BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 228, 586–595 (1996) ARTICLE NO. 1702 Mutated Atf4 Suppresses c-Ha-ras Oncogene Transcript Levels and Cellular Transformation in NIH3T3 Fibroblasts Lawrence M. Mielnicki,* ,1 Robert G. Hughes,* Pierre M. Chevray,² and Steven C. Pruitt* ,2 *Department of Molecular and Cellular Biology, Roswell Park Cancer Center, Elm & Carlton Streets, Buffalo, New York 14263; and ² Department of Molecular Biology and Genetics, John Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205-2185 Received September 19, 1996 A frameshift mutation is present in one allele of the Atf4 gene in genomic DNA from F9 embryonal carcinoma stem cells. The mutation results in the fusion of a short 5 open reading frame to the coding sequence of Atf4, replacing the first 18 N-terminal amino acids with 50 amino acids encoded by the upstream open reading frame. The ability of both normal and mutated Atf4 gene products to influence cell growth was tested using an NIH3T3 cell transformation assay. Overexpression of mutant Atf4 suppresses ras-induced transformation in this assay. In G418 r cell lines derived from parallel co-transfections, expression of transfected mutant Atf4 mRNA correlates with a loss of transformed morphology and a reduction in ras mRNA levels. Transient cotransfection assays in NIH3T3 cells demonstrate that wild type Atf4 is able to inhibit transcription directed by the human c-Ha-ras1 promoter and that this effect is increased by the mutation.  1996 Academic Press, Inc. Activating transcription factor 4 (Atf4) is a member of the ATF/CREB sub-family of basic region/leucine zipper (bZIP) containing, DNA binding transcription factors (1; reviewed in 2) and is located on mouse chromosome 15 (3). Atf4 interacts with a variety of bZIP proteins (4-7) and binds several different cellular and viral DNA regulatory elements (1, 5-8), in vitro. Dimerization of Atf4 with JUN or FOS family members alters the DNA binding specificity of JUN/FOS from preference for an AP-1 site to an almost exclusive preference for the CRE site (5). Atf4 can both activate and repress transcription from different CRE containing reporter genes (7, 9). Atf4 is constitutively expressed in a variety of adult murine tissues, uninduced P19 and F9 EC cells, macrophages, human T- and B-cells and a number of human tumor cell lines (4, 6-9 and Mielnicki and Pruitt, unpublished data). The ability of Atf4 to interact with proteins and DNA elements known to be involved in the regulation of growth in many different cell types suggests that Atf4 may also have a role in the control of this process. MATERIALS AND METHODS Plasmid DNAs. Atf4 cDNAs and the activated ras expression plasmid (pEJ) have been described previously (4, 10, 11). Wild type (Atf4 wt ) and mutated Atf4 (Atf4 F9MT ) expression plasmids construction details are available upon request. Genomic DNA analysis. NIH Swiss 3T3 cells, F9, OTF9 and P19 EC stem cells were grown under standard conditions (R. G. Hughes, unpublished and 12). The genomic DNA isolation procedure has been described previously 13). Genomic DNAs were isolated from NIH3T3 cells, G418 r cell lines derived from NIH3T3 cells, F9 cells, OTF9 cells, P19 cells, strain 129/J mice, a mouse strain 129/SV genomic DNA library in lambda-Fix II (Stratagene) and a Balb/c genomic DNA library (4) and Southern blot analysis was performed as described previously (14). 1 Present address: Dept. of Experimental Pathology, Science Bldg. Rm 605, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. 2 To whom correspondence should be addressed. Fax: (716)845-8126. 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 586