On the basis of preliminary data indicating a critical role for polyamines (PA) (putrescine, spermidine, spermine) in breast cancer cell proliferation, we propose that constitutive activation of the PA pathway may lead to the acquisition of aggressive phenotypic characteristics by breast cancer cells. We plan to test the direct role of PA in tumor biology by evaluating the influence of constitutive over-expression of key PA biosynthetic enzymes (ornithine decarboxylase [ODC], S- adenosylmethionine decarboxylase [SAM-DC]) on critical biologic features of breast cancer cells, including proliferative activity, sensitivity to antiestrogens and estrogens, invasive capacity and metastatic potential. To this end, we will transfect hormone-responsive MCF-7 breast cancer cells with ODC and/or SAM-DC cDNAs to induce activation of PA biosynthesis. In addition, we will generate MCF-7 cells with increased ODC activity induced by selective pressure with alpha- difluoromethylornithine (DFMO) which will be evaluated for biologic properties. The potential cooperativity between the PA pathway and the ras signalling system in the acquisition of the aggressive breast cancer phenotype will be tested by transfecting MCF-7 cells over-expressing ODC and/or SAM-DC activity with wild-type and mutated v-ras oncogene and studying their phenotypic features. Current evidence indicates that the 5'-untranslated region (5'UTR) of the ODC gene plays a critical role in message translation. ODC and SAM-DC cDNAs with complete and incomplete or absent 5'UTR will be subcloned in a metallothionein-driven expression vector and transfected into MCF-7 cells. This approach will allow us to test the importance of this segment of the genes in determining the levels of enzymatic expression and their hormonal regulation. Finally, we will measure the levels of ODC and SAM-DC activities in human breast cancer specimens and relate them to their hormone receptor status. We will determine whether increases in enzymatic activities are due to gene amplification, increased message levels or increased translation. In the latter case, using PCR and density gradient gel electrophoresis followed by sequencing, we will investigate whether deletions/mutations of the 5'UTR of the genes accounts for increased message translation.
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