The biology of bone metastasis for breast cancer in expression of growth factor and receptor

1.人乳腺癌骨转移裸鼠模型的建立：为了研究人乳腺癌骨转移的生物学特性，我们建立了8周龄裸鼠动脉内注射MDA-MB-231细胞(10^6个细胞)的动物模型。注射4周后，所有动物(20/20)都发生了BM，只有一只动物有肺转移。用另一种转移潜能增强的b-成纤维细胞生长因子(MKL-4)细胞系，20%(1/5)发生骨髓，每周X线检查观察骨髓进展至8周。肿瘤的生长可能首先受到骨髓微环境的调节。我们注意到TGFβ是骨基质中含量最丰富的因子，它是由骨吸收释放的。最近有报道称，P53基因调控肿瘤细胞的生长与转化生长因子β相关。在免疫组织化学研究中，转移部位的MDA-MB-231细胞高表达P53蛋白，而MKL-4细胞不表达。两者均不表达转化生长因子β。我们还从裸鼠的肺自发转移灶中培育出一种具有高转移潜能的变异型细胞株MDA-MB-231。2.生长因子及其受体在尸检标本骨髓中的表达：c-met、ER和c-erbB-2在27例尸检标本中的表达分别为87%、52%和43%。这些结果与原发肿瘤是一致的。现在，我们从尸检标本的石蜡包埋块中提取DNA，用于检测基因扩增。在动物模型中，比较不同转移潜能的乳腺癌细胞，用免疫组织化学方法检测激活和失活的TGFbeta的表达，用原位杂交法定位TGFbeta的表达，用PCR-SSCP方法检测p53点突变。

1. Establishment of bone metastasis (BM) for human breast cancer in a nude rat model : To investigate the biology of BM,we have developed an animal model using 8 week aged nude rats with intra-aoric injection of MDA-MB-231 cells (10^6 cells). Four weeks after the injection, all animals (20/20) developed BM,and only one animal with lung metastasis. Using another cell line (MKL-4) transfected b-FGF with enhanced metastatic potential, 20% (1/5) developed BM.Progression of BM was observed by the X-ray examination every week up to 8 weeks after. The growth of tumor may be regulated by the microenvironment in the bone marrow when trapped first. We noticed TGFbeta which is the most abundant factor in bone matrix and released by the bone resorption. Recently, it has been reported the P53 gene regulates the growth of tumor cells related to TGFbeta. In immunohistochemical study, MDA-MB-231 cells in metastatic site overexpressed p53 protein, but MKL-4 did not. TGFbeta was not expressed in both. We have also developed a variant cell line of MDA-MB-231 with highly metastatic potential from spontaneously metastasizing foci of the lung in nude mice. 2. Expression of growth factor and receptor in BM of autopsy specimens : C-met, ER,and c-erbB-2 expressions were observed in 87%, 52%, and 43% of 27 specimens. These results were consistent with primary tumors. Now, we extracted DNA from the paraffin embedded blocks of autopsy specimens for detect of gene amplification. In an animal model, breast cancer cells with different metastatic potentials will be compared by the expression of activated and in-activated TGFbeta with immunohistochemistry, location of the TGFbeta mRNA with in situ hybridization, and p53 point mutation with PCR-SSCP.