Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4442-4446, May 1995 Cell Biology

ETS1 suppresses tumorigenicity of human colon cancer cells () HIROAKI SUZUKI*t, VINCENZO ROMANO-SPICA*, TAKIS S. PAPASt, AND NARAYAN K. BHAT*§ *Laboratory of Molecular Oncology, National Cancer Institute, P.O. Box B, Frederick, MD 21702-1201; *Center for Molecular and Structural Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425-2213; and §Program Resources, Inc./DynCorp, Frederick Cancer Research and Development Center, P.O. Box B, Frederick, MD 21702-1201 Communicated by Robert C. Gallo, National Cancer Institute, Bethesda, MD, January 3, 1995 (received for review June 24, 1994) ABSTRACT We have ectopically expressed transcription 202 (Glu202 to Gly202) and 212 (Asp212 to His212) is capable of factor ETS1 in two different highly tumorigenic human colon binding to ETS1-binding sequence (EBS) motifs in DNA but cancer cell lines, DLD-1 and HCT116, that do not express lacks transcriptional activity. Overexpression of mutant ETS1 endogenous ETS1 and have obtained several indepen- protein in DLD-1 cells did not reduce their tumorigenic dent clones. The expression ofwild-type ETS1 protein in these potential. Since DLD-1 cells express copious amounts of other colon cancer cells reverses the transformed phenotype and ETS-related (9), results presented in this paper tumorigenicity in a dose-dependent manner. By contrast, demonstrate that reduction in tumorigenicity appears to be expression in DLD-1 cells of a variant form of ETS1, lacking due to overexpression of wild-type ETS1. transcriptional activity, did not alter the tumorigenic prop- erties of the cells, suggesting that the reduction in tumorige- nicity in these clones was specific for the wild-type ETSI MATERIALS AND METHODS products. Since these colon cancer cells have multiple genetic Plasmid DNAs. A 1.95-kb full-length human ETS1 cDNA alterations, the system described in this paper could be a good (ref. 18; gift ofJ. M. Leiden, University of Chicago) was cloned model to study the suppression of tumorigenicity at a tran- into the HindIII site of pcDM7 and pcDNAI (Invitrogen). scriptional level, which could lead to the design and develop- pEBSCAT plasmid DNA has been described (25). ment of novel drugs for cancer treatment. Isolation of cDNA Coding for Mutant ETS1. Total genomic DNA from DE1-2-3 cells (1.2 ,ug) was used for polymerase ETS1 is a member of the ets gene family and is a cellular chain reaction (PCR) with sense (5'-ggattcCACCATGAAG- counterpart of the v-ets oncogene of the avian erythroblastosis GCGGCCGT-3'; positions -4 to 14) and antisense (5'- virus E26'(1-3). The ETS1 gene is expressed at high levels in caagcttgTCAGTGCCATCACTC-3'; positions 1321-1335) lymphoid cells (4-9) and in astrocytes and endothelial cells ETS1 primer pairs to clone integrated ETS1 cDNA as de- (10-12). ETS1 is a nuclear phosphoprotein (13-15) that binds scribed (26). to purine-rich DNA sequences and functions as a transcription Cell Culture and Transfection. The human colon carcinoma factor (1-3, 16-19). The DNA-binding domain is localized at cell lines DLD-1 (27) and HCT116 (28) were transfected with the carboxyl-terminal region (20-22), and the transactivation human ETS1 cDNA expression vectors along with pSV2neo by domain is localized in the protein domain encoded by exons 5 use of Lipofectin reagent (GIBCO/BRL). Transfectants were and 6 ofETS1 (23). The ETS1 protein binds to its target DNA selected with G418 (400 ,ug/ml). DE-1-1, DE1-1-7, and sequences present in the transcriptional regulatory regions of DE1-3-2 are DLD-1 transfectants obtained with pcDM7 ETS1 a number of "housekeeping" , as well as certain types of expression vector, whereas DE1-2-3, DE1-2-4, and DE1-2-8 tissue-specific genes, and regulates their transcription (1-3). are DLD-1 transfectants obtained with pcDNAI ETS1 expres- Thus, it is possible that the aberrant expression of the ETS1 sion vector. DME1-1, DME1-9, DME1-12, DME1-21, and gene may result in the alteration of growth. The expression of DME1-26 are DLD-1 transfectants obtained with PRC cyto- the ETS1 gene is induced at late stages of thymocyte differ- megalovirus mutant ETS1 expression vector. HE1-2 and entiation (7). In resting T cells, the ETS1 gene is expressed at HE1-3 are HCT116 transfectants obtained with PRC cyto- high levels, and upon activation of T cells, the ETS1 gene megalovirus ETS1 expression vector. product is decreased to low levels (8). ETS1 Cell Labeling, Immunoprecipitation, Polyacrylamide Gel is also induced during differentiation of P19 murine embryonal Electrophoresis, and Electrophoretic Mobility-Shift Assay. carcinoma cells (24). These results suggest that ETS1 plays a These were done as described (7, 9, 29-31). role in induction of cellular differentiation and suppression of cell growth. To identify whether ETS1 has tumor-suppressive activities, RESULTS AND DISCUSSION we have ectopically expressed ETS1 in two types of human Generation of DLD-1 Cells Expressing ETS1 Proteins. We colon cancer cell lines, DLD-1 and HCT116, that do not expressed the full-length ETS1 protein in DLD-1 and HCT116 express endogenous ETS1 protein. We have obtained several cells under the control of the cytomegalovirus promoter and independent clones expressing different amounts offull-length obtained several independent cell lines. These cell lines were ETS1 protein. Overexpression of ETS1 in these colon cancer derived from single cell clones. DNA blot analyses of trans- cells did not change their anchorage-dependent cell growth. fectants digested with multiple restriction enzymes confirmed However, rates of anchorage-independent growth were re- that these transfectants truly represented independent clones duced in a dose-dependent manner. Cells expressing the (data not shown). ETS1 protein formation was examined by highest levels of ETS1 made smaller and fewer colonies in soft radioimmunoprecipitation using ETS1-specific monoclonal agar and showed reduced tumor incidence in nude mice. A In DLD-1 transfectants mutant form of ETS1 with amino acid substitution at codon antibody (13, 31). (DE1-1-1, DE1-1-7, Abbreviations: CAT, chloramphenicol acetyltransferase; EBS, ETS1- The publication costs of this article were defrayed in part by page charge binding sequence. payment. This article must therefore be hereby marked "advertisement" in tPresent address: Department of Pathology, Hokkaido University, accordance with 18 U.S.C. §1734 solely to indicate this fact. School of Medicine, Sapporo 060, Japan. 4442 Downloaded by guest on September 26, 2021 Cell Biology: Suzuki et at Proc. Natl. Acad. Sci. USA 92 (1995) 4443

(-) neo-1 0 DLD-1 (-) e' rt't'n o'onGo o ,n ...I...I bo D S Q0 O a n a c (- r r I I .: . :' I'- '' '''"' - - - - "':;.-.''.. + + ++ + + ; r *;, ,,I,':-...... , ''':;

-1 *. ''s E.p51-,,..:,~ i:,:'. ;_*.: ...*l _..ETS1 p42 -.- 1-1 (+I 2-4 (+) 2-3 (+ + +,*Mut)

FIG. 1. ETS1 protein in DLD-1 transfectants. Cells were labeled with [35S]methionine (100 ,uCi/ml) for 1 hr and were immunoprecipi- tated with the human ETS1-specific monoclonal antibody E44 in the absence (-) or presence (+) of cognate peptide (31). In T cells, the two ETS-1 isoforms detected (p51 and p42) are the products of full-length and alternatively spliced forms of ETS1 mRNA (13). DE1-2-3, DE1-2-4, DE1-2-8, and DE1-3-2), ETS1 protein 1-7 (+ + + + +) 2-8 (+ + +) 3-2(+ +) (p51) is encoded by exogenous ETS1 (Fig. 1), because (i) it is undetectable in parental cells (DLD-1) as well as in cells transfected with pSV2neo alone (DLD-1-Neol, -Neo2, and -Neo3), (ii) it is immunoprecipitated by two different ETS1 antibodies and the immunoprecipitation can be blocked by cognate (13, 30, 31), (iii) the levels of p51 correlated well with the levels of ETS1 mRNA initiated from the cyto- megalovirus promoter (data not shown), and (iv) p51 is similar in size to the endogenous full-length proteins detected in FIG. 2. Soft-agar colony formation assays of the ETS1 transfec- T-lymphoma cells (Fig. 1) (8, 13, 30). Also, we have previously tants. Cells were plated in 0.33% noble agar (Difco) in RPMI 1640 in DE1-1-7 with 15% fetal bovine serum at 104 cells per 60-mm plate and stained shown that the exogenous ETS1 protein expressed after 10 of in the nucleus and binds to the with p-iodonitrotetrazolium violet (1 mg/ml; Sigma) days cells is localized purine-rich incubation. ETS-1 protein expression: (-), negative; (+)-(+ + + + +), DNA sequences; further, this product is similar in biochemical relative levels of ETS1 in various DLD-1 ETS1 transfectants in cells expressed properties to the ETS1 protein expressed lymphoid as shown in Fig. 1. Wild-type and mutant (*Mut) ETS1 are expressed (29). However, we observed that ETS1 migrated as a doublet at similar levels. (p51/p50) in extracts derived from DE1-2-3 cells but as a single species (p51) in all other ETS1 transfectants examined (Fig. 1). ETS1. We found no substantial differences in their growth Formation of p50 ETS1 protein appears to be due to mutation rates (Table 1), suggesting that ETS1 expression did not affect in ETS1 (for details, see Fig. 4). The significance of p50 ETS1 the anchorage-dependent growth rates of the DLD-1 trans- protein is discussed below. fectants and was not toxic to these cells. On the other hand, Overexpression of ETS1 in DLD-1 Cells Reduces Their anchorage-independent growth rates, indicated by the colony- Ability to Form Colonies in Soft Agar and to Form Tumors in forming ability in soft agar, were reduced in ETS1 transfec- Nude Mice. To determine whether overexpression of ETS1 has tants. The ability to form colonies in soft agar was dependent any effect on cell proliferation, we studied the growth prop- on the level ofwild-type ETS1 expression (Table 1 and Fig. 2). erties of the DLD-1 transfectants expressing different levels of Not only the efficiencies but also the sizes of soft agar colonies Table 1. Growth and tumorigenicity of ETS1 transfectants TumorTumor incidence Tumor Doubling Soft-agar ETS1 i nude mice diameter,$ time,§ colonies, Cell line expression* 2 wk 3 wk 4 wk mm hr % Controls DLD-1 - 3/3 3/3 3/3 13.2 21.5 30.5 DLD-1 neo-1 - 5/6 5/6 6/6 9.4 19.0 20.9 DLD-1 neo-2 - 5/6 5/6 5/6 10.0 18.0 24.0 DLD-1 neo-3 - 4/5 5/5 5/5 11.0 24.2 ND ETS1 transfectants Wild-type DE-1-1 + 5/7 7/7 6/7 12.8 22.0 13.2 DE1-2-4 + 2/5 3/5 5/5 5.3 24.5 17.5 DE1-3-2 ++ 2/6 4/6 4/6 4.4 39.0 3.4 DE1-2-8 +++ 0/4 1/4 2/4 3.8 21.0 10.4 DE1-1-7 +++++ 0/9 0/9 3/9 3.7 19.5 7.4 Wild-type and mutant DE1-2-3 ++++ll 1/5 3/5 4/5 7.1 23.2 31.4 *-, Undetectable; +, low; + + + + +, high (comparable to the level in lymphoid cells as shown in Fig. 1). tCells (2 x 106) of each cell line were injected subcutaneously into the right anterior flank area of 6- to 8-week-old female nude mice. tMean diameter of the tumors in nude mice 4 weeks after injection. §Cells were plated at 2 x 105 in RPMI 1640 medium containing 15% fetal bovine serum. ¶Percent soft-agar colonies per 104 cells plated, scored after 10 days of incubation. ND, not done. IIWild-type and mutant ETS1 proteins are expressed at similar levels. Downloaded by guest on September 26, 2021 4444 Cell Biology: Suzuki et at. Proc. Natl. Acad Sci USA 92 (1995) were suppressed by transfection of ETS1 cDNA (Fig. 2); i.e., tumors in all nine nude mice studied within the same obser- cells expressing higher levels of ETS1 (DE1-1-7 and DE1-2-8) vation time (Table 1). However, in transfectants expressing showed a reduction in the number of colonies in soft agar, as abundant ETS1, the tumor incidence was delayed by 3 weeks well as in their colony size. The reduction in soft-agar colony and the tumors were smaller (Table 1). Three other ETS1 number was noticeable only when ETS1 expression occurred transfectants (DE1-2-8, -3-2, and -2-4) also showed reduced at moderate levels, suggesting that the ETS1 gene product(s) tumor incidences, but at different levels. However, DE1-2-3 may be competing with other gene products which are involved cells, expressing the p51/p50 ETS1 doublet, did not show a in anchorage-independent growth. The DE1-3-2 cells showed decrease in the number of colonies formed in soft agar (Fig. less colony-forming ability in soft agar than the DE1-2-8 cells, 2) or in the incidence of tumor formation in nude mice which express higher levels of ETS1 protein; this observation (Table 1). is consistent with the longer doubling time exhibited by the Characterization of Mutant ETS1. In DE1-2-3 cells, in DE1-3-2 cells. addition to p51, a faster migrating 50-kDa ETS1 protein (p50) The parental and neo transfectants (controls) all formed was specifically recognized by three different ETS1 antibodies tumors in nude mice within 3 weeks. In contrast, DE1-1-7 cells, raised against the amino-terminal (13, 31), middle (30), and expressing high levels of the ETS1 protein, produced no carboxyl-terminal regions (15) of ETS1 in both radioimmu- A

1 2 3 4 5 6 7 8 9 10 11 12 ETS1 [ p50 - P:i

B TA Domain DNA BD x Helix RVPS 1 EDE D · c( Helix Basic Domain ~~I KA M ' E=77\l 1GH ETS1 GH KA p -- - --7\ mETS1 C ETS2 1*8 gatcGAGACCGGAAGTGGGG D ETS2 1*8M gatcGAGACAACAAGTGGGG o 1 2 3 1 2 3 1 2 3 con- w (Jo w ETS21.8 - - -+ + + - - - E ETS218M + + + a a ] ETS1

1 2 3 ETS1 14 E 12 O. 0 10 8 0 x 6 4 I- 0(J 2 -FP 0 2 3

FIG. 3. (A) (Left) Characterization of the mutant p50 ETS-1 protein. Cells were labeled and lysates were immunoprecipitated with anti-ETS-1 monoclonal antibody E44. Bound antigens were released as described (30) and were used for immunoprecipitation with either E44 (lanes 1, 3, and 5) or G19E (ref. 15) (lanes 2, 4, and 6) antibody. Lanes: 1 and 2, DLD-1; 3 and 4, DE1-1-7; 5 and 6, DE1-2-3. (Right) Clone 0-15 encodes mutant ETS1 protein. Plasmid DNAs encoding mutant or wild-type ETS1 were used to generate proteins in TNT reticulocyte lysate (Promega). Total 35S-labeled translation product with no added DNA (lane 10) or with wild-type (lane 11) or mutant ETS1 (lane 12) cDNA and the ETS1 immunoprecipitates from DE1-2-3 cell extract (lane 7) or from mutant (lane 8) or wild-type (lane 9) translation products were analyzed. (B) Schematic representation of wild-type (ETS1) and mutant (mETS1) proteins and designated motifs: RVPS (Arg-Val-Pro-Ser), phosphorylation domain; TA domain, transactivation domain; DNA BD, DNA-binding domain. The amino acid substitutions from Glu (E) to Gly (G) and Asp (D) to His (H) in mutant ETS1 protein and their location are indicated. (C) Mutant ETS1 protein binds to DNA. Sequences of an ETS1-binding oligonucleotide (ETS2 1-8) and a mutant form (ETS2 1-8M) to which ETS1 does not bind (29) are shown at the top. Aliquots representing equal amounts of wild-type (lanes 2) and mutant (lanes 3) ETS1 proteins were mixed with 32P-labeled ETS2 1-8M oligonucleotide and the protein-DNA complexes were analyzed. Lanes 1, translation product without any added plasmid DNA. For competition, a 50-fold molar excess of nonradioactive ETS2 1-8 or ETS2 1-8M oligonucleotide was used. Positions of ETS1-DNA complex and free probe (FP) are indicated at right. (D) Wild-type ETS1, but not mutant ETS1, activates transcription through the EBS motif. DLD-1 cells were transfected with an optimum amount ofwild-type and mutant ETS1 expression vectors along with pEBSCAT plasmid DNA. Forty-eight hours after transfection, chloramphenicol acetyltransferase (CAT) activity was measured by diffusion assay (NEN kit). (Inset) Expression of ETS1 protein was measured by radioimmunoprecipitation 48 hr after transfection of DLD-1 cells without any added DNA (lane 1) or with mutant (lane 2) or wild-type (lane 3) ETS1 expression plasmid. Downloaded by guest on September 26, 2021 Cell Biology: Suzuki et al. Proc. Natl. Acad. Sci. USA 92 (1995) 4445

noprecipitation and Western blot analyses. As shown in Figs. , , 1 and 3A both p50 and p51 ETS1 were expressed at similar _ - _ _ _ levels and recognized by antibodies raised against peptides ._w derived from epitope regions encoded by exons 6, 7, 8, and 9 -uJ ,- u]ETS1 and from the carboxyl terminus of the human ETS1 protein (13, 15, 30, 31), indicating that p50 formation was not due to *e 4I truncation of the protein but appeared to be due to some other in ETS1. mutation(s) B (D'7 Mutant ETS1 Protein Has Amino Acid Substitutions. To C4 co - characterize the the ETS1 DNA in the I- mutation, integrated IllI UJltI I genome of DE1-2-3 cells (following transfection of exogenous 'r ' I ETS1 cDNA) was isolated from genomic DNA by use of PCR 68- and characterized according to the size of the ETS1 protein in an in vitro translation produced transcription-coupled sys- *"4'l|H| -, ETS1 tem. The protein immunoprecipitated from the in vitro trans- " lation products generated from clone 0-15 was very similar to 43 . the p50 ETS1 protein seen in DE1-2-3 cell extracts (Fig. 3A), suggesting that clone 0-15 encodes p50 ETS1. DNA sequence analyses of clone 0-15 revealed that it was identical to the ETSI FIG. 4. Expression of ETS1 protein in DLD-1 and HCT116 colon sequence (26), except for four nucleotide substitutions (at cancer cell lines. (A) DLD-1 cells were transfected with an expression codons and in its entire frame. vector carrying mutant ETS1 cDNA and independent clones express- 202, 208, 212, 296) open reading ing mutant ETS1 proteins were selected for further analysis. The ETS1 Two substitutions in codons 208 (GTC to GTT) and 296 (CCC transfectants and control cells were metabolically labeled with to CCT) did not effect any change in its amino acid residues. [35S]methionine for 1 hr and ETS1 proteins were detected by mono- However, substitutions in codons 202 (GAG to GGG) and 212 clonal antibody E44. Transfectants expressing wild-type ETS1 (DE1- (GAC to CAC) did change Glu202 to Gly202 and Asp212 to 1-7 and DE1-2-8) and mutant ETS1 (DME1-1, DME1-9, DME1-12, His212, respectively (Fig. 3B). Substitution of neutral amino DME1-21, and DME1-26) are shown. (B) HCT116 cells were trans- acid residues for acidic residues could account for the observed fected with an expression vector carrying wild-type ETS1 cDNA and faster migration of this mutant ETS1 protein (p50), similar to independent clones expressing wild-type ETS1 were selected for the mutation (Glu6 to Val6) in sickle cell hemoglobin (32). further analysis. The ETS1 transfectants and parental HCT116 cells Mutant ETS1 Protein Binds to DNA But Lacks were metabolically labeled with [35S]methionine and ETS1 proteins Transcrip- were detected by immunoprecipitation as in A. ETS1 proteins ex- tional Activity. Structure function analysis of ETS1 has shown pressed in DLD-1 (DE1-1-7) and HCT transfectants (HE1-2 and that the minimal DNA-binding domain consists of 85 aa and HE1-3) are shown. Size markers (68 and 43 kDa) are at left. is localized at the carboxyl-terminal region (21, 22). The transactivation domain maps to the protein domain encoded , DCC, MCC/APC, as well as activation of the oncogenes by exons 5 and 6 of the ETS1 gene (23), a region not well and Ki-ras (34, 35). DLD-1 cells contain one normal and conserved among other members of the ets family of genes. one mutant Ki-ras allele (Gly13 to Asp13), as well as point Exon 6 of human ETS1 encodes aa 177-243 (33); therefore, the mutations in to We observed no mutations observed in p50 appear to be clustered around the p53 (Ser241 Phe241) (36). Both changes in the level of wild-type or mutant p53 proteins putative transactivation domain of ETS1 (Fig. 3B). between the DLD-1-ETS-1 transfectants and control cells mutant and ETS1 were of to DNA by wild-type capable binding not Further- mutant ETS1 was unable to trans- radioimmunoprecipitation analysis (data shown). (Fig. 3C). However, (p50) of DLD-1 cells could be activate CAT gene expression through EBS, whereas wild-type more, tumorigenicity suppressed by (p51) ETS1 transactivated the EBS-CAT gene by 2- to 3-fold knocking out the mutant Ki-ras allele (36), suggesting that the (Fig. 3D). The failure to transactivate EBS-CAT gene expres- activated Ki-ras gene product plays a dominant role in tumor sion by mutant ETS1 was not due to transfection efficiency or formation. Activated Ras proteins have been shown to activate expression of p50 ETS1, because similar levels of p50 and p51 the MAP kinase signal transduction pathway, thereby inducing ETS1 were expressed in DLD-1 cells (Fig. 3D). These results are consistent with the finding that there is no amino acid Table 2. Tumorigenicity of DLD-1 and HCT116 substitution in the DNA-binding domain and suggest that the ETS1 transfectants mutation in the putative transactivation domain affects tran- Tumor incidence in nude scriptional activation. mice Mutant ETS1 Protein Has Lost the Ability to Reduce ETS1 Tumorigenicity of DLD-1 Cells. We expressed mutant ETS1 Cell line expression 2 wk 3 wk 4 wk protein in DLD-1 cells and obtained several independent Wild-type ETS1 transfectants expressing different amounts of mutant ETS1 DE1 1-7 +++++ 0/25 4/25 6/25 protein (Fig. 4A). DME-1, DME1-9, and DME1-21 cells DE1 2-8 +++ 0/10 3/10 6/10 express mutant ETS1 comparable to wild-type ETS1 expressed Mutant ETS1 in DE1-2-8 transfectants. Transfectants expressing mutant DME1-1 ++ 1/9 4/9 6/9 ETS1 proteins were more tumorigenic than wild-type ETS1 DME1-9 ++ 2/9 7/9 8/9 expressing transfectants (Table 2). These results further con- DME1-12 + 6/9 9/9 9/9 firm that the reduction in tumorigenicity of colon cancer cells DME1-21 +++ 2/10 8/10 8/10 appears to be due to overexpression of wild-type ETS1. DME1-26 + 2/5 5/5 5/5 Overexpression of Wild-Type ETS-1 in HCT116 Cells Also Parental HCT116 - 7/17 15/17 17/17 Reduces its Tumorigenicity. We have also obtained HCT116 Wild-type ETS1 transfectants expressing various amounts ofETS1 protein (Fig. HCT-HE1-2 + 8/18 16/17 16/17 4B) and found that the transfectants expressing higher levels HCT-HE1-3 +++ 0/18 9/18 13/18 ofwild-type ETS1 protein exhibited reduced tumorigenicity in Tumor growth was assayed as described in Table 1. Cells (2 x 106) nude mice (Table 2). were injected subcutaneously into the right anterior flank area of 6- to Colon tumor formation is associated with multiple genetic 8-week-old female nude mice (three to five animals per experiment) changes, including inactivation of the tumor-suppressor genes and tumor formation was examined on a weekly basis. Downloaded by guest on September 26, 2021 4446 Cell Biology: Suzuki et ail Proc. Natl. Acad. Sci USA 92 (1995) many target genes, including those encoding Ets family of 12. Wernert, N., Raes, M. B., Lassalle, P., Dehouclr, M. P., Gosselin, transcription factors (1-3, 37). In fact, overexpression of B., Vandenbunder, B. & Stehelin, D. (1992) Am. J. Pathol. 140, truncated ETS2 protein containing the DNA-binding domain 119-127. has been shown to block the myc gene expression mediated 13. Koizumi, S., Fisher, R. J., Fujiwara, S., Jorcyk, C., Bhat, N. K., through the Ras signal transduction pathway (38). It is also Seth, A. & Papas, T. S. (1990) Oncogene 5, 675-681. interesting that DLD-1 wild-type ETS1 transfectants express 14. Fujiwara, S., Fisher, R. J., Seth, A., Bhat, N. K., Showalter, S. D., indicates that the Zweig, M. & Papas, T. S. (1988) Oncogene 2, 99-103. the same levels of ETS2 proteins, which 15. Fisher, R. J., Koizumi, S., Kondoh, A., Mariano, J. M., Mavroth- exogenous expressed ETS1 protein does not target the ETS2 alassitis, G., Bhat, N. K. & Papas, T. S. (1992) J. Biol. Chem. 267, promoter. Though DLD-1 and HCT116 cells have multiple 17957-17965. genetic alterations leading to tumor formation, the expression 16. Gunther, C. V., Nye, J. A., Bryner, R. S. & Graves, B. J. (1990) of wild-type ETS1 may be able to overcome these changes and Genes Dev. 4, 667-679. suppress tumorigenicity by either blocking the downstream 17. Wasylyk, B., Wasylyk, C., Flores, P., Begue, A., Leprince, D. & targets for ETS2 gene products or by inducing new gene Stehelin, D. (1990) Nature (London) 346, 191-193. products required for tumor suppression. In fact, we have 18. Ho, I. C., Bhat, N. K., Gottschalk, L. R., Lindsten, T., Thompson, shown before that a 54.5-kDa protein is enhanced in DE1-7 C. B., Papas, T. S. & Leiden, J. M. (1990) Science 250, 814-818. cells compared with parental or neo-transfected cell lines (29). 19. Bosselut, R., Duvall, J. F., Gegonne, A., Bailly, M., Hemar, A., It is also possible that the ETS1 gene products may be able to Brady, J. & Ghysdael, J. (1990) EMBO J. 9, 3137-3144. modify the preexisting proteins ofa specific signal transduction 20. Boulukos, K. E., Pognonec, P., Rabault, B. & Ghysdael, J. (1989) pathway involved in colon tumor cell formation. More direct Mol. Cell. Biol. 9, 5718-5721. substantiate these The 21. Wang, C. Y., Petryniak, B., Ho, I. C., Thompson, C. B. & Leiden, evidence is needed to points. approach J. M. (1992) J. Exp. 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