Identification of genes differentially expressed in association with metastatic potential of K-1735 murine melanoma by messenger RNA differential display.

Abstract

To identify genes differentially expressed in association with the metastatic potential of K-1735 mouse melanoma cells, the mRNA differential display method was applied to compare mRNAs from high- and low-metastatic K-1735-derived cells. Three of the high- and three of the low-metastatic clones were used to reduce the false positives in the initial screening, and Southern blot screening against reverse transcription-PCR products was used to confirm that cDNA fragments detect differential expression between high- and low-metastatic cells. By using 256 different combinations of modified long arbitrary primers which provide broad screening of expressed genes, approximately 12,000 cDNA fragments were amplified from mRNA of each cell line. Among them, eight genes were identified as being expressed in either high- or low-metastatic cells using Northern blot analysis. Integrin alpha6 and two unknown genes were expressed in high-metastatic cells, whereas beta-tropomyosin, macrophage colony-stimulating factor, inhibin/activin betaB subunit, and two unknown genes were expressed in low-metastatic cells. These results indicate that the acquisition of metastatic potential in tumor cells was regulated by activation and/or inactivation of several specific genes, such as those for cell adhesion molecule, cytoskeletal protein, and growth factors.