Mechanisms of Prostate Tumorigenesis Using Genetically Engineered Mouse Models

细胞类型易感性和雄激素非依赖性：pRb肿瘤抑制途径在人前列腺癌中经常改变。我们以前在多种细胞类型中的研究已经证明了pRb在抑制肿瘤发生中的重要性。我们已经开发了一种策略，通过细胞特异性表达的SV 40 T抗原结构域（T121），结合并灭活所有三种蛋白质，显性干扰pRb和潜在的补偿相关蛋白p107和p130。我们利用角蛋白调节来产生在前列腺上皮中具有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，而管腔细胞表达K8和K18。最近，已经描述了具有K5/18和其他角蛋白如K15、17和19的共表达的中间细胞。为了探索易感细胞靶标，我们使用隔室特异性角蛋白基因转录信号来驱动条件性T121等位基因的表达，所述条件性T121等位基因由报告基因eGFP基因和终止序列组成，所述终止序列侧翼为T121编码序列上游的loxP位点。具体地，选择K5、K18和K19基因分别靶向PE中的基底细胞、腔细胞和中间细胞。为了获得最佳的转基因表达，我们选择利用细菌人工染色体（BAC）窝藏角蛋白基因，以产生转基因小鼠。BAC-转基因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。eGFP终止盒和T121基因将使用Neal Copeland及其同事开创的重组工程技术插入BAC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（通过体细胞病毒递送或组织特异性转基因）将去除终止序列并诱导T121表达。 已成功产生所有三种KeGT 121转基因创始小鼠品系（K5 eGT 121、K18 eGT 121和K19 eGT 121）。F1已用于小鼠品系表征。还将小鼠与PbCre 4杂交以激活T121表达。为了确定肿瘤表型是否可以在Pten缺陷背景下加速，我们将小鼠在条件Pten背景下杂交。2008年，货车戴克实验室从北卡罗来纳大学教堂山分校过渡到北卡罗来纳大学弗雷德里克分校。小鼠群已从鹿特丹运往NCI-Frederick。我们正在将所有的单一菌株重新衍生到无帮助的设施中。这些菌株是：K5 eGT 121（4个株系）、K18 eGT 121（2个株系）、K19 eGT 121（2个株系）、PbCre 4（1个株系）、条件Pten（1个株系）。目前，所有实验小鼠仍处于隔离状态。解剖具有肿瘤的小鼠，并在10%福尔马林、OCT或-80 ℃中收集组织。我们正在收集组织学数据。 在APT 121小鼠模型：APT 121; p53 cf/+;FSPCreER、APT 121; p53 cf/f;FSPCreER和相对对照（例如p53 cf/f;FSPCreER、p53 cf/+;FSPCreE和APT 121）小鼠中，造口对前列腺肿瘤进展的贡献已成功产生。对于p53，我们在2月龄时连续5天腹膜内注射他莫昔芬（lmg/小鼠/天）或油作为对照。小鼠被老化到不同的阶段。收获组织用于组织学评价和分子分析。2008年，货车戴克实验室从北卡罗来纳大学教堂山分校过渡到了北卡罗来纳大学弗雷德里克分校。小鼠群已从鹿特丹运往NCI-Frederick。目前，所有实验小鼠仍处于隔离状态。解剖具有肿瘤的小鼠，并在10%福尔马林、OCT或-80 ℃中收集组织。我们正在老化小鼠，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.