(CANCER RESEARCH 48. 6999-7003. December IS, 1988] Loss of Growth Factor Dependence and Conversion of Transforming Growth Factor-/?i Inhibition to Stimulation in Metastatic H-ras-transformed Murine Fibroblasts1

Lois C. Schwarz, Marie-Claude Gingras,2 Grant Goldberg, Arnold H. Greenberg,3 and Jim A. Wright4

Manitoba institute of Cell Biology, and Departments of Biochemistry [L. C. S., J. A. W.] and Immunology [M. C. G., A. H. G.J, The University of Manitoba, 100 Olivia Street, Winnipeg, Manitoba K3E OV9, Canada

ABSTRACT transfected cells are hypersensitive to TGF-0 (12), EGF, and bFGF (13). Furthermore, the effects of TGF-/3 on cells trans Cell lines with varying tumorigenic and metastatic potentials have been fected with the myc-oncogene are dependent on the entire set obtained by transformation of KIT1•¿fibroblastsusing radiation or trans- of GFs present at a given time (14). In contrast, ros-transfected fection with 1-24 H-ras. We have observed an inverse relationship cells can become insensitive to the effects of PDGF, EGF, between metastatic potential and dependence on serum for growth. The TGF-0 (12, 15), and bFGF (13), but are hypersensitive to the effects of basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, and transforming growth factor-/?i (TGF-0i) on effects of insulin-like GFs (13). It was postulated that ras- these lines were then examined to determine if the changes in the serum transfected cells secrete their own GFs leading to autocrine dependence of metastatic cells may be due to altered responsiveness to stimulation of growth (16). Ultimately, the secretion of GFs specific growth factors (GFs). Cells were grown in monolayer culture and may lead to down regulation of receptors for GFs making such DNA synthesis was measured by |CH3-3H]thymidine incorporation ex cells insensitive to exogenous GFs. /îas-transformed cells ex periments. Both metastatic and nonmetastatic cells were shown to be hibit enhanced metastatic potential (17-20), suggesting a direct equivalent in their diminished responsiveness to basic fibroblast growth role for the ras-oncogene in the development of métastases(17). factor, platelet-derived growth factor, and epidermal growth factor as compared to their nontransformed, parental KIT1.•cells.However, a In this study, we tested the hypothesis that the enhanced met astatic potential of a series of ros-transfected cells may be unique response of metastatic cells to TGF-/?i was identified. While TGF- ß,inhibited DNA synthesis in lOT'/i cells and a nonmetastatic tumor, partially due to altered growth factor sensitivity. cells with intermediate to high metastatic ability were stimulated up to 5.8-fold by TGF-01. Interestingly, epidermal growth factor abrogated the TGF-01 inhibition of the parental 101' 2 cells, but had no effect on the MATERIALS AND METHODS TGF-01 response of any metastatic line. Therefore, metastatic but not Cells and Culture Media. The selection and properties of the cell nonmetastatic cells, demonstrated a dramatically altered sensitivity to lines used in this study have been described elsewhere (17). In brief, TGF-01, a response which may be important in determining metastatic lOTVi mouse cells were transfected with T-24 H-ras. After transfection, potential. three cell lines, Cl, C2, and C3 were morphologically transformed while the two cell lines NR3 and NR4 were morphologically nontrans formed. NR3.1LA and NR3.1LB were derived from experimental lung INTRODUCTION métastases,and the NR3.4 cell line was obtained from a nonregressing GFs5 have been categorized into competence and progression S.C.tumor of NR3 cells. The MDS.R1 and MDS.R5 cell lines were clones of radiation-transformed lOT'/z cells (21). Both MDS.R1 and families. Competence factors such as PDGF or bFGF stimulate MDS.R5 cells were tumorigenic but nonmetastatic (17). quiescent BALB/C-3T3 cells to enter Gì(1, 2). Progression All cell lines were maintained at 37°Con 100-mm plastic tissue factors such as EGF and insulin, IGF-I or IGF-II, promote the culture plates (Falcon) in culture medium containing minimal essential transit of BALB/C-3T3 cells through G, and their entry into medium («-MEM) (Flow Laboratories) supplemented with antibiotics the S phase of the cell cycle (1, 3, 4). Competence factors act and 10% (V/V) FBS (GIBCO). A serum-free medium was also used before progression factors in BALB/C-3T3 cells. which contained 0.4 mg transferrin (Sigma Chemical Co.) and 0.2 mg insulin (Sigma Chemical Co.) in 100 ml of oc-MEM. In some experi GFs from the competence and progression families exert ments EGF (1 ^g/100 ml) (Collaborative Research) was added to the their effects by stimulating the expression of a discrete set of serum-free medium. genes which are important for the regulation of cellular prolif Assay for DNA Synthesis. To initiate experiments, medium was eration (5-11). GFs not only regulate the expression of genes removed when the cells were subconfluent. The cells were then washed involved in growth, but enhanced expression of such genes can with PBS. Medium containing 10% FBS, or serum-free medium with alter the responsiveness of cells to GFs. For example, myc- or without EGF, was added to the cells for 24 h. Following this incubation, the cells were always subconfluent and growing exponen Received 3/3/88; revised 7/21/88; accepted 9/16/88. tially. Cells were then removed with buffered trypsin solution (Sigma 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 Chemical Co.) and centrifuged. The pellet was suspended in the same accordance with 18 U.S.C. Section 1734 solely to indicate this fact. medium which was added to the cells 24 h before their removal with 1Supported by the National Cancer Institute of Canada and the Medical buffered trypsin solution. Cell number was then determined using a Research Council of Canada. 2Recipient of a Postdoctoral Studentship from the Manitoba Health Research model ZBI Coulter electronic particle counter (Coulter Electronics) and was found to range between 4 and 7x10* cells/plate. Cells were then Council. 3Terry Fox Cancer Research Scientist of the National Cancer Institute of seeded at various densities into 6-mm microtiter plates (Nunc). PDGF, Canada. TGF-/SI (R & D Systems Inc.) or bFGF (Collaborative Research) were 4 Senior Research Scientist of the National Cancer Institute of Canada. To added at various concentrations to triplicate cultures and the micro- whom requests for reprints should be addressed, at Manitoba Institute of Cell plates were incubated at 37°Cin the presence of 5% CC«2for42.5 h. Biology, 100 Olivia Street, Winnipeg, Manitoba, Canada R3E OV9. *The abbreviations used are: GFs, growth factors; <*-MEM, ex-minimal essen Cells were pulsed with [CH3-3H]thymidine (50 i/Ci/ml) for 2.0 or 24.0 tial medium; bFGF. basic fibroblast growth factor; Cl, CIRAS-1; C2, CIRAS-2; h before harvesting as indicated in the text. Growth medium was then C3, CIRAS-3; DM. defined or serum-free medium; EGF, epidermal growth factor; FBS, fetal bovine serum; IS. index of stimulation; IGF-I or -II, insulin-like growth removed and 0.3% buffered trypsin solution was added to the wells for factor I or II; PDGF, platelet-derived growth factor; TGF-«,transforming growth 15 min at 2 PC. Ice-cold trichloroacetic acid was then added to a final factor-oc; TGF-/3, transforming growth factor-/3. concentration of 10% for at least 30 min at 4°C.Cells were harvested 6999

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. TGF-tf, STIMULATION OF METASTATIC FIBROSARCOMAS using a 12-well harvester (Skatron Inc.) attached to a source of water for washing. Filters were dried and placed into 7.0 ml of Aquasol-2 100.0 C2 C3 (New England Nuclear). Radioactivity was determined by liquid scin tillation spectroscopy using a scintillation counter (Model LS 7800, NR3.1LA Beckman). An IS was calculated as: dpms from cells treated with GF/ i NR3.4 dpms from cells grown in the absence of GF as described by Schwarz NR3.1LB et al. (22). An IS of 2.0 or <0.5 was significant at P < 0.05 for individual •¿C1 10.0 values of the IS. Measurement of Doubling Time. All cell lines were maintained in 10% FBS. A constant number of cells (2.5 x IO4)was then transferred M to 60-mm plates (Falcon) in 2 ml Dulbecco's modified Eagle's medium CO M CO and nutrient F-12 mixture (GIBCO Laboratories) containing 0.5% FBS l NR4 (GIBCO). Proliferation of cells was followed each day by counting cells 1.0 removed with trypsin solution from duplicate plates using a /.»,Coulter electronic particle counter (Coulter Electronics). Growth curves were HI then prepared and doubling times were calculated from the linear portion of the curves (23). o> a x UJ 0.1 l NR3 RESULTS

Reduced Serum Dependence of Metastatic Cells. The met- •¿ astatic properties of lOT'/z cell lines which were transfected MDS.RI MDS.R5 10T1/2 with T-24 H-ras, or were radiation-transformed have been 20 30 40 50 90 100 110 120 described (17). A summary of the metastatic properties of these lines is presented in Table 1. It is important to note that the Doubling Time (hours) in 0.5% FBS 10T'/2 parental cells are not tumorigenic, whereas the MDS.R1 Fig. 1. The relationship between metastatic potential and serum requirements. Cells (2.5 x IO4) were plated onto 60-mm dishes in 2 ml Dulbecco's modified and MDS.R5 cell lines are fully tumorigenic but nonmetastatic. Eagle's medium/nutrient F-12 mixture containing 0.5% FBS. Proliferation of All the other cell lines demonstrated increasing potential for cells was followed each day by counting cells removed with trypsin solution from metastatic behaviour as listed in Table 1. duplicate plates using a Coulter counter. Doubling time was calculated from the linear portion of growth curves. Growth factor autonomy was initially characterized by deter mining their ability to grow under reduced serum concentra tions, in medium containing 0.5% FBS (Fig. 1). As cells became the metastatic cells demonstrated a reduced requirement for more metastatic their requirements for serum declined, re serum, it was next determined whether this was related to a flected by their decreased doubling times. For example, the loss of responsiveness to specific GFs. The effects of various most metastatic cells, C2 and C3, had the shortest doubling concentrations of bFGF and PDGF were determined on: non- times (23 and 26 h, respectively). In contrast, MDS.R5 and transformed parental cells (lOT'/z), tumorigenic but nonmetas MDS.R1 which are fully tumorigenic but nonmetastatic, re tatic cells (MDS.R5), cells of intermediate metastatic potential quired 93 and 45 h, respectively, to transit one cell cycle. These (NR4), and cells of high metastatic potential (C3). The effects doubling times more closely resembled the 101 h of the parental of bFGF and PDGF were examined using three different media: JOT1/:cells than the doubling times found for C2 and C3 cells. (a) oc-MEM containing 10% FBS; (b) serum-free medium This relationship between metastatic potential and doubling which contained 0.4 mg transferrin and 0.2 mg insulin in 100 times in 0.5% FBS was also maintained following standardi ml oc-MEM; (c) serum-free medium as in (b) but in the presence zation of doubling times, accomplished by comparing the of 1 tig EGF per 100 ml oc-MEM (Table 2). growth of each cell line in 10% FBS to that in 0.5% FBS (data Two competence growth factors, bFGF (20 ng/ml) and not shown). PDGF (100 ng/ml) had little effect on lOT'/z, MDS.R5, NR4, Responsiveness of Metastatic Cells to Growth Factors. Since and C3 cells in the presence of 10% FBS (Table 2). However, DNA synthesis in lOT'/i cells was stimulated by bFGF and Table 1 Tumorigenic and metastatic properties ofT24-H-ras-lransfected ¡OT^h PDGF in the serum-free medium (Table 2). The progression flbroblasts factor, EGF (10 ng/ml) further enhanced this response (Table The data was summarized from previously reported results (17). 2). In contrast to lOT'/z cells, all three tumor lines, regardless Experimental métastases' of metastatic potential, exhibited relatively little response to No. of lung bFGF or PDGF, in serum-free medium with or without EGF nodules (Table 2). Tumorigenicity (mean ± Cell line frequency" Frequency SE) The effects of TGF-/3, (10 ng/ml) on DNA synthesis were TWMDS.R5MDS.RINR3NR4ClNR3.4NR3.1LBNR3.1LAC2C30/125/55/56/810/1013/135/55/55/511/1111/110/120/60/31/1312/1920/274/46/66/68/814/140000.1determined in initial experiments using nontransformed, paren tal 10T'/2 cells and highly metastatic C3 cells (Fig. 2). lOT'/i cells were inhibited by TGF-/3, at all cell densities, and were ±0.12.0 ±0.514±516±540 inhibited or did not respond to TGF-/S| in the presence of EGF (Fig. 2). The metastatic C3 cells demonstrated a dramatically different response to TGF-/3i, which was a stimulation of DNA ±1849 ±7118 synthesis at all cell densities, with or without EGF (Fig. 2). ±6121 Since the growth response differences between lOTVi and C3 ±20 " Tumorigenicity was determined following s.c. injections of 3 x 10s cells, cells were similar at all cell densities, 15,000 cells/well was * Injections were performed using 3x10* cells except for ththe MDS.R5 and chosen for future experiments. To extend this study to include MDS.RI cells, where 10' cells were injected. other cell lines, the effects of TGF-/3i on a large number of 7000

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. TGF-01 STIMULATION OF METASTATIC FIBROSARCOMAS

Table 2 The effects ofbFGF (20.0 ng/ml) and PDGF (100.0 ng/ml) on DNA synthesis in lOT'h fibrosarcomas cultured in fetal bovine serum (FBS) or defined medium (DM) Parental, nontransformed 10TV: cells, transformed, nonmetastatic MDS.RS cells, cells of intermediate metastatic potential (NR4), or cells of high metastatic potential (C3) were exposed for 24 h to one of three different media. They were: 10% FBS in a-MEM, DM containing a-MEM plus insulin (2.0 jig/ml) and ; 6.0 tmuster riii (4.0 Mg/ml) or DM plus EGF (10.0 ng/ml). Cells were then removed I 50 with buffered trypsin solution and plated at 5000 cells/well. Either bFGF or 3 4.0 PDGF were then added and 42.5 h later, cells were pulsed with [3H]thymidine ° 3.0 <5>Ini1 (4>' for 2.0 h. 5 2.0 E i (7) (7) (91 (9)191 I : i 1.0 ~cr TIT GrowthlinebFGF factor Cell (meanno.)002121002121Media10% 0.75 lOT'/ibFGF FBSDM*DM 0.50 +EGF10% 0.25 O IOTl/2 MDS.RS NR3 NR4 CI C2 C3 NR3.4 NR3.ILA MDS.RSbFGF FBSDMDM Celi Lines +EGF10% Fig. 3. Effects of TGF-/3, (10.0 ng/ml) on DNA synthesis in fibroblast cell NR4bFGF FBSDMDM lines with or without EGF (10.0 ng/ml). Cells were grown to a density between 4 and 7 x 10' cells/100 mm plate in serum-free medium with (D) or without (D) +EGF10% EGF for 24 h. Subconfluent lOT'/z cells in serum-free medium were not stimulated by TGF-/SI at any cell density. However, cells which were stimulated by TGF-/3i C3PDGF FBSDMDM were most responsive to TGF-|Si within the range between 4 and 7x10* cells/ plate after the 24 h. Cells ( 15,000 cells/well) were then plated in the presence or +EGF10% absence of TGF-/3, for 42.5 h. Cells were then pulsed with [3H)thymidine for 2.0 h and an index of stimulation was determined as described in "Materials and 10T'/2PDGF FBSDMDM Methods." An IS value of 1.0 represents no effect. The number of experiments performed is indicated in parentheses. The SE was <10% for each experiment. +EGF10%

MDS.RSPDGF FBSDMDM response to TGF-ßiin the presence or absence of EGF (Fig. 3). In sharp contrast to lOT'/z or MDS.RS cells, lines which +EGF10% demonstrated moderate to high metastatic ability were stimu NR4PDGF FBSDMDM lated by TGF-/3i in all cases. This stimulation was statistically significant in the presence or absence of EGF (Fig. 3). Similar +EGF10% differences in TGF-01 response among the various cell lines C3Métastases FBSDMDM were also observed following a longer pulse of [3H]thymidine

+ EGFIS"2.519.0107.11.01.30.90.62.66.90.83.86.90.952.067.81.50.80.70.81.10.70.97.02.6for 24 h (data not shown). "The Index of Stimulation refers to the cpm's in the presence of GF/cpm's in the absence of GF. The effects ofbFGF were determined in 4 separate experiments using triplicate cultures for all cell lines in each experiment. The effects of PDGF DISCUSSION were determined in 2 separate experiments using triplicate cultures for all cell lines in each experiment. The SE was <10% in each experiment. * DM, defined medium, i.e., serum-free medium. In this report we have examined the effects of serum, bFGF, PDGF, EGF, and TGF-/3i on the growth of metastatic fibro- sarcomas in monolayer culture. This is the first demonstration of a correlation between the metastatic properties of cells and a reduced dependence on serum for cell proliferation. This decreased requirement for serum was also reflected in the poor responsiveness to specific serum growth factors such as bFGF, PDGF, and EGF. These growth factor responses did not distin guish between transformed metastatic and nonmetastatic tu mors. Indeed, the responses of these cell lines were similar to those reported for other ros-transformed cells (13, 15, 24, 25). This suggests that altered responsiveness of cells to some GFs 1 2 3 4 5 6 7 8 9101112131415 reflects the tumorigenic rather than the metastatic phenotype, Cell Number (x103) since the reduced responsiveness of all transformed cells oc curred independent of their mode of transformation and their Fig. 2. The effects of cell density on the responsiveness of fibroblast cell lines to TGF-/SI (10.0 ng/ml). Parental, nontransformed lOT'A cells ( ) or highly metastatic properties. metastatic C3 cells ( ) were used. Cells were equilibrated for 24.0 h in medium On the other hand, TGF-01 which was growth factor inhibi containing * -MEM plus insulin (2.0 ng 'ml) and transferrin (4.0 fig/ml), in the tory for nonmetastatic cell lines, was growth stimulatory for all presence or absence of EGF (10.0 ng/ml). Cells were then removed and replated at densities of 1,000, 5,000, or 15,000 cells/well. TGF-0, was then added to cells metastatic tumors examined. This altered response clearly dis growing in the presence (•)or absence (•)ofEGF for 42.5 h. Cells were pulsed with [3H]thymidine during the last 2.0 h. An index of stimulation (IS) was then tinguished cells of intermediate to high metastatic potential calculated as described in "Materials and Methods." An IS value of 1.0 represents from normal or very poorly metastatic cells. Normal parental no effect. Each point is the average of triplicate cultures. The SE was <10%. lOT'/z fibroblasts were also significantly inhibited by TGF-/3, (Fig. 3). This latter response is similar to that previously ob lines, which varied in their metastatic properties, were deter served for various normal epithelial cells such as those derived mined in several experiments. TGF-01 inhibited DNA synthesis from kidney (26), bronchus (27), skin (28), intestine (29), liver in MDS.RS cells which are tumorigenic but nonmetastatic. (30), and mammary gland (31). Cells of mesenchymal origin This inhibition occurred in the presence or absence of EGF such as fibroblasts from late gestational stage embryos are also (Fig. 3). Very poorly metastatic NR3 cells exhibited no obvious inhibited by TGF-/S (32). The lack of effect of TGF-fr on 7001

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. TGF-0, STIMULATION OF METASTATIC FIBROSARCOMAS tumorigenic but poorly metastatic fibrosarcomas in our study nism might be the result of modified responses to growth factors is similar to the response of epithelial cells following transfor produced by oncogene-induced changes at the DNA level. The mation. For example, TGF-/3 inhibited DNA synthesis in nor metastatic ra.v transformed cells used in this study have been mal human bronchial epithelial cells, however no effect of TGF- shown to have significantly enhanced levels of endogenous c- ßwasobserved in human lung carcinoma cells (27). In addition, myc (46). The c-myc gene is known to alter many growth factor although isolated normal hepatocytes are very sensitive to the induced responses (15) including TGF-ß(12, 14). One interest antiproliferative effect of TGF-ß,transfection of these cells ing observation, which is relevant to the present discussion is with the H-ras gene also confers resistance to this growth factor that c-myc transfection of PC 12 pheochromocytoma cells con (33). Effects of TGF-0 following transformation have not been verts a nerve growth factor-induced differentiation to a prolif previously determined on mesenchymal cells which are nor erative response (47). TGF-0 normally has the ability to induce mally inhibited by this growth factor. To our knowledge this is differentiation in a number of cell lines (27). This differentiation the first demonstration that TGP-ß\can stimulate metastatic is usually accompanied by inhibition of growth and the induc cells while inhibiting the parental line. There has been only one tion of a number of markers of differentiation, and in some other demonstration of a similar stimulatory response of trans cases altered morphology (48,49). For example, TGF-/3 inhibits formed cells in culture using lymphocytes derived from adult growth of endothelial cells while inducing their differentiation T-cell leukemias (34). All other reports have demonstrated a (40, 49, 51). However, the metastatic cell may now utilize the lack of response rather than a stimulatory effect of TGF-ßon TGF-0 as a growth factor instead of a differentiation signal as DNA synthesis following transformation. It should also be a consequence of the transformation events leading to metas noted that the inhibitory effect of TGF-0! on parental lOT'/z tasis formation. This proliferative response could clearly lead cells and the nonmetastatic tumor MDS.R5 cells was partially to an autocrine pathway that would promote the growth of abrogated with EGF. EGF did not significantly alter the re metastatic cells upon entry into secondary tissue sites. Since sponsiveness of any metastatic cells to TGF-01. The reasons for virtually all transformed cells have increased secretion of TGF- the lack of effect of EGF on metastatic cells are not known. ß(38, 43), this type of autocrine regulation may have general However, cells transfected with viruses harboring the ras-on significance for the aggressiveness of metastatic disease. cogene have been shown to secrete increased levels of TGF-oc In conclusion, we have shown that moderately or highly (35-38), which binds to the same receptor as EGF (39). The metastatic fibrosarcomas are growth stimulated by TGF-/S] continued secretion of endogenous TGF-oc and its subsequent while nonmetastatic transformed cells of identical cell lineage binding to the EGF receptor may prevent the occupancy of this were growth inhibited, similar to the nontransformed parental receptor by exogenous EGF and render the cells insensitive to cell lines. Future experiments will be directed towards deter added EGF. mining the mechanisms responsible for the altered responsive The molecular mechanisms responsible for the conversion of ness of metastatic cells to TGF-/3|. an inhibitory to a stimulatory signal by TGF-ßiare unknown. However, transformation by H-ras may affect TGF-0 respon siveness in several ways that might explain this observation: the Note Added in Proof type of TGF-/3 secreted, TGF-0 receptor expression, and mod ification of TGF-/3 signals at the DNA level, could all alter the A recent report by D. Chadwick and A. Lagarde (JNCI 80: 318, 1988) confirms the growth factor independence of metastatic tumors. response to exogenous TGF-ßi.TGF-/3 is produced in at least two forms, TGF-|Si and TGF-/32 (40, 41), and the cellular responses to these types of TGF are not identical. Indeed, there REFERENCES is some evidence that tissue specific responses are unique to each type. Xenopus mesenchymal differentiation is responsive 1. Pledger, W. J., Stiles, C. D., Antoniades, H. N., and Scher, C. D. An ordered only to TGF-fo (42). Since an increased secretion of TGF-0 is sequence of events is required before BALB/C-3T3 cells become committed to DNA synthesis. Proc. Nati. Acad. Sci. USA, 75: 2839-2843, 1978. ubiquitous to virtually all transformed cells (38, 43), it is 2. Lobb, R. R., and Fett, J. W. Purification of two distinct growth factors from possible that altered secretion rates of one type of TGF-/3 may bovine neural tissue by heparin affinity chromatography. Biochemistry, 23: 6295-6299, 1984. occur which would then modify the response to exogenous 3. Leof, E. B., Wharton, W., Van Wyk, J. J., and Pledger, W. J. Epidermal TGF-01. That is, the combination of enhanced secretion of one growth factor (EGF) and somatomedin C regulate d progression in com type of TGF-/3 by metastatic cells plus exogenous TGF-01 may petent BALB/C-3T3 cells. Exp. Cell Res., 141: 107-114, 1982. 4. Campisi, J., and Pardee, A. B. Post-transcriptional control of the onset of lead to alterations in the ratio of TGF-ßito TGF-/32 available DNA synthesis by insulin-like growth factor. Mol. Cell. Biol., 4:1807-1814, to the cell during the period of the assay. The second mechanism 1984. 5. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. Cell-specific regulation of altered response to TGF-/3 by metastatic cells could be an of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. alteration in receptor expression for TGF-0] and TGF-fo- Loss Cell, 35:603-610,1983. of TGF-j3i growth inhibitory responses has been suggested as a 6. Cochran, B. H., Zullo, J., Verma, I. M., and Stiles, C. D. Expression of the c-fos gene and of a ybs-related gene is stimulated by platelet-derived growth mechanism for promoting the transformation event (33), and factor. Science (Wash, DC), 226: 1080-1082, 1984. retinoblastoma cell lines have been shown to lose their TGF-0 7. Greenberg, M. E., and /ill. E. B. Stimulation of 3T3 cells induces transcrip tion of the c-fos proto-oncogene. Nature (Lond.), 311:433-437, 1984. receptor (44). Since metastatic cells acquire a proliferative 8. Rollins, B. J., Morrison, E. D., and Stiles, C. D. A cell-cycle constraint on response rather than simply losing a growth inhibitory re the regulation of gene expression by platelet-derived growth factor. Science sponse, the loss of TGF-/3 receptor is not a likely explanation. (Wash, DC), 238: 1269-1271, 1987. 9. Bouche, G., Gas, N., Prats, H., Baldin, V., Tauber, J.-P., Teissie, L, and One must postulate the appearance of a new type of receptor Amalric, F. Basic fibroblast growth factor enters the nucleolus and stimulates which is capable of exhibiting a modified response. Since TGF- the transcription of ribosomal genes in ABAE cells undergoing G0/Gi trans ition. Proc. Nati. Acad. Sci. USA, 84: 6770-6774, 1987. ß,and & likely do not have the identical receptor (45), it is 10. Takehara, K., LeRoy, E. C., and Grotendorst, G. R. TGF-/3 inhibition of possible that either the acquisition of a new receptor type or endothelial cell proliferation: alteration of EGF binding and EGF-induced increased receptor numbers and affinity associated with conver growth-regulating (competence) gene expression. Cell, 49: 415-422, 1987. 11. Leof, E. B., Proper, J. A., Goustin, A. S., Shipley, G. D., DiCorleto, P. E., sion to the metastatic phenotype may lead to a qualitatively and Moses, H. L. Induction of c-sis mRNA and activity similar to platelet- altered response to exogenous TGF-/3i. Finally, a third mecha derived growth factor by transforming growth factor ft: a proposed model for 7002

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. TGF-S, STIMULATION OF METASTATIC FIBROSARCOMAS

indirect mitogenesis involving autocrine activity. Proc. Nati. Acad. Sci. USA, 31. Silberstein, G., and Daniel, C. Reversible inhibition of mammary gland 83: 2453-2457, 1986. growth by transforming growth factor-beta. Science, 237: 291-293, 1987. 12. Leof, E. B., Proper, J. A., and Moses, H. L. Modulation of transforming 32. Hill, D. J., Strain, A. J., Elstow, S. F., Sweene, I., and Milner, R. D. G. Bi growth factor type ßactionby activated ras and c-myc. Mol. Cell. Biol., 7: functional action of transforming growth factor-beta on DNA synthesis in 2649-2652,1987. early passage human fetal fibroblasts. J. Cell. Physiol., ¡28:322-328, 1986. 13. Balk, S. D., Riley, T. M., Günther,H. S., and Morisi, A. Heparin-treated, v- 33. Houck, K. A., Strom, S. C., and Michalopoulos, G. K. Resistance to the /mr transformed chicken heart mesenchymal cells assume a normal mor growth inhibitory effect of transforming growth factor beta (TGF-beta) is phology but are hypersensitive to epidermal growth factor (EGF) and brain induced by transfection of an activated H-ras oncogene into rat liver epithelial fibroblast growth factor (bFGF); cells transformed by the v-H-ros oncogene cells (RLEC). Proc. Am. Assoc. Cancer Res., 28:64, 1987. are refractory to EGF and bFGF but are hypersensitive to insulin-like growth 34. Niitsu, Y., Urushizaki, Y., Koshida, Y., Temi, K., Mahara, K., Kohgo, Y., factors. Proc. Nati. Acad. Sci. USA, 82: 5781-5785, 1985. and Urushizaki, I. Expression of TGF-beta gene in adult T cell leukemia. 14. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. Blood, 71: 263-266, 1988. F., and Spom, M. B. Type ßtransforming growth factor; a bifunctional 35. DeLarco, J. E., and Todaro, G. J. Growth factors from murine sarcoma regulator of cellular growth. Proc. Nati. Acad. Sci. USA, 82:119-123,1985. virus-transformed cells. Proc. Nati. Acad. Sci. USA, 75:4001-4005, 1978. 15. Stern, D. F., Roberts, A. B., Roche, N. S., Sporn, M. B., and Weinberg, R. 36. Kaplan, P. L., Anderson, M., and Ozanne, B. Transforming growth factor! s) A. Differential responsiveness of myc- and roj-transfected cells to growth production enables cells to grow in the absence of serum: an autocrine system. factors: selective stimulation of myc-transfected cells by epidermal growth Proc. Nati. Acad. Sci. USA, 79:485-489, 1982. factor. Mol. Cell. Biol., 6: 870-877, 1986. 37. Ozanne, B. R., Fulton, R. J., and Kaplan, P. L. Kirsten murine sarcoma 16. Todaro, G. J., and DeLarco, J. E. Growth factors produced by sarcoma virus- virus transformed cell lines and a spontaneously transformed rat cell line transformed cells. Cancer Res., 38:4147-4154, 1978. produce transforming growth factors. J. Cell. Physiol., 105: 163-180, 1980. 17. Egan, S. E., McClarty, G. A., Jarolim, L., Wright, J. A., Spiro, I., Hager, 38. Jakowlew, S. B., Kondaiah, P., Flanders, K. C., Thompson, N. L., Dillard, G., and Greenberg, A. H. Expression of H-ras correlates with metastatic P. J., Sporn, M. B., and Roberts, A. B. Increased expression of growth factor potential: evidence for direct regulation of the metastatic phenotype in lOT'/z mRNAs accompanies viral transformation of rodent cells. Oncogene Res., 2: and NIH 3T3 cells. Mol. Cell. Biol., 7: 830-837, 1987. 135-148, 1988. 18. Greig, R. G., Koestler, T. P., Trainer, D. L., Corwin, S. P., Miles, L., Kline, 39. Massague, J. Epidermal growth factor-like transforming growth factor. II. T., Sweet, R., Yokoyama, S., and Poste, G. Tumorigenic and metastatic Interaction with epidermal growth factor receptors in human placenta mem properties of "normal and ros-transfected NIH/3T3 cells. Proc. Nati. Acad. branes and A431 cells. J. Biol. Chem., 258: 13614-13620, 1983. Sci. USA, 82: 3698-3701, 1985. 40. Seyedin, S. M., Thomas, T. C., Thompson, A. Y., Rosen, D. M., and Piez, 19. Liona, L. A. Tumor invasion and metastasis-role of the extracellular matrix. K. A. Purification and characterization of two cartilage-inducing factors from Cancer Res., 46: 1-7, 1986. bovine demineralized bone. Proc. Nati. Acad. Sci. USA, 82: 2267-2271, 20. Muschel, R. J., Williams, J. E., Lowy, D. R., and Liotta, L. A. Harvey ras 1985. induction of metastatic potential depends upon oncogene activation and the 41. Seyedin, S. M., Segarmi, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., type of recipient cell. Am. J. Pathol., 121: 1-8, 1985. and Graycar, J. Cartilage-inducing Factor B is a unique protein structurally 21. Raaphorst, G. P., Vadasz, J. A., Azzam, E. I., Sargent, M. D., Borsa, J. and and functionally related to transforming growth factor-/?. J. Biol. Chem., Einspenner, M. Comparison of heat and/or radiation sensitivity and mem 262: 1946-1949,1987. brane composition of seven x-ray-transformed C3H lOT'/j cell lines and 42. Rosa, F., Roberts, A. B., Danielpour, D., Dart, L. L., Sporn, M. B., and normal C3H 10TO cells. Cancer Res., 45: 5452-5456, 1985. Dawid, I. B. Mesoderm induction in amphibians: the role of TGF-ft-like 22. Schwarz, L. C., Makowka, L., Falk, J. A„andFalk, R. The characterization factors. Science (Wash, DC), 239: 783-785, 1988. and partial purificai ion of hepatocyte proliferation factor. Ann. Surg., 202: 43. Anzano, M. A., Roberts, A. B., DeLarco, J. E., Wakefield, L. M., Assoian, 296-302, 1985. R. K., Roche, N. S., Smith, J. M., Lazarus, J. E., and Sporn, M. B. Increased 23. Ceri, H., and Wright, J. A. A correlation between concanavalin A resistance secretion of type ßtransforming growth factor accompanies viral transfor and specific alterations in growth and surface membrane-associated proper mation of cells. Mol. Cell. Biol., 5: 242-247, 1985. ties. Exp. Cell Res., 114: 217-227, 1978. 44. Kimchi, A., Wang, X.-F., Weinberg, R. A., Cheifetz, S., and Massague, J. 24. Kalekar, A., and Cole, M. D. Immortalization by c-myc, H-ras and Eia Absence of TGF-/3 receptors and growth inhibitory responses in retinoblas- toma cells. Science (Wash, DC), 240: 196-199, 1988. induces differential cellular gene expression and growth factor responses. Mol. Cell. Biol., 7: 3899-3907, 1987. 45. Cheifetz, S., Weatherbee, J. A., Tsang, M. L.-S., Anderson, J. K., Mole, J. E., Lucas, R., and Massague, J. The transforming growth factor ,¡system, a 25. Tubo, R. A., and Rheinwald, J. G. Normal human mesothelial cells and complex pattern of cross-reactive ligands and receptors. Cell, 48: 409-415, fibroblasts transfected with the EJ nu oncogene become EGF-independent, but are not malignantly transformed. Oncogene Res., /: 407-421, 1987. 1987. 46. Egan, S. E., McClarty, G. A., Broere, J., Jarolim, L., Wright, J. A., and 26. Tucker, R. F., Shipley, G. D., Moses, H. L., and Holley, R. W. Growth inhibitor from BSC-1 cells closely related to platelet type .¡transforming Greenberg, A. H. Enhanced expression of complementary oncogenes in growth factor. Science (Wash, DC), 226: 705-707, 1984. highly metastatic cells. Fourth Annual Meeting on Oncogenes, p. 214, 1988. 47. Maruyama, K., Schiavi, S. C., Huse, W., Johnson, G. L., and Ruley, H. E. 27. Masui, T.. Wakefield, L. M., Lechner, J. F., La Veck, M. A., Sporn, M. B., Myc and I.In oncogenes alter the responses of PC 12 cells to nerve growth and Harris, C. C. Type ßtransforming growth factor is the primary differ factor and block differentiation. Oncogene Res., /: 361-367, 1987. entiation-inducing serum factor for normal human bronchial epithelial cells. 48. Heine, U. L, Muñoz,E. F., Flanders, K. C., Ellingsworth, L. R., Lam, H.-Y. Proc. Nati. Acad. Sci. USA, 83:2438-2442, 1986. P., Thompson, N. L., Roberts, A. B., and Sporn, M. B. Role of transforming 28. Shipley, G. D.. Pittelkow, M. R., Wille, J. J., Scott, R. E., and Moses, H. L. growth factor-/? in the development of the mouse embryo. J. Cell Biol., 105: Reversible inhibition of normal human prokeratinocyte proliferation by type 2861-2876, 1987. ßtransforming growth factor-growth inhibitor in serum-free medium. Cancer 49. Madri, J. A., Pratt, B. M., and Tucker, A. Phenotypic modulation of Res., 46:2068-2071, 1986. endothelial cells by transforming growth factor-J depends upon the compo 29. Kurokowa, M., Lynch, K., and Podolsky, D. K. Effects of growth factors on sition and organization of the extracellular matrix. J. Cell Biol., 106: 1375- an intestinal epithelial cell line: transforming growth factor beta inhibits 1384, 1988. proliferation and stimulates differentiation. Biochem. Biophys. Res. Com 50. Prater-Schroder, M., Muller, G., Birchmeier, W., and Bohlen, P. Transform mun., 142: 775-782, 1987. ing growth factor-beta inhibits endothelial cell proliferation. Biochem. Bio 30. McMahon. J. B., Richards. W. L., del Campo, A. A., Song, M.-K., and phys. Res. Commun., 137: 295-302, 1986. Thorgiersson, S. Differential effects of transforming growth factor-/? on 51. Muller, G., Behrens, J., Nussbaumer, U., Bohlen, P., and Birchmeier, W. proliferation of normal and malignant rat liver epithelial cells in culture. Inhibitory action of transforming growth factor-/? on endothelial cells. Proc. Cancer Res., 46:4665-4671, 1986. Nati. Acad. Sci. USA, 84:5600-5604. 1987.

7003

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. Loss of Growth Factor Dependence and Conversion of Transforming Growth Factor- β1 Inhibition to Stimulation in Metastatic H- ras-transformed Murine Fibroblasts

Lois C. Schwarz, Marie-Claude Gingras, Grant Goldberg, et al.

Cancer Res 1988;48:6999-7003.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/24_Part_1/6999

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/48/24_Part_1/6999. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research.