SPECIFIC ROLES FOR D-CYCLINS IN ONCOGENESIS

D型细胞周期蛋白（cyclin D1，D2和D3）是所有促有丝分裂和致癌信号的最终受体。 在许多人类恶性肿瘤中发现了D-细胞周期蛋白的异常表达，并且几乎所有人类癌症都含有影响D-细胞周期蛋白的途径中的病变。我们已经产生敲除小鼠品系缺乏细胞周期蛋白D1，D2或D3使用同源重组胚胎干细胞。 所有三种菌株都是活的，并显示出集中的组织特异性表型。 在拟议的工作中，我们将使用这些小鼠作为一种工具来研究D-细胞周期蛋白在肿瘤发生中的作用。 在第一个具体目标中，我们将询问细胞周期蛋白D1的缺失是否会使小鼠对各种癌基因诱导的乳腺癌产生抗性。 细胞周期蛋白D1在大多数人乳腺癌中过表达。 重要的是，细胞周期蛋白D1的过度表达被认为在肿瘤发生中起着致病作用，因为中和细胞周期蛋白D1功能的药物在体外显示出关闭乳腺癌细胞的增殖。 细胞周期蛋白D1通常在几乎所有的人体组织中表达，直到最近，被认为是所有细胞系增殖所必需的。 然而，我们意外发现，在成年细胞周期蛋白D1-/-小鼠中，细胞周期蛋白D1消融的后果仅限于乳腺上皮细胞，这提高了一种特异性抗细胞周期蛋白D1治疗人类乳腺癌的可能性，这种治疗可能在关闭肿瘤细胞增殖同时保留其他组织方面具有高度选择性。 作为实现这一目标的第一步，我们希望测试细胞周期蛋白D1的消融是否可以预防体内癌症的形成。 在第二个具体目标中，我们将利用缺乏细胞周期蛋白D1，D2或D3的成纤维细胞，在细胞和分子水平上研究各种致癌信号转导途径对不同D-细胞周期蛋白的依赖性。 在第三个具体目标中描述的研究利用了最近产生的细胞周期蛋白E大于D1“敲入”小鼠。 在该菌株中，我们删除了细胞周期蛋白D1的编码外显子，并将其替换为细胞周期蛋白E cDNA。 对这些小鼠的分析表明，细胞周期蛋白E可以取代细胞周期蛋白D1驱动正常细胞增殖和拯救细胞周期蛋白D1缺陷的表型表现。 在这个特定的目标，我们将问是否细胞周期蛋白E可以取代细胞周期蛋白D1驱动癌细胞的致癌生长。

D-type cyclins (cyclin D1, D2 and D3) are the ultimate recipients of all mitogenic and oncogenic signals. Aberrant expression of D-cyclins is seen in many human malignancies and virtually all human cancers contain lesions in pathways impacting on D-cyclins. We have generated knockout mouse strains lacking cyclin D1, D2 or D3 using homologous recombination in embryonal stem cells. All three strains are viable and display focused, tissue-specific phenotypes. In the proposed work we will use these mice as a tool to study the role of D-cyclins in tumorigenesis. In the first Specific Aim we will ask whether the loss of cyclin D1 renders mice resistant to breast cancers induced by various oncogenes. Cyclin D1 is overexpressed in the majority of human breast cancers. Importantly, overexpression of cyclin D1 is believed to play a causative role in tumorigenesis, as agents that neutralize cyclin D1 function were shown to shut off the proliferation of breast cancer cells in vitro. Cyclin D1 is normally expressed in nearly all human tissues and, until recently, was thought to be indispensable for proliferation of all cell lineages. However, our unexpected finding that in adult cyclin D1-/- mice the consequences of cyclin D1-ablation are restricted to mammary epithelium, raises the possibility that a specific, anti-cyclin D1 therapy for human breast cancers might be highly selective in shutting off the proliferation of tumor cells while sparing other tissues. As a first step towards this goal we wish to test if the ablation of cyclin D1 prevents cancer formation in vivo. In the second Specific Aim we will take advantage of fibroblasts lacking cyclin D1, D2 or D3 to study the dependence of various oncogenic signal transduction pathways on different D-cyclins at the cellular and molecular level. The studies described in the third Specific Aim take advantage of recently-generated cyclin E greater than D1 "knock-in" mice. In this strain we have deleted the coding exons of cyclin D1 and replaced them by cyclin E cDNA. Analyses of these mice have demonstrated that cyclin E can replace cyclin D1 in driving normal cell proliferation and rescue the phenotypic manifestations of cyclin Dl-deficiency. In this Specific Aim we will ask whether cyclin E can replace cyclin D1 in driving oncogenic growth of cancer cells.