Mechanisms of Prostate Tumorigenesis Using Genetically Engineered Mouse Models

细胞类型易感性和雄激素依赖性：pRb肿瘤抑制通路在人类前列腺癌中经常发生改变。我们之前对多种细胞类型的研究已经证明了pRb在抑制肿瘤发生中的重要性。我们已经开发出一种策略，通过SV40 T抗原结构域（T121）的细胞特异性表达，结合并灭活这三种蛋白，从而主要干扰pRb和潜在的代偿性相关蛋白p107和p130。我们利用角蛋白调控在前列腺上皮中产生雄激素非依赖性上皮亚型靶向表达T121的转基因小鼠。在前列腺中，基底细胞表达K5和K14，管腔细胞表达K8和K18。最近，中间细胞被描述为K5/18和其他角蛋白如K15、17和19的共同表达。为了探索易感细胞靶点，我们使用区室特异性角蛋白基因转录信号驱动条件T121等位基因的表达，该等位基因由报告基因eGFP基因和T121编码序列上游loxP位点两侧的停止序列组成。具体来说，K5、K18和K19基因分别靶向PE的基底细胞、腔细胞和中间细胞。为了获得最佳的转基因表达，我们选择利用含有角蛋白基因的细菌人工染色体（BAC）来产生转基因小鼠。BAC-trangene的使用最大限度地减少了与标准转基因相关的位置效应，并增加了所有必要调控元件存在的可能性。利用Neal Copeland及其同事首创的重组技术，eGFP停止盒和T121基因将被插入BACs中，这样角蛋白基因的调节因子也将驱动条件T121等位基因的表达。随后引入Cre重组酶（通过体细胞病毒传递或组织特异性转基因）将去除停止序列并诱导T121表达。K5eGT121、K18eGT121和K19eGT121三株转基因创鼠均已成功培育。f1已用于小鼠品系表征。将小鼠与PbCre4杂交以激活T121的表达。为了确定Pten缺乏背景下是否可以加速肿瘤表型，我们对条件Pten背景的小鼠进行了杂交。2008年，范戴克实验室从北卡罗来纳大学教堂山分校过渡到nci弗雷德里克分校。老鼠群已经从北卡罗来纳大学运到腓特烈中心。我们正在将所有的单菌株重新导出到无幽门螺杆菌的设备中。这些菌株分别是：K5eGT121（4株）、K18eGT121（2株）、K19eGT121（2株）、PbCre4（1株）、条件Pten（1株）。所有实验小鼠目前仍在隔离中。解剖肿瘤小鼠，在10%福尔马林、OCT或-80℃中收集组织。我们正在收集组织学资料。APT121小鼠模型中气孔对前列腺肿瘤进展的贡献：APT121；p53cf/+；FSPCreER APT121; p53cf / f;FSPCreER和相对对照（如p53cf/f、FSPCreER、p53cf/+、FSPCreE和APT121）小鼠已成功生成。为了使p53失活，我们在小鼠2月龄时连续5天ig注射他莫昔芬（1mg/只/天）或油作为对照。老鼠被衰老到不同的阶段。采集组织用于组织学评估和分子分析。2008年，Van Dyke实验室从北卡罗来纳大学教堂山分校过渡到nci -弗雷德里克分校。老鼠群已经从北卡罗来纳大学运到腓特烈中心。所有实验小鼠目前仍在隔离中。解剖肿瘤小鼠，在10%福尔马林、OCT或-80℃中收集组织。我们正在让老鼠衰老，并收集组织样本。

Cell type susceptibility and androgen independence: The pRb tumor suppressor pathway is frequently altered in human prostate cancer. Our previous studies in multiple cell types have demonstrated the importance of pRb in suppressing tumor initiation. We have developed a strategy to dominantly interfere with pRb and potentially compensatory related proteins p107 and p130 by cell specific expression of the SV40 T antigen domain (T121) that binds to and inactivates all three proteins. We utilized keratin regulation to generate transgenic mice with androgen-independent epithelial subtype- targeted expression of T121 in prostate epithelium. In prostate, basal cells express K5 and K14, while luminal cells express K8 and K18. More recently, intermediate cells have been described with co expression of K5/18 and other keratins such as K15, 17 and 19. To explore the susceptible cell target(s), we used compartment-specific keratin gene transcriptional signals to drive expression of a conditional T121 allele composed of a reporter eGFP gene and stop sequences flanked by loxP sites upstream of T121 coding sequences. Specifically, K5, K18, and K19 genes were chosen to target basal cells, luminal cells, and intermediate cells in PE, respectively. To obtain optimal transgene expression, we have chosen to utilize a bacterial artificial chromosome (BAC) harboring the keratin gene to generate transgenic mice. The use of BAC-trangene minimizes position effects associated with standard transgenes and increases the likelihood that all necessary regulatory elements will be present. The eGFP stop cassette and T121 gene will be inserted into the BACs using recombineering technology pioneered by Neal Copeland and colleagues, such that regulators of the keratin gene will also drive the expression of a conditional T121 allele. The subsequent introduction of Cre recombinase (via somatic viral delivery or a tissue-specific transgene) will remove the stop sequences and induce T121 expression. All three strains of KeGT121 transgenic founder mice have been successfully generated (K5eGT121, K18eGT121, and K19eGT121). F1s have been used for mouse line characterization. Mice were also crossed to PbCre4 to activate T121 expression. To determine if tumor phenotype can be accelerated on Pten deficient background, we have crossed the mice on conditional Pten background. Year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. We are in the processing of rederiving all the single strains to helicobacter free facility. These strains are: K5eGT121 (4 lines), K18eGT121 (2 lines), K19eGT121 (2 lines), PbCre4 (1 line), conditional Pten (1 line). All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of histology data collection. Stomal contribution to prostate tumor progression in APT121 mouse model: APT121;p53cf/+;FSPCreER, APT121;p53cf/f;FSPCreER, and relative control (e.g. p53cf/f;FSPCreER, p53cf/+;FSPCreE, and APT121) mice have been successfully generated. To inactivate p53, we i.p. injected the mice with tamoxifen (1mg/mouse/day) or oil as control at 2 months of age for 5 consecutive days. Mice are aged to various stages. Tissues are harvested for histological evaluation and molecular analysis. In year 2008, Van Dyke lab was in a transition from UNC-Chapel Hill to NCI-Frederick. Mouse colony has been shipped from UNC to NCI-Frederick. All the experimental mice are still in quarantine currently. Mice with tumors have been dissected and tissues were collected in either 10% formalin, OCT, or -80C. We are in the process of aging the mice, and collecting tissue samples.