Cancer Letters,51(1990)

Elsevier Scientific Publishers

221

221-225 Ireland

Transforming growth factor-beta and ovarian carcinoma cells: regulation of proliferation and surface antigen expression C. Marth, T. Lang, A. Koza, I. Mayer and G. Daxenbichler Department ofObstetricsand (Received 1 February 1990) (Revision received 20 February (Accepted 20 February 1990)

Gynecology,

Innsbruck

UnioersityClinic, Anichstrasse

35, A-6020 Innsbruck,

(Austria)

1990)

Summary Transforming growth factor-beta (TGFj3) is a multifunctional peptide regulating several processes in ovarian cells. The growth of ovarian carcinoma cell lines (OVCAR-3, HTB77, 2780 and CRL-1572) was reduced by TGF$ in a dose related manner. The antiproliferatiue activity was not improued by combination with other biological response modifiers Treatment with TGF$ augmented the expression of interferon-y induced class I and 11antigens of the major histocompatibilty complex The presentation of another antigen namely the tumor marker CA-125 on the cell surface was markedly reduced by TGF-fi.

must be considered as a general mediator of regulation in the cell and one of special importance for negative control of cell growth. For a wide variety of cells these pleiotropic effects

have been described. Also cells from the ovary are targets for TGF-/I, it regulates e.g., hormone production and differentiation of granulosa cells [l]. Moreover, TGF-fi has been found to have structure homology with other important gonadal regulatory peptides i.e., inhibin and the miillerian inhibiting substance [ 151. It is the objective of the studies reported herein to further elucidate the potential relevance of TGF-/3 in regulating proliferation and antigen presentation of cultured ovarian carcinoma cells. Materials and methods

Keywords: transforming growth ovarian cancer; interferon-gamma.

factor

/3;

Introduction The TGF-/3 is a multifunctional peptide acting by both autocrine and paracrine mechanisms [5,15,16]. It can either stimulate or inhibit proliferation, differentiation and other crucial cellular functions. Therefore, the TGF$ Correspondence to: C. Marth

0304-3835/90/$03.50 0 1990 El sevier Scientific Publishers Published and Printed in Ireland

Reagents

The TGF-/32 was derived from human recombinant DNA and kindly provided by Dr. Ch. Huber, Innsbruck University Clinic; mouse monoclonal antibody OC- 125 against the ovarian carcinoma marker CA-125 was purchased from CIS (Gif-Sur-Yvette, France) ; the anti-HLA-DR and anti-class I MHC were from Becton-Dickinson, Mountain View, CA; 1251labeled anti-mouse immunoglobulin F(ab’), fragment from sheep was obtained from

Ireland Ltd.

222

Amersham International (Buckinghamshire, U.K.) Minimal essential Medium (MEM) and fetal bovine serum (FBS) were from Biological Industries, Kibbutz Beth Haemek, Israel. The tissue culture plastic ware was obtained from Falcon Plastics (Oxnard, CA. The interferon-y and tumor necrosis factor alpha (TNF) were derived from human recombinant DNA and kindly provided by Dr. G. Adolf; E. Boehringer Institute, Vienna, Austria. The 1,25-dihydroxy-cholecalciferol (1,25(0H),-vitamin DJ and all-trans-retinoic acid were obtained from the Hoffmann-La Roche, Basel. Cell culture The human ovarian carcinoma cell lines OVCAR-3, HTB-77, 2780, and CRL-1572 were kindly provided by Dr. T. Hamilton, NIH, Bethesda and Dr. Ch. Dittrich, Vienna, Austria, respectively, and were cultured under usual conditions as described recently [ll]. The cells were cultured in 24-well tissue culture plates in presence of different concentrations of TGF-/3 and enumerated using an electronic particle counter after 7- 10 days of treatment. Expression

of surface antigens

The presentation of either HLA-DR or CA125 on the cell membrane was detected by a living cell radioimmunoassay according to Greiner et al. [6,7] as described recently [lo].

number of cells in per cent 1051

0

1

10

100

TGF-8 (ng/ml) Fig. 1.

Effects of TGF-fl on proliferation. About 50,000 cells of the different lines were cultured for 7- 10 days in the presence of TGF-f3 as indicated. Thereafter the cells were harvested and their number was determined using an electronic particle counter. Each point represents the median cell number of 6 wells counted in percent of control. The coefficient of variation was always below 10% and is not shown for reasons of clarity.

Statistics

Data were analyzed using non parametric statistics and differences in median cell number or antibody bound to the cell surface were tested according to Wilcoxon-Mann-Whitney 1141. Results Proliferation

The growth of the human ovarian carcinoma cell lines was reduced by a treatment with recombinant TGF-fi in a dose related manner (P < 0.01 for OVCAR-3 and HTB77, P < 0.05 for 2780 and CRL 1572, Fig. 1). The half maximal effect was achieved for

OVCAR3 and HTB-77 cells at about 2 ng/ml which is in agreement with Roberts et al. [13]. These authors reported recently that for several human tumor cell lines the ED,, for reduction of anchorage-independent growth ranges between 0.1 and 2 ng/ml of TGF-/3. To reduce the proliferation of CRL-1572 and 2780 cells higher concentrations of TGF-P were necessary (Fig. 1). Biological response modifiers are involved in a complicated network and their combination leads frequently to a synergistic interaction. Especially for interferons interactions with TNF or retinoic acid have been described recently [9,12,17]. For the TGF-p,

223

however, we did not observe an amplification of the anti-proliferative action when combining it with other biological response modifiers (Table 1). Antigen modulation Interferons are known to be a potent inducer of the antigens of the major histocompatibility complex (MHC). Interferon-y in particular induces both class I (HLA-A,B,C) and II (HLA-DR) MHC on the majority of cells [4]. TGF+ is able to antagonize the interferon-y mediated induction of HLA-DR on melanoma cells [3]. The aim of our study was to find out whether this observation is generally valld. OVCAR-3 ovarian carcinoma cells were treated with interferon-y either alone or in combination with TGF-/3. The expression of HLA-DR on the tumor cell surface was not detected in untreated control cells but was induced in a dose related manner by interferon-y (Fig. 2). TGF$ alone did not induce this antigen but in combination with interferony we observed consistently a weak increase in HLA-DR presentation (p < 0.05 compared to interferon-y alone). Class I antigens of the MHC were constitutively expressed on OVCAR-3 cells and interferon-y augmented

Table 1.

Interaction of TGF-/3 with other biological response modifiers on proliferation of OVCAR-3 ovarian carcinoma cells. Substance

Concentration”

Effectb

Interferon-a Tumor necrosis factor-a

O.l-long/ml lo- 100 ng/ml

Additive Additive

Retinoic acid 1,25 (OH),-vitamin

0.1-l O.lpM

Additive Additive

D,

PM

aTheOVCAR-3 cells were counted treatment with TGF-/I (0.1-10

after 7-10 days of ng/ml) alone or in in the dose range indi-

combination with the substance cated, bThe effect of the combined treatment was classified as synergistic, additive or antagonistic applying the law of probability as recently described (Denz, Marth)

bound antibody kpm in thourandr)

0

0.1

Control

1

interferon-gamma

-6-

TGF-6dnglml) 10

100

(ng/ml)

Fig. 2. Modulation of HLA-DR expression by interferon-y and TGF-/.? on OVCAR-3 cells. The cells were cultured in the presence of interferon-y for 3 days (as indicated on the abscissa) with or without TGF-/3 (1 ng/ml) . The cell surface presentation of the HLA-DR was determined using the living cell radioimmunoassay. Each point represents the median cpm of bound antibody measured in 4 wells. The coefficient of variation was always below 15% and is not shown for reasons of clarity.

their cell surface concentration (Fig. 3). Also for this antigen TGF-fi supported the interferon-y action in a moderate way (P < 0.05). A similar influence of TGF-/3 only in combination with interferongamma on either class I or II MHC antigens was also found for SKBR-3 human mammary carcinoma cells (data not shown). In the follow-up of patients with ovarian carcinoma CA-125 has become a widely used tumor marker, since an increase in its serum concentration has been shown to be associated with tumor progression [2]. We analyzed whether TGF$ is able to modulate the expres-

224 bound antibody 91

(cpm

in thouaandr) 6000

OC-126 bindin

/

kpm)

OVCAR-3

-

control

-El- TQF-6 tlnglml)

/

I

/

0.1

1

10

100

30001 0

’

’ “/“”

i k-8

interferon-gamma (ng/ml)

’ ’ ““”

10

’

’ ’ iiJsi’ 100

(ng/ml)

Fig. 3. Modulation of the expression class I antigens of the MHC by interferon-y and TFG-fi on OVCARS cells. The cells were cultured in the presence of interferon-y for 3 days (as indicated on the abscissa) with or without TGFfi (1 ng/ml). The cell surface presentation of the class I antigens of the MHC was determined using the living cell radioimmunoassay. Each point represents the median cpm of bound antibody measured in 4 wells. The coefficient of variation was always below 15% and is not shown for reasons of clarity.

Fig. 4.

sion of this important tumor marker on cultured ovarian carcinoma cells and observed a significant reduction in CA125 surface concentration of OVCAR-3 cells in a dose-related manner (P< 0.01, Fig. 4).

more than 30%. One possible explanation for this limited activity could be the large amounts of TGF-fl present in the FBS. Experiments with reduced serum concentration in the culture medium did not result in an increased efficacy of a treatment with TGF-p (data not shown). This may exclude a significant role of the PBS as a source of TGF-fi. Biological response modifiers are involved in a complicated network of interactions and administered together they can either amplificate or inhibit their biological effects. The combination of TGF-fl with other biological response modifiers, however, did not result in a synergy regarding the growth inhibitory

Discussion The role of TGF-/3 in ovarian physiology is poorly elucidated, whereas in vitro it clearly reduced the proliferation of 4 carcinoma cell lines. Although this action was statistically significant, the growth retardation was weak and even at a concentration of 100 ng/ml of TGF-fi the number of cells was not reduced

CA-125 surface presentation after TGF-/3 The cells were cultured in the presence of TGF-f3 for 3 days (as indicated on the abscissa). The cell surface presentation of the tumor marker CA-125 was determined using the living cell radioimmiunoassay. Each point represents the median cpm of bound antibody measured in 4 wells. The coefficient of variation was always below 15% and is not shown for reasons of clarity. treatment.

225

action. Despite the independent regulation of proliferation by TGF$ and interferon-y an interaction on antigen presentation is known. Czamiecki et al. recently showed on Hs294T melanoma cells that TGF-fl suppresses the interferon-y induced expression of class II MHC antigens [3]. We could not confirm this neither on OVCAR-3 ovarian carcinoma nor on SKBR-3 mammary carcinoma cells. On the contrary, TGF+ augmented synergistically the interferon effect on both class I and II MHC antigens. This action is similar to the synergy between TNF and interferon-y. The latter combination exerts, however, also a synergistic anti-proliferative activity. This difference between TGF-/3 and TNF suggests that proliferation and HLA-DR are regulated independently and that in contrast to TNF, TGF-fl does not amplify all interferon-induced effects. Although serum determinations of the tumor marker CA-125 are valid in the followpatients the up of ovarian carcinoma regulation of this antigen is widely unknown. Interferons have recently been shown to augment the CA-125 concentration intracellularly, on the cell surface and in the supernatant culture medium [lo]. Karlan et al. were able to decrease CA-125 expression on OVCA 433 ovarian carcinoma cells by glucocorticoids [8]. TGF-fi is to our knowledge the third naturally occurring substance which modulates this tumor marker. The importance of the pieiotropic actions of TGF-j3 which can be observed under experimental conditions for the in vivo situation remains a matter of future studies. References Adashi, E.Y., Resnick C.E., Hernandez, E.R., May, J.V., Purchio, A.F. and Twardztk D.R. (1989) Ovarian transforming growth factor-8 (TGF/3): cellular site(s) and mechanism(s) of action. Mol. Cell Endocrinol., 61, 247256. Bast, C.R., Klug, T.L., John, ES., Jenison, E., Niloff. J.M., Lazarus, H., Berkowitz, R.S., Leavitt, T., Griffiths, C.T., Parker L., Zurawski, V.R. and Knapp, R.C. (1983) A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer. New Engl. J. Med., 309.883-887.

Czarniecki, C.W., Chiu, H.H., Wong, G.H.W., McCabe, SM. and Palladino, M.A. (1988) Transforming growth facto+ modulates the expression of class II histocompatfbility antigens on human cells. J. fmmunol., 140, 42174223. 4 Gasth, G. and Huber, C.H. (1988) The biology of interferon acttons. Blut, 56, 193-199. 5 Got&in, A.S., Leof, E.B., Shipley, G.D. and Moses, H.L. (1986) Growth factors and cancer. Cancer Res., 46, 1015 -1929. 6 Greiner, J.W., Hand, P.H., Wunderlich, D. and Colcher, D. (1986) Radioimmunoassay for detection of changes in cell surface tumor antigen expression induced by interferon. Methods Enzymol., 119,682-688. 7 Greiner, J.W. (1986) Recombinant human leukocyte interferon induces alterations in the antigen phenotype of human breast carcinoma cells. Anticancer Res., 6, 653658. 8 Karlan, B.Y., Amin, W., Casper, S.E. and Littlefield, B.A. (1988) Hormonal regulation of CA 125 tumor marker expression in human ovarian carcinoma cells: inhibition by glucocorttcoids. Cancer Res., 45,3502-3506. 9 Marth, C., Daxenbichler, G. and Dapunt, 0. (1986) Synergistic antiproliferative effects of human recombinant interferons in combination with rettnoic acid in cultured breast cancer cells. J. Natl. Cancer Inst., 77, 1197-1202. 10 Marth, C., Fuith, L.C., Bock, G., Daxenbichler, G. and Dapunt, 0. (1989) Modulation of ovarian carcinoma tumor marker CA-125 by interferon-y. Cancer Res., in press. 11 Marth, C., Helmberg, M., Mayer, I., Fuith, L.C., Daxenbichler G. and Dapunt, 0. (1989) Effects of biological response modiiiers on ovarian carcinoma cell lines. Anticancer Res., 9,461-468. 12 Marth, C., Zech, J.. Bock, G., Mayer I. and Daxenbichler, G. (1987) Effects of retinoidr and interferon-y on cultured breast cancer cells in comparison with tumor necrosis factor alpha. Int. J. Cancer, 40,840-845. 13 Roberts, A.B., Anzano, M.A., Wakefield, L.M., Roche, N.S., Stern, D.F. and Spom, M.B. (1985) Type /I transforming growth factor: A bifunctional regulator of cellular growth. Proc. Natl. Acad. Sci., 82, 119-123. 14 Sachs, L. (1978) Angewandte Statistik. Springer, Berlin, Heidelberg, New York. 15 Sporn, M.B., Roberts, A.B., Wakefield, L.M. and Assoian, R.K (1986) Transforming growth factor-b: Biological function and chemical structure. Science, 233, 532 -534. 16 Spom, M.B., Roberts, A.B., Wakefield, L.M. and De Crombrugghe, B. (1987) Some recent advances in the chemistry and biology of transforming growth factor-beta. J. Cell Biol., 105, 1039-1045. 17 Williamson, B.D., Carswell, E.A., Rubin, B.Y., Prendergast, J.S. and Old, L.J. (1983) Human tumor necrosis factor produced by human B-cell lines: synergistic cytotoxic interaction with human interferon. Proc. Natl. Acad. Sci., 80,5397-5401. 3