MECHANISMS OF METASTASIS IN EXPERIMENTAL PROSTATE CANCER

转移是一个复杂的生物学过程，对于它来说， 在分子和细胞水平上的理解。虽然很差 了解，转移通常被描述为 恶性进展通常是癌症死亡的直接原因 播散性转移细胞的生长。这种深刻的影响 转移性疾病显然是前列腺癌的情况，它将是 1994年在20万名美国男性中确诊，在3.8万名美国人中导致死亡 男人。 最近，我们改进了小鼠前列腺重建(MPR)模型 一种使用来自P53基因敲除小鼠的尿生殖窦组织作为 Ras和myc癌基因引入的目标。在中使用此功能 体内模型系统中，我们研究了ras+myc的致瘤作用。 在野生型P53、杂合型P53突变体或 纯合子p53突变体。我们证明了ras+myc启动的野生型 129/Sv小鼠以低频率增殖为主 (约10%)局灶性非转移性癌症。相比之下，两者都 杂合子P53和纯合子P53 MPR均可发生转移 近100%的病例为癌。Ras+myc+杂合子的研究进展 P53基因敲除的MPR与完全丢失、部分丢失或 野生型等位基因表达缺失。总体而言，我们的数据显然 提示P53功能完全丧失会导致该肿瘤的转移 实验性小鼠前列腺癌模型。有趣的是，这种模式 转移与人类前列腺癌非常相似，包括广泛的 转移至肺、肠系膜淋巴结、骨，偶见 肝脏。我们已扩展这一模式，以方便在 分子和细胞水平，通过产生一系列细胞系 来源于原发部位癌和转移性疾病 来自不同的器官部位。到目前为止，我们已经证明了TGFbeta1 抑制ALL(n=6)原发部位肿瘤来源细胞的体外生长 品系(8天后平均抑制率为45+/-9%)和对照 泌尿生殖窦上皮(CUGE)细胞，但仅抑制了6个中的3个 肺转移癌来源的细胞系(敏感的平均抑制 20%+/-1%)。对转化生长因子-β1的类似差异反应也是如此 观察集落形成和运动试验。结果是 具有特异性的转化生长因子-β1的交联性研究 所有原发部位肿瘤中L、2型和3型转化生长因子-β受体的结合 而转移衍生的细胞株表明这些转化生长因子-β1的丢失 在转移衍生细胞系中的诱导反应是在 受体结合。我们建议使用这种新的前列腺癌转移 建立模型系统来研究遗传机制造成的损失 TGFbeta1反应及其与转移进展的关系。在……里面 此外，与以下相关的纯合缺失的特定区域 转移进展将使用以下方法进行识别和克隆 代表性差异分析与杂合性缺失区域 在体内转移过程中发生的将通过使用 微卫星探测器。从过去使用MPR模型系统的研究来看，我们 可以预测，这些研究的结果也将与 人类疾病。

Metastasis is a complex biological process for which there is minimal understanding at the molecular and cellular level. Although poorly understood, metastasis is often described as the most significant step in malignant progression as cancer mortality in general results directly from the growth of disseminated metastatic cells. This profound impact of metastatic disease is clearly the case in prostate cancer which will be diagnosed in 200,000 US males in 1994 and cause mortality in 38,000 US men. Recently, we have modified the mouse prostate reconstitution (MPR) model system to use urogenital sinus tissue derived from p53 "knock-out" mice as target for the introduction of the ras and myc oncogenes. Using this in vivo model system, we have studied the tumorigenic effects of ras+myc initiation in a background of wild-type p53, heterozygous p53 mutants or homozygous p53 mutants. We demonstrated that ras+myc-initiated wild-type 129/Sv mice produce predominantly hyperplasia with low frequency (approximately 10%) of focal nonmetastatic cancer. In contrast, both heterozygous p53 as well as homozygous p53 MPRs produce metastatic carcinoma in nearly 100% of the cases. Progress of ras+myc + heterozygote p53 "knock-out" MPRs correlated with either complete loss, partial loss or loss of expression of the wild-type allele. Overall, our data clearly indicate that complete loss of p53 function leads to metastasis in this experimental mouse prostate cancer model. Interestingly, the pattern of metastasis closely mimics that seen in human prostate including widespread metastasis to the lung, mesenteric lymph nodes, bone and occasionally liver. We have extended this model to facilitate mechanistic studies at the molecular and cellular level, by generating a series of cell lines derived from both primary site carcinomas as well as metastatic disease from different organ sites. We have thus far demonstrated that TGFbeta1 inhibited growth in vitro in all (n=6) primary site tumor-derived cell lines (mean inhibition of 45+/- 9% after 8 days) as well as control urogenital sinus epithelial (CUGE) cells but only inhibited 3 out of 6 cell lines derived from lung metastases (mean inhibition in sensitive lines 20 +/- 1%). Similar differential responses to TGF-beta1 were also observed for colony formation and motility assays. The results of crosslinking studies for TGF-beta1 which demonstrated specific TGF-beta1 binding for type l, 2 and 3 TGF-beta receptors in all primary site tumor- and metastasis-derived cell lines suggest that loss of these TGF-beta1 induced responses in metastasis-derived cell lines is downstream of receptor binding. We propose to use this novel prostate cancer metastasis model system to investigate the genetic mechanisms which underlie the loss of TGFbeta1 response and its relationship to metastatic progression. In addition, specific regions of homozygous deletion associated with metastatic progression will be identified and cloned using representational difference analysis and regions of loss of heterozygosity that occur during metastasis in vivo will be determined using microsatellite probes. From past studies using the MPR model system we can predict that the results of these studies will also be relevant to the human disease.