Transformed cells trigger induction of their own apoptosis in coculture with normal cells

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1 ONCOLOGY REPORTS 3: 27-31, 1996 Transformed cells trigger induction of their own apoptosis in coculture with normal cells KLAUS HACKENJOS, CHRISTIANE LANGER, SUSANNE ZABEL and GEORG BAUER Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Str. 11, D Freiburg, Germany Received September 20, 1995; Accepted November 6, 1995 Abstract. In vitro, transforming growth factor type betal triggers normal cells to induce apoptosis in transformed cells. We show that in the absence of exogenous transforming growth factor type betal, induction of apoptosis of transformed cells in coculture with normal cells is dependent on the number of transformed cells per assay and is abrogated by antibodies against TGF-ßl. Therefore, transforming growth factor type betal produced by transformed cells seems to be responsible for triggering a mechanism that leads to the induction of their apoptosis. This mechanism may be crucial for the control of carcinogenesis in vivo. Introduction Cocultivation of transformed and normal fibroblasts in the presence of exogenous TGF-ß leads to the specific elimination of transformed cells (1,2). This novel potential control step in carcinogenesis is due to the induction of apoptosis in transformed cells, as indicated by membrane blebbing, chromatin condensation and DNA fragmentation (2). It is dependent on the concentration of TGF-ß applied and on the density of normal cells (1), but not on direct cellto-cell contact between normal and transformed cells (3). In the presence of optimal concentrations of TGF-ß, all cells within a population of normal cells are able to exert induction of apoptosis, indicating that this ability is not the property of a defined subpopulation of normal cells (4). However, the threshold level for TGF-ß for induction of apoptosis is different for individual cell clones of normal cells. Sensitivity against the elimination process seems to be a regular feature associated with the transformed phenotype of fibroblasts, as cells transformed by different transformation principles are equally sensitive against TGF-ß-induced apoptosis. (2). Correspondence to: Dr Georg Bauer, Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Hermann- Herder-Str. 11, D Freiburg, Germany Key words: transformed cells, apoptosis Antioxidants and radical scavengers were shown to interfere with elimination of transformed cells by normal cells (3,5). Separation of the in vitro system in distinct phases revealed that reactive oxygen species are specifically involved in the interaction between TGF-ß and normal cells and during the transmission of an apoptosis-inducing signal between TGF-ß-treated normal cells and transformed cells (6). When normal and transformed cells are cocultured in vitro, exogenous TGF-ß has to be added for a maximal induction of apoptosis. In the absence of exogenously added TGF-ß, cocultures of normal and transformed cells exert a 'basal eliminative activity' that increases with the passage number of normal cells (4). We studied whether, in the absence of exogenously added TGF-ß, transformed cells are themselves involved in the induction of a process in normal cells that finally leads to their death and elimination. Materials and methods Cell culture. The C3H 10T1/2C18 mouse fibroblast cell line was obtained from Dr U. Rapp, NIH, Bethesda, Maryland. The spontaneously transformed Swiss 3T3 MxCll cell line was isolated by cloning Swiss 3T3 MxA cells in soft agar. It shows criss-cross morphology and colony formation in soft agar. The nontransformed Swiss 3T3 MxA cell line (7) was provided by Dr O. Haller, Abteilung Virologie, Freiburg, Germany. The transformed cell line HSP 111/1 was established by transformation of NRK 536 cells by Herpes simplex virus type 1 (HSV-1) temperature-sensitive in the UL9 reading frame. The cells do not contain HSV-1 DNA (8). Cells were kept in Eagle's minimal essential medium, containing 5% fetal calf serum, that had been heated for 30 min at 56 C prior to use. Medium was supplemented with penicillin (40 U/ml), streptomycin (50 J,g/ml), neomycin (10 (Xg/ml), moronal (10 U/ml) and glutamine (280 ig/ml). Cell culture was performed in plastic tissue culture flasks. G 418 resistant cell lines were kept in the presence of 500 ig/ml G418. Elimination in cocultures without cell-to-cell contact. For cocultivation of cells without cell-to-cell contact, cell culture clusters with inserts [pore-size 0.45 J,m (Falcon), distance between cell layers approximately 2 mm] were used. Transformed target cells were clonally seeded in the tissue culture clusters in the presence of 15% conditioned medium.

2 28 HACKEN JOS et ai. TRANSFORMED CELLS AND APOPTOSIS Figure 1. Induction of apoptosis by increasing numbers of transformed cells. A, B: 7,000 normal C3H 10 Tl/2 cells (passage number 30) were seeded in Costar 24-well tissue culture clusters (total volume 0.5 ml). After they had attached, the indicated numbers of HSP III/l (A) or HSP III/2 (B) were added. [HSP III/l and HSP III/2 cells originate from NRK 536 cells transformed by HSV-1 (8)]. Assays under A contained 0%, 0.5 % or 1% DMSO, as indicated. Assays under B obtained 1 ng/ml TGF-ßl or no TGF-ßl, as indicated in the figure. After 6 days of coculture, assays were stained and scored for intact and substantially damaged foci. C: 7,000 C3H 10 Tl/2 cells were seeded in Costar 24-well tissue culture clusters. After the cells had attached to the surface, 4,000 transformed HSP III/2 cells were added. In addition, assays contained 50 or 150 ii.g/ml of neutralizing antibody against TGF-ßl (closed circles) or normal chicken IgG (open circles). After 6 days of coculture, the percentage of intact and substantially damaged foci was determined. Parallel control assays showed that 150 (lg/ml of anti-tgf-ßl were sufficient to neutralize the apoptosing-inducing effect of 1 ng/ml exogenous TGF-ßl completely, whereas 50 u.g/ml neutralized 75% of the effect of 1 ng/ml TGF-ß 1. Each assay contained about 100 clones. Normal cells (effector cells) were seeded into the inserts (4xl0 4 cells per insert) with or without the indicated number of transformed inducer cells. Control assays were pretreated with TGF-ßl (standard concentration 10 ng/ml or as indicated) for 2 days (or as indicated). Immediately before transfer of the inserts, medium was renewed in the clusters containing the transformed cell clones. Medium was removed from the inserts and these were washed with medium from both sides. After the indicated time of coculture, inserts were removed and the clones were checked for apoptotic cells using phase contrast microscopy as recently described (2). Control experiments ensured that membrane blebbing was paralleled by chromatin condensation. Three categories of clones were defined: (i) less than 10% apoptotic cells per clone; (ii) more than 10 but less than 50% apoptotic cells; (iii) more than 50% apoptotic cells. All other procedures are described in the respective figure legends. TGF-ß. TGF-ßl was purified from the extract of human platelets as described elsewhere (9). Neutralizing antibody against TGF-ß (rabbit) was purchased from Fa. H. Biermann, Bad Nauheim. Results To study whether transformed cells are able to trigger induction of their own apoptosis, normal rat fibroblasts were overlaid with increasing numbers of transformed fibroblasts in the absence of exogenous TGF-ßl. Analysis after 6 days of coculture revealed that the percentage of foci of transformed cells that showed substantial apoptosis was dependent on the number of transformed cells originally seeded (Fig. 1A and B). High numbers of transformed cells seeded per assay led to the same efficiency of induction of apoptosis as addition of exogenous TGF-ßl (Fig. IB), indicating that transformed cells were able to functionally substitute for exogenous TGF-ßl. Triggering of induction of apoptosis by transformed cells was inhibited by antibodies against TGF-ßl and by the radical scavenger DMSO (Fig. 1A and C). In the experiments performed so far, the inducing and affected cells were the same. Therefore, measurable apoptosis was at the same time dependent on the potential of the cells to induce apoptosis and on their sensitivity. In order to differentiate between the inducing potential of transformed cells and their sensitivity for induction of apoptosis, survival of G 418-resistant transformed target cells cocultured with G 418-sensitive inductor cells and G 418-sensitive normal effector cells was quantitated. A constant number of transformed Swiss 3T3 Mx Cll cells were used as target cells and were seeded in the absence or presence of variable numbers of normal fibroblasts. In addition, the assays obtained increasing numbers of transformed HSP III/l cells as potential inductor cells for the process of apoptosis. Fig. 2 shows that in the presence of normal cells, the decrease of rescuable G 418-resistant clones (indicating elimination of the target cells) was both dependent on the number of normal cells and the number of transformed inductor cells initially seeded. The presence of 32,000 normal cells and 4,000

3 ONCOLOGY REPORTS 3: 27-31, Figure 2. Quantitation of induction of apoptosis using G 418-resistant transformed indicator cells and G 418-sensitive transformed inducer cells. 500 transformed Swiss 3T3 MxACll cells (G 418-resistant, indicator cells) were seeded together with the indicated numbers of normal C3H 10 T1/2 cells (G 418- sensitive) and transformed HSP III71 cells (G 418-sensitive, inductor cells). Assays were performed in duplicate. After 4 days of coculture, assays were washed and fresh medium, containing 1 mg/ml G 418 was added. After 5 days the number of surviving clones of Swiss 3T3 MxCll cells were determined. Parallel control assays (performed in duplicate and not shown in the graph) ensured that 2.5 ng/ml TGF-ßl in the presence of 32,000 C3H 10 Tl/2 cells and the absence of HSP III/l cells induced elimination of the transformed Swiss 3T3 MxCll cells to less than 5% of the original' number of clones seeded Catechol (1 ug/ml) added to the coculture of 32,000 C3H 10 Tl/2 cells, 500 Swiss 3T3 MxA Cll cells and 10,000 HSP III/l cells completely abrogated the apoptosis-inducing effect of the HSP III/l cells. inductor cells or 16,000 normal cells and 10,000 inductor cells initially caused nearly complete elimination and thus resembled the control in the presence of exogenous TGF-ß. Addition of the antioxidant catechol to assays containing 32,000 normal cells and 10,000 inductor cells completely abrogated their apoptosis-inducing effect, indicating the role of reactive oxygen species during this process. Coculture of inductor and target cells in the absence of normal cells did not cause detectable loss of target cells, indicating that the decrease of G 418-resistant cells was not due to a negative interaction between the two types of transformed cells, but that normal cells are required for elimination of transformed cells. The next experiment was aimed to the spatial separation of the induction process and apoptosis induction in target cells. Normal cells and increasing numbers of transformed cells were seeded together in tissue culture inserts and incubated for two days. The inserts were then washed and set above tissue culture clusters containing clones of transformed cells. After 3 days of coculture, inserts were removed and the percentage of clones with substantial apoptosis was determined 24 hours later. As can be seen in Fig. 3, preincubation of normal cells with 10,000 transformed cells induced a signal that was transferred to the transformed target cells over distance and there induced substantial degree of apoptosis. These data show that transformed cells induce the production of an apoptosis-mediating signal in normal cells which is transmitted to the transformed cells and induces their apoptosis. Discussion Our data show that increasing numbers of transformed fibroblasts in coculture with normal fibroblasts trigger induction of their apoptosis without addition of exogenous TGF-ß. Large numbers of transformed cells thus substitute for exogenous TGF-ß that is required for maximal induction of apoptosis in the presence of smaller numbers of transformed cells in our typical elimination system in vitro. The inducing effect of transformed cells is abrogated by neutralizing antibody against TGF-ß. This finding and the fact that induction of apoptosis was dependent on the number of transformed cells, indicates that TGF-ß released by the transformed cells is responsible for induction of apoptosis. The sensitivity of this process against radical scavengers and antioxidants reveals that the process induced by the transformed cells is biochemically related or identical to the one induced by exogenous TGF-ß (3). Abrogation of apoptosis by antioxidants and radical scavengers also shows that apoptosis is caused by specific effects and is not due to unspecific effects, such as depletion of the medium. Release of biologically active TGF-ß by transformed cells seems to be responsible for induction of 'basal elimination' (i.e. in the absence of exogenously added TGF-ß) of normal cells (1,4). Several distinct approaches have been used in this study to quantitate triggering of apoptosis by transformed cells. Cocultivation of normal and transformed cells and evaluation of substantial signs of apoptosis within the arising foci allowed demonstration of the inducing activity of transformed

4 30 HACKENJOS et al: TRANSFORMED CELLS AND APOPTOSIS Figure 3. Spatial separation of inducer and normal cells from transformed indicator cells. A, B: Tissue culture inserts contained either 4X10 4 C3H 10 Tl/2 cells, or 2x1c 4 Swiss 3T3 MxCll cells, or 4x10 4 C3H 10 Tl/2 cells plus 10 4 Swiss 3T3 MxCll cells, or 4xl0 4 C3H 10 Tl/2 cells plus 2x10" Swiss 3T3 MxCll cells, or 4x10* C3H 10 Tl/2 cells plus 10 ng/ml TGF-ßl, as indicated in the figure. After two days of coculture, the inserts were washed and transferred to Costar 6 well tissue culture clusters containing approximately 100 clones of Swiss 3T3 MxCll cells (clones consisting of cells each). After 3 (A) or 4 days (B) of coculture, the clones were categorized according to the degree of apoptosis. Categories: (a) less than 10% apoptotic cells; (b) more than 10 and less than 50% apoptotic cells; (c) more than 50% apoptotic cells. C, D: The experiment was performed as described under A and B, except that HSP III/l cells were used instead of Swiss 3T3 MxCll cells. C: quantitation of apoptotic clones after 3 days; D: quantitation after 4 days; categories as described under A and B. The use of tissue culture inserts for the study of induction of apoptosis has been recently described (3). The essential modification in the experiment described here was the use of clones of transformed cells rather than of a random population as described previously. Analysis of clones enhances the sensitivity of the assay. In order to get clones of transformed target cells (approximately 100 clones per assay), cells were seeded sparsely in the presence of 15% conditioned medium and cultivated until each clone consisted of 25 to 50 cells. cells, the role of TGF-ß released by them and the action of reactive oxygen species. In this experimental system, both the inducing potential of the cells and their sensitivity define the final result. Therefore, this approach does not permit to specifically measure and compare the inducing potential of different transformed cell lines. The use of G 418-resistant target cells and G 418-sensitive inducer cells allowed separation of the influences of induction and sensitivity and furthermore enabled precise quantitation by determining the number of surviving colonies. Using the same target cells, this assay should permit a comparison of the inducing potential of different transformed cell lines. It therefore represents a quantitative test for the release of biologically active TGF-ß by different cell lines. Coculture of normal and transformed cells in tissue culture inserts, followed by spatially separated coculture with transformed indicator cells enables the study of the induction process independent of the processes following, i.e. transmission of the apoptosis-inducing signal and the process of apoptosis of transformed indicator cells. The efficiency of induction of apoptosis in the experimental system of spatial separation confirms that transformed cells induce normal cells to transmit an apoptosis-inducing signal, independent of direct cell-to-cell contact. Induction of apoptosis by transformed cells cocultured with normal cells in vitro in the absence of exogenous TGF-ß can be measured when relatively high numbers of transformed cells are supplied. For an extrapolation for the relevance of this finding to the situation in vivo, the different geometric situations in vitro versus in vivo have to be taken into account. In vitro, the thin layer of cocultured cells is overlaid with a vast excess of medium, leading to a strong dilution of TGF-ß released by transformed cells. Therefore, the maximal effect requires addition of exogenous TGF-ß. In the in vivo situation, arising transformed cells are surrounded by normal cells and a relatively small volume of intercellular space. It is fair to assume that under these conditions dilution of TGF-ß is more than ten thousand-fold less compared to

5 ONCOLOGY REPORTS 3: 27-31, in vitro conditions. Thus release of TGF-ß from individual transformed cells surrounded by normal cells should be sufficient to trigger the production of apoptosis-inducing factors in normal cells and subsequently induce apoptosis of transformed cells. TGF-ß produced by transformed cells and required for the maintenance of their transformed state (10) might therefore prove to be an obstacle for their survival. Induction of this novel elimination mechanism of transformed cells by surrounding normal cells may represent an efficient control step early in carcinogenesis (11). Cells transformed in vitro are generally sensitive for induction of apoptosis by TGF-ß-treated normal cells, independent of the transformation principle that originally caused their transformation (2). Most transformed cell lines studied have been shown to release TGF-ß (12-18). Release of active TGF-ß by transformed cells and their sensitivity for induction of apoptosis by TGF-ß-stimulated cells may represent the key for their efficient elimination in vivo. Survival and expansion of transformed cells, leading to tumor formation, may therefore require either the action of substances interfering with this process, acquisition of resistance against induction of apoptosis or the inability of the surrounding tissue to produce apoptosis-inducing factors (11). Acknowledgements We thank Drs J. M. Jûrgensmeier and C. Weiand for critical comments on ehe manuscript. This work was supported by the Deutsche Krebshilfe (grant W 58/94 Ba2). References 1. Höfler P, Wehrle I and Bauer G: TGF-ß induces an inhibitory effect of normal cells directed against transformed cells. Int J Cancer 54: , Jürgensmeier JM, Schmitt CP, Viesel E, Höfler P and Bauer G: TGF-ß-treated normal fibroblasts eliminate transformed fibroblasts by induction of apoptosis. Cancer Res 54: , Jürgensmeier JM, Höfler P and Bauer G: TGF-ß-induced elimination of transformed fibroblasts by normal cells: independence of cell-to-cell contact and dependence on reactive oxygen species. Int J Oncol 5: , Picht G, Hundertmark N, Schmitt CP and Bauer G: Clonal analysis of the effect of TGF-ß on the apoptosis-inducing activity of normal cells. Exp Cell Res 218: 71-78, Schaefer D, Jürgensmeier J and Bauer G: Catechol interferes with TGF-ß-induced elimination of transformed cells by normal cells: implications for the survival of transformed cells during carcinogenesis. Int J Cancer 60: , Langer C, Jürgensmeier JM and Bauer G: Reactive oxygen species act both at TGF-ß-dependent and -independent steps during induction of apoptosis of transformed by normal cells. Exp Cell Res (In press). 7. Pavlovic J, Zürcher T, Haller O and Staeheli P: Resistance to influença virus and vesicular stomatitis virus conferred by expression of human MxA protein. J Virol 64: , Bauer G, Kahl S, Singh Sawhney I, Höfler P, Gerspach R and Matz B: Transformation of rodent fibroblasts by herpes simplex virus: Presence of morphological transforming region 1 {mtr\) is not required for the maintenance of the transformed state. Int J Cancer 51: , Bauer G, Götschl M and Höfler P: Tumor-promoting activity of Epstein-Barr-virus-inducing factor/transforming growth factor type beta (EIF/TGF-ß) is due to the induction of irreversible transformation. Int J Cancer 47: , Wehrle I, Jakob A, Höfler P and Bauer G: Transformation of murine fibroblasts by UV light and TGF-ß: establishment of an autocrine TGF-ß loop. Int J Oncol 5: , Bauer G: Resistance to TGF-ß-induced elimination of transformed cells is required during tumor progression (Review- Hypothesis). Int J Oncol 6: , Roberts AB and Sporn MB: The transforming growth factorbetas. In: Peptide Growth Factors and their Receptors. Sporn MB and Roberts AB (eds). Springer-Verlag, Heidelberg, Barnard JA, Lyons RM and Moses HL: The cell biology of transforming growth factor beta. Biochim Biophys Acta 1032: 79-87, Heldin CH and Westermark B: Growth factors as transforming proteins. Eur J Biochem 184: , Browder TM, Dunbar CE and Nienhuls AW: Private and public autocrine loops in neoplastic cells. Cancer Cells 1:9-17, Lang RA and Burgess AW: Autocrine growth factors and tumorigenic transformation. Immunol Today 11: , Nugent MA, Lane EA, Keski-Oja J, Moses HL and Newman MJ: Growth stimulation, altered regulation of epidermal growth factor receptors, and autocrine transformation of spontaneously transformed normal rat kidney cells by transforming growth factor ß. Cancer Res 49: , Jennings MT, Maciunas RJ, Carver R, Bascom CC, Juneau P, Misulis K and Moses HL: TGFßl and TGFß2 are potential growth regulators for low-grade and malignant gliomas in vitro: evidence in support of an autocrine hypothesis. Int J Cancer 49: , 1991.