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Neoplastic Transformation Inactivates Specific Trans-Acting Factor(S

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Neoplastic Transformation Inactivates Specific Trans-Acting Factor(S Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1744-1748, March 1988 Biochemistry Neoplastic transformation inactivates specific trans-acting factor(s) required for the expression of the thyroglobulin gene (differentiation/DNA-protein interaction/thyroglobufln) V. ENRICO AVVEDIMENTO*tt, ANNAMARIA MUSTI*, ALFREDO Fusco*, MARCO J. BONAPACE*, AND ROBERTO Di LAURO§¶ *Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerce, Dipartimento di Biologia e Patologia Molecolare, II Facolta di Medicina e Chirurgia, Universita di Napoli, Naples, Italy; and fLaboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Maxine F. Singer, October 5, 1987 (receivedfor review February 18, 1987) ABSTRACT The expression of rat thyroglobulin gene is sible for the block of the thyroglobulin expression in trans- repressed following the transformation ofrat thyroid cells with formed thyroid cells. Kirsten murine sarcoma virus. The expression of a reporter We report here data on the expression of the reporter gene gene fused to the thyroglobulin promoter is down-regulated in for chloramphenicol acetyl transferase fused to the Tg pro- transformed thyroid cells in transient or in stable transfection moter, designated Tg P-CAT gene, in transformed cells assays. DNase and exonuclease HI cleavage-protection analysis either in transient or in stable transfection assays. We show: reveals that a promoter binding activity located at -60 base (i) that this marker is down-regulated in transformed thyroid pairs from the transcription start site is substantially reduced cells; (ii) that a specific Tg promoter binding activity is in transformed thyroid cells. The repression in the trans- substantially reduced upon transformation; and (iii) that the formed cells of the reporter gene joined to the thyroglobulin Tg P-CAT gene in transformed cells can be reactivated by promoter can be reversed by fusion with normal differentiated fusion with a normal differentiated thyroid cell. Fusion to thyroid cells. Fusion of transformed thyroid cells to liver cells liver cells does not reactivate Tg P-CAT gene in transformed does not reactivate the reporter under control of the thyro- thyroid cells. On the basis of these data we propose that globulin promoter. transformation inactivates one or more positive trans-acting factor(s) required for the expression of the Tg gene. Cellular transformation produces important changes in the biosynthetic pattern of cellular proteins. Rous sarcoma virus MATERIALS AND METHODS (RSV) transformation represses the expression of collagen type I and fibronectin genes in chicken embryo fibroblasts Cell Lines. Two differentiated untransformed rat thyroid (1, 2) and the v-mos oncogene product has a similar effect on cell lines were used in this study. FRTL (herein called TL) transcription of the collagen type I gene in mouse 3T3 and PC are cloned thyroid cells growing in a chemically fibroblasts (3). In several instances the differentiation pro- defined medium (7, 8); these cell lines were transformed with gram is disrupted by transformation, suggesting that the Kirsten murine sarcoma virus (9), yielding lines TL-KM and changes in the transcription specificity of a variety of cells PC-KK, respectively. Buffalo rat liver cell line BRL 3A was are a general phenomenon induced by this event (4, 5). a gift from H. Coon (National Institutes of Health, Bethesda, Recently it has been shown that the transfection of mouse MD). muscle cells with Nras and Hras alleles completely sup- Plasmids. Plasmids containing the Tg promoter with the presses expression of muscle specific genes (6). reporter gene for CAT will be described elsewhere (12). Thyroid cells represent a good model system to study how Briefly, the Sac I site at + 60 from the transcription start site transformation interferes with the differentiation program. of the Tg gene in exon I (11) was removed by BAL-31 These cells (TL or PC) possess three unique differentiation digestion up to position +20, and a HindIII linker was markers: thyroglobulin synthesis, thyrotropin-dependent added. The CAT gene coding sequence was linked at the growth, and active iodine uptake from the medium (7). HindIII site (13). The plasmid structures were confirmed by Chemical, physical, or virus-induced transformation blocks DNA sequence analysis. the expression of these markers (8-10). TL cells, trans- Cell Growth and Transfection. Approximately 12-16 hr formed with a mutant p21 temperature-sensitive Kirsten prior to transfection, 106 cells were plated onto 10-cm dishes murine sarcoma virus at a permissive temperature, are in F-12 Coon's modified medium containing 5% (vol/vol) unable to recover the differentiated phenotype at 390C, the calf serum and six growth factors (7, 8). Cells were refed 2-4 nonpermissive temperature for transformation. This sug- hr prior to the transfection. Calcium phosphate precipitates gests that an irreversible change must have occurred at the were prepared with 10 j.g of plasmid DNA per 10-cm plate time of the transformation (9). (14-16). Cells were incubated with the precipitate for 2 hr. We have cloned the rat thyroglobulin gene Tg (11). The The precipitate was removed, and the cells were incubated promoter region has been defined; all of the DNA sequence for 1 min in Hepes-buffered saline containing 15% (vol/vol) elements required for the tissue-specific expression of the glycerol at 370C, after which the cells were incubated for gene in thyroid cells have been mapped to a region extending another 48 hr in normal growth medium. In some experi- 170 base pairs (bp) 5' from the transcription start site (12). The Tg promoter fused to a testable marker provides a Abbreviations: CAT, chloramphenicol acetyl transferase; RSV, powerful tool to analyze the molecular mechanism respon- Rous sarcoma virus. tPresent address: Institute of Cancer Research, Columbia Univer- sity, 701 West 168th Street, New York, NY 10032. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" Present address: European Molecular Biology Laboratory, Heidel- in accordance with 18 U.S.C. §1734 solely to indicate this fact. berg, F.R.G. 1744 Downloaded by guest on September 23, 2021 Biochemistry: Avvedimento et al Proc. NatL Acad. Sci. USA 85 (1988) 1745 ments the efficiency of transfection was calculated by count- RESULTS AND DISCUSSION ing the number ofclones containing the marker [resistance to G418 in the case of the RSV promoter fused to the gene for Transient Expression of Tg P-CAT Gene in Differentiated resistance (RSV P-neo)] per 1 x 106 cells per pug Untransformed and Transformed Thyroid Cells. Two un- neomycin transformed, differentiated thyroid cell lines (TL and PC) of RSV P-neo DNA. and the same cells transformed with Kirsten murine sarcoma CAT Assay. The cell extracts were prepared 48 hr after the virus (TL-KM and PC-KK) were compared throughout this transfection, and the CAT assays were performed as de- study. The transformed cell lines do not express Tg at a scribed (13) in a total volume of 0.15 ml containing 250 mM significant level (9). The block in expression is at the Tris-HCl (pH 7.8), 4.4 mM acetyl coenzyme A, 0.2 AtCi of transcriptional level (V.E.A., unpublished data). In the [14C]chloramphenicol (40-60 Ci/mmol, New England Nu- experiments reported here, three fragments containing the 5' clear; 1 Ci = 37 GBq), and cell extract containing 50 Ag of flanking sequences of Tg, extending from positions -800, protein. The reactions were allowed to proceed up to 2 hr at -300, or -170 to + 20 bp from the transcription start site of 37TC. The analysis of the reaction was performed as de- Tg, were separately fused to the bacterial gene for CAT and scribed (13). The CAT activity was quantitated by counting introduced into the four lines described above by the calcium regions of the chromatograms in a liquid scintillation spec- phosphate (14, 15) or DEAE-dextran methods (16). All three trometer. plasmid constructs contain the sequences responsible for the Fusion of Untransformed and Transformed Ceils. Trans- tissue-specific expression of Tg in the thyroid cells (12). The formed [TL-KM (see below)] and untransformed (PC) cells long terminal repeat of RSV linked to the CAT gene was were fused by using the Sendai virus technique. The cells transfected into the thyroid cell lines as a reference pro- were washed three times in a serum-free medium, mixed, moter, RSV P-CAT. and incubated for 10 min in 1 ml of serum-free medium Fig. 1 shows the result of a transient expression assay. containing 1500 units of inactivated Sendai virus at 40C. The expression of the CAT gene directed by the Tg 5' Fresh serum-free medium (10 ml) was added, and the cells flanking sequences is reported as a percentage of the expres- were incubated 1 hr at 37TC. At the end ofthe incubation, the sion driven by the RSV promoter. The CAT expression cells were centrifuged, resuspended in the normal medium, driven by the 5' flanking fragments of Tg-800, 300, and 170 and plated at 5 x 105 per 100-mm dish. The CAT activity bp long (Tg P800, Tg P300, and Tg P170)-appeared to be was measured 48 hr after the cell fusion. Fusion was also consistently reduced in transformed cells, independently of carried out by the PEG technique (17). the technique of transfection used [Fig. LA Left (calcium A Promoter PC T L TL-KM PC-KK PC TL TL-KM PC-KK Tg P800 32 30 3 3 80 80 5 6 Tg P300 22 28 3 2 70 50 4 7 Tg P170 30 22 3 2 80 70 5 6 1 1.5 1 - 2 - 3 - B D.
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