Jun 23, 1986 - Angelika Gebhardt, John C.Bell1 and J.Gordon Foulkes. Laboratory of ... evidence for non-autocrine mechanisms (Cook et al., 1985; Pierce.
The EMBO Journal vol.5 no.9 pp.2191-2195, 1986
Abelson transformed fibroblasts lacking the EGF receptor tumourigenic in nude mice
Angelika Gebhardt, John C.Bell1 and J.Gordon Foulkes Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK, and 'Department of Biochemistry, McGill University, Montreal, Quebec, H36 1Y6, Canada Communicated by R.R.Friis
Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor oaTGF. caTGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by aTGF as opposed to the v-abl-encoded proteintyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild-type fibroblast lines. However, in contrast to the wild-type lines, v-abl-transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for aTGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas. Key words: Abelson murine leukaemia virus/cell transformation/EGF receptor/tumourigenicity/tumour growth factors Introduction A classical parameter of transformed cells in culture is their ability to grow in the presence of reduced amounts of exogenous growth factors. In 1980, Sporn and Todaro proposed that transformed cells might produce their own growth factors which would act back on the secreting cell. This was termed the autocrine hypothesis (Spom and Todaro, 1980). Since that time a variety of such factors, termed tumour growth factors (TGFs), have been isolated (for review, see Goustin et al., 1986). A number of TGFs have been shown to be related to normal peptide hormones. Thus, aTGF shows homology to the epidermal growth factor (EGF) (Todaro et al., 1976; Marquardt et al., 1983, 1984), while the product of the sis oncogene is homologous to the platelet-derived growth factor (PDGF) (Doolittle et al., 1983; Waterfield et al., 1983). The addition of TGFs to normal cells induces a variety of properties which are characteristic for the transformed phenotype, including an altered cell morphology, the loss of contact inhibition and the ability to grow in semi-solid media (Proper et al., 1982). Several reports have demonstrated the importance of autocrine stimulation in cell transformation. For example, antibodies to PDGF can revert transformation by the v-sis oncogene (Johnsson et al., 1985) while antibodies to the growth hormone bombesin block the ability of a human small cell lung carcinoma to form tumours in nude mice (Cuttitta et al., 1985). Previously we have focused our attention on the oncogene of the Abelson murine leukaemia virus, v-abl, which encodes a protein-tyrosine kinase (Witte et al., 1980; Foulkes et al., 1985), © IRL Press Limited, Oxford, England
are
not
an activity which correlates closely with the ability of v-abl to transform cells (Prywes et al., 1985). Cells transformed by the v-abl kinase also produce a number of TGFs, including ceTGF (Twardzik et al., 1982). Thus, the phenotype of v-abltransformed cells may reflect the combined action of the intracellular v-abl kinase and secreted TGFs. There is no evidence, however, that autocrine stimulation is essential for transformation by v-abl. On the contrary, three recent publications give evidence for non-autocrine mechanisms (Cook et al., 1985; Pierce et al., 1985; Oliff et al., 1985). In all three cases, cell lines dependent on interleukin 3 (IL-3) for growth in tissue culture lose their IL-3 requirement after infection with the Abelson virus. These cell lines neither secrete IL-3 like growth factors nor do they show a significant change in the number of IL-3 receptors. These data point to an interaction of the v-abl kinase with the IL-3-stimulated pathway independent of the action of secreted IL-3-like TGFs. However, this does not exclude an important role for other types of TGFs. To delineate events in v-abl-transformed fibroblasts mediated by aTGF, we have employed a variant of the Swiss 3T3 cell line, NR6 (Pruss and Herschman, 1977). NR6 cells do not express the EGF receptor and are unable to respond therefore to either EGF or aTGF (Schneider et al., 1986). In this paper we describe the establishment of a v-abltransformed NR6 cell line. A striking difference was found in the tumourigenicity of these v-abl-transformed NR6 cells in comparison with two v-abl-transformed wild-type lines.
Results Transformation of NR6 cells by v-abl NR6 and wild-type Swiss 3T3 cells (3T3K) were tested for their ability to be transformed using virus prepared from a v-abltransformed NIH3T3 cell line. This producer line, AB14(2)/NIH, was obtained by co-transfection of a plasmid coding for the 14 N-terminal amino acids of the gag sequence coupled to the minimal fibroblast transforming region of the v-abl gene (Prywes et al., 1983, 1985) with the murine leukaemia helper virus plasmid pZAP (Goff et al., 1982). Foci of morphologically transformed cells could be detected in both cell lines with the same frequency after 3 -4 days. After 3 weeks, transformed cells were washed off the dish and grown to mass culture. Transformation parameters Abelson-infected NR6 cells [AB14(2)/NR6] show the same rounded morphology as the tranformed wild-type lines AB14(2)/NIH (Figure 1) and AB14(2)/3T3K (not shown). All three lines express a similar amount of the active Abelson kinase, as determined by immunoprecipitation (Figure 2), in vitro kinase activity using angiotensin II as a substrate (40 pmol 32P/min/mg cell protein) and the level of intracellular protein-bound phosphotyrosine (1-2% of total phosphoamino acids). A reduction in the level of cell surface fibronectin was observed in all three transformed cell lines [Figure 3A-D; data not shown for AB14(2)/3T3K]. Furthermore, the cellular protein p53, which 2191
A.Gebhardt, J.C.Bell and J.G.Foulkes
Fig. 1. Morphology of v-abl-transformed wild-type and variant fibroblasts. (A) NIH 3T3; (B) NR6; (C) AB14(2)/NIH; (D) AB14(2)/NR6.
is essential for the tumourigenicity of v-abl-induced lymphoid tumours (Wolf et al., 1984) is elevated in each of our transformed lines [Figure 3E/F; only NIH 3T3 and AB14(2)/NIH cells are shown as representative lines]. In addition, their cloning efficiency in semi-solid media, which is widely regarded as the best in vitro criteria for tumourigenicity in vivo (Shin et al., 1975), is virtually identical ( -30%). Hence by a variety of in vitro criteria, we have been unable to distinguish v-abl-transformed NR6 cells from the corresponding wild-type lines. These results demonstrate that the stimulation of the EGF receptor by caTGF is not required for the transformation of cells by v-abl in tissue culture. Tumourigenicity of v-abl-transformed NR6 cells A striking difference between Abelson-transformed NR6 cells and transformed wild-type fibroblasts was revealed when we analysed their ability to induce tumours in athymic nude mice. Whereas 2.5 x 105 AB14(2)/NIH or AB14(2)/3T3K cells injected s.c. into the inguinal region induced tumours in NIMR:nu/nu mice after only 10 days, no tumours could be detected in these mice after 90 days following injection of up to 5 x 106 AB14(2)/NR6 cells (Table I). This suggests that the inability of v-abl-transformed NR6 cells to respond to oTGF/EGF results in the failure of these cells to establish tumours in vivo. In only two cases tumours arose as a consequence of the injection of AB14(2)/NR6 cells after 86 and 90 days, respectively. In contrast to all the other tumours, these tumours, termed T2 and T4, formed not at the site of injection but in the musculature of the hind leg in the region of the lower lumber vertebrae. Taken into culture, these cells were found to express v-abl but proved to be highly tumourigenic when injected back into nude mice. Similarly, untransformed NR6 cells led to 2192
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Fig. 2. Immunoprecipitation of the Abelson protein. Lysates from [32p]orthophosphate-labelled NIH 3T3 (lane 1), AB14(2)/NIH (lane 2), AB14(2)/NR6 (lane 3) and AB14(2)/3T3K (lane 4) were precipitated with anti-Abelson serum as described in Materials and methods.
Role of EGF receptor in v-abl-induced tumours
Fig. 3. Immunofluorescence of normal and transformed cells. NIH 3T3 (A,E), AB14(2)/NIH (B,F), NR6 (C) and AB14(2)/NR6 cells (D) were permeabilised with Triton X-100 and treated with antibodies against either fibronectin (A-D) or p53 (E,F) as described in Materials and methods.
tumour formation in two cases after a prolonged period. In contrast to v-abl, NR6 cells tranformed by the activated N-ras oncogene were highly tumourigenic when injected into NIMR:nu/nu mice (Table I).
Discussion Cells transformed by the v-abl oncogene, like many other transformed cells, produce a number of tumour growth factors, including aTGF (Twardzik et al., 1982). Addition of medium conditioned by v-abl-transformed cells to normal fibroblasts induces a variety of properties characteristic of the transformed phenotype, including an altered cell morphology, the loss of contact inhibition, as well as the ability to grow in semi-solid media (Gebhardt, unpublished observations). The various alterations occurring in the process of transformation by the v-abl oncogene may, in part, be due to autocrine responses to these secreted growth factors.
To delineate events in v-abl-transformed fibroblasts mediated by ctTGF, we have employed the Swiss 3T3 variant cell line NR6, which lacks the EGF receptor and is unable to respond therefore to either EGF or aTGF (Pruss and Herschman, 1977; Schneider et al., 1986). The comparison of v-abl-transformed NR6 and two transformed wild-type fibroblast lines revealed no difference in a variety of in vitro transformation parameters. Firstly, all three lines expressed the same amount of active Abelson kinase, as determined by immunoprecipitation of 32P-labelled protein (Figure 1), in vitro kinase assays and the level of intracellular protein-bound phosphotyrosine. Secondly, all three transformed lines showed the same reduced level of fibronectin (Figure 3A-D) as well as elevation of the cellular protein p53 (Figure 3E,F), which as been shown to be essential for the tumourigenicity of v-abl-induced lymphoid tumours (Wolf et al., 1984). Finally, their cloning efficiency in semi-solid media, which is widely regarded as the best in vitro criteria for tumourigenicity in vivo
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A.Gebhardt, J.C.Bell and J.G.Foulkes Table I. Tumourigenicity of fibroblasts in NIMR:nu/nu mice Final tumour size/time of sacrifice
Cell type
Cell no./site
No. of sites
No. of tumours
Latency
NIH 3T3 AB14(2)/NIH AB14(2)/NIH AB14(2)/NIH AB14(2)/NIH
x 106 1 2.5 X 105 X 105 S 1.25 x 106 2.5 x 106
4 4 4 8 4
0 4 4 8 4
>90 days 10 days 8 days 8 days 8 days
(Swiss 3T3K) AB14(2)/3T3K AB14(2)/3T3K
X 106 1 2.5 X 106 S x 106
4 6 2
0 6 2
>90 days 8 days 8 days
-
NR6 NR6 AB14(2)/NR6
5 1 5 1
x 105 x 106 x 105 X 106 1.25 x 106 2.5 x 106 x 106 5 2.5 x 106 2. 5 x 106 2.5 x 106
4 4 2 4 4 8 2 4 4 4
1 1 0 1 0 1 0 4 4 4
90 days 90 days >90 days 86 days >90 days 90 days >90 days 8 days 8 days 8 days
0.25 cm/109 days 0.25 cm/109 days
AB14(2)/NR6 AB14(2)/NR6 AB14(2)/NR6 AB14(2)/NR6 AB14(2)/NR6-T2 AB14(2)/NR6-T4 NR6/ras
(Shin et al., 1975), was virtually identical for all three transformed cell lines. However, we observed a striking difference between our different v-abl-transformed lines when they were tested for their ability to induce tumours in athymic nude mice. Whereas the transformed wild-type lines induced tumours after only 8 days, no tumours could be detected within 90 days after injection of up to 10 times more AB14(2)/NR6 cells (Table I). After 90 days, two tumours appeared in mice injected wth v-abl-transformed NR6 cells. However, we also observed tumour formation in two mice after a prolonged period following injection of untransformed NR6 cells. This demonstrates that NR6 cells can undergo changes leading to a tumourigenic phenotype. There are a number of possible explanations for the lack of tumourigenicity of v-abl-transformed NR6 cells. Firstly, this phenomenon may reflect the different availability of growth factors present in vivo versus in vitro, i.e. in the effective absence of aTGF stimulation, the in vivo milieu could act to inhibit the growth of v-abl-transformed NR6 cells. For example, depending on the balance between different growth factors, ,BTGF can act as bi-functional regulator to either accelerate or inhibit cell growth (Anzano et al., 1982; Tucker et al., 1984; Roberts et al., 1985). Alternatively, certain cell types may require an activated EGF receptor for the establishment of tumours in vivo. This hypothesis is supported by the observation that tumour formation by several epidermal cell lines can be blocked by monoclonal antibodies against the EGF receptor (Masui et al., 1984). There is also data correlating expression of the EGF receptor with the metastatic potential of human breast (Sainsbury et al., 1985) and bladder cancer (Neal et al., 1985). Thus it is tempting to speculate that stimulation of the EGF receptor by ctTGF fulfils an essential function in the establishment of fibrosarcomas involving the v-abl oncogene, e.g. in angiogenesis (Folkman, 1985), by changes in extracellular matrix proteins (Chen et al., 1977; McCarthy et al., 1985; Skantze et al., 1985) or via stimulating the secretion of plasminogen activator (Lee and Weinstein, 1978) or other proteinases (Eaton and Baker, 1983). If aTGF acts by stimulating the kinase activity of the EGF receptor, one would predict the existence of certain phosphotyrosyl proteins in v-abl-transformed cells induced by aTGF. We have isolated phosphotyrosyl pro-
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1.5 1.5 1.5 1.5
cm/ cm/ cm/ cm/
21 21 21 21
days days days days
1.5 cm/ 42 days 1.5 cm/ 42 days
-
1.5 cm/109 days (termed T2) -
1.5 cm/109 days (termed T4) -
1.5 cm/ 21 days 1.5 cm/ 21 days >1.5 cm/ 21 days
teins from both v-abl-transformed NIH 3T3 and NR6 cells but to date have failed to observe any significant differences. Although NR6 cells were selected specifically using EGF and colchicine (Pruss and Herschman, 1977), it remains a theoretical possibility that NR6 cells contain a second mutation and that the lack of v-abl NR6 tumourigenicity is unrelated to the EGF receptor lesion. Formal proof for the involvement of this receptor would be the re-introduction of the cloned EGF receptor gene. To date, however, it has not proved possible to obtain expression of this gene in NR6 cells (M.Waterfield, personal communication). Given that NR6 cells containing the activated N-ras oncogene are tumourigenic in nude mice (Table I and MaKay et al., 1986), it will be interesting to analyse whether cells transformed by other members of the protein-tyrosine kinase family have a requirement for an activated EGF receptor in tumour formation. Finally, this work provides another example of the way several oncoproteins interact along the multistep pathway leading to the appearance of the malignant phenotype.
Materials and methods Cells and viruses
NIH 3T3, 3T3K and all transformed lines were maintained in Dulbecco's modified Eagles medium (DMEM), containing 10% newborn calf serum. NR6 cells were cultured in MEM Alpha medium, supplemented wth 10% newborn calf serum. For transformation of NR6 and 3T3K cells with Abelson virus, 5 x I05 cells were subcultured onto a 10 cm dish, and infected 24 h later with 2 ml of filtered supernatant (0.45 ltm) from the Abelson producer cell line AB14(2)/NIH. Virus was allowed to absorb for 2 h before a change of medium. Fresh medium was added to the cultures every second day for 3 weeks at which time transformed cells were washed off, grown to mass culture and used for all subsequent experiments. Immunoprecipitation of the Abelson transforming protein Confluent dishes were labelled for 3 h with 200 ,Ci/ml [32P]orthophosphate in phosphate-free medium, washed once with phosphate-buffered saline (PBS) and lysed for 2 min on ice in RIPA [20 mM Na-phosphate, pH 7.2, 50 mM NaF, 5 mM EDTA, 100 ,uM Na-vanadate, 100 mM NaCl, 0.1 % SDS, 1 % Na-deoxycholate, 1% Triton X-100, 2 mM phenylmethylsulfonylfluoride, 100 kallikrein units of Aprotinin (Sigma) per ml]. Lysates were cleared by centrifugation, normalised for trichloroacetic acid-precipitable counts and pre-cleared with 20 M1 of normal rabbit serum (NRS) and 200 i1 of 10% Staphylococcus aureus bacteria (Kessler, 1975). The subsequent precipitations were performed with 5 $1 of antiAbelson serum and 25 A1 of bacterial suspension. Immunocomplexes were washed
Role of EGF receptor in v-abl-induced tumours
four times with RIPA and boiled for 1 min in sample buffer (80 mM Tris/HCI, pH 6.8, 2% SDS, 10% glycerol). SDS-polyacrylamide gel electrophoresis in a 10% slab gel was performed as described (Laemmli, 1970) and the dried gel subjected to autoradiography. v-abl protein-tyrosine kinase activity The v-abl kinase activity in cell extracts was determined as described previously (Foulkes et al., 1985; Fry et al., 1985). Two-dimensional phosphoamino acid analysis was performed by the method of Hunter and Sefton (1980). Immunofluorescence Cells grown on coverslips were fixed with ice-cold methanol for 10 min, permeabilised with I % Triton X-100 in PBS for 30 min and then washed with several changes of PBS. The first antibody directed towards either fibronectin or p53 was slotted onto the appropriate coverslip and incubated at room temperature for I h. Coverslips were washed three times for 5 min each and then incubated for I h with FITC-conjugated second antibody (sheep anti-rabbit IgG). Coverslips were washed repeatedly with PBS and then mounted in 40% glycerol before viewing with epifluorescence optics. Cloning of cells in soft agarose 103 cells were suspended into 4 ml of medium containing 0.15% agarose and plated onto a layer of 4 mnl of medium containing 0.3 % agarose. Foci were scored after 3 weeks. Assay for in vivo tumourigenicity The indicated numbers of cells in Table I were injected into each of two inguinal s.c. sites of NIMR:nu/nu mice.
Acknowledgements We wish to thank Drs I.McKay and A.Hall (Institute of Cancer Research, London, UK) for their N-ras transformed NR6 line. We thank Drs A.I.Magee, R.Morris (NIMR) and D.Lane (ICRF) for their gifts of antibodies against fibronectin and p53, and FITC-conjugated sheep anti-rabbit IgG, respectively. This work was supported by the Medical Research Council, UK. A.G. is a recipient of a fellowship from the Deutsche Forschungsgemeinschaft. J.C.B. is supported by a fellowship from the Canadian National Cancer Institute.
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