Role of transforming growth factor-β in chronic lymphocytic leukemia

Leukemia Research Vol. 17, No. 1, pp. 81-87, 1993.

(1145-2126/93 $6.0(I + .0(1 © 1993 Pergamon Press lad

Printed in Great Britain.

ROLE OF TRANSFORMING GROWTH FACTOR-fl IN CHRONIC LYMPHOCYTIC LEUKEMIA L. G. ISRAELS,S. J. ISRAELS,A. BEGLEITER,L. VERBURG,L. SCHWARTZ,M. R. A. MOWAT and J. B. JOHNSTON Manitoba Institute of Cell Biology, Manitoba Cancer Treatment and Research Foundation, 100 Olivia Street, Winnipeg, Canada, R3E 0V9

(Received 10 April 1992. Revision accepted 5 September 1992) Abstract--TGF-/3 is an important immunoregulator as it suppresses proliferation and function of Band T-lymphocytes. In the present study we have examined the cellular localization and secretion of TGF-fl in B-cells from normal donors and patients with CLL and have assessed the influence of TGFfll on DNA synthesis in these cells. Using anti-LC(1-30)--a polyclonal anti-TGF-fll antibody--TGFfl was localized to discrete sites within the cytoplasm of both normal and malignant lymphocytes. These areas co-localized with areas detected by an antigranule antibody (D545), suggesting that TGFfl may be stored within cytoplasmic secretory vesicles. Both normal B- and CLL cells contained low or undetectable levels of TGF-fl mRNA and secreted low and equivalent amounts of TGF-fl. Compared to untreated cells, DNA synthesis was reduced by TGF-/31 to a mean +- S. E. of 0.84 -+ 0.07 in CLL cells and this was significantly less (p < 0.001) than that observed in normal B-cells (mean -+ S. E. of control, 0.12 +- 0.02). In 3 of the 18 patients, TGF-flI stimulated DNA synthesis. The reduced inhibition of leukemic cell DNA synthesis by TGF-fll in CLL may provide these cells with a growth or survival advantage over normal lymphocytes and contribute to their selective accumulation.

Key words: Transforming growth factor-fl, DNA synthesis, chronic lymphocytic leukemia.

TGF-fl has been shown to be an important immunoregulatory protein for human T- and B-lymphocytes [5]. TGF-/3 activity is found in human tonsil T-cell supernatants and is synthesized and secreted by interleukin-2-activated T-cells in concentrations capable of inhibiting mitogen-induced proliferation of these cells [6]. The secreted TGF-fl is primarily TGF-fl~, and T G F - ~ is not usually produced by these cells [5]. TGF-fl also inhibits the proliferation of ConA-, interleukin-1- or phytohemagglutinin-stimulated murine thymocytes, where both TGF-fll and TGF-fl2 are equipotent [7, 8]. Like T-cells, human tonsil Blymphocytes also secrete TGF-fl and have high affinity TGF-fl receptors, and both the amount of TGF/3 secreted and the number of receptors increase six-fold on in vitro stimulation [9]. TGF-/3 inhibits mitogen-induced proliferation and differentiation of these cells [6], with both TGF-/31 and TGF-fl2 having equivalent activities [10]. TGF-/3 also inhibits the proliferative response of human peripheral blood Blymphocytes to B-cell growth factor [10, 12]. TGF-/3 is secreted by T- and B-cells in the latent form, but is converted by the lymphocytes to the active form which acts in an autocrine manner to suppress cell growth [10, 12]. These observations suggest that

INTRODUCTION TGF-fl is a 25 kD polypeptide which binds to a unique receptor, and was first identified by its ability to induce a transformed phenotype in fibroblasts, allowing them to undergo anchorage-independent growth [1]. However, subsequent studies have demonstrated that TGF-/3 has multifunctional properties and can either stimulate or inhibit cellular proliferation or differentiation, depending on the cell type and the presence of other growth factors [1]. Additionally, TGF-/3 is a potent inducer of collagen synthesis by fibroblasts [2] and plays a major role in tissue repair and embryogenesis [1]. The three isoforms TGF-/31, -/32, and -/33, have an overall sequence homology of approximately 70% [1] and, although their receptor affinities appear different, in general they have similar biological activities [3, 4].

Abbreviations: TGF-fl, transforming growth factor-beta; CLL, chronic lymphocytic leukemia; TBS, Tris-buffered saline; BSA, bovine serum albumin; HBSS, Hank's balanced salt solution. Correspondence to: L. G. Israels, Manitoba Cancer Treatment and Research Foundation, 100 Olivia St., Winnipeg, Manitoba, Canada, R3E 0V9. 81

L.G. ISRAELSet al.

82

TGF-fl may be an important autoregulatory lymphokine, which limits normal B- and T-cell clonal expansion, and that loss of this negative growth control might permit the d e v e l o p m e n t of lymphoid malignancies. Indeed, the growth of E p s t e i n - B a r r transformed B-cells [9,13], and the leukemic B-cells from 4 of 5 patients with hairy-cell leukemia were not influenced by TGF-fl [11]. TGF-fl actually stimulated the proliferation of leukemic cells in adult T-cell leukemia [14] and, as these cells also secrete very high levels of TGF-fl [14, 15], TGF-fll may act in an autocrine fashion to p r o m o t e t u m o r growth. However, loss of the growth inhibitory effects of TGF-fl is not a universal finding in lymphoid malignancies as TGF-fl inhibits the proliferation of nonHodgkins l y m p h o m a cells to the same extent as normal B-cells [16]. Like hairy-cell leukemia, C L L is a chronic B-cell disorder in which there is expansion of a monoclonal B-lymphocyte malignant clone, with suppression of normal T- and B-cells and function [17]. In the present study, we have examined the cellular localization and secretion of TGF-fl in normal B- and C L L cells. Additionally, the effects of TGF-fll on D N A synthesis in B-lymphocytes from normal individuals and C L L patients have been examined.

MATERIALS AND METHODS

Patients Heparinized blood was obtained from 7 normal donors and 18 patients with CLL. The CLL patients had Rai stage 0--IV disease [18], and had not received treatment for a minimum of one month. Isolation of lymphocytes Mononuclear cells were obtained from the peripheral blood of CLL patients using a Ficoll-Hypaque gradient [19]. These cells were assumed to be predominately CLL cells as >90% were lymphocytes. B-cells were obtained from normal controls using magnetic beads (DYNABEADS) coated with mouse IgG directed against CD19 (Dynal, Great Neck, NY). Following incubation at 4°C for 30 min, the CD19 cells were removed with a magnet and the beads were then removed from the cells by DETACHaBEAD (Dynal, Great Neck, NY). Cellular localization of TGF-fl TGF-/~ was localized in permeabilized cells using antiLC(1-30), which is a polyclonal antibody raised in rabbits by incubating with the first 30 amino acids of TGF-fll [20]. Cells, on ice, were fixed by the addition to the cell suspension of an equal volume of 2% paraformaldehyde/ 0.2% glutaraldehyde in 0.2 M cacodylate buffer [21]. After I h incubation, unreacted aldehyde was neutralized by three washes with TBS (50mmoi/L Tris, 100mmol/L NaCl, pH 7.5). The cells were permeabilized by exposure to 0.1% Triton X-100 for 3 min and again washed three

times with TBS/0.1% BSA. For staining, cells were incubated with anti-LC(1-30) for 30 rain, then washed three times with TBS/0.1% BSA. In some experiments the incubation included the presence of the LC(1-30) peptide as well as the primary antibody. The second antibody, biotinylated goat anti-rabbit, was applied in TBS for 2 h and then washed. Fluorescein-avidin was then incubated with the cells for 30 min in complete darkness. In double-label experiments the cells were also incubated with D545 [22], followed by biotinylated goat anti-mouse antibody and rhodamine-avidin. D545 is a monoclonal antibody to platelet dense granule membrane (D545) which recognizes a 40 kD protein in platelet dense granule membranes and also cross-reacts with membrane proteins in neutrophils [22]. As controls, the primary antibodies were replaced by non-immune rabbit serum or non-immune mouse ascites (Cappel Laboratories, Malvern, PA). Final washing with TBS/0.1% BSA was followed by resuspending the cells in TBS/glycerol and mounting the suspension on a glass slide with coverslip. Ceils were allowed to settle overnight before viewing. Ceils were viewed under a Zeiss Axiovert 35 mm fluorescent photo-microscope with green and blue excitation range filters and FITC- and TRITC-selective observation filters. Fields were photographed using Kodak TriX Pan film (ASA 400), and were exposed for 30 s.

Conditioned media for TGF-fl Normal B- or CLL cells were washed in phosphatebuffered saline (pH 7.4) and suspended (107cells/ml) in serum-free o~-MEM in siliconized tubes at 37°C for 24 h. The ceils were removed by centrifuging at 250 × g for 5 min, and 1 ~tg/ml of aprotinin (Sigma Chemical Co., St Louis, MO) and 0.5 ~tg/ml of both leupeptin and pepstatin A (Sigma Chemical Co.) were added [2]. Aliquots of the conditioned media were frozen at -80°C in siliconized cryovials until assayed for TGF-/~ activity. CCL-64 mink lung growth inhibition assay for TGF-[3 The assay for TGF-fl was carried out with modifications of a previously described assay [2]. CCL-64 mink lung epithelial cells were maintained in o:-MEM with 10% fetal calf serum. Near-confluent cells were used for the assay and cells were trypsinized, washed twice in o~-MEM with 0.2% bovine calf plasma and plated at 2 × 104 cells/0.2 ml in Corning 96-well flat-bottomed microtitre plates (Corning Glass Works, Corning, NY). Cells were incubated at 37°C for 3 h and aliquots of conditioned media (neutral or acidified) were added. After 24 h incubation, the cells were pulsed with 0.2 ~tCi [methyl-3H]thymidine (specific activity, 20--40 Ci/mmole) for 3 h. Media was removed and the cells were washed with Hanks balanced salt solution (HBSS) and were incubated for 15 min at 37°C in HBSS containing 0.53 mM EDTA and 0.05% trypsin. The cells were harvested onto glass-fibre discs and radioactivity determined by liquid scintillation counting. Quantitation of TGF-/3 was by comparison to a standard growth inhibition curve using porcine TGF-fll (R&D Systems, Minneapolis, MN). AntiTGF-fl neutralizing antibody (R&D Systems, Minneapolis, MN) was added to the standards and sample wells to confirm that growth inhibition was TGF-fl related. mRNA extraction and Northern analysis Total RNA was isolated from lymphocytes using acid guanidinium thiocyanate-phenol-chloroform [23]. Twenty microgram samples of RNA were then resolved on a 1% agarose gel containing formaldehyde and transferred to

TGF-fl in chronic lymphocyticleukemia

83

FIG. 1. Immunolocalization of TGF-fl in CLL cells. CLL cells were permeabilized with Triton X-100 and the same ceils were treated with fluorescent-labelled anti-TGF-fl~ antibody [anti-TGF-fl(1-30)] (a) and fluorescent-labelled anti-dense granule antibody (D545) (b). TGF-fl is detected in multiple discrete sites within the cytoplasm which colocalize with sites detected by the anti-granule antibody. Controls using non-immune rabbit serum or non-immune mouse ascites showed no fluorescence. a Zeta-probe nylon membrane (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Pre-hybridization, hybridization and washings were performed using the method of Church and Gilbert [24]. A porcine TGF-fll cDNA was used which contained the entire coding sequence except that cysteines 223 and 225 were changed to serines [25]. [3:p]labelled cDNA was prepared by oligolabelling with an oligolabelling kit (Pharmacia, Baie d'Urfe, Quebec, Canada).

Effect of TGF-fll on DNA synthesis Normal B- or CLL cells were plated in 96-well microtitre plates at a concentration of 2 x 105 cells/well in 200 ~tl of RPMI 1640 (Gibco Laboratories, Grand Island, NY), supplemented with 2 ~tg/ml insulin, and 4 ~tg/ml human transferrin (Sigma Chemical Co., St Louis, MO). TGF-fll of porcine platelet origin (R&D Systems, Minneapolis, MN) was added to test wells at a final concentration of i ng/ml, while control wells received the same volume of BSA (1 mg/ml), the TGF-fl carrier. The plates were incubated for 48 h at 37°C under 5% CO2, 95% air, at which time 5 ~tCi [3H]thymidine (specific activity 20--40 Ci/ mmole) (ICN Biomedicals Inc., Irvine, CA) was added to each well, and the plates re-incubated for an additional 24 h. The cells were harvested with water using a Skatron harvester (Skatron Inc., Sterling, VA) and counted by liquid scintillation techniques. For each assay the inhibition or stimulation of DNA synthesis by TGF-fll was assessed by the incorporation of [3H]thymidine into cells treated with TGF-fll, compared with the incorporation of [3H]thymidine into untreated cells from the same individual. The results are presented as the ratio of [3H]thymidine incorporation in cells treated with TGF-fl~ compared to untreated cells. Statistical significance was determined using two-tailed t-tests. RESULTS

Cellular localization of TGF-fl Figure l(a) shows the localization of TGF-fl in

permeabilized C L L cells as detected by fluorescent labelled antibody to TGF-fl~ [anti-LC(1-30)] and demonstrates that TGF-/3 is located in discrete sites within the cytoplasm. Non-permeabilized cells did not show evidence of staining. Studies of cells incubated with anti-LC(1-30) in the presence of the LC(130) peptide showed that this peptide blocked the staining by anti-LC(1-30). This blocking effect was specific and did not affect staining by D545, the antigranule antibody. Figure l(b) shows the same cells treated with a fluorescent-labelled (D545), a monoclonal antibody directed against platelet dense granules, which cross-reacts with neutrophil granules [22]. The anti-granule antibody was distributed in multiple sites in the cytoplasm of the cells and some, but not all, of these sites co-localized with the areas detected by the TGF-/3 antibody. Similar findings were obtained with normal B-cells. These data suggest that TGF-fl may be stored within secretory vesicles in the cytoplasm of B-cells.

TGF-fl mRNA in lymphocytes To determine if CLL cells contain higher TGF-/3 than normal B- or T-cells, the TGF-/3 m R N A was determined in eight patients and compared with the level of m R N A in T- or Bqymphocytes pooled from normal individuals (Fig. 2). As previously observed [5], the level of TGF-fl m R N A in the unstimulated B- and T-cells was very low. A similar pattern was observed in CLL cells, where the m R N A was either undetectable or present at low levels.

TGF-~ secretion by lymphocytes Table 1 shows the secretion of TGF-fl by normal

84

L . G . ISRAELSet al.

FIG. 2. TGF-fl m R N A expression in normal B- and Tlymphocytes and CLL cells by Northern blot analysis. Twenty /~g of R N A was loaded in each lane and the presence of equivalent quantities of RNA in each lane was confirmed by examining the gels under UV light following staining by ethidium bromide. The normal lymphocyte R N A represents RNA isolated from the pooled B- or Tcells from 6 to 10 normal individuals. The CLL RNA represents RNA isolated from leukemic cells of individual patients. Probing was carried out with a TGF-fll cDNA, as described in Materials and Methods.

TABLE 1. TGF-fl SECRETION BY NORMAL B-CELLS AND

CLL CELLS TGF-fl secretion (fmoles/107 cells/24 h) Normal Normal Normal Normal Normal Normal Normal CLL CLL CLL -E~L CLL CLL CLL CLL CLL

1 2 3 4 5 6 7

patient patient patient patient patient patient patient patient patient

ND a ND 5.3 -+ 1.0 6.2 - 1.6 ND 5.4 - 1.0 ND 1 2 3 8 11 13 14 15 18

7.5 - 1.0 ND ND 9.7 --+2.0 2.8 - 0.2 12.0 --- 1.0 10.1 : 2.1 ND 6.8 -+ 1.3

a N D ~ n o n e detected. Normal B- or CLL cells were incubated in tr-MEM (107 cells/ml) at 37°C for 24 h. Aliquots of the conditioned media were assayed for TGF-fl activity as described in the text. Results represent the mean +-- S. E. of measurements done in triplicate on two separate occasions.

B- and C L L cells following 24 h incubation in serumfree media. B o t h cell types secreted equivalent a m o u n t s of TGF-fl, which was either undetectable or else was secreted in very low concentrations.

Effect of TGF-fll on DNA synthesis Baseline D N A synthesis, as assessed by the incorporation of [3H]thymidine into acid-insoluble macromolecules, in u n t r e a t e d n o r m a l B-cells and C L L

TABLE 2. EFFECT OF TGF-fll ON [3H]THYMIDINE INCORPORATION IN NORMAL B-LYMPHOCYTES

Normal Sex 1 2 3 4 5 6 7

F F F F M M M

Control DPM

TGF-/~: control (DPM ratio)

p

13,645 _ 3273 9221 --- 621 8931 -+ 1314 4344 -+-886 19,956_+ 1999 14,588 _+ 645 18,874 _+ 687

0 . 0 5 6 - 0.010 0.061 --- 0.010 0.129 - 0.013 0.217 - 0.041 0.127-+ 0.035 0.159 -+ 0.019 0.098 --- 0.014

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

B-lymphocytes (2 × 105) were incubated in 200 ~tl of media/well and were treated with TGF-fll (1 ng/ml), as described in Materials and Methods. After 48 h, cells were pulse-labelled with [3H]thymidine and cell proliferation assessed 24 h later by the incorporation of [3H]thymidine into acid-insoluble macromolecules. The mean-+ S. E. [3H]thymidine incorporation in seven normals was 12,794--+ 2134 dpm/well. The results are expressed as a ratio of [3H]thymidine incorporation by TGF-fll treated cells, compared to untreated cells, and represent the mean-+ S.E. of 2-3 separate experiments. Cell proliferation in treated and untreated cells was compared statistically by a two-tailed t-test comparing the significance of the difference of the mean [3H]thymidine incorporation.

cells, are shown in Tables 2 and 3. Baseline D N A synthesis was lower in the C L L cells ( m e a n ± S. E . , 2078 ± 490 dpm/well) than in n o r m a l B-cells (mean --- S. E., 12,794 ± 2134 dpm/well). T h e effect of TGF-fl~ on D N A synthesis in n o r m a l peripheral Blymphocytes is shown in Table 2. TGF-fll significantly inhibited [3H]thymidine i n c o r p o r a t i o n in cells f r o m all seven normals, with an overall m e a n ± S. E . , 0.12 ± 0.02 of control levels. T h e influence of T G F fll on C L L cells f r o m 18 patients with C L L is shown

TGF-/3in chronic lymphocyticleukemia

85

TABLE 3. EFFECT OF TGF-fl~ ON [3H]THYMIDINE INCORPORATION IN CHRONIC LYMPHOCYTIC LEUKEMIA

Patient

Sex

Lymphocyte count (cells/mm 3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

F M M M M M M F F M F M M F M M M M

106,000 68,000 74,000 67,000 51,000 70,000 27,000 45,000 21,000 66,000 20,000 33,000 41,000 34,000 288,000 59,000 66,000 39,000

Rai stage a

Control DPM

TGF-fl: control (DPM ratio)

p

0 IV I I III IV III I I II IV II II I IV III IV I

453 + 30 551 +- 59 1082 - 51 977 +- 48 1887 -+ 134 3263 -+ 76 1834 +- 49 2678 +- 61 1362 -+ 46 2526 -+ 49 2411 -+ 137 2329 -+ 39 1907 - 33 845 - 25 3078 -+ 132 3082 +- 92 3655 - 88 3479 -+ 277

1.397 -+ 0.079 1.370 +- 0,121 1.200 - 0.027 0.975 +- 0.058 0.914 +- 0.028 0.863 +--0.013 0.855 -+ 0.024 0.845 -+ 0.011 0.822 +- 0.019 0.804 -+ 0.018 0.797 -+ 0.028 0.795 -+ 0.021 0.761 -+ 0.026 0.722 -+ 0.032 0.722 +- 0.014 0.431 -+ 0.010 0.419 -+ 0.010 0.353 -+ 0.016

<0.001 <0.01 <0.001 NSh <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Rai staging (2) stage 0, lymphocytes > 15,000/~tl; stage I, lymphocytosis and lymphadenopathy; stage II, lymphocytosis and splenomegaly; stage III, lymphocytosis and anemia (hemoglobin < 100 g/ixl); and stage IV, lymphocytosis and thrombocytopenia (platelets < 100,000/

~tl). h NS---not significant. CLL cells (2 × 105) were incubated in 200 Ixl of media/well and were treated with TGF-fl~ (1 ng/ ml), as described in Table 2. The mean - S. E. [3H]thymidine incorporation in 18 CLL patients was 2078 +- 490 dpm/well. The results are expressed as a ratio of [3H]thymidine incorporation by TGFfll treated ceils, compared to untreated cells, and represent the mean +- S.E. of 2-3 different experiments. Cell proliferation in treated and untreated cells was compared statistically by a twotailed t-test comparing the significance of the difference of the mean [3H]thymidine incorporation.

in Table 3. The overall mean ___S.E. of thymidine incorporation for the 18 C L L patients was reduced to 0.84-+ 0.07 of control levels, and this was significantly less inhibition than in normals (p < 0.001) (Fig. 3). The lymphocytes of patients 1-3, in fact, showed stimulated thymidine incorporation by TGF/51- There was no apparent correlation between the effect of TGF-fll on D N A synthesis in the C L L patients and the age, sex, Rai stage or peripheral blood lymphocyte counts of these patients. DISCUSSION TGF-/5 is believed to have an important role in immune modulation, in that it is secreted by both Band T-cells and inhibits the proliferation of mitogen stimulated lymphocytes [5-11, 16]. In the present study we have demonstrated, using fluorescent-labelled antibody to TGF-fll [20], that TGF-fl is present in discrete areas in the cytoplasm of C L L and normal B-cells (Fig. 1). These areas were found to co-localize with sites detected by an antigranule antibody [Fig.

l(b)] [22], although morphologically no granules were observed in the cells. Thus, as TGF-fl is located in the granules in platelets [26], this cytokine is also probably stored in secretory vesicles in the cytoplasm of lymphoid cells. Studies have demonstrated that TGF-fl arrests cell cycling of lymphocytes in late G1 [16], perhaps through reduction of phosphorylation of the retinoblastoma gene product [27]. As the secretion of T G F fl by these cells is increased following exposure to mitogens which induce cell proliferation [6, 9], T G F /5 may function as an autocrine growth factor limiting clonal expansion [10, 12]. It has thus been suggested that a breakdown in this regulatory pathway could be involved in the pathogenesis of lymphoid malignancies and this may be the case in acute adult T-cell leukemia [14, 15]. In this disorder, the leukemic cells produce high levels of TGF-/51 [14, 15], as the infecting human T-lymphotropic virus 1 (HTLV-1) Tax gene product activates the gene for TGF-/5 t [15] and in contrast to normal T-cells, TGF-/5~ stimulated proliferation of the leukemic cells. In the present

86

L.G.

ISRAELS et al.

fll on C L L cells as compared with normal B-cells could be related to changes at the post-receptor level [29]. TGF-fl induces TNF-tr secretion by monocytes [30], and TNF-a~ has been shown to stimulate the proliferation of C L L cells whilst inhibiting the growth of normal B-cells [31, 32]. In summary, the results of the present study demonstrate that TGF-fl is located in discrete sites in the cytoplasm of CLL and normal B-cells, which may represent a storage form of TGF-fl in these cells. Unstimulated normal B- and CLL cells both contain similar low levels of TGF-fl m R N A and secrete low levels of this cytokine and, while TGF-fll produces marked inhibition of D N A synthesis in normal Bcells, this effect is significantly less in C L L cells. This latter feature may provide CLL cells with a growth advantage over normal lymphocytes in oivo. Further studies are required to determine whether this effect is related to alterations in the CLL cells at the receptor or post-receptor level.

1.0

..J O l,-z o o qa.

I LI_ Op.

0.5

0 0.0 NORMAL

CLL

FIG. 3. Inhibition of DNA synthesis by TGF-fll in normal B- and CLL cells. Normal B- or CLL cells were incubated alone or with TGF-ftI (1 ng/ml) and DNA synthesis measured, as described in Tables 2 and 3. Results represent the fraction of DNA synthesis in TGF-ftl-treated cells compared to untreated cells. Points represent the mean -+ S. E. for 18 CLL patients and 7 normals.

study we have demonstrated that like normal Bcells, unstimulated C L L cells contain very low or undetectable levels of TGF-fl m R N A and this is paralleled by the quantity of TGF-fl secreted. In contrast, the inhibition of D N A synthesis by TGF-fl~ was significantly less marked in C L L cells than in normal B-cells. A similar finding has been observed in cells in hairy cell leukemia [11] and transformed lymphoid cell lines in culture [10, 28]. The degree of inhibition of D N A synthesis in C L L cells by TGF-fl~ did not correlate with the age, sex, lymphocyte count or Rai staging of the patient. The reduced effect of TGF-fll on D N A synthesis in CLL cells compared with normal B-cells could be related to differences in TGF-fl receptor numbers and type as has been shown for Epstein-Barr infected B-cells [9] and other transformed B- and T-cell lines [28]. In some of these lines, stimulation of the cells with phorbol myristate acetate can induce the expression of TGF-fl receptors causing the cells to once again respond to growth suppression by TGFfl [28]. These findings suggest that loss of the receptors for TGF-fl may allow tumor cells to escape the growth inhibiting effects of TGF-/3 and thus provide them with a growth advantage over normal lymphocytes. Alternatively, the differing effect of TGF-

Acknowledgements--This study was supported by a grant from the Medical Research Council of Canada. Dr James B. Johnston is a Portney Research Scientist of the Manitoba Cancer Treatment and Research Foundation. The authors thank Dr Michael Sporn for providing the anti-LC(1-30) antibody, Eileen MacMillan for technical assistance, and Joan Powell for typing this manuscript.

REFERENCES 1. Roberts A. B. & Sporn M. B. (1990) The transforming growth factors-ft. In Handbook of Experimental Pharmacology, Vol. 95/1. Peptide Growth Factors and their Receptors, Sporn M. B. & Roberts A. B. (Eds), p. 419. Springer, Heidelberg. 2. Khalil N., Bereznay O., Sporn M. & Greenberg A. H. (1989) Macrophage production of transforming growth factor ft and fibroblast collagen synthesis in chronic pulmonary inflammation. J. exp. Med. 170, 727. 3. Segarini P. R., Roberts A. B., Rosen D. M. & Seyedin S. M. (1987) Membrane binding characteristics of two forms of transforming growth factor-ft. J. biol. Chem. 262, 14,656. 4. Seyedin S. M., Segarini P. R., Rosen D. M., Thompson A. Y., Bentz H. & Graycar J. (1987) Cartilage-inducing Factor-B is a unique protein structurally and functionally related to Transforming Growth Factor-ft. J. biol. Chem. 262, 1946. 5. Kehrl J. H., Taylor A., Kim S.-J. & Fauci A. S. (1991) Transforming growth factor-ft is a potent negative regulator of human lymphocytes. Ann. N Y Acad. Sci. 628, 194. 6. Kehrl J. H., Wakefield L. M., Roberts A. B., Jakowlew S., Alvarez-Mon M., Derynck R., Sporn M. B. & Fauci A. S. (1986) Production of transforming growth factor ft by human T-lymphocytes and its potential role in the regulation of T-cell growth. J. exp. Med. 163, 1037. 7. Ristow H.-J. (1986) BSC-1 growth inhibitor/type B

TGF-fl in chronic lymphocyticleukemia transforming growth factor is a strong inhibitor of thymocyte proliferation. Proc. natn. Acad. Sci. U.S.A. 83, 5531. 8. EUingsworth L. R., Nakayama D., Segarini P., Dasch J., Carrilo P. & Waegell W. (1988) Transforming growth factor-fls are equipotent growth inhibitors of interleukin 1-induced thymocyte proliferation. Cell. lmmun. 114, 41. 9. Kehrl J. H., Roberts A. B., Wakefield L. M., Jakowlew S., Sporn M. B. & Fauci A. S. (1986) Transforming growth factor fl is an important immunomodulatory protein for human B-lymphocytes. J. Immun. 137, 3855. 10. Kehrl J. H., Taylor A. S., Delsing G. A., Roberts A. B., Sporn M. B. & Fauci A. S. (1989) Further studies of the role of transforming growth factor-fl in human B-cell function. J. Immun. 143, 1868. 11. Petit-Koskas E., Genot E., Lawrence D. & Kolb J.-P. (1988) Inhibition of the proliferative response of human B-lymphocytes to B-cell growth factor by transforming growth factor-beta. Eur. J. Immun. 18, 111. 12. Lucas C., Bald L. N., Fendly B. M., Mora-Worms M., Figari I, S., Patzer E. J. & Palladino M. A. (1990) The autocrine production of transforming growth factor-fl during thymocyte activation. A study with a monoclonal antibody-based ELSA. J. lmmun. 145, 1415. 13. Blomhoff H. K., Smeland E., Mustafa A. S., Godal T. & Ohlsson R. (1987) Epstein-Barr virus mediates a switch in responsiveness to transforming growth factor, type beta, in cells of the B-cell lineage. Eur. J. lmmun. 17, 299. 14. Niitsu Y., Urushizaki Y., Koshida Y., Terui K., Mahara K., Kohgo Y. & Urushizaki I. (1988) Expression of TGF-beta in adult T-cell leukemia. Blood 71,263. 15. Kim S.-J., Kehrl J. H., Burton J., Tendler C. L., Jeang K.-T., Danielpour D., Thevenin C., Kim K. Y., Sporn M. B. & Roberts A. B. (1990) Transactivation of the transforming growth factor/~1 (TGF-fll) gene by human T-lymphotropic virus type 1 Tax: a potential mechanism for the increased production of TGF-fll in adult T-cell leukemia. J. exp. Med. 172, 121. 16. Smeland E. B., Blomhoff H. K., Holte H., Ruud E., Beiske K., Funderud S., Godal T. & Ohlsson R. (1987) Transforming growth factor type fl (TGF fl) inhibits G1 to S transition, but not activation of human Blymphocytes. Expl Cell Res. 171,213. 1I. Foon K. A., Rai K. R. & Gale R. P. (1990) Chronic lymphocytic leukemia: new insights into biology and therapy. Ann. Intern. Med. 113, 525. 18. Rai K. R., Sawitsky A., Cronkite E., Chanan P. A., Levy R. N. & Pasternak B. S. (1975) Clinical staging of chronic lymphocytic leukemia. Blood 46, 219. 19. Boyum A. (1976) Isolation of lymphocytes, granulocytes, and macrophages. Scand. J. Immun. 5 (Suppl. 5), 9. 20. Flanders K. C., Thompson N. L., Cissel D. S., Van Obberghen-Schilling E., Baker C. C., Kass M. E., Ellingsworth L. R., Roberts A. B. & Sporn M. B.

87

(1989) Transforming growth factor-~]l: histochemical localization with antibodies to different epitomes. J. Cell Biol. 108, 653. 21. Wencel-Drake J. D., Plow E. F., Zimmerman T. S., Painter R. G. & Ginsberg M. H. (1984) Immunofluorescent localization of adhesive glycoproteins in resting and thrombin-stimulated platelets. A m . J. Path. 115, 156. 22. Gerrard J. M., Lint D., Sims P. J., Wiedmer T., Fugate R. D., McMillan E., Robertson C. & Israels S. J. (1991) Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome. Blood 77, 101. 23. Chomczynski P. & Sacchi N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Ann. Biochem. 162, 156. 24. Church G. M. & Gilbert W. (1984) Genomic sequencing. Proc. natn. Acad. Sci. U.S.A. 81, 1991. 25. Brunner A. M., Marquardt H., Malacko A. M., Lioubin M. N. & Purchio A. F. (1989) Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor fll precursor. J. biol. Chem. 264, 13,660. 26. Fava R. A., Casey T. T., Wilcox J., Pelton R. W., Moses H. L. & Nancy L. B. (1990) Synthesis of transforming growth factor-ill by megakaryocytes and its localization to megakaryocyte and platelet or-granules. Blood 76, 1946. 27. Laiho M., De Capio J. A., Ludlow J. W., Livingston D. M. & Massague J. (1990) Growth inhibition of TGF-fl linked to suppression of retinoblastoma protein phosphorylation. Cell 62, 175. 28. Sing G. K., Ruscetti F. W., Beckwith M., Keller J. R., Ellingsworth L., Urba W. J. & Longo D. L. (1990) Growth inhibition of a human lymphoma cell line: induction of a transforming growth factor-fl-mediated autocrine negative loop by phorbol myristate acetate. Cell Growth Differ. 1, 549. 29. Moses H. L., Yang E. Y. & Pietenpol J. A. (1990) TGF-fl stimulation and inhibition of cell proliferation : new mechanistic insights. Cell 63, 245. 30. Wahl S. M., Hunt D. A., Wong H. L., Dougherty S., McCortney-Francis N., Wahl L. M., Ellingsworth L., Schmidt J. A., Hall G., Roberts A. B. & Sporn M. B. (1988) Transforming growth factor-~ is a potent immunosuppressive agent that inhibits IL-l-dependent lymphocyte proliferation. J. lmmun. 140, 3026. 31. Cordingley F. T., Hoffbrand A. V., Heslop H. E., Turner M., Bianchi A., Reittie J. E., Vyakarnam A., Meager A. & Brenner M. K. (1988) Tumor necrosis factor as an autocrine growth factor for chronic B-cell malignancies. Lancet I, 969. 32. Digel W., Stefanic M., Schoniger W., Buck C., Raghavachar A., Frickhofen N., Heimpel H. & Porzsolt F. (1989) Tumor necrosis factor induces proliferation of neoplastic B-cells from chronic lymphocytic leukemia. Blood 73, 1242.