment

Hialeah, FL). After 7 days, and daily thereafter, quadruplicate dishes from each control and test condition were sacrificed for cell counts. Simultaneously, the monolayer from a fifth dish was fixed in formalin (10%) and stained with methylene blue for correlation.

Soft Agar Colony Formation (Total TGF Activity). This assay was performed as previously described (1-3). Clone 49F NRK cells were plated in 1 ml 0.35% Noble's agar (3 x 103 cells/35-mm scored dish). The cell-containing top agar layer was plated over a 1-ml 0.5% agar layer in complete medium. Test materials were added to the upper layer. Controls were complete medium and complete medium supple- mented with EGF (6 ng/dish). Test dishes were treated with 100, 10, and 1 #g urine protein. All conditions were cultured in duplicate. In- cubation was at 37"C with 100% humidity. A viable single-cell suspen- sion was identified with an inverted microscope on day 1 using trypan blue dye exclusion. Colonies (>10 cells) were looked for weekly. All plates were scored weekly for 3 weeks. Results were expressed as the mean colonies achieved at 14 days +_ SEM.

Epithelial Cell DNA Synthesis Inhibition (TGFB Activity). This as- say was performed as previously described (3). Mink lung epithelial cells (CCL-64) were plated into the microtiter wells of a flat-bottomed 96-well plate (104 cells/well in 1% fetal calf serum). Triplicate test wells were used for each test condition; after 24 h, the cells were at a stable rate of DNA synthesis (~30,000 cpm). A 12-point titration curve of TGFB~ was constructed, and the result was used to measure TGFB~ inhibitory activity (0-240 pM. [3H]Thymidine (0.5 #Ci; New England Nuclear, Boston, MA) was added to each well for 16 h, and the cells were collected on glass filters, dried, and counted in a scintillation counter with Hydrofluor scintillation fluid (National Diagnostics, Man- ville, N J). Native urine proteins were tested by dissolving in IMDM and filtrating (0.2 urn). Urine proteins were also acidified (1 M acetic acid followed by neutralization with 1 M sodium bicarbonate) to determine the role of in vitro activation (7).

Primary Antibodies

Anti-TGF~ is a polyclonal rabbit antibody (R & D Systems, Inc.) prepared by injection of highly purified TGF/3~ from porcine platelets. The antiserum is purified by Staph A chromatography and has been shown to cross-react with TGF/3~ in immunoblotting (3, 10). This an- tibody is nonneutralizing in bioassay and cross-reacts with TGF~2 but not with acidic or basic FGF or EGF (10). Anti-Hodgkin's TGF~ (T 1A5) is a monoclonal IgG 1 murine antibody prepared against HMW- TGFI3 (11). This monoclonal antibody partially neutralizes Hodgkin's TGF~ from L-428 Reed-Sternberg cells (11) and from Ki-l-positive lymphoma cells (12). Immunoblot and enzyme-linked immunoabsor- bent assay indicate that T1A5 cross-reacts with a unique epitope on Hodgkin's TGF~ distinct from TGF~I (11).

Immunoblot Detection of Urinary TGFB

Unreduced urinary colloids (l mg/20 #L) were solubilized in 2% SDS electrophoresis buffer (0.01 M Tris-HCI-0.001 EDTA, pH 8.0). Elec- trophoresis was performed using 4-30% polyacrylamide gradient gels (Pharmacia, Piscataway, N J) and a modification of the Laemmli

a S. R. Newcom, unpublished observations.

method (l 3). Electrophoresed proteins were blotted onto nitrocellulose paper using a 24-h transfer (80 V) with a cooling coil (4~ Complete transfer was documented by staining control and electroeluted gels (0.1% Coomassie blue). Immunoblotting was performed according to the directions of the manufacturer (R & D Systems, Inc). Immunoblots were blocked for 2 h using 1% bovine serum albumin in buffer (500 mM NaCI-20 mM Tris-HCl-0.05% Tween 20, pH 7.2). Primary antibodies were applied for 2 h at 20~ [rabbit polyclonal anti-TGF~31 from R & D Systems (l: 1000) or T 1 A5 murine monoclonal IgG 1 anti-HMW-TGFfl (11) (10 ug/ml)]. Reactions of primary antibodies were detected using a 1:2000 dilution of a biotin-conjugated goat anti-mouse IgG purified by immunoaffinity chromatography (Pierce, Rockford, IL). Biotin was de- tected using alkaline phosphatase-conjugated avidin and enzymatic color development of nitroblue tetrazolium. Control blots were rou- tinely performed to detect nonspecific reactions caused by bridging of the second antibody or endogenous enzymes.

Molecular Weight of TGFB Activity from Hodgkin's Urine. Urine proteins (HDU 3A, CU 13, CU 14) were solubilized in 2% SDS-elec- trophoresis sample buffer and electrophoresed into 7% polyacrylamide gels. Gels were sliced horizontally at 8-ram intervals, and protein from each slice was electroeluted into dialysis tubing bags (Mr 3500 cut-off). Each slice fraction was tested for TGF/3 activity after equilibration in IMDM (PD-10 column; Pharmacia), concentration using an Amicon concentration chamber (YM2 membrane), and filtration through a 0.2-#m filter.

R E S U L T S

Study Pa t ien ts and Controls . Each H o d g k i n ' s pa t ien t had a lymph node biopsy d e m o n s t r a t i n g nodu la r sclerosis. Ini t ial

25_

2O ...... t Hodgkin's C'M

/::5:/Z;%: ..... _.-~" , z Control

~_.Z- L ......

/ ~ HDU 4"

5 "#"':" / 0 I I' ~ ' I

0 6 7 8 10 Day

Fig. 1. Effect on NIH/3T3 cells of urine HMW-TGF~ from 4 patients with active nodular sclerosing Hodgkin's disease. All media were renewed weekly. HDU and FGF were added every other day (200 ng FGF/35-mm dish and 10 ug HDU protein/35-mm dish). Serum-free conditioned medium from a Hodgkin's lymph node cell suspension was added at 50% concentration once weekly. All cell counts were in quadruplicate. FGF and Hodgkin's conditioned medium (CM) produced a doubling of the cell count. HDU proteins did not change the prolif- eration of 3T3 cells.

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URINARY TGFd IN HODGKIN'S DISEASE

urine specimens were collected prior to therapy. Follow-up specimens were collected at the patient's home using containers provided by the investigators. Similarly, control samples were obtained from 12 healthy volunteers (100-500 ml). The clinical status of Hodgkin's disease existing at the time each of the 12 HDUs were collected is shown in Table 1.

Fibroblast Monolayer Proliferation. The absence of FGF- like biological activity in HDU is shown in Fig. 1. HDUs did not promote 3T3 cell proliferation. Data from 4 different ex- periments are pooled and show no statistically significant dif- ference between the growth in complete medium and that sup- plemented with HDU proteins. A doubling of cell number was measured at day 8 and day 10 when 3T3 cells were treated with either FGF (200 ng/ml every other day) or 50% nodular scle- rosing Hodgkin's disease serum-free conditioned medium.

Fibroblast Colony Formation in Soft Agar. Colony forma- tion was induced by all HDUs studied (range, 16-63 colonies/ 35-mm dish). Similar to previous reports (8, 9), there was no statistically significant difference between the TGF activity of control urine samples from healthy subjects (69.78 _+ 35.4 colonies/35-mm dish) versus samples from Hodgkin's patients (44.8 _+ 22.9, P < 0.1). The data are summarized in Table 2.

Immunoblot Detection of Hodgkin's Urinary HMW-TGFB. HDU 1 was not tested by immunoblotting because of lack of material. Fig. 2 demonstrates the HMW-TGFt3 detected in samples from the untreated HDU 2 and HDU 5 patients. After 6 months of MOPP chemotherapy (14), both patients had achieved a complete clinical remission. Follow-up specimens demonstrated clearing of the HMW-TGF/3.

Fig. 3 demonstrates the serial measurements for HDU 3. This 15-year-old female had a large tumor burden with medi- astinal, liver, and retroperitoneal lymph node involvement. A year of alternating MOPP/ABVD chemotherapy (15) was giv- en. After 6 and 8 months of therapy, restaging indicated a persistently elevated erythrocyte sedimentation rate (60 mm/h). Residual liver disease was found in liver biopsy tissue at 6 months. The HMW-TGF/3 remained detectable at the same time. In the last urine specimen obtained prior to completion of therapy (10 months), the HMW-TGF/3 had cleared. Results of an abdominal computed tomographic scan, liver function tests, and erythrocyte sedimentation rate had also returned to normal. A follow-up liver biopsy was refused by the patient.

Fig. 4 demonstrates the positive urine obtained from patient HDU 4 prior to treatment and the negative urine after complete remission had been obtained. After 6 months of therapy, a faint TGFI31 band persisted. A follow-up urine sample 3 months later revealed complete clearing without further therapy. These

Table 2 TGF activity for NRK fibroblasts in soft agar Colony counts were determined at 14 days when all colonies containing > 10

cells were noted. The difference between the means is not significant (t = 2.002, P < 0.1).

HDU Colonies/35-mm CU Colonies/35-mm sample no. dish sample no. dish

Medium only 0 EGF (6 ng/dish) 269 +_ 5

1 41_+4 2 63_+5 3 72_+ 10 4 32 -+ 20 5 16_+2

Mean _ SEM 44.8 _ 22.9

1 46_+3 2 67_+7 10 52_+6 11 82.5 _+ 15 12 78 -+ 22 13 43.5 _+ 26 14 124 _+ 33 15 109 -+ 19 16 26 -+ 12

69.78 _+ 35.4

1 2 3 4

HMW-TGF{J- 200 kDA �9

94 kDA �9

67 kDa �9

43 kDa�9

30 kDa�9

20 kDa,

Fig. 2. Immunoblot of HDU 2 and HDU 5. Electrophoresed native proteins were detected with monoclonal anti-Hodgkin's HMW-TGF~ (T1A5). Lane 1, HDU 2A, prior to treatment; lane 2, HDU 2B, after induction of complete clinical remission; lane 3, HDU 5B, after induction of complete clinical remission; lane 4, HDU 5A, prior to treatment. There is complete clearing of Hodgkin's HMW- TGF~ in both patients after successful treatment of their Hodgkin's disease with MOPP chemotherapy. Partial reduction of HMW-TGF~ yields an Mr 90,000 molecule that is seen in lane 1.

1 2 3 4 5 6 ! !:

H M W - T G F I ~

94 kDA

67 kDa

43 kDa~

30 kDa,

20 kDa ~,

14 kDa i,

Fig. 3. Immunoblot of HDU 3. Electrophoresed native proteins were detected with monoclonai anti-Hodgkin's HMW-TGF/~. Lane 1, HDU 3D, after 10 months of chemotherapy and in complete clinical remission; lane 2, HDU 3C, after 8 months of treatment; lane 3, HDU 3B, after 6 months of treatment and pathological evidence of persistent disease; lane 4, HDU 3A, prior to treatment; lane 5, blank, lane 6, control urine 9. The patient had detectable Hodgkin's TGF~ prior to treatment and failed to clear completely after 6 and 8 months of treatment with alternating MOPP/ABVD. This finding correlated with clinical evidence of persistent Hodgkin's disease. By 10 months of treatment, there was complete clearing of TGFB and complete remission of measurable Hodgkin's disease.

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A

M kDA �9

1 2 3 4 5 6

URINARY TGF~ IN HODGKIN'S DISEASE

purification is the inhibitory activity of the native HMW-TGF(3 measurable using the CCL-64 assay.

HMW.TGFB-* 200 kDA �9

67 kDa�9

43 kDa�9

30 kDa�9

20 kDa�9

HMW-TGFB-* 200 kDA )

[ 3 I 2 3 4 5 6

9 4 k D A �9

67 kDa �9

43 kDa�9

30 kDa �9

20 kDa �9

14 kDa �9

Fig. 4. lmmunoblots of HDU 4. Electrophoresed native proteins were detected with anti-TGFB1 (,4) and anti-Hodgkin's HMW-TGF/3 (TIA5) (B). Lane 1, con- trol urine 10; lane 2, blank; lane 3, HDU 4A, prior to treatment; lane 4, HDU 4B, after 6 months of treatment; lane 5, HDU 4C at 3 months of follow-up without treatment and 9 months after diagnosis; lane 6, blank. The patient was secreting a HMW-TGFB prior to treatment that cross-reacted with both anti-Hodgkin's TGFB (T1AS) and anti-TGF~l. There was complete clearing 3 months after treatment ended. Partial reduction has yielded Mr 90,000 (anti-TGF~l) and 68,000 (anti-Hodgkin's TGF~) proteins.

findings correlated with a durable remission. Fig. 4 also dem- onstrates the cross-reactivity of the HMW-TGFB in the urine of Hodgkin's patients with both anti-TGFB1 and anti-Hodgkin's H M W - T G F B (T 1A5).

Twelve control urine specimens were tested using this method, and no HMW-TGF/3 band was identified (for an ex- ample, see Fig. 5). Nei ther the TGF~3~ antigen nor the T1A5 antigen was identified in any of the control specimens.

Measurement of Molecular Weight of H D U TGFB Biological Activity. No CCL-64 TGFB activity was measured in either H D U 2-5 or 12 CU specimens using native urine protein con- centrat ions of 100, 10, 1, and 0.1 ug/ml. Following acid acti- vation trace amounts of TGFB activity (2-30 pM) were measur- able in all specimens. Purif ication of native proteins from H D U 3A, C U 13, and C U 14 demonstra ted physiologically active H M W - T G F ~ in H D U but not C U (Fig. 6). These results indi- cate that the activity of native urinary H M W - T G F ~ at physio- logical pH is masked in the CCL-64 assay by the coexcretion of s t imulatory factors such as E G F and T G F a (8, 9). Only by

D I S C U S S I O N

TGF~ is a member of a family of mult i funct ional polypep- tides that induce anchorage-independent growth of nontrans- formed fibroblasts (7, 16, 17) and inhibit the proliferation of many cells, including epithelial cells (18, 19) and lymphocytes (20, 21). It has recently been established that there are at least three TGFB mammal ian proteins. Each TGFB is encoded by a different gene (22).

TGFB is important in l imiting the expansion of nonmalig- nant activated lymphocytes (20, 21). TGF/3 suppresses D N A

1 2 3 4 5 6

200 kDA )

94 kDA )

67 kDa)

43 k D a )

30 k D a )

20 kDa)

14 kDa~

TGF#I

200 kDA

94 kDA )

67 kDa)

43 kDa)

30 kDa)

20 k D a )

1 2 3 4 5 6

Fig. 5. Immunoblot of 5 control urine specimens and TGF~I. Porcine platelet TGF~! (333 ng) was dissolved in 10 uL SDS sample buffer. Dialyzed and iyo- philized colloid from control urine specimens were dissolved in SDS sample buffer (1 mg/ml filtered through a 0.2-um filter prior to use). The urine sample (10 ~g/10 ~L) and TGFfl~ sample were loaded into respective wells of a 4-30% polyacrylamide gradient gel. Electrophoresed native proteins were electrophoret- ically blotted onto nitrocellulose paper and detected with anti-TGFB1 (,4) and anti-Hodgkin's TGFB (TIA5)(B). Lanes 1-5 show that CU 11, 12, 13, 14, and 15, respectively, were negative for TGFB. The control lane (lane 6) shows the Mr 25,000 TGF/~I molecule purified from porcine platelets. Anti-Hodgkin's TGF~ (TIAS) was negative against all control urine samples and TGF~I. Faint bands in B, lane 2 (CU 14), are seen at Mr 100,000 and 60,000; the significance is un- known.

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URINARY TGF9 IN HODGKIN'S DISEASE

50.

�9 - - 45,

g 40.

as.

n ~ 30.

25.

20,

~ 15.

~ 10.

o

M r x 10-3

I I I I I I I

0 8 16 24 32 40 48 56 64 72 80

SDS Gel Slices (mm)

Fig. 6. Determination of molecular weight of HDU 3A TGFB activity. Poly- acrylamide gels were sliced at 8-ram intervals, and the protein from each slice was electroeluted for TGFB bioassay. The activity peak was measured at Mr 350,000- 300,000. Control urine specimens (CU 13 and CU 14) had no significant HMW- TGFB activity. Plating medium (1% fetal calf serum) had control cpm of 23,117 + 3,999. A 12-point TGFB~ titration control curve from 0-240 pM was con- structed (e.g., 240 pM TGFBI gave cpm of 155 _+ 24 or 99.2% DNA synthesis suppression)

DNA synthesis suppression (%) = Test cpm - Control cpm

• 100. Control cpm

The identification of HMW-TGF/~ in active nodular scleros- ing HDU and the demonstration of clearing after treatment suggests that the source is the Reed-Sternberg cell. Confirma- tion of this conclusion would require specific in vivo labeling of Reed-Sternberg cell-derived HMW-TGF/~, a procedure not yet available.

Study of experimental glomerulonephritis induced in rabbits (28) and rats (29) suggests that TGF/~ plays a central role in the accumulation of pathological extracellular matrix in this lesion. The TGF~ identified in rabbit antiglomerular basement mem- brane disease and rat antithymocyte glomerulonephritis is physiologically active without acidification (28, 29). However, the native molecular weight of glomerulonephritis-derived TGF/~ has not been determined, nor has the cross-reactivity with anti-TGF~l and anti-Hodgkin's TGF/3 been measured. Circulating immune complexes have been demonstrated in 50-88% of serum specimens of patients with Hodgkin's dis- ease (30-33) and, rarely, the nephrotic syndrome occurs in Hodgkin's disease (34-36). None of our patients had nephrotic syndrome or even significant proteinuria.

The data presented here show that HMW-TGF/~, identical with that purified from cloned Reed-Sternberg cells, is secreted in the urine of patients with untreated nodular sclerosing Hodgkin's disease and disappears with successful treatment. Although immune complex disease or reactive cells could be sources of this TGF~, the high molecular weight and antigenic- ity indicate that the likely source is the Reed-Sternberg cell.

synthesis by 60-80% in T-lymphocytes (20) and 65% in B-lym- phocytes (21). The Reed-Sternberg cell from nodular sclerosis, which has many features of a malignant activated lymphocyte (23-25), secretes up to 1 fM TGF~/day but is unresponsive to the inhibitory effects of this growth regulator (12, 26).

HMW-TGFB from the L-428 Reed-Sternberg cell has been characterized (3). This molecule, in its native state, has the biological features of TGF~ , including inhibition of epithelial cell proliferation, induction of AKR-2B and NRK fibroblast colonies in soft agar, and competition with T G F ~ for its mem- brane receptor (3). Hodgkin's HMW-TGF~ contains the T G F ~ epitope which is demonstrated by the cross-reactivity of Hodgkin's TGF~ with anti-TGF~t antibody, the identification of the Mr 25,000 T G F ~ molecule after partial reduction of Hodgkin's TGF~, and the affinity of HMW-TGF/~ for the T G F ~ receptor (3). After disruption of disulfide bonds and vigorous boiling, the Mr 12,500 TGF/~j monomer can be iden- tified (12). In addition, Hodgkin's HMW-TGF~ contains unique epitopes recognized by specific monoclonal antibodies that do not cross-react with TGF/~ (1 l). Physiologically active HMW-TGF~ has been measured in the supernatants of 8 pri- mary nodular sclerosing Hodgkin's disease cell suspensions (l), the Ki-l-positive lymphoma cell line, Mac-1 (26), in cut sec- tions of lymph nodes replaced by nodular sclerosing Hodgkin's disease (27), and in the supernatants of cloned nodular scleros- ing cell lines, L-428 (3) and HDLM. 4 HMW-TGF~ has not been found in supernatants from mixed cellularity Hodgkin's disease (1, 2), the KMH2 Reed-Sternberg cell line from mixed cellularity Hodgkin's disease, 4 or supernatants from 12 control cultures of closely related lymphoproliferative disorders (2). In the present study, we demonstrated the secretion of HMW- TGFB in the urine of 4 patients with active nodular sclerosing Hodgkin's disease and clearing after the eradication of Reed- Sternberg cells by combination chemotherapy.

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1992;52:6768-6773. Cancer Res   Samuel R. Newcom and Krishna K. Tagra  DiseaseExcreted in the Urine in Active Nodular Sclerosing Hodgkin's

IsβHigh Molecular Weight Transforming Growth Factor

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