71

Cancer Letters, 67 (1992) 71- 78 Elsevier Scientific Publishers Ireland Ltd.

C-ets-1 protooncogene expression immortalized rat fibroblasts L.Z. TopolaPb, A.G. Tatosyanb, A. Seth” ‘Laboratory Center,

R. Ascionea,

of Molecular Oncology, National

alters the growth properties

D.M. Thompson”,

Cancer Institute,

Moscow (Russ/a) and ‘Centre for Early Human

D.G. Blair”, I. Kola’ and

Frederic/c, MD, 21702-1201

Development,

Clayton,

of

(USA),

bCancer Research

Victoria (AustraliaJ

(Received 29 May 1992) (Revision received

11 September

(Accepted September

1992)

14 1992)

Summary Ets family genes have been cloned and characterized from a variety of species ranging from human to Drosophila. The ets proteins encode transcript.ion factors that activate transcription via specific binding to GGAA core

sequence enhancers.

present

in

various

promoter/

To investigate the role of ets protooncogene expression on the growth properties of rat embryo fibroblasts (REF), we constructed and introduced ets expression vectors into primary, (IS well as immorfalized REF cells. The transfected cells contained multiple copies of the vector DNA, and the Northern blot analysis demonstrated overexpression of the c-ets-l-specific mRNA. Although the expression of the ets genes was unable to immortalize primary rat embryo Fbroblasts, the expression of e&-l in REF-1 cells enabled their growth in serum-.free medium and effected tumorigenic activit,y in nude mice.

Correspondence to: L.Z. Topol, Laboratory cology, National Cancer Institute, Frederick, USA. 0304s3835/92/$05.00 Printed and Published

of Molecular OnMD, 21702-1201,

0 1992 Elsevier Scientific Publishers in Ireland

Keywords: c-ek- 1 protooncogene; rat embryo fibroblasts; immortalization; transformation Introduction Mutations, amplification, rearrangements and overexpression of cellular protooncogenes have been found in a wide variety of tumor cells and tissue [1,2]. Neoplastic transformation can be caused by either expression of a cellular protooncogene product at inappropriately high levels, or the expression of an altered form of such protein [3,4]. In an attempt to understand the role of overexpression of protooncogenes in cellular growth and tumorigenesis, a variety of different protooncogenes have been tested for their ability to immortalize primary cells, as well as to transform various established cell lines [2]. Several protooncogenes encode nuclear factors, which may act as activators of the transcription of genes that mediate transformation [5,6]. Although v-jun and v-fos oncogenes transform established cell lines, the latter oncogene is unable to immortalize primary cells [7 - 91. In addition, established fibroblast cell lines, such as rat-1 and NIH3T3, Ireland Ltd.

72

have been shown to be transformed by overexpression of nuclear protooncogenes, in particular, c-myc, N-myc, c-jun, c-fos, c-e&-l and c-ets-2 [3,6,10 - 141. Unlike transformation, immortalization of primary cells can be achieved by overexpression of a single nuclear protooncogene product, such as c-myc and c-fos [9,X2]. However, other nuclear protooncogenes, such as c-jun, are incapable of immortalizing primary cells [6]. Although overexpression of the e&-l and ets-2 protooncogene products in NIH3T3 cells has been shown [13,14] to induce foci of transformed cells, no transformation or immortalization of primary cells caused by overexpression of the e&l or ets-2 product has been reported however. Moreover, it is not clear whether the overexpression of the ets-1 or ets2 product can transform cells other than NIH3T3 mouse fibroblasts. This study investigates the ability of ets protooncogenes to immortalize primary rat embryo fibroblasts, as well as to transform primary and established rat embryo fibroblasts (REF- 1). Our results demonstrate that the overexpression of c-ets-1 gene does indeed alter the growth properties

of REF-1 established ceils by abolishing their serum requirement for growth; it also induces tumors in nude mice. However, the overexpression of the e&l or ets-2 gene product is unable to either immortalize or transform primary rat embryo fibroblasts. Materials and Methods Cells

Primary rat fibrobiasts were obtained from 18 - 23 day embryos (strain, Fischer). The rat immortaliied REF-1 cells have been described previously [17]. Cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO) with 10% fetal calf serum (Hyclone), in low serum (0.5%) or serum-free defined medium QBSF51 (Quality BiologicaI, Gaithersburg, MD). DNA transfection DNA transfection of REF-1 cells was performed by the calcium phosphate DNA precipitate technique [16]. Cells were seeded at a density of 3 x lo5 cells per 60-mm dish and were transfected with 5 pg of plasmid DNA and colonies resistant to G-418

F&. 1. ~ons~uction of the e&-l expression vectors (pCEGV-1A and pCEGV-1C). A DNA fragment containing chicken ets-1 cDNA (1.6 kb) was cloned into the Born HI site of vector, pFpgv-1 (5.0 kb, ref. 15). The vector pCEGV-1A (6.6 kb) contains ets-1 in correct orientation and the pCEGV-IC (6.6 kb) contains efs-1 in reverse orientation.

73

(Geneticin, 400 pglml; Gibco Labs.) were pooled after 14 days of incubation. TS-42 cells were derived from the pCEGV-1C vector and the TS-43 cells were derived from the pCEGV1A vector. Growth properties of TS-42 and TS-43 were investigated in a defined medium (QBSF-51) containing either low or no serum. Plates were incubated at 37OC for 14 - 21 days with changes every 4 days, foci of densely-growing cells appeared after 10 days. Co~s~~ctju~ of the e&f expression vectors A 1.6-kb DNA fragment that contained the entire coding region from the chicken e&l gene 1131 was cloned at the Bum HI site of pFpgv-l (Fig. 1). The pFpgv-1 vector contains the entire MSV LTR with the promoter, the SV40 virus pol~~adenylation signals and neomycin gene to confer resistance against the drug, G418 [15]. Positive clones with the ets-1 gene in correct (pCEGV-1A) and reverse (pCEGV-1C) orientation were identified by restriction enzyme analysis of ‘mini’ prep DNA. Southern and northern blot affa~ys~s High molecular weight DNA was prepared and digested with restriction enzymes as described [ZO] .The digest was run on a 0.8% agarose gel fol1~owed by transfer onto nitrocellulose [20]. The nitrocellulose blot was hybridized with a “P-nick-~anslated ets- 1 probe. For Northern blot analysis, total cytoplasmic RNA was isolated and run on a formaldehydecontaining agarose gel followed by transfer onto a nitrocellulose paper [21]. The nitrocellulose blot was probed with the chicken e&-l cDNA probe. Detailed conditions for hybridisation and washing have been described [22]. Tumorigenicjty of ~?~-~-~ransfectedcefl lines in nude mice Nude mice weire injected subcutaneously with 3 x lo6 cells which were expanded from foci grown in serum-free or low serum containing medium [23]. The mice were examined every week up to 6 months for tumor induc-

tion. EJ-ras transforming cells were used as a positive control. Results Co~str~c~o~ of the ets-1 expression uecfors fpCEGV-IA and pCECV-lC) A 1.6-kb DNA fragment containing the complete coding region of the c-e&-l (chicken) gene was cloned into the Barn HI site of the pFpgv-1. This vector contains the MSV LTR, SV40, polyadenylation signals and a neo gene for drug selection (see Ref. 15; Materials and methods). Two ets- 1-containing expression vectors, pCEGV-1A (correct orientation) and pCEGV-lC, (opposite orientation) were isolated and used for transfection studies (Fig. 1). Ets-l expression does not immortalize primary rut embryo fibroblasts To study whether ets family genes are able to immo~alize primary cells, we transfected e&-l (pCEGV-1) and ets-2 ~pMME-18, ref. 14) expression vectors into rat embryo fibroblasts (REF) using the calcium phosphate precipitation technique [ 161. The G41W selected colonies expressed ets mRNA, but were unable to proliferate and senesce (data not shown). Although nuclear transcription factors, such as c-myc and c-fos, have been shown to immortalize REF cells [9,12], the e&-l and ets-2 ~ansfectants did not appear to possess this activity. E&-l transforms established rat embryo fibrobtasts Previously, it has been shown that the c-e&1 protooncogene ~ansforms NlH3T3 cells [13]. In this study, we investigated whether the e&-l gene product is able to transform other established cell lines, such as REF-1. This cell line had been established from primary rat embryo fibroblasts by transfection with the early regions of the simian adenovirus SA7 genome 1171. The REF-1 cell line was chosen because it possesses many of the normal cellular phenotypic characteristics, such as: (a) flat

phenotype; (b) anchorage-dependence; (c) requirements for high concentrations (10%) of serum growth factors; (d) being nontumorigenic in nude mice; and (e) ability to be transformed by oncogenes; collectively, these properties make this cell line an appropriate recipient for transfection assays in order to study the transforming activity of oncogenes and protooncogenes [ 181. To investigate whether the ets-1 is able to transform this cell line (REF1) and alter its growth properties, we introduced our expression vectors containing the ets gene in both forward (pCEGV-1A) and reverse (pCEGV-16) orientation by the calcium phosphate DNA transfection method [ 161. Both vectors induced foci of transformed cells when the G418@ colonies were grown in a serum-free or low serum (0.5%) containing medium (Fig. 2b). However, we detected much larger foci when cells were transfected with pCEGV-1A (Fig. 2b) as compared to those transfected with the pCEGV-1C (Fig. 2~). Moreover, for pCEGV-lA, the proliferative activity in this serum-deficient media was also much greater than with the pCEGV1C vector. No transformed foci were detected in cells transfected with the vector that lacks the ets-1 gene, suggesting that the expression of ets-1 is required to morphologically alter the REF-1 cells (Fig. 2a).

Ets-1 is expressed at high levels in transformed

Fig. 2. Morphological phenotypes of REF-1 cells transformed with c-ets-1 protooncogene grown in serumm-free medium. Panel: (a) control REF cells; (b) cells transformed with pCEGV-1A; (c) cells transformec I with pCEGV-IC.

cells To investigate whether morphological changes in these cells is mediated by ets-1 excell pression, we expanded two transformed lines, TS-42 and TS-43 and examined for the ets vector DNA integration and its RNA expression. High molecular weight DNA was prepared from these cell lines, digested with Barn HI and performed Southern blot transfers using an ets- l-specific probe. Figure 3A shows 1.6-kb fragment, common the expected for both the expression vectors (lanes 2 and 3). No such band was detected in the control REF cells (lane 1). The larger sized bands (minor) are most likely due to the presence of

75

B 1

2

3

M -

23.0

-

2.3

-

2.0

M

18s’

0

Fig. 3.

A. Integration of c-e&l sequences into the genome of REF-1 cells. DNA was extracted from TS-42, TS-43 and control cells and digested with Barn HI. The digest was analyzed by Southern blots using an ek-l-specific probe. Lane 1, REF-1 transfected with pSV2neo; lane 2, TS-42 cell line carrying pCEGV-1A; lane 3, TS-43 cell line carrying pCEGV-1C. B. Expression of c-e&l-specific mRNA in transformed REF-1 cells. Total cellular RNA was isolated from TS-42, TS-43 and normal REF-1 cell lines. Lanes 1, TS-42 cell line; lane 2, TS-43 cell line; lane 3, REF-1.

endogenous ets-1 sequences or gene rearrangement. To examine whether the transformed cells are able to express the ets- 1 gene, total cellular RNA was prepared and analyzed by Northern blots. The TS-42 cell line, which was transformed by pCEGV-1A containing the ets-1 gene in its correct orientation, expresses high levels of a 5.6-kb sense mRNA (Fig. 3B, lane 1). In contrast, the TS-43 cell line that was transformed by the pCEGV-1C vector containing the ets-1 gene in an opposite orientation, expresses high levels of a 4.2-kb antisense RNA (Fig. 3B, lane 2). In addition, the TS-43 cell line also produ8ces low levels of the 5.6-kb sense RNA, which may also explain the low transforming ability of this cell line. Endogenous e&l mRNA was not detectable because of its low level of expression (Fig. 3B, lane 3).

Ets 1 transformed REF-1 cells can grow in serum-free medium The role of c-ets-1 expression on the growth properties of cell lines TS-42 and TS-43 was tested in a serum free and low serum (0.5%) containing defined media. Both the cell lines were able to grow for an extended period of time; however, the growth rate for the TS-42 cell line expressing high levels of 5.6-kb sense RNA, was 2.5 times higher than the TS-43 cell line that expresses extremely low levels of the 5.6-kb ets-1 message (Fig. 4). The parental cell line (REF-1) , lacking ets-1 mRNA, was unable to grow in this low-serum or serum-free medium. Moreover, after 10 days, TS-42 and TS-43 cell lines showed foci of denselygrowing and morphologically-altered cells on top of the flat monolayer present in serum-free medium (Fig. 2). Although both the cell lines

76 lo-

%-

l

OO

I 1

I 2

I 3

I 5

I 4

1 6

FIEF-1 (Control)

I 7

Days Fig. 4. Growth curve of the c-ets-l-transformed cells. Cells were seeded at a density of 1.5 DMEM containing 10% fetal calf serum. After 16 h the media was changed to a defined medium the cells were counted every day.

show foci of transformed cells, the TS-43 cell line always grew slower than the TS-42 cell line. No such foci were observed in the control cells (Fig. Za). These results suggest that the expression of the e&l gene markedly alters the cellular morphology and abolishes the serum dependence required for growth of these cells. Ets-1 expressing REF-1 cells are tumorigenic in nude mice We also examined the tumorigenicity of normal (REF-1) and c-ets-1 transformed cell lines Table 1. Tumorigenicity lines.

of the c-ets-1-transfected

Cell lines

Tumor production

Time in weeks

REF-1 TS-42 TS-43 TS-44 (EJ-ras)

o/3 3/3 l/3 3/3

24 8 10 1

cell

The cell line TS-42 and TS-43 were grown in serum-free medium and injected subcutaneously into athymic nude mice (3 x lo6 cells/mouse). Mice were monitored weekly for tumor formation.

x

lo5 cells/plate without serum

in

and

(TS-42 and TS-43) in nude mice. The c-ets-ltransformed cells were grown in serum-free medium for 2 weeks and then injected subcutaneously into nude mice. Both the cell lines induced tumors in 2 months (Table I). However, again the efficiency of tumor induction was higher for the cell line that produces higher levels of 5.6-kb ets-1 mRNA than for the cell line that produces lower levels of the 5.6-kb e&l mRNA. In contrast, the control REF-1 cells did not show any tumor formation for a long period. The efficiency of tumor induction and growth capacity in serum-free medium for both the cell lines (TS-42 and TS43) correlates with the level of expression of the 5.6-kb e&l mRNA. Discussion We have investigated the effects of c-ets-1 protooncogene on the morphology and growth properties of primary and immortalized rat embryo fibroblasts. The expression of the et%-1 or ets-2 genes were unable to either immortalize or transform primary rat embryo fibroblasts. However, expression of the c-ets-1 gene transforms immortalized REF-1 cells and abolishes their serum requirement for growth.

77

The ets-l-transfected REF-1 cells formed foci similar to those observed with the c-ets-1 transfected NlH3T3 cells in a defined medium containing low serum [13]. In this medium the normal cells grow only after addition of epidermal growth factor or fibroblast growth factor [19]. The immortalized REF-1 cells were selected for our study because their morphology and growth characteristics are near to normal and they do not show spontaneous transformation during long-term (more than 6 months) growth in vitro and in vivo. Transformation of the REF-1 cell line correlates with the presence of multiple copies of integrated ets-1 vector DNA and efficient transcription of 5.6-kb c-ets-1 mRNA. The TS-42 cell line proliferates efficiently in serum-free medium and is highly tumorigenic in nude mice, which correlates with the overexpression of ets-specific 5.6-kb sense mRNA. On the other hand, the TS-43 cell line grows slowly and is weakly tumorigenic and expresses only low levels of the 5.6-kb sense mRNA. We have also shown that the foci of morphologically transformed cells appear rapidly within the population of proliferating cells and they are the result of growth of existing cells with definite genetic: changes, but not the result of selection of spontaneously occurring genetic variants. The expre’ssion of c-ets- 1 did not alter the morphology and the growth rate of the REF-1 cell line in DMEM containing 10% serum medium; however, it did drastically change its growth properties in serum-free medium, suggesting that such cells undergoing transformation initially remain sensitive to the inhibitory effects of neighboring contactinhibited non-transformed cells. Taking into consideration the :nuclear localization of the ets-1 protein, as well as its transcriptional activation activity, it is quite possible that the activation of certain cellular genes by e&l might lead to cell proliferation. The cells isolated from foci that appe’ared in serum-free medium is able to induce tumor formation within 2 months of inoculatison into nude mice. The cell line (TS-43) expressing low levels of 5.6-kb mRNA gave only l/3 tumors as compared to

3/3 by a cell line (TS-42) that is expressing high levels of 5.6-kb mRNA. Further, no tumors were detected in mice injected with control cell lines for a long period of time, suggesting that the ets-1 expression plays a role in tumor induction. Thus, our data demonstrates that the c-e&-l expression is required to render REF-1 cells capable of growing in the absence of serum growth factors. It is, however, also possible that additional genetic events may have taken place among the cells which have acquired the ability to divide in the absence of serum. These additional events may directly or indirectly depend on the transcriptional activation activity of c-ets-1. Therefore, these ets-1 expressing cell lines should prove useful in identifying cellular genes activated as a consequence of c-ets- 1 expression. Acknowledgments We thank Julie Olson for technical assistance; Karen Cannon and Lisa Virts for preparing the manuscript. References Cooper,

J.M.

(1991)

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I.M. (1984) c-fos pro-

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C-ets-1 protooncogene expression alters the growth properties of immortalized rat fibroblasts.

Ets family genes have been cloned and characterized from a variety of species ranging from human to Drosophila. The ets proteins encode transcription ...
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