ROLE OF POLYAMINES IN BREAST TUMOR MITOGENESIS

根据初步数据， 乳腺癌细胞中的多胺（PA）（腐胺、亚精胺、精胺） 增殖，我们认为PA通路的组成性激活 可能导致获得侵略性的表型特征， 乳腺癌细胞 我们计划测试PA在肿瘤中的直接作用 生物学通过评估组成型过度表达的影响， 关键的PA生物合成酶（鸟氨酸脱羧酶[ODC]，S- 腺苷甲硫氨酸脱羧酶[SAM-DC]）对关键生物学特征的影响 乳腺癌细胞，包括增殖活性，敏感性 抗雌激素和雌激素、侵袭能力和转移潜力。 为此，我们将检测对乳腺癌有反应的MCF-7乳腺癌， 用ODC和/或SAM-DC cDNA诱导PA活化的细胞 生物合成 此外，我们将产生MCF-7细胞， 选择性压力诱导的ODC活性 二氟甲基鸟氨酸（DFMO），将进行生物学评价 特性. PA通路与细胞凋亡之间的潜在协同性是一个研究热点。 ras信号系统在侵袭性乳腺癌发生中的作用 将通过检测过表达ODC的MCF-7细胞来检测表型 和/或SAM-DC活性与野生型和突变的v-ras癌基因， 研究它们的表型特征 目前的证据表明， ODC基因的5 '-非翻译区（5' UTR）在以下方面起关键作用： 信息翻译 ODC和SAM-DC cDNA具有完整和不完整的 或缺失的5 'UTR将在金属硫蛋白驱动的表达中亚克隆 载体并转染MCF-7细胞。 这种方法将使我们能够 测试这段基因在决定 酶表达水平及其激素调节。 最后， 我们将测量人乳腺中ODC和SAM-DC的活性水平， 癌症标本，并将它们与激素受体状态联系起来。 我们 将确定酶活性的增加是否是由于基因 放大，增加的信息水平或增加的翻译。 在 后者采用PCR和密度梯度凝胶电泳， 通过测序，我们将研究是否缺失/突变的基因， 基因的5 'UTR解释了增加的信息翻译。

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.