PTH-RELATED PEPTIDE AND VIT D IN PROSTATE CANCER GROWTH

前列腺癌是美国男性癌症死亡的最大原因。前列腺癌细胞产生多种自分泌生长因子，其中包括甲状旁腺激素相关肽（PTHrP）。该项目的第一个目标将使用两种方法来检查PTHrP对前列腺细胞生长的影响：建立PTHrP过表达或过低表达的细胞系，并用重组PTHrP处理细胞。因此，我们可以区分肽的自分泌/旁分泌（细胞表面）与分泌内（核）效应。维生素D会减少前列腺癌细胞的生长，流行病学研究表明，缺乏维生素D是前列腺癌的一个危险因素。我们研究的长期目标是确定维生素D或其类似物是否可以作为前列腺癌的化学预防或化学治疗药物。PTHrP基因在前列腺癌细胞中被1,25-二羟基维生素D (1,25(OH)2D)（维生素D的激素活性形式）下调。因此，维生素D不仅具有抗增殖作用，还能降低前列腺细胞中PTHrP的分泌。该项目旨在确定1,25(OH)2D单独或与9-顺式维甲酸联合抑制正常和癌前列腺细胞系中PTHrP基因表达的机制。人类PTHrP基因是一个复杂的转录单位，至少有三个不同的启动子。不同的细胞系和组织表现出不同的启动子利用模式。每个启动子对1,25(OH)2D的响应性将使用瞬时转染测定法进行评估。在一个特定的启动子中，精确的序列元件赋予对1,25(OH)2D的响应性，将通过缺失映射来定位。与这些启动子元件相互作用的反式核蛋白将通过凝胶阻滞和核酸酶保护试验来表征。迄今为止，125 (OH)2D本身的高钙血症作用阻碍了其预防和治疗用途，但最近合成了各种非高钙血症维生素D类似物。这些类似物中的一些将被测试其下调PTHrP基因表达和减少PTHrP分泌的能力，从而确定这些衍生物是否发挥与母体化合物相同的净理想效果。因此，这些研究应该为使用非高钙维生素D类似物作为针对pthrp介导的前列腺癌细胞增殖和恶性肿瘤的体液性高钙血症的化学预防和化疗药物提供分子基础。

Prostate cancer is the largest cause of cancer death among men in the United States. Prostate cancer cells produce a variety of autocrine growth factors, among them the parathyroid hormone- related peptide (PTHrP). The first aim of this project will use two approaches to examine the effects of PTHrP on prostate cell growth: establishing cell lines over- or under-expressing PTHrP, and treating cells with recombinant PTHrP (1-139). Thus we can distinguish between the autocrine/paracrine (cell surface) vs. intracrine (nuclear) effects of the peptide. Vitamin D decreases prostate cancer cell growth and epidemiological studies have shown that vitamin D deficiency is a risk factor for prostate cancer. The long-term objective of our research is to determine whether vitamin D or its analogues are useful as chemopreventative or chemotherapeutic agents in prostate cancer. The PTHrP gene is down-regulated by 1,25-dihydroxyvitamin D (1,25(OH)2D), the hormonally active form of vitamin D, in prostate cancer cells. Therefore, vitamin D not only has antiproliferative effects, but decreases PTHrP secretion in prostate cells. This project seeks to define the mechanism(s) by which 1,25(OH)2D, alone and in combination with 9-cis-retinoic acid, represses PTHrP gene expression in normal and cancerous prostate cell lines. The human PTHrP gene is a complex transcriptional unit with at least three different promoters. Different cell lines and tissues exhibit different promoter utilization patterns. The responsiveness of each promoter to 1,25(OH)2D will be assessed using transient transfection assays. The precise sequence elements conferring responsiveness to 1,25(OH)2D within a particular promoter will be located by deletion mapping. Trans-acting nuclear proteins interacting with these promoter elements will be characterized by gel retardation and nuclease protection assays. The hypercalcemic effects of 1,25(OH)2D itself have thus far prevented its prophylactic and therapeutic use, but various non-hypercalcemic vitamin D analogues have recently been synthesized. Some of these analogues will be tested for their ability to down-regulate PTHrP gene expression and decrease PTHrP secretion, thereby addressing whether these derivatives exert the same net desirable effects as the parent compound. These studies should thus provide a molecular basis for use of non-hypercalcemic vitamin D analogues as chemopreventive and chemotherapeutic agents targeted at both PTHrP-mediated prostate cancer cell proliferation and humoral hypercalcemia of malignancy.