[CANCER RESEARCH 52, 6877-6884, December 15, 1992] Regulation of Smooth Muscle a-Actin Promoter in ras-transformed Cells: Usefulness for Setting Up Reporter Gene-based Assay System for Drug Screening C. Chandra Kumar, ~ Pierre Bushel, Sheela Mohan-Peterson, 2 and Fernando Ramirez Department of Tumor Biology, Schering-Plough Research Institute, Bloomfield, New Jersey 07003 ABSTRACT cancer have impaired GTPase activity, are insensitive to GAP, and remain bound to GTP leading to uncontrolled cell growth Oncogenic activation of ras results in changes in the transcription of (6). In addition, a number of posttranslational modifications of several genes leading to uncontrolled cell growth. In this paper, we ras such as palmitoylation, farnesylation, proteolytic cleavage, demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle a-actin pro- and carboxymethylation have been found to be essential for moter. Transient transfection analysis of plasmids containing the 5' biological activity of ras (9-11). upstream region of the human a-actin gene fused to human growth A number of approaches are being taken to find drugs that hormone or bacterial chloramphenicol acetyltransferase coding se- inhibit the accumulation of the GTP-bound form of the ras p21 quences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that protein. These include assay systems designed to identify drugs a-actin promoter is repressed in ras-transformed cells. In addition, that inhibit the nucleotide exchange activity of ras, or stimulate stable rat fibroblast cell lines expressing human growth hormone or the GTPase activity of mutant ras, or inhibit ras:GAP interac- B-galactosidase under the control of ~,-actin promoter exhibit repressed tion, etc. (11). Recently, the enzymes involved in the modifica- reporter gene activity following transformation by the ras oncogene. tion of ras proteins have also been targeted for new drug dis- a-Actin promoter-driven B-galactosidase activity is derepressed in re- vertants of ras-transformed stable cell lines. This revertant cell line covery (11). Most of these assay systems are in vitro based, and expresses elevated levels of ras p21 protein and is resistant to retrans- the lead compounds in these assay systems have to be evaluated formation by Ki and Ha-ras oncogenes. The revertant may have either in secondary cell-based assay systems to determine their effi- a defective target protein whose activity is essential for the transforming cacy. In this paper, we describe a general cell-based assay sys- activity of ras or an activated tumor suppressor gene which can suppress tem capable of detecting compounds that inhibit the transform- the activity of ras. These results indicate that smooth muscle a-actin ing activity of ras, either directly or indirectly. promoter activity is a sensitive marker to follow phenotypic changes Previous studies have shown that human sm forms of myo- following transformation by ras and subsequent reversion. The advan- sin light chain 2 (MLC-2) and a-actin mRNA and protein tages of this a-actin promoter-reporter gene assay system to screen for levels are repressed in ras-transformed fibroblast cells (12, 13). drugs that inhibit the transforming activity of ras, either directly or These changes are part of the cytoskeletal changes that occur indirectly, are discussed. following neoplastic transformation of fibroblast cells (14). We have also shown that revertants of ras-transformed cells show INTRODUCTION normal levels of MLC-2 gene expression, suggesting that the expression of these cytoskeletal markers, such as sm MLC-2 ras proteins play a central role in neoplasia and growth con- and sm a-actin, is modulated by the ras-transformed pheno- trol. Activated ras oncogenes have been implicated in the onset type (12). In this paper, using plasmids containing 5' upstream of 20% of human cancers and greater than 90% of pancreatic sequences of the human a-actin gene fused to different reporter and colon carcinomas (1-3). Hence, drugs that interfere with genes, we first demonstrate that a 906-base pair fragment is the function of ras, either directly or indirectly, will be useful in the treatment of a number of cancers. A great deal of effort has sufficient and necessary to confer responsiveness to the ras oncogene. Using stable rat fibroblast cell lines that express been focused in understanding the biochemical and structural characteristics of the ras p21 protein (4, 5). The mechanisms by LacZ reporter gene activity from the a-actin promoter, we which ras can induce transformation of cells have also been further show that revertants of ras-transformed cells exhibit studied in great detail (6). ras p21 protein exhibits GTPase derepression of promoter activity. The revertant cell line ex- activity and exists either in GTP-bound activated form or in presses elevated levels of the ras p21 protein and is resistant GDP-bound inactive form. Proteins that modulate the activity to retransformation by Ki and Ha-ras oncogenes. The useful- of ras consist of (a) a nucleotide exchange factor that facilitates ness of this promoter system to identify agents that can inhibit the exchange of GTP/GDP and thus promotes the activation of the transforming activity of ras, either directly or indirectly, is ras (7) and (b) a protein that acts as a GAP 3 and promotes discussed. inactivation of ras (8). Mutant forms of ras found in human MATERIALS AND METHODS Received 6/19/92; accepted 9/30/92. Cell Culture and Transfections 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 accord- Rat-2 fibroblast cells obtained from the American Type Culture Col- ance with 18 U.S.C. Section 1734 solely to indicate this fact. lection (CRL 1764) and stably transfected derivatives were grown in 1To whom requests for reprints should be addressed. 2 Present address: DNAX Research Institute, Palo Alto, CA 94304. DMEM supplemented with 2 mM glutamine and 10% (vol/vol) fetal 3 The abbreviations used are: GAP, GTPase-activating protein; sm, smooth bovine serum. Rat-6 fibroblasts, obtained from Dr. Bernard Weinstein muscle; DMEM, Dulbecco's modified Eagle's medium; TK, thymidine kinase; at Columbia University, were grown in DMEM supplemented with 2 PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; hGH, human growth hormone; X-gal, 5-bromo-7-chloro-3-indolyl-~-i)-galactopyrano- mM glutamine and 10% bovine calf serum. To derive stable transfor- side; ONPG, O-nitrophenyl-/3-o-galactopyranoside; FITC, fluorescein isothiocyo- mants, cells were transfected with 10:1 mixtures of the reporter gene nate; IgG, immunoglobulin G; CHP, 4-cis-hydroxy-L-proline. plasmid (see below and Fig. 1) and plasmid pIBW DNA containing a 6877 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1992 American Association for Cancer Research. REGULATION OF SMOOTH MUSCLE a-ACTIN PROMOTER IN ras-TRANSFORMED CELLS neomycin resistance marker fused to the herpes simplex virus TK pro- were washed extensively with PBS solution and photographed using a moter. Briefly, 5 x 105 cells, plated in 100-mm dishes, were incubated Zeiss IM-35 fluorescent microscope equipped with a 200-W mercury for 6 h with calcium phosphate DNA precipitates. Calcium phosphate light source. precipitates were prepared with 20 ~tg of plasmid DNA per 10-cm plate (15). The transfection was stopped by replacing the medium with the Isolation and Characterization of Revertant Cell Line standard culture medium after a brief rinse with PBS. Twenty-four h later, transfected cultures were split 1:10 and grown in the presence of To isolate the revertant cell line D-3, Rat-6 (2S-HO6) cells were the neomycin analogue G418 (Sigma) at 400 #g/ml. After 10 days, seeded at a density of 1 x 106 cells/100-mm dish and treated with 7 resistant clones were isolated using cloning cylinders. ~g/ml of 5-aza-cytidine for 24 h. After a recovery period of an addi- tional 48 h, cells were plated at a density of 1 x 105 cells for each 100-mm dish in the growth medium containing 200/~g/ml of CHP Construction of Plasmids (23, 24). The plates were fluid changed with DMEM containing CHP, The EcoRI-DraIII fragment consisting of-894 to + 12 base pairs of twice weekly for 3 to 4 wk, after which colonies made up of apparently the human a-actin gene was isolated from the paAS plasmid (16) and nontransformed cells were marked and isolated using cloning cylinders. ligated into the HindIII site of plasmids pCH 126 (17), pOGH (18), and Cells were further subcloned using the limiting dilution technique. pBasic (19) to derive paAP126, paAPGH, and paAPCAT plasmids, To determine the susceptibility of cells for transformation by retro- respectively. The structure of the plasmids was confirmed by DNA viruses containing Kirsten and Harvey ras oncogenes, Rat-6 (2S) and sequence analysis, pBasic and pOGH are promoterless plasmids con- D-3 revertant cells were plated at a density of 2 x 105 ceils in 100-mm taining CAT and hGH coding sequences, respectively (18, 19). Plasmid dishes, and virus stocks (a gift from Dr. Bassin, National Cancer Insti- pHO6T1 containing the T24 Ha-ras oncogene was a kind gift from Dr. tute) diluted with the growth medium were added to the cells in the Earl Ruley (Massachusetts Institute of Technology) (20). presence of 200 ug/ml of DEAE-dextran. Following infection for 2 h, the medium containing the virus was aspirated and replaced with fresh medium. The cells were fed with fresh medium at 3-day intervals, and Enzyme Assays transformed foci were scored after 12 days by fixing the cells in meth- /~-Galactosidase Assay. To identify stable fibroblast cell lines ex- anol and staining with Giemsa reagent as described (24).