All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH2-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.
S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the synthesis of polyamines essential for cell growth and proliferation. Its overexpression induces the transformation of murine fibroblasts in both sense and antisense orientations, yielding highly invasive tumors in nude mice. These cell lines hence provide a good model to study cell invasion. Here, the gene expression profiles of these cells were compared with their normal counterpart by microarray analyses (Incyte Genomics, Palo Alto, CA, and Affymetrix, Santa Clara, CA). Up-regulation of the actin sequestering molecule thymosin beta4 was the most prominent change in both cell lines. Tetracycline-inducible expression of thymosin beta4 antisense RNA caused a partial reversal of the transformed phenotype. Further, reversal of transformation by dominant-negative mutant of c-Jun (TAM67) caused reduction in thymosin beta4 mRNA. Interestingly, a sponge toxin, latrunculin A, which inhibits the binding of thymosin beta4 to actin, was found to profoundly affect the morphology and proliferation of the AdoMetDC transformants and to block their invasion in three-dimensional Matrigel. Thus, thymosin beta4 is a determinant of AdoMetDC-induced transformed phenotype and invasiveness. Up-regulation of thymosin beta4 was also found in ras-transformed fibroblasts and metastatic human melanoma cells. These data encourage testing latrunculin A-like and other agents interfering with thymosin beta4 for treatment of thymosin beta4-overexpressing tumors with high invasive and metastatic potential.
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