Partial transformation of mouse fibroblastic and epithelial cell lines with the v-myc oncogene

Experimental

Cell Research 168 (1987) 273-284

Partial Transformation of Mouse Fibroblastic and Epithelial Cell Lines with the v-myc Oncogene GERMANA FALCONE, IAN C. SUMMERHAYES, HUGH PATERSON, CHRISTOPHER J. MARSHALL and ALAN HALL* Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, England

To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v-myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse tibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v-myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors. v-myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v-myc-transformed tibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v-myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2-3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v-myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v-myc oncogene induces morphological changes and anchorage independence in immortal mouse tibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic. @ 1987 Academic press, IIK.

The v-myc oncogene was first identified in the avian myelocytomatosis virus MC29 as a viral sequence not required for replicative functions [ 11.The discovery of a cellular counterpart, the proto-oncogene c-myc, in the genome of a wide variety of vertebrates [2], indicated that the viral gene originated from transduction of the cellular gene by the retrovirus. In vivo MC29 causes myelocytomatosis, carcinomas and sarcomas in chicken [3] and is capable of transforming several cell types in vitro, i.e. macrophages, fibroblasts and myoblasts [4, 51. Chicken and quail cells have proved to be very susceptible to transformation by MC29 virus, although the transformed cells do not generate tumours when injected into nude mice [6,7]. The mechanism by which v-myc transforms cells is far from clear. The viral myc protein is known to be localized in the nucleus of infected cells [8, 91. It has been shown to bind to DNA in vitro [8] and to be * To whom offprint requests should be sent. Address: Chester Beatty Laboratories, Institute of Cancer Research, Fulham Road, London SW3 6JB, London. Copyright IQ 1987 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827/87 503.a)

274 Falcone et al. associated with the nuclear matrix in vivo [lo], but so far no function for the myc protein has been identified. Many mouse and human B-cell-derived lymphomas have been shown to contain a translocated c-myc gene [ 11, 12, 131and c-myc and related genes have been found amplified in a variety of human tumours [14, 15, 16, 171. In order to understand the role of the myc gene in mammalian cell transformation, several groups have attempted to introduce v-myc and c-myc genes into rodent fibroblasts either by viral infection or DNA-mediated gene transfection. It has been reported that transformation of mammalian cells by infection or transfection with the MC29 virus is very inefficient [18, 191. However, mammalian tibroblasts and macrophages have been successfully transformed by an experimentally derived murine retrovirus encoding the avian v-myc oncogene [20]. In this case the transformed cells exhibited morphological changes and acquired the ability to grow in agar suspension. We show here that v-myc is capable of inducing morphological transformation of both mouse fibroblastic and epithelial cell lines, but only under conditions in which neighboring normal cells are removed. v-myccontaining cells are not directly tumorigenic and further selective events are necessary for tumour formation.

MATERIALS Plasmid Construction

AND METHODS

and Transfection

The MC29-LTR-G2 plasmid was constructed by subcloning the 5.5 kb EcoRI fragment from pMCV38 [21] into the EcoR I site of pSV2-neo [22]. The orientation was such that transcription from the MC29 fragment was towards the SV40 terminator. The MMTV-LTR-G4 plasmid was constructed in two steps. First, the 1.4 kb Hind III/BamH I fragment containing the MMTV-LTR [23] was inserted into pSV2-neo between EcoR I and BamH I sites. The blunt-ended 3 kb BstE II/Sph I fragment from pMCV38 was then inserted into the blunt-ended BamH I site of this plasmid, downstream from the MMTV-LTR. The RSV-LTR-GS plasmid was obtained by inserting the blunt-ended 3.9 kb Bst II/ EcoRI fragment from pMCV38 into a vector already containing the RSV-LTR and the SV40 polyadenylation signal. pEJ-ras was a gift of R. A. Weinberg. Plasmid DNAs were transfected (I ug per plate) or co-transfected with pSV2-neo (0.1 ug per plate) into NIH-3T3, BBN3 or BBN7 cells in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% calf serum (NIH-3T3) or 10% fetal calf serum (FCS) (BBN3 and BBN7) using the calcium phosphate co-precipitation technique as described by Wigler et al. [24]. High molecular weight (HMW) mouse liver DNA was used as a carrier. Microinjection was performed as described by Graessmann & Graessmann [25]. Cells were fed and maintained in selection media containing 1 mg/ml G418 (Gibco) the day following transfection or microinjection. G418-resistant colonies were scored and isolated after 2 weeks.

Plating Efficiency

Experiments

For assaying anchorage-independent growth, v-myc-containing NIH-3T3, BBN3 and BBN7 cells and controls were seeded at 10’ in 0.25 % agar over a 0.5 % lower layer in 50-mm culture dishes. Plates were scored for colonies 4 weeks following seeding. For the focus formation assay, v-myccontaining NIH-3T3 cells and controls were seeded at 16 and 10’ viable cells together with 1.3~ 10’ NIH-3T3 per 60-mm dish and maintained by feeding with DMEM containing 5 % calf serum every 3-4 days. Epidermal growth factor (EGF, BRL) at concentrations of 0.6 rig/ml (0.1 nM) and 1.8 @ml (0.3 nM) was added to parallel cultures. Foci were scored 14 days after seeding. Exp Cell Res 168 (1987)

Transformation of mammalian cells with the v-myc oncogene E

AT0 4tw

myc

Aan”

-*

..c?

SV40-ori

Swo-mr

MC2S-LTR

Y YYTV-

E

Swo-ter

LTR

~NEjATG

pJ RSV-LTR

[EM

Agag

myc ‘3.“”

*i-l

i

SV40-ter

&

-+

i

275

Fig. 1. v-myc constructs. For details of the construction, see Materials and Methods. (A) MC29-LTRG2; (B) MMTV-LTR-G4; (C) RSV-LTR-GS construct. E, EcoR I; H, Hind III; B, BamH I; BE, BstE II; SP, Sph I; XH, Xho I; neoR, G418 resistance gene; ATG, start codon for translation; SW0 ori, SV40 enhancer and promoter; SWOter, sv40 polyadenylation signal; LTR, long terminal repeat.

[ ‘251]EGF Binding Murine EGF was iodinated and used a binding assay following the procedure described by Marshall et al. [26].

Immunojluorescence Staining Cells growing on coverslips were rinsed with phosphate-buffered saline (PBS), fixed for 10 min in 3.7% formaldehyde, washed in PBS for 2-5 min and then permeabilized in 0.5 M ‘Ris-HCl, pH 7.5, 0.5% ‘Biton X-100 (TTX) for 30 min. TTX buffer was also used for all the subsequent washes and antiserum incubations. PllW~g~mycprotein was stained by indirect immunofluorescence using rabbit anti-gag antiserum (1: 30), a genereous gift from M. Hayman, and then a 1: 15 dilution of isothiocyanate-conjugated goat anti-rabbit antiserum (Miles).

Southern and Northern Blots Digested DNAs were electrophoresed on 0.8 % agarose gels and transferred to nitrocellulose paper essentially as described by Southern [27]. Total RNA was extracted and then electrophoresed and blotted to nitrocellulose as described by Thomas [28]. Hybridization was carried out at 42°C in a buffer containing 50% formamide, 3xSSC, 10% dextran sulfate and lo6 cpm/ml of nick-translated probe. The 1.5 kb Pst I v-myc internal fragment was used as a probe [21]. Filters were washed with 0.1 XSSC at 60°C.

RESULTS v-myc Constructs The first construct, MC29-LTR-G2, represents the whole MC29 viral genome with its original Long Terminal Repeat (LTR) sequence inserted into the pSV2neo plasmid (fig. 1A); transcription is expected to start at the MC29 LTR and terminate at the SV40 polyadenylation signal. The second construct, MMTVLTR-G4, is essentially the same as the first, except that the original MC29 LTR has been replaced with the Mouse Mammary Tumor Virus (MMTV)-LTR promoter that should initiate transcription only when activated by glucocorticoid hormones [29] (fig. 1B). In the third construct, RSV-LTR-G5, the gag-myc-env Erp Cell Res I68 (1987)

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Falcone et al.

portion of the MC29 genome has been placed under the control of the Rous Sarcoma Virus (RSV)-LTR (fig. 1 C), which has been reported to be a potent promoter in mammalian cells. Each of these constructs contains a portion of pBR322 carrying the ampicillin resistance gene for selection in bacteria. Establishment

of v-myc Expressing

Clones

Two different mouse bladder epithelial cell lines, BBN3 and BBN7, and the mouse libroblast cell line NIH-3T3 [30, 311were used as recipients for transfected v-myc genes. The two epithelial cell lines share similar characteristics to NIH3T3 in that they are contact-inhibited and anchorage-dependent for growth. However, unlike NIH-3T3 cells, they show no spontaneous transformation or tumorigenicity after prolonged passaging in vitro [32]. The v-myc constructs were introduced into NIH-3T3 cells alone or with pSV2neo where necessary, by calcium-phosphate-mediated DNA transfection or by microinjection. In the case of NIH-3T3 cells, no foci could be observed on confluent monolayers, even after transfecting up to 10 ug/plate of DNA. However, after G418 selection, 20-30% of the resistant colonies were morphologically transformed. It appears that the vmyc-containing cells are unable to overgrow surrounding normal cells. Similar results were obtained after transfection of the MC29-LTR-G2 construct into the epithelial BBN3 and BBN7 cells. Unfortunately, we were unable to obtain colonies with an inducible morphological phenotype after transfection with the MMTV-LTR-G4 construct. Properties of v-myc Transformed

NIH-3T3

cells

Although varying degrees of morphological transformation could be observed among the v-myc-expressing clones, most cells underwent a dramatic morphological change from the typical fibroblast-elongated shape (fig. 2A) to a more rounded or polygonal shape with very pronounced nucleoli (fig. 20. The transformed cells were more refractile and poorly adherent to plastic, sometimes needing a collagen coating to help adherence. They reached a higher saturation density than control cells. It is worth noting that the morphology of these cells is completely different from that of NIH-3T3 cells transformed by the activated Nras genes (fig. 2B): the latter look more elongated and grow in a disorderly crisscross pattern. Several clones were analysed by the Southern blot technique to determine the number of integrated v-myc genes (see for example fig. 3A) and all the clones analysed contained at least one copy of the transfected gene. In Northern blots, one size mRNA (6 kb) was found in most of the clones containing the MC29LTR-G2 construct (fig. 3B), suggesting that transcription of the integrated gene(s) terminated, as expected, at the SV40 polyadenylation signal; however, in some clones aberrant transcripts were found, presumably originating from rearranged genes. Most morphologically transformed clones analysed expressed high levels of the PllWng-myc fusion protein as detected by indirect immunofluoresExp Cell Res 168 (1987)

Transformation of mammalian cells with the v-myc oncogene 277

Fig. 2. Morphology of v-myc and N-ras transformants and anti-gag immunofluorescence. Phasecontrast pictures of clones (A) neo 9; (B) N-ras 149169; (C) v-myc NIH-3T3 clone G4/3; (E) BBN7

neo; Q BBN7 v-myc A. (0) Immunofluorescence staining of clone G4/3 using anti-gag antibodies.

cence staining using anti-gag antibody (fig. 20), in which the fluorescence was mainly nuclear and nucleoli appeared unstained. Although the most morphologically transformed cells also typically expressed the highest levels of protein, this correlation was not always observed. The v-myc-expressing clones were tested for their ability to grow in soft agar. It was found that most were anchorage-independent, but only a few grew as well as the activated N-ras-transformed cells used as positive controls (table 1). Under the same conditions, untransfected NIH-3T3 cells or cells transfected with Exp Cell Res 168 (1987)

278

Falcone et al. Kb

A

12346

1234567

Fig. 3. Analysis of DNA and RNA from v-myc-transfected cells. (A) Cellular DNA from NIH-3T3

transfectants was cleaved with Hind111 and analysed on Southern blots by hybridization with a 32Plabelled v-myc probe; lanes: I, neo 9; 2, G4/3; 3, G2/3; 4, G2/3-T2; 5, G2l3-T3. (B) Total RNA was analysed on Northern blots with a 32P-labelled v-myc probe; lanes: 1, BBN7-neo; 2, v-myc NIH-3T3 clone G2/3; 3, BBN7 v-myc B; 4, BBN7 v-myc A; 5, BBN3 v-myc B; 6, BBN3 v-myc C; 7, v-myc NIH-3T3 clone G2/12.

pSV2neo alone did not form colonies. There is no clear correlation between the level of expression of v-myc protein and the ability to grow in soft agar: for example, as shown in table 1, clone G4/1 expresses a high level of protein but grows poorly in soft agar. Cells Transformed by v-myc Are Only Poorly Tumorigenic

NIH-3T3 cells transfected with each of the three constructs were tested for tumorigenicity in nude mice (table 1). No tumours appeared from v-myc or Table 1. Properties of transformed Cell line

Anti-gag fluorescence

v-myc NIH-3T3

clones

% PE in agar

Foci on NIH-3T3 monolayers

Tumorigenicity (latency)

N-rus

-

Neo-2

-

2 0

40 0

6/6 (2 wks) 4/24 (g-16 wks)

G2ll G2l3 G4/1 G4/3 GYIO

+ ++ ++ ++ ++

0 0.6 0.2 1.5 ND

ND 0.5 0 2 0

ND 3/12 (8 wks) ND 2/6 (7 wks) O/6

G2/3-T2 G2/3-T3

++

0.1 2.5

6 5

G2, G4, G5 refer to morphologically transformed clones derived from the respective constructs (see fig. 1). N-rus refers to NIH-3T3 transformed with a mutant N-rus gene. Plating efficiencies in soft agar and foci on a monolayer of NIH-3T3 were determined as described in Materials and Methods. T2 and T3 are two independent tumours isolated from inoculation of G2/3 cells into nude mice. Foci formation is expressed as percentage of seeded cells capable of forming foci. Tumorigenicity assays were performed by injecting subcutaneously lo6 viable cells per site into 6-week-old nude mice. Data are expressed as number of tumours observed per number of injections. PE, Plating efficiency; ND, not done. Exp Cell Res 168 (1987)

Transformation of mammalian cells with the v-myc oncogene 279 pSV2neo-transfected cells within 5 weeks after injection, whereas 1 cm turnout-s were present in mice injected with N-ras transfected cells after 10 days. After a significantly longer period of time, occasional turnouts appeared but from only a minority of sites inoculated with v-myc-transfected cells. Tumours also appeared at a similar incidence for pSV2-neo-transfected controls. Representative tumours were taken from the mice; they exhibited a very ‘wild’ transformed phenotype, quite different from the morphology of the parental cells. Cells from one tumour (G2/3-T3) were found to have preserved the v-myc gene and to express the pl lo@w-mY~ protein at a level comparable to that of the parental cells (G2/3) (table 1, fig. 3A). However, their growth properties were different from those of the parental cells; G2/3-T3 tumour cells showed a 4-fold higher plating efficiency in soft agar and a better ability to form foci when co-cultivated with NIH-3T3 (table 1). However, cells from another tumour (G2/3-T2) were no longer resistant to G418 and Southern blot analysis and immunofluorescence with anti-gag antibody showed that they had lost the transfected gene (table 1, fig. 3A). Growth Factor Requirements of v-myc-Containing NIH-3T3 Cells In order to assay the growth factor requirements of the v-myc-containing cells, growth-curve experiments were performed in low serum and serum-free medium. v-myc-containing cells, ras-containing cells and parental normal cells showed similar growth curves in low serum (0.5-2%). In contrast, when cultured in serum-free medium, v-myc-containing cells and the parental cells died within 5-6 days, while ras-containing cells continued to grow normally (data. not shown). Clearly, v-myc- and ras-containing cells have different requirements for exogenous growth factors. It is known that NIH-3T3 cells transformed with an activated ras gene produce transforming growth factor a (TFGa) [26] and possibly other growth factors. Since TGFa and EGF both bind to the EGF receptor, production of TGFa would result in blockage of the receptor and this can be measured by reduced binding of exogenous [1251]EGF [33]. We found no inhibition of binding of [‘251]EGF to vmyc-containing cells compared with control NIH-3T3. Therefore, it appears that v-myc-transformed NIH-3T3 cells do not make TGFa. In order to test whether the inability of v-myc-transformed cells to form foci is related to the lack of production of an EGF-like growth factor, transformed clones were co-cultivated with normal NIH-3T3 cells in the presence of varying amounts of EGF. In the absence of EGF, ras-transformed cells, as expected, form foci with a very high efficiency (40% or more, see table l), whereas only two out of eight v-myc-transformed clones tested showed any ability to form foci, and at an efficiency at least 20-fold lower than ras-containing cells (table 1). With the addition of EGF to the medium, v-myc-transformed clones tested showed a four- to ninefold increase in the number of foci, though the efficiency is still well below that of ras-transformed cells (table 2). Addition of high levels of EGF (2 nM) causes normal NIH-3T3 cells to overcome contact inhibition of growth and Exp Cell Res 168 (1987)

280

Falcone et al.

they form foci by themselves (data not shown). The observation that v-myccontaining NlH-3T3 require a lower concentration of EGF than normal NIH-3T3 to form foci suggests that v-myc sensitizes the cells to this growth factor. Properties of v-myc Transformed Epithelial

Cells

Transfection of the MC29-LTR-G2 construct into epithelial cells followed by selection in G418 resulted in a range of morphologically altered colonies after about 2 weeks (see fig. 2). Colonies of BBN3 and BBN7 expressing v-myc protein were distinct from their EJ-ras-transfected counterparts [32]. They were characterized by the disorganized array of cells at the periphery, in contrast to the smooth colony edges presented by neo-transfected controls, and a loss of well defined cell borders within a colony (fig. 2 E, F). Colonies selected for further study showed intense nuclear staining with a-gag antibodies, whereas nucleoli remained unstained. A uniform staining intensity was observed throughout all the cells within a clone. Further confirmation of expression of the v-myc construct was shown by Northern blotting, where the major transcript was detected at 6 kb (fig. 3B, lanes 3-7). Transformed colonies picked after transfection and grown into mass culture displayed similar saturation densities to that of parental lines and no tendency to pile up in contrast to the behaviour of EJ-ras transfectants. In addition, no giant cells, another characteristic of EJ-ras-transfected epithelial cells, were observed in myc-transfected cells. All the v-myc transfectants tested consistently displayed anchorage-independent growth, albeit at a very low efficiency, ten- to twentyfold lower than that observed for EJ-ras transfectants (table 3). Tumorigenicity assays in nude mice resulted in rapidly growing tumours within 2 weeks after the injection of EJ-rastransfected BBN3 or BBN7. However, no tumours were detectable in the corresponding v-myc transfectants after 2 months. As shown in table 3, rare tumours appeared in BBN3 v-myc cells after prolonged latency periods (3-4 months) and, in contrast to NIH-3T3 transfectants, cells isolated from these tumours all retained the v-myc construct as assessed by immunofluorescence and G418

Table 2. Znjluence of EGF on foci formation Percentage focus formation Cell line

-EGF cw

0.1 nM EGF (%I

0.3 nM EGF (%I

N-rll.9 neo 9 G2/3 G4/3

42 0 0.6 2

36 0 3.1 9

30 0 5.4 8.3

The experiments were performed as described in Materials and Methods. At 2 nM EGF, parental NIH-3T3 cells lost contact inhibition of growth and began to form foci. Exp Cell Res 168 (1987)

Transformation of mammalian cells with the v-myc oncogene

281

resistance. No spontaneous background has ever been detected with pSV2neotransfected parental lines. Tumour-derived BBN3 v-myc cells were morphologically similar to cells of the original inoculum but, when they were assayed in C57BL/6J syngeneic hosts for tumorigenic potential, no tumours were detected even after 4 months. All EJ-ras-transfected cells produced tumours in syngeneic mice after very short latency periods.

DISCUSSION Previous transfection experiments using rat embryo fibroblasts have shown that cooperation between the ras and myc oncogenes results in the formation of transformed and tumorigenic permanent cell lines [34]. The fact that the action of the ras oncogene can also be complemented by polyoma large-T and adenovirus early region 1A functions [35], both of which have been implicated in cell immortalization, led to the hypothesis that the myc oncogene helps provide an establishment/immortalization function required for full cell transformation. However, the data presented here show that the introduction of the v-myc gene into already immortalized cells induces some important phenotypic alterations. After transfection of v-myc-containing constructs with a neo-resistance gene followed by selection in G418, morphologically transformed colonies can be obtained which are capable of anchorage-independent growth. However, transformation is incomplete when compared with the more dramatic changes induced by an activated ras gene. In particular, v-myc-transformed NIH-3T3 cells cannot

Table 3. Transformed properties clones

and tumorigenicity

Anti-gag

of v-myc BBN3 and BBN7 epithelial

Tumorigenicity

Cell line

Fluorescence

Morphology

% PE in agar

BBN7 pEJ-ras

-

T

2.1

818 (2 wks)

8/8 (2 wks)

BBN7 neo

-

N

0

O/8

O/8

BBN7 v-mycA BBN7 v-mycE BBN7 v-mycG

T T T

0.2 0.2 0.1

O/8 O/8 018

O/8 O/8 018

BBN3 pEJ-ras

++ ++ + -

T

2.7

8/8 (2 wks)

8/8 (2 wks)

BBN3 neo

-

N

0

O/8

O/8

BBN3 BBN3 BBN3 BBN3

+ + + +

T T T T

0.1 0.1 0.2 0.1

l/8 (3 mths) 2/8 (3 mths) O/8 l/8 (4 mths)

018 O/8 018 O/8

v-mycB v-mycC v-mycG v-mycE

Nude mice

Syngeneic mice

BBN3 and BBN7 cells were transfected only with the MC29-LTR-G2 construct. performed as described in table 1. Syngeneic mice were C57 BLW.

Tumorigenicity

assays were

Exp Cell Res 168(1987)

282

Falcone et al.

be selected as transformed foci after transfection. It appears that the cells are not capable of growing over the normal surrounding NIH-3T3 cells. However, we have shown that if clonal populations of v-myc-transformed NIH-3T3 cells are isolated and then co-cultivated with normal cells, some of the clones can form foci, though only inefficiently. A possible explanation for the differences in the ability of ras- and myc-transformed cells to form foci lies in their growth factor requirements. It is known that ras-transformed NIH-3T3 cells produce TGFa [26]. We have observed that addition of EGF to the medium increases the efficiency of foci formation when v-myc-transformed NIH-3T3 cells are co-cultivated with normal NIH-3T3 cells. These observations suggest that it may be the lack of TGFa production by the v-myc-transformed cells that is, at least in part, responsible for their inability to form foci. However, v-myc-containing NIH-3T3 cells are sensitized to EGF; they form foci in response to lower levels @-lo-fold) of EGF than are required for stimulation of confluent monolayers of normal cells to overcome contact inhibition. This sensitization to growth factors may also account for the low level of anchorage-independent growth found in v-myc-containing cells, since others have shown that EGF alone can induce colony formation in c-myctransfected rat fibroblasts [36]. In both fibroblasts and epithelial cells, a striking aspect of the incomplete transformation induced by the v-myc oncogene is the inability to form tumours in nude or syngeneic mice. The inefficiency of the v-myc gene in inducing tumours in syngeneic animals had previously been reported for MC2Ptransformed chicken fibroblasts [6]. More recently, v-myc-transformed quail fibroblasts have been found incapable of inducing tumours when injected into nude mice [7]. We have found that v-myc-transformed NIH-3T3 cells only rarely produce tumours in nude mice and even then only after a relatively long latency. When these tumours are analysed, they have different properties from the parental cells, and since occasional tumours also appeared from neo-resistant controls after a comparable period of time, a secondary neoplastic event has probably occurred. Whether vmyc has any effect on the frequency of this second event is difficult to determine in NIH-3T3. However, the observation that BBN3 v-myc cells gave a low incidence of tumours, while BBN3-neo controls gave no tumours, suggests that the presence of v-myc does enhance the generation of tumorigenic cells. The results on tumorigenicity obtained by us and others using v-myc contrast with experiments by Keath et al. [37], in which NIH-3T3 and rat-2 fibroblasts transfected with a mouse c-myc gene under the control of the MLV promoter have been shown to rapidly induce tumours in nude mice and syngeneic animals. Although it is possible that v-myc and c-myc behave differently in mammalian cells, we prefer the alternative explanation that the NIH-3T3 cells used by Keath et al. [37] differ from ours and already contain the additional genetic change required for tumorigenicity after introduction of myc. In conclusion, we have shown that v-myc induces partial transformation of Exp Cell Res 168 (1987)

Transformation

of mammalian

cells with the v-myc oncogene

283

both mouse fibroblasts and epithelial cells and that, even with high expression, neither cell type is directly tumorigenic. Moreover, the ras and the myc oncogenes have different effects on these cells, both on morphology and growth alterations. We propose that differences in growth factor production of myc- and ras-transformed cells could account at least in part for their different transformed properties. ras-Transformed NIH-3T3 cells grow in serum-free medium and are known to produce TGFa, whereas the v-myc cells do neither. However, it has been reported by others that cells constitutively expressing a c-myc gene have a diminished requirement for PDGF [38] and we have shown here that v-myctransformed NIH-3T3 cells are sensitized to lower levels of EGF. It appears then that v-myc-transformed NIH-3T3 cells still have a requirement for certain growth factors but that this is reduced. This may account for the morphological transformation and anchorage independence observed. Experiments are currently in progress to determine whether v-myc-transformed epithelial cells have become independent or have a reduced requirement for any specific growth factors. G. F. was the recipient of a post-doctoral fellowship from the Istituto Pasteur-Fondazione Cenci Bolognetti. The work was supported jointly by the Medical Research Council (UK) and the Cancer Research Campaign (UK).

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Received April 9, 1986 Revised version received June 26, 1986

Exp Cell Res 168 (1987)