Expression in rat fibroblasts of a human transforming growth factor-α cDNA results in transformation

Cell, Vol. 46, 301-309,

July 18, 1986, Copyright

0 1986 by Cell Press

Expression in Rat Fibroblasts of a Human Transforming G rowth Factor-a cDNA Results in Transformation Arnon Rosenthal, Patricia B. Lindquist, Timothy S. Bringman, David V. Goeddel, and Rik Derynck Department of Molecular Biology Genentech, Inc. 460 Point San Bruno Boulevard South San Francisco, California 94080

Summary Acquisition of the ability to produce and respond to a growth factor may result in increased cellular proliferation and could lead to malignant transformation. The fact that a large variety of tumor cells secrete transforming growth factor-a (TGF-a) suggests involvement of TGF-a in cellular transformation and provides supporting evidence for the autocrine stimulation model. In order to determine directly the role of TGF-a in tumorigenicity, we introduced a human TGF-a cDNA expression vector into established nontransformed Fischer rat fibroblast (Rat-l) cells. Synthesis and secretion of human TGF-a by these cells results in the loss of anchorage-dependent growth and induces tumor formation in nude mice. Anti-human TGF-a monoclonal antibodies prevent TGF-a expressing Rat-l cells from forming colonies in soft agar. Introduction Human TGF-a is a 50 amino acid peptide (Marquardt et al., 1983) derived from a 160 amino acid precursor that is encoded by a 4.5-4.8 kb mRNA (Derynck et al., 1984). The peptide is structurally related to both epidermal growth factor (EGF) (Marquardt et al., 1983, 1984) and Vaccinia virus growth factor (Stroobant et al., 1985; Brown et al., 1985), and its effects are thought to be mediated through binding to the EGF receptor (Carpenter et al., 1983; Massague, 1983b). Interaction of either TGF-a or EGF with this receptor leads to an activation of receptor-associated tyrosine kinase (Pike et al., 1982) and to a mitogenic response. Both peptides trigger many similar biological effects, but some differential activities are apparent in, for example, the much stronger potency of TGF-a to promote calcium release from fetal rat long bones (Stern et al., 1985; lbbotson et al., 1986) and to induce angiogenesis (Schreiber et al., 1986). Exogenously added TGF-a or EGF are equally efficient in enabling anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar, and their ability to promote anchorage-independent growth is potentiated by transforming growth factor-8 (TGF-8) (Anzano et al., 1983; Massague, 1983a; Derynck et al., 1984). TGF-a is synthesized in embryos during early fetal development (Twardzik, 1985; Lee et al., 1985a), in several cell lines transformed by Moloney (De Larco and Todaro, 1978) or Kirsten murine sarcoma virus (Ozanne et al., 1980), simian virus 40 (SV40) (Kaplan et al., 1981), polyoma virus

(Kaplan and Ozanne, 1982), and in a variety of human tumors (Ft. Derynck, D. V. Goeddel, A. Ullrich, J. U. Gutterman, Ft. D. Williams, T. S. Bringman, and W. H. Berger, submitted). The synthesis of TGF-a by a variety of tumor cells and its ability to promote mitogenic stimulation and anchorage-independent growth implies that it may be involved in malignant transformation through an autocrine stimulation of growth. The concept of autocrine stimulation of cellular proliferation originally postulated that a tumor cell could gain growth autonomy by acquiring the ability to produce, secrete, and respond to a given growth factor (Todaro et al., 1977; Sporn and Todaro, 1980). Similar growth autonomy might also be acquired by abnormal activation of any modulator of signal transmission pathways that control cell proliferation or by diminished response to growth inhibitors (Sporn and Roberts, 1985). As predicted by the autocrine model a variety of tumor cells produce and secrete growth factors for which they bear receptors. Fibroblasts transformed by Moloney or Kirsten murine sarcoma virus or by SV40 express, in addition to TGF-a, a plateletderived growth factor (PDGF) related peptide (Dicker et al., 1981; Bowen-Pope et al., 1984) and TGF-8 (Anzano et al., 1985). Human osteosarcoma and glioma cells produce a peptide related to PDGF (Heldin et
Cell 302

1000

t

/*I. 1' TGF-a

+ TGF-0

(2 6ngIml)

A AA

I’

: pMLl ,’ or,

Figure 2. Schematic Vector

TGF-a Concentration (rig/ml) Figure 1. Formation of Colonies in Soft Agar by Rat Fibroblasts sponse to Exogenously Added TGF-a The assay was performed in the presence (solid line) of 2.6 Kg/ml TGF-8.

in Re-

(dashed line) or absence

locyte-macrophage colony stimulating factor (GM-CSF) cDNA (Lang et al., 1985). To determine the role of TGF-a in the acquisition of a malignant phenotype, we introduced a plasmid that directs the constitutive synthesis of human TGF-a into established nontransformed Fischer rat (Rat-l) fibroblasts. Expression and subsequent secretion of TGF-a resulted in a loss of anchorage dependence, as indicated by the ability of the expressing cells to form colonies in soft agar. These cells also displayed reduced contact inhibition, as indicated by the formation of foci in monolayer, and were able to form tumors when injected into nude mice. The ability of anti-TGF-a monoclonal antibodies to prevent colony formation in soft agar indicates that the hormone is secreted and then mediates its activities through receptors on the cell surface, as postulated originally in the autocrine hypothesis (Todaro et al., 1977; Sporn and Todaro, 1980). Since TGF-a is expressed in a wide variety of human solid tumors (Derynck et al., submitted), our results suggest that TGF-a could be involved in an autocrine fashion in the development of malignancies in vivo.

TGF-a Induces Anchorage-Independent Growth of Rat-l Fibroblasts TGF-a was originally characterized by its ability to induce reversible mitogenic activity, morphological changes, and anchorage-independent growth of NRK-49F cells (De Larco and Todaro, 1978; Anzano et al., 1983) through interaction with the EGF receptor (Carpenter et al., 1983; Massague, 1983b). Further purification of the original TGF-a

Diagram

of the pMTE4E

TGF-a

Expression

The SV40 promoter segments with the direction of early transcription are indicated with black arrows. The coding sequences transcribed from the SV40 promoter elements are boxed. Within the TGF-a precursor coding sequence, the dotted box represents the signal sequence, and the dashed area corresponds to the 50 amino acid TGF-a. Some marker restriction sites are shown. The distances in kilobases are marked in the center. HBsAg 3’UT, hepatitis B surface antigen gene 3’ untranslated sequence: DHFR, dihydrofolate reductase.

Table 1. Formation Rat-l Clones

of Colonies

in Soft Agar by TGF-a Expressing

Clone Number

Concentration of TGF-a Secreted after 24 hr

Rat-l 5 41 45 47 43 16 42 T24 c-Hams

0 nglmt 0.2 nglml 0.2 nglml 0.3 nglml 0.4 nglml 0.7 nglml 0.7 nglml 0.95 nglml Not determined

Number of Colonies - TGF-8 3

+ TGF-8 (2.6 nglml) 5

1

8

3 2 20 72 120 133 ,-“5000

6 4 37 154 385 180 ~8000

Formation of colonies in soft agar by Rat-l clones expressing different levels of human TGF-a with (+ TGF-8) or without (- TGF-8) the addition of 2.6 nglml human TGF-8. The cells were grown to confluency in monolayers in 24 well plates and the secretion of TGF-a in 1 ml of medium was measured by ELISA after 24 hr.

preparations revealed that TGF-a and the structurally unrelated polypeptide TGF-j3 are both required for efficient phenotypic transformation of the NRK cells (Anzano et al., 1983). NRK-49F cells form colonies in soft agar about loto %-fold more efficiently in the presence of TGF-a and TGF-9 than in the presence of TGF-a alone (Anzano et al., 1983; Derynck et al., 1984). The potentiation of the effects of either TGF-a or EGF by TGF-fi may be partially attributed to the ability of TGF-f3 to induce an increase in the number of EGF receptors on the cell surface (Assoian et al., 1984a). The Rat-l cells we chose as a model system for our study were evaluated for the effects of TGF-a and

TGF-a Expression 303

Transforms

Rat Fibroblasts

TGF-a - 16

TGF-,B - 16

IGF-II - 16

c-myc - 16

TGF-8 on the formation of colonies in soft agar. In the presence of serum, TGF-a induces anchorage-independent growth in a dose-dependent manner between 0.1-10 nglml (Figure 1). TGF-6 by itself does not induce colony formation of Rat-l cells in soft agar (data not shown) but increases the number of anchorage-independent colonies in response to TGF-a approximately 2-fold (Figure 1). Expression of a Human TGF-a cDNA in Rat Flbroblasts An expression vector was constructed in which the cDNA sequence encoding the human TGF-a precursor protein (Derynck et al., 1984) was placed downstream of the SV40 early promoter. The proximal part of the natural TGF-a 3’ untranslated region is followed by a segment of the 3’ untranslated region of the Hepatitis B surface antigen (HBsAg) gene, which provides the transcription termination and polyadenylation signal sequences. This plasmid also contains the sequences coding for aminoglycosyl phosphotransferase II as a selectable marker (Southern and Berg, 1982) and for dihydrofolate reductase, thus enabling the plasmid to be amplified in mammalian cells (Alt et al., 1978). The latter two genes are also preceded by separate SV40 early promoter elements and followed by the polyadenylation signal sequences of the HBsAg gene. The plasmid pMTE4E (Figure 2) was introduced into the Rat-l fibroblasts using the calcium phosphate transfection method (Wigler et al., 1979) and clones resistant to 400 rrg/ml of the antibiotic Geneticin G-418 were picked, expanded, and analyzed for their ability to secrete human TGF-a into the culture medium. As judged by a sensitive ELISA, using recombinant human TGF-a as a standard, approximately 40 out of the 100 clones analyzed secreted human TGF-a at concentrations ranging between 0.1 and

c-ras - I6

Figure 3. Detection of Specific mRNAs in the Transfected Cells Clone 16 and pSVENeoBal6 (Seeburg et al., 1964) transfected Fischer rat fibroblasts (-) were analyzed by Northern blot hybridization using as a probe human TGF-a cDNA, murine TGF-P cDNA, human IGF-II DNA, human c-rnyc gene and human c-Ha-ras cDNA.

1 rig/ml per lo5 cells per 24 hr. Seven clones expressing varying levels of TGF-a were chosen for turther studies (Table 1). Northern analysis reveals an expected predominant human TGF-a mRNA of about 2.3 kb (Figure 3) consistent with the efficient use of the Hepatitis 6 surface antigen gene polyadenylation sequence. The larger endogenous rat TGF-a mRNA (Lee et al., 1985b) was not detected. Increased levels of transcripts for the cellular counterparts of the oncogenes c-myc and c-fos have been observed in serum- or PDGF-treated cells (Kelly et al., 1983; Greenberg and Ziff, 1984). We therefore examined the level of transcripts for these and several other proteins in TGF-a-expressing Rat-l cells. As shown in Figure 3, the TGF-a expressing fibroblasts had a 2-told higher level of c-myc mRNA than the parent cells, but no change in the level of c-fos mRNA was detected (not shown). Likewise, no changes in the level of transcripts for TGF-8, insulin-like growth factor II (IGF-II), or for the c-ras oncogene were observed (Figure 3). The levels of these individual mRNAs in the TGF-a transfected cells was similar to the levels observed in Rat-l cells grown in the presence of exogenously added TGF-a or EGF (data not shown). TGF-a Expressing Rat-l Fibroblasts Are Transformed The acquisition of anchorage independence is generally considered a property concomitant with the transformed phenotype. The transfected Rat-l fibroblasts were therefore examined for their growth characteristics. The parental Rat-l cells grew on a solid support in a monolayer and displayed contact inhibition. The three clones (clones 16, 42, and 43) that express high levels of TGF-a grew in culture plates to a higher density and displayed a tendency to pile up (Figure 4). Clones synthesizing TGF-a at levels

Cell 304

Figure 4. Morphology

of Monolayer

Cell Cultures

Normal Rat-l cells are shown on the left, and the TGF-a-expressing clone 16 is shown on the right. Confluent cultures were stained with crystal violet.

of 0.4 nglml or higher also acquired the ability to form colonies in soft agar (Table 1). The number of colonies correlates roughly with the level of TGF-a secreted and corresponds to the number of colonies obtained with similar concentrations of exogenously added TGF-a. Clones expressing low levels of TGF-a as well as the parental Rat-l cells do not show any anchorage-independent growth. The number of colonies formed in soft agar increased approximately e-fold when human TGF-p was added at a concentration of 2.6 nglml (Table l), indicating that TGF-p can potentiate the effect of endogenously produced TGF-a. No colony formation was observed when these assays were performed in serum-free conditions (data not shown). Soft agar colony formation by Rat-l cells in the presence of saturating levels of exogenously added TGF-a (Figure 1) or by clones producing the hormone (Table 1) is 20- to 30-fold less efficient than for Rat-l cells transformed by a c-Ha-rasl T24 bladder oncogene under transcriptional control of the SV40 early promoter (Seeburg et al., 1984; Table 1). This result indicates that an activated ras oncogene is more potent than TGF-a in transforming Rat-l cells. Interestingly, exogenously added TGF-P still potentiates the efficiency of colony formation in soft agar by c-Ha-ras-transformed Rat-l fibroblasts (Table 1). Anti-TGF-a Antibodies Revert Transformed Phenotype of TGF-a Expressing Rat-l Fibroblasts The hypothesis that autocrine growth factors are first secreted and subsequently act through cell-surface receptors suggests that the growth of tumor cells might be controlled by extracellular antagonists for either the autocrine peptides or their receptors. In order to test this possibility, we analyzed the ability of antibodies raised against human TGF-a to prevent colony formation in soft agar by rat fibroblasts expressing human TGF-a. The monoclonal antibody preparation does not inhibit the growth of TGF-aexpressing clones in monolayer (data not shown) nor does it inhibit colony formation in soft agar by c-Ha-ras-trans-

formed Rat-l fibroblasts. This monoclonal antibody has a very low affinity for rat TGF-a. It is therefore unclear whether endogenous synthesis of TGF-a by the c-Ha-rasl-transformed cells contributes to the transformed phenotype. The monoclonal antibody does prevent colony formation by normal Rat-l cells in response to exogenously added human TGF-a and by the TGF-a-expressing clones (Figure 5, Table 2). In both cases colony formation was not inhibited by an anti-human growth hormone (hGH) monoclonal antibody. Thus, an autocrine growth-stimulation system has been experimentally generated in which TGF-a is first secreted and subsequently exerts its effects through receptors on the cell surface. Colony formation was inhibited to a large extent by anti-human TGF-a antibodies, implying an extracellular interaction of TGF-a with the EGF receptor. TGF-a-Expressing Rat-l Cells Are Tumorigenic in Nude Mice The data presented here indicate that production of TGF-a enables nontransformed Rat-l fibroblasts to acquire the transformed phenotype in culture via an autocrine mechanism of growth stimulation. In order to assess the tumorigenicity of these cells in vivo, 1 x lo6 or 5 x lo6 cells from two clones that express high levels of TGF-a were injected subcutaneously into nude mice, and the resulting tumors were scored. As shown in Table 3, mice injected with clones 16 and 42 displayed a much higher frequency of tumor formation, as compared with mice injected with nontransfected Rat-l cells. All animals injected with the Rat-l cells that express TGF-a had developed palpable tumors after 4 weeks. But as judged by latency period and tumor size, the c-Ha-ras-transformed Rat-l cells are much more tumorigenic than the TGF-a-producing Rat-l cells in this in vivo system. Discussion Unrestricted growth that leads to malignant transformation might begin with acell acquiring the ability to produce and respond to growth peptides (Todaro et al., 1977; Sporn and Todaro, 1980; Sporn and Roberts, 1985). If true, this hypothesis implies that expression of an introduced growth factor-coding sequence in cells that bear its receptors could lead to or contribute to their malignant transformation. It also implies that an extracellular antagonist for the autocrine peptide or its receptor could reverse this growth factor-mediated phenotypic transformation. We tested this hypothesis directly by introducing and expressing a human TGF-a cDNA in normal rat fibroblasts, thus experimentally generating a TGF-a-mediated autocrine stimulation system. As predicted by the autocrine hypothesis, synthesis and secretion of human TGF-a results in transformation of Rat-l cells. The cells show anchorage-independent growth and efficient tumor formation in nude mice. The transformed phenotype in culture could be reversed by anti-TGF-a monoclonal antibodies, indicating that TGF-a is secreted and then exerts its effects through receptors on the cell surface. We also examined the effect of the TGF-a-induced

TGF-a Expression 305

Transforms

Rat Fibroblasts

Anti hTGF-a Ab

Figure 5. Anti-TGF-a Antibodies

Inhibit Anchorage

Anti hGH Ab

Independence

TGF-a-producing clones were assayed for soft agar colony formation in the presence and absence of anti-human TGF-a monoclonal antibodies (AntihTGF-a Ab). The frames marked Rat-l contain nontransfected Rat-l cells in the presence of 10 nglml exogenously added TGF-a, while the frames marked T-24 RAS contain the c-Ha-ras-transfected Rat-l cells. A monoclonal antibody raised against human growth hormone (Anti-hGH Ab) was used as negalrve control.

transformation on the levels of mRNA for TGF-6, IGF-II, myc, and Ha-raas. It has previously been shown that several sarcoma virus-transformed fibroblasts have significantly greater TGF-b expression than their nontransformed counterparts (Anzano et al., 1985; Derynck et al., 1986). Our results indicate that the TGF-f3 mRNA level in the TGF-a-transformed cell line is not altered in comparison with Rat-l cells. Therefore, increased TGF-f3 expression is presumably not a prerequisite for transformation. Likewise, the mRNA levels for IGF-II, which are elevated

in many tumors (A. Ullrich, personal communication), were not affected by transformation with the TGF-a cDNA. In contrast, the c-myc mRNA levels were about 2-fold higher in the transformed cells, as well as in the parental Rat-l cells that had been grown in the presence of TGF-a or EGF, than in untransformed Rat-l cells. Treatment of fibroblasts with PDGF has previously been shown to result in greatly elevated c-myc mRNA levels (Kelly et al., 1983; Greenberg and Ziff, 1984). Experimental reconstitution of autocrine systems has

Cell 306

Table 2. Inhibition of Colony Formation by anti-TGF-a Monoclonal Antibodies

Two separate instance8 of inhibition of autocrine growth stimulation by extracellular antagonists have been reported. The growth of a human small cell lung carcinoma that produces the growth factor bombesin can be inhibited by anti-bombesin monoclonal antibodies (Cuttita et al., 1985). The growth of virally transformed chicken hematopoietic cells that produce macrophage growth factor (MGF) can be inhibited by anti-MGF polyclonal antibodies (Adkins et al., 1984). The difference in the ability to control autocrine effects of growth factors with extracellular antagonists in the above examples may reflect differences in either the mechanism of hormone-receptor interactions, in the cell dependence on a particular growth factor for maintenance of the transformed phenotype, or on differences in the affinity and neutralizing ability of the antibodies used. Our results and other experimental studies discussed above thus indicate that expression of an introduced growth factor gene in established cell lines can result in the acquisition of the transformed phenotype via an autocrine mechanism. The inability of TGF-a-expressing Rat-l cells and many other transformed cells to grow in soft agar under conditions that do not supply exogenous growth factors, might indicate that in addition to TGF-a, other growth factors are required for the induction and maintenance of the observed transformed phenotype. This is supported by the finding that mitotic stimulation of fibroblasts by IGF-II is required for phenotypic transformation by TGF-8 (Massague et al., 1985). Possible synergistic actions between several growth factor8 might also explain the efficient anchorage-independent growth of nontransformed rat fibroblasts in the presence of EGF, TGF-8 and PDGF (Assoian et al., 1984b). Constitutive synthesis of TGF-a may be of significance for the development of malignancies in vivo. We have previously examined the expression of TGF-a mRNA in a large variety of human tumors and tumor cell lines (Derynck et al., submitted). TGF-a mRNA was found in many types of solid tumors, especially squamous, renal, and mammary carcinomas, and tumors of neuroectodermal origin such as melanomas. All of the tumors that con-

in Soft Agar

Clone Number

Number of Colonies in the Presence of 100 nglml Control Monoclonal Antibody

Number of Colonies in the Presence of 100 pg/ml anti-TGF-a Monoclonal Antibody

Rat-l (+lO nglml TG F-c) Clone 16 Clone 42 T24 c-Ha-ras

800 290 195 ~5000

18 20 20 -5000

Inhibition of colony formation in soft agar of TGF-a expressing clones by anti-human TGF-a monoclonal antibodies. Anti-human growth hormone (hGH) antibodies were used as negative controls.

been reported previously for two other growth factors, PDGF and GM-C%. Expression of the normal PDGF 6 chain gene (Gazit et al., 1984) or of its acquired viral counterpart, the ~28”+~s gene of SSV (Huang et al., 1984; Johnsson et al., 1985) in nontumorigenic cells results in malignant transformation. Anti-PDGF antiserum partially inhibits the growth of SSV-transformed mouse NIH 3T3 cells and of several SSV-transformed fibroblast cell lines (Huang et al., 1984; Johnsson et al., 1985), but not of a SSV-transformed marmoset monkey fibroblast line (Huang et al., 1984) or of PDGFsecreting osteosarcoma cells (Betsholtz et al., 1984). The fact that only a small fraction of the PDGF produced by SSV-transformed cells is secreted (Robbins et al., 1985) combined with very limited growth inhibition of these cells by antisera to PDGF, led to the proposal that the growth factor may be able to interact and activate its receptor in an intracellular compartment. This may offer an explanation for the inability of extracellular PDGF antagonists to inhibit transformation completely. Similarly, nontumorigenic hematopoietic cells can be converted into transformed leukemic cells following expression of an introduced human GM-CSF gene. The inability of anti-GM-CSF antiserum to prevent transformation implies that at least part of the GM-CSF receptor interaction is intracellular (Lang et al., 1985).

Table 3. Tumor

Formation

in Nude Mice by TGF-u-Expressing

3 Weeks

4 Weeks

Animals with Tumors

Animals with Tumors

Animals with Tumors

Animals with Tumors

Tumor Size

114 4/4 4/4

0.2 x 0.3 cm 0.3 x 0.4 cm 0.3 x 0.3 cm

Experiment

Clone No.

1

Rat-l Clone 16 Clone 42 Rat-l Clone 16 T-24 c-Ha-ras

5 5 5 1 1 1

10s 106 106 10s 10s lo6

014 114 114 014 114 4/4

Rat-l Clone 18 Clone 42

5 x 10s 5 x 10s 5 x 10s

115 415 515

2

Clones

2 Weeks

No. Cells Injected per Mouse x x x x X x

Rat-l

1 Week

Tumor Size

Tumor Size

0.3 x 0.4 cm 1.8 x 1.4 cm

014 3/4 2/4 114 314 4/4

0.2 0.2 0.2 0.4 2.8

0.4 x 0.4 cm 0.4 x 0.4 cm 0.4 x 0.4 cm

115 4/5 5/5

0.2 x 0.3 cm 0.3 x 0.3 cm 0.4 x 0.4 cm

0.2 x 0.3 cm 0.2 x 0.2 cm

x x x x x

0.3 0.3 0.2 0.5 2.3

cm cm cm

cm cm

Tumor Size

o/4 4/4 2/4 114 4/4 3/4 114

0.3 x 0.3 x 0.3 x 0.3 x dead 2.5 x

2/5 5/5 5/5

0.4 x 0.4 cm 0.3 x 0.3 cm 0.4 x 0.3 cm

0.4 0.3 0.4 0.5

cm cm cm cm

1.8 cm

This table gives the incidence of tumor formation and the width and length of the tumors at the end of each week. Where more than one animal had tumors, the tumor size is expressed as an average.

TGF-a ExpressIon 307

Transforms

Rat Fibroblasts

tained TGF-a mRNA also contained mRNA for the EGF receptor, supporting the possibility that autocrine action of TGF-a may play a physiological role in tumor development. Both TGF-a and EGF mediate their activity through the same receptor and are equally effective at inducing mitosis and anchorage-independent growth of NRK fibroblasts. However, TGF-a synthesis is far more prevalent than EGF synthesis in tumors. EGF expression has been demonstrated in only a single salivary gland adenocarcinoma cell line (Sato et al., 1985). It is conceivable that synthesis and proper processing of the EGF precursor could trigger transformation in vitro in a manner analogous to that observed with TGF-a. However, no experimental data are available to support this proposal. The differential expression in vivo of these related growth factors could reflect differences in their mechanisms of gene regulation, whereby the TGF-a gene can be more easily activated during the transformation process. The expression of TGF-a in naturally occurring tumors may also be related to significant differences in biological activities between TGF-a and EGF. TGF-a has indeed been shown to be superior to EGF in the induction of bone resorption in vitro (Stern et al., 1985; lbbotson et al., 1988) and of angiogenesis (Schreiber et al., 1986), activities that may play key roles in the development of malignancies in vivo. The studies described here support the notion that TGF-a synthesis could result in tumor formation under physiological conditions. Furthermore, the susceptibility of the TGF-aexpressing Rat-l cells to the anti-TGF-a antibodies raises the intriguing possibility of inhibiting the growth of TGF-aexpressing human tumors with extracellular antagonists. Experimental Procedures Plasmid pMTE4E Construction The final mammalian expression vector pMTE4E was constructed by ligation of the following fragments. Starling from the Sall site and proceeding clockwise, the various segments are as follows: The Sall-Clal fragment corresponds to the Sall-EcoRI fragment of plasmid pML-1 (Lusky and Botchan, 1981), containing the pBR322 origin of replication and the /3-lactamase gene fused at its EcoRl site to the Hincll site of the 600 bp Hincll-Hindlll fragment of SV40 (Fiers et al., 1978), which contains the 72 bp repeat and early promoter. The Clal site is derived from a polylinker that was inserted downstream from the Hindlll site and contains the sequence AAGCTTATCGATTCTAGA, which consecutively contains a Hindlll, Clal, and Xbal site. The TGF-a transcription unit is derived as follows. The Xbal site of the above-described linker was fused to the Bgll site, which is 62 bp upstream of the ATG of the TGF-a precursor-coding sequence (Derynck et al., 1984). The TGF-a stop codon is followed by 140 bp of 3’ untranslated sequence ligated to a 957 bp EcoRV-Bglll fragment in the 3’ untranslated region of the HBsAg gene (Crowley et al., 1983). The neomycin-resistance transcription unit is derived from pSVENeoBal6 (Seeburg et al., 1984) and contains the neomycin resistance gene isolated from Tn5 (Southern and Berg, 1982) under transcriptional control of the SV40 early promoter (a 348 bp Pvull-Hindlll fragment; Fiers et al., 1978). The neomycin-resistance coding sequence is followed by 741 bp from Tn5, which are followed by a 585 bp BamHI-Bglll fragment in the S’untranslated region of the HBsAg gene (Crowley et al., 1983), thus providing the polyadenylation signal. A synthetic EcoRl recognition site was introduced immediately downstream. The dihydrofolate reductase transcription unit extends from the EcoRl to Sal1 Site. The dihydrofolate reductase coding sequence is preceded by the SV40 early promoter (the 600 bp Hincll-Hindlll fragment; Fiers et al., 1978) and followed by part of its natural 3’ untrans-

lated region fused to the 585 bp BamHI-Bglll fragment of gene 3’ untranslated region. The latter segment is followed bp BarnHI-Sall sequence in pBR322. This dihydrofolate transcription unit has been derived from plasmid pFD11 and Levinson, 1983).

the HBsAg by the 275 reductase (Simonsen

Transfection of Rat Fibroblssts Rat-l fibroblasts (1 x 10s cells) in 100 m m dishes containing 10 ml of complete culture medium (Hams F12 and DMEM 50:50 with 10% fetal calf serum [Gibco]) were transfected with 20 pg of pMTE4E as a calcium phosphate precipitate (Wigler et al., 1979). Cells were incubated for 6 hr, the medium was removed, and 1 ml of 20% glycerol was added for 1 min. The glycerol was removed, the cells were grown in culture medium for 24 hr, and then were incubated in selective medium, that is, complete medium plus 400 pglml Geneticin (G-418, Gibco). Antibiotic resistant clones were visible after approximately 1 week in selective medium and were transferred into 24 well plates. The supernatant medium (1 ml) from confluent wells was harvested after 24 hr and analyzed for the presence of human TGF-a using an ELISA. RNA Extraction and Northern Hybridizations All RNA preparations were made from cells harvested at early confluency. The cells were lysed in RSB (10 m M Tris-HCI. pH 7.5; 10 m M NaCI; and 1.5 m M MgCl$ containing 1% NP-40 and 200 &ml heparin. The nuclei were removed by centrifugation in a Sorvall SS-34 rotor for 5 min at 3000 rpm. The supernatant was then adjusted to 1% SDS, 0.5 M NaCI, extracted with phenol and chloroform, and the RNA was precipitated in ethanol. The polyadenylated mRNA fraction was isolated by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972). Three micrograms of polyadenylated RNA was electrophoresed into a formaldehyde-1.2% agarose gel (Dobner et al., 1981) and was blotted onto nitrocellulose (Thomas, 1980). The nitrocellulose filters were hybridized with 32P-labeled (Taylor et al., 1976) cDNA probes in 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 m M sodium phosphate (pH 6.8), 2x Denhardt’s solution, 10% dextran sulfate at 42OC for 15-20 hr. Extensive washings were done in 0.2x SSC and 0.1% SDS at 50DC. The following DNA probes were used for Northern hybridizations. The human TGF-a probe consists of the cDNA insert contained in pTGF-Cl (Derynck et al., 1984) and comprises the complete coding sequence. The murine TGF-a probe was isolated from a genomic DNA library and is a 1300 bp SauJAl fragment comprising the last 5 bp of the sequence coding for the TGF-a precursor and a large section of the 3’ untranslated region (Derynck, unpublished). The levels of TGF-a mRNA were probed using the 1650 bp cDNA insert of plasmid pMuro2, which contains the complete sequence coding for the murine TGF-g precursor (Derynck et al., 1986). The human c-myc probe is the 1150 bp Smal-Sacl fragment, which comprises the first coding exon (Colby et al., 1983). The c-Ha-ras cDNA probe was excised from plasmid pGAtrp (McGrath et al., 1984) as a 700 bp EcoRI-Pstl fragment. The fos mRNA was detected using a 950 bp Pstl fragment from pfos-1 (Curran et al., 1982), and IGF-II mRNA levels were measured using a 1.5 kb human IGF-II cDNA (Dull et al., 1984). Anti-TGF-a Antibodies and TGF-a ELISA The rabbit polyclonal antiserum 34D and the murine monoclonal antibody TGF-al were raised using the 50 amino acid human TGF-a, purified from recombinant E. coli cultures (Derynck et al., 1984) as antigen. Purified IgG fractions were prepared by adsorption to Protein-A Sepharose. The double sandwich ELISA consists of microtiter wells coated with the monoclonal antibody TGF-al and used the rabbit polyclonal antiserum 34D and goat anti-rabbit horseradish peroxidaseconjugated antibodies as detecting antibodies. Human EGF (Amgen) and murine EGF are negative in this assay to a concentration of 10 pglml. The assay values are based on the EGF radioreceptor-binding activity of HPLC purified, refolded, E. coli-derived recombinant TGF-a, which is used as the assay standard. The characterization of the antibodies and the ELISA will be described elsewhere (Bringman et al., unpublished). Soft Agar Colony Assay Two milliliters of McCoy’s 5A (Gibco) medium plus 10% fetal calf serum (FCS) and containing 0.5 percent Noble agar (Difco) were added to

Cell 308

each well of a 12 well plate. Cells were trypsinized, counted, and suspended in the same medium containing 0.3% Noble agar, then plated in duplicate at a density of 2 x 104 cells per well. Human TGF-a, TGF-8, or anti-TGF-a antibodies were added to the cell suspension before they were plated. These cultures were incubated at 37oC in a 5% Cop atmosphere for 5-7 days. Viable colonies were stained by addition of 1 ml/well iodonitrotetrazolium stain (50 mg/lOO ml; Sigma), which was incubated with the cultures overnight at 37oC. Dark brown colonies of more than ~30 cells were counted and photographed using a Nikon Diaphot inverted microscope. Antibody Toxicity in Monolayer Cultures The cells were plated in 12 well plates in Dulbecco’s MEMHam’s F12 (50:50) supplemented with 10% FCS at a density of lo4 cells per 4 ml per well. The monoclonal antibody TGF-al was added at appropriate dilutions, and the cultures were incubated at 37oC in a 5% COs atmosphere for 6 or 7 days. Cells were washed with PBS, fixed, and stained in a 3:l mixture of methanol-glacial acetic acid containing 0.1 percent crystal violet. Nude Mouse Injections Groups of four or five female BALBlc nude mice were injected subcutaneously into the lateral abdomen with 5 x 10s cells (Rat-l, clone 16, clone 42) or with 1 x lo6 cells (Rat-l, clone 16 and T-24 c-Ha-rastransformed Rat-l; Seeburg et al., 1984). Mice were monitored every other day for the appearance of solid tumors. The length and the width of the tumors were measured twice a week for 4 weeks. Cells were prepared for injection by trypsinization, multiple washes with PBS, and resuspension in 0.5 ml per sample of serum-free Hepes-buffered DMEM (Gibco). Acknowledgments We are grateful to Bill Lagrimas, Mike Cagle and Dietrich Crase for their help with the nude mice experiments, to Art Levinson for the gift of the c-myc and c-H-ras plasmids and mammalian cell expression vectors, and to Axel Ullrich for the gift of an IGF-II cDNA plasmid. lnder Verma (Salk Institute, La Jolla, CA) allowed the use of plasmid pv-fos-1, while Mike Sporn and Anita Roberts (Laboratory of Chemoprevention, N.I.H.) provided theTGF-8. We thank Jeanne Arch for typing the manuscript. 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 accordance with 18 USC. Section 1734 solely to indicate this fact. Received April 4, 1986; revised May 7, 1986.

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