To better understand the molecular mechanism of multistage carcinogenesis, we have attempted to identify putative oncogenes and/or tumor suppressor genes involved in preneoplastic-to-neoplastic progression of mouse epidermal JB6 variants. The JB6 variants consist of P- (promotion resistant), P+ (promotion sensitive), and Tx [transformed; both apoptosissensitive (As) and apoptosis-resistant (Ar)] cells, representing progression from early to late stages of carcinogenesis. By using the newly developed differential mRNA display technique, we have isolated five clones from these JB6 variants. The isolated clones were uniquely expressed either in P-/P+ cells or in Tx (As/Ar) cells or showed highly differential expression among the variants. The expression pattern shown by differential mRNA display was confirmed by Northern blot analysis. DNA sequencing followed by computer search against Genbank and EMBL DNA databases indicates that three clones are novel and two have high homology with recorded genes. One of the clones (Cl.14), which detects expression in preneoplastic not neoplastic JB6 cells, was used as a probe for complementary DNA library screening. The corresponding gene, named sun for specifically unexpressed in neoplastic JB6 cells, was isolated and sequenced. The longest open reading frame of the sun clone predicts a peptide showing 96% amino acid sequence identity to the recorded sequence of human tissue inhibitor of metalloproteinases-3, one of a family of genes implicated in tumorigenesis and tumor invasion. This is the first report, to our knowledge, of the simultaneous display of mRNAs of four phenotypically distinct cell variants and of the isolation of five clones which may be associated with specific stages of tumor promotion and/or progression and apoptosis.

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