In an attempt to isolate novel regulatory and/or tumor suppressor genes, we identified cDNAs whose abundance is low in NIH 3T3 cells and further decreased following the expression of the activated oncogene, v-src. The transcription of one such gene, 322, is suppressed at least 15-fold in src-, ras-, and fos-transformed cells and 3-fold in myc-transformed cells but is unaffected in raf-, mos-, or neu-transformed cells. Activation of a ts-v-src allele in confluent 3Y1 fibroblasts resulted in an initial increase in 322 mRNA levels after 1 to 2 h followed by a rapid decrease to suppressed levels after 4 to 8 h. The inactivation of several tumor suppressor gene families (e.g., those encoding p53, Rb, and APC) as a result of mutation is acknowledged to contribute to the oncogenicity of several types of human cancers (21). Many of these so-called class I tumor suppressor genes (20) were identified and isolated following cumbersome pedigree and cytogenetic analyses (29). Recently, another class of genes (class II) whose expression is known to be down-regulated in tumor cells has been shown by gene transfer techniques to encode potential tumor suppressors. These include nonmuscle ␣-actinin, tropomyosin I, CLP, retinoic acid receptor , and interferon regulatory factor 1 (14,17,18,23,26). Additional tumor suppressor gene families, such as the maspin gene, rrg, and N03 (4, 25, 34), were isolated by subtractive hybridization techniques designed to identify down-regulated genes. These findings indicate a widening definition of tumor suppressor genes and strongly suggest that additional suppressor genes could be identified in gene populations whose expression is down-regulated in response to oncogenic stimuli such as activation of oncogenes. The ability of these gene families to reverse an array of oncogenic phenotypes following gene transfer and overexpression supports the possibility for novel therapeutic modalities for cancer.We previously reported using a novel PCR-based subtractive hybridization method to generate expressed sequence tags (EST) representing genes expressed at low basal levels in NIH 3T3 cells and at suppressed levels in v-src-transformed NIH 3T3 cells (10). We envisioned that the derived EST might contain potential mitogenic regulators or tumor suppressors. Of the nine distinct cDNAs identified in this manner, four were identical or highly similar to known genes, although none had been previously shown to be down-regulated by v-src. These known genes include genes for helix-destabilizing protein A1 (hnRNP A1), CTLA-2␣ cysteine protease, and cytochrome c oxidase VIc subunit and gravin. An additional EST was highly homologous to a randomly cloned human cDNA (clone A7C09; GenBank accession no. Z25236). Four other EST were not similar to published sequences.In this study, we have characterized one such EST, initially identified as clone 3.2.2, whose steady-state level of mRNA expression in NIH/v-src cells is Ͼ15-fold less than that in NIH 3T3 controls. An initial GenBank search showed a significant homology t...