ROLE OF POLYAMINES IN BREAST TUMOR MITOGENESIS

根据初步数据显示， 乳腺癌细胞中的多胺(PA)(腐胺、亚精胺、精胺) 增殖，我们认为PA通路的结构性激活 可能导致通过以下途径获得攻击性表型特征 乳腺癌细胞。我们计划测试PA在肿瘤中的直接作用 通过评估结构性过度表达对生物学的影响 PA生物合成的关键酶(鸟氨酸脱羧酶)，S 腺苷蛋氨酸脱羧酶[SAM-DC])对关键生物学特性的影响 乳腺癌细胞，包括增殖活性，对 抗雌激素和雌激素、侵袭能力和转移潜能。 为此，我们将转染激素反应性乳腺癌MCF-7 含有ODC和/或SAM-DC cDNA的细胞诱导PA激活 生物合成。此外，我们还将生成MCF-7细胞， α-半乳糖选择性加压诱导的ODC活性 将对二氟甲基鸟氨酸(DFMO)进行生物学评价 属性。纤溶酶原激活物途径与蛋白水解酶的潜在协同性 RAS信号系统在获得侵袭性乳腺癌中的作用 将通过转染过表达ODC的MCF-7细胞来测试表型 和/或野生型和突变的v-ras癌基因的SAM-DC活性和 研究它们的表型特征。目前的证据表明， ODC基因5‘-非翻译区(5’-UTR)在骨肉瘤中起重要作用 消息翻译。具有完整和不完全的ODC和SAM-DC cDNA 或缺失5‘非编码区将在金属硫蛋白驱动的表达中亚克隆 并将其导入MCF-7细胞。这种方法将使我们能够 测试这段基因在确定 酶的表达水平及其激素调节。最后， 我们将测量人类乳房中ODC和SAM-DC的活性水平 并将其与荷尔蒙受体状态联系起来。我们 将确定酶活性的增加是否是由于基因 扩大、增加消息级别或增加翻译。在 后一种情况下，使用聚合酶链式反应和密度梯度凝胶电泳 通过测序，我们将调查该基因的缺失/突变 基因的5‘非编码区解释了信息翻译的增加。

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.