Zhang Jian, Lu Yi, Dai Jinlu, Yao Zhi, Kitazawa Riko, Kitazawa Sohei, Zhao Xinping, Hall Daniel E, Pienta Kenneth J, Keller Evan T
Department of Pathology and Unit for Laboratory Animal Medicine, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Prostate. 2004 Jun 1;59(4):360-9. doi: 10.1002/pros.20019.
Current animal models of prostate cancer (CaP) bone metastasis do not allow measurement of either tumor growth in bone over time or activation of gene promoters in intraosseous tumors. To develop these methods, we used bioluminescent imaging (BLI) to determine if expression of receptor activator of NF-kappaB ligand (RANKL), a pro-osteoclastogenic factor that promotes CaP bone metastases, is modulated by the bone matrix protein transforming growth factor-beta (TGF-beta) in vivo.
C4-2B human CaP cells were treated with TGF-beta in vitro and RANKL mRNA and protein production were measured by polymerase chain reaction (PCR) and ELISA, respectively. Then C4-2B cells stably transfected with the RANKL promoter driving luciferase (lux) were injected intra-tibially into severe combined immundeficient (SCID) mice. Tumors were subjected to BLI every 2 weeks for 6 weeks and serum prostate specific antigen (PSA) was measured using ELISA. Vehicle (V), 1,25 dihydroxyvitamin D (VitD), or TGF-beta was administered to mice with established tumors and BLI to measure RANKL promoter activity was performed. Tumors were then subjected to immunohistochemistry for lux and assayed for RANKL mRNA levels.
TGF-beta induced RANKL protein and mRNA expression and activated the RANKL promoter activity in a dose-dependent manner in vitro. BLI demonstrated an increase in intraosseous tumor size over time, which correlated with serum PSA levels. Administration of TGF-beta and VitD to mice with established intraosseous tumors increased lux activity compared to V. Intratibial tumor RANKL mRNA expression paralleled the increased promoter activity. Immunohistochemistry confirmed the presence of lux in the intraosseous tumors.
These results demonstrate the ability to measure intraosseous tumor growth over time and gene promoter activation in an established intraosseous tumor in vivo and also demonstrate that TGF-beta induces activates the RANKL promoter. These results provide a novel method to explore the biology of CaP bone metastases.
目前的前列腺癌（CaP）骨转移动物模型无法测量骨内肿瘤随时间的生长情况，也无法测量骨内肿瘤中基因启动子的激活情况。为了开发这些方法，我们使用生物发光成像（BLI）来确定核因子κB受体活化因子配体（RANKL）的表达是否在体内受到骨基质蛋白转化生长因子-β（TGF-β）的调节，RANKL是一种促进破骨细胞生成的因子，可促进CaP骨转移。
体外将C4-2B人CaP细胞用TGF-β处理，分别通过聚合酶链反应（PCR）和酶联免疫吸附测定（ELISA）测量RANKL mRNA和蛋白产量。然后将稳定转染了驱动荧光素酶（lux）的RANKL启动子的C4-2B细胞经胫骨内注射到严重联合免疫缺陷（SCID）小鼠体内。每2周对肿瘤进行一次BLI检查，持续6周，并使用ELISA测量血清前列腺特异性抗原（PSA）。对已形成肿瘤的小鼠给予赋形剂（V）、1,25-二羟基维生素D（VitD）或TGF-β，并进行BLI以测量RANKL启动子活性。然后对肿瘤进行lux免疫组织化学染色并检测RANKL mRNA水平。
TGF-β在体外以剂量依赖性方式诱导RANKL蛋白和mRNA表达并激活RANKL启动子活性。BLI显示骨内肿瘤大小随时间增加，这与血清PSA水平相关。与V相比，对已形成骨内肿瘤的小鼠给予TGF-β和VitD可增加lux活性。胫骨内肿瘤RANKL mRNA表达与启动子活性增加平行。免疫组织化学证实骨内肿瘤中存在lux。
这些结果证明了能够在体内测量已形成的骨内肿瘤随时间的生长情况以及基因启动子激活情况，并且还证明了TGF-β诱导激活RANKL启动子。这些结果提供了一种探索CaP骨转移生物学的新方法。
