225 Subtractive cDNA Cloning and Characterization of Genes Induced by All-Trans Retinoic Acid YACHI CHEN AND DAVID A. TALMAGE a Institute of Human Nutrition and Department of Pediatrics, Columbia University, New York, New York 10032, USA Retinoids, a group of natural and synthetic analogs of vitamin A, have proven effective in preventing tumor formation in animal studies and clinical trials 1 (see ref. 2 for reviews). Of all retinoids, all-trans retinoic acid (RA) is the most biologically active vitamin A metabolite. Using an experimental model involving F111/RARα cells (parental F111 rat fibroblasts do not express detectable RARα, β, or γ, whereas F111/RARα cells express RARα and are therefore RA responsive) and polyoma virus middle T antigen as an oncogenic agent, our laboratory has demonstrated that RA, acting via RARα, suppresses mT-induced transformation by blocking transcription of the c-fos gene whose product is a component of the transcription factor AP-1. 3,4 Fur- ther study indicated that the observed inhibitory effect of RA on transformation in- duced by mT resulted from RA preventing mT signaling to the c-fos promoter via the phosphatidylinositol 3-kinase (PI3-kinase)/Rac/Jun N-terminal kinase (JNK) signal- ing cascade. 5 We think that RA blocks this pathway by inducing expression of some inhibitory proteins that act on the components of this pathway (FIG. 1). Our hypothe- sis is supported by several observations: (1) activation of RARα, itself a nuclear tran- scription factor, is required for inhibition of mT-induced transformation; (2) analysis of RA repression of c-fos transcription demonstrated that pretreatment of cells with RA for >6 hours was required and that the observed RA effect on c-fos expression was blocked by the protein synthesis inhibitor cycloheximide; and (3) no RARE has been found in the c-fos promoter and therefore RAR is unlikely to inhibit c-fos ex- pression directly. A full understanding of the mechanism of the anticarcinogenic ef- fect of RA requires identification of these RARα-regulated inhibitory genes. RESULTS With the polymerase chain reaction (PCR)-select cDNA subtraction method (Clontech Laboratories, Inc.), seven genes expressed after induction by RA were ob- tained (TABLE 1). These genes include two novel genes whose sequences do not match with sequences present in the Genbank or EMBL database, and they are des- ignated as M33 and M90. The five known genes are: M61, which is homologous to a Corresponding author: Dr. David A. Talmage, Institute of Human Nutrition, Columbia Uni- versity, 701 West 168 th Street, Hammer Health Science Bldg. Room 5-503, New York, New York 10032. Phone, 212/305-5342; fax, 212/305-3079. e-mail, dat1@columbia.edu