Department of Virology, Lapeyronie Hospital, University Medical Center of Montpellier, Montpellier Cedex 5, France.
Breast Cancer Res. 2009;11(3):R39. doi: 10.1186/bcr2326. Epub 2009 Jun 23.
We evaluated whether CK19, one of the main cytoskeleton proteins of epithelial cells, is released as full-length protein from viable tumor cells and whether this property is relevant for metastatic progression in breast cancer patients.
EPISPOT (EPithelial ImmunoSPOT) assays were performed to analyze the release of full-length CK19 by carcinoma cells of various origins, and the sequence of CK19 was analyzed with mass spectrometry. Additional functional experiments with cycloheximide, Brefeldin A, or vincristine were done to analyze the biology of the CK19-release. CK19-EPISPOT was used to detect disseminated tumor cells in bone marrow (BM) of 45 breast cancer patients who were then followed up over a median of 6 years.
CK19 was expressed and released by colorectal (HT-29, HCT116, Caco-2) and breast (MCF-7, SKBR3, and MDA-MB-231) cancer cell lines. The CK19-EPISPOT was more sensitive than the CK19-ELISA. Dual fluorescent EPISPOT with antibodies against different CK19 epitopes showed the release of the full-length CK19, which was confirmed by mass spectrometry. Functional experiments indicated that CK19 release was an active process and not simply the consequence of cell death. CK19-releasing cells (RCs) were detectable in BM of 44% to 70% of breast cancer patients. This incidence and the number of CK19-RCs were correlated to the presence of overt metastases, and patients with CK19-RCs had a reduced survival as compared with patients without these cells (P = 0.025, log-rank test; P = 0.0019, hazard ratio, 4.7; multivariate analysis).
Full-length CK19 is released by viable epithelial tumor cells, and CK19-RCs might constitute a biologically active subset of breast cancer cells with high metastatic properties.
我们评估了细胞角蛋白 19（CK19）作为上皮细胞主要细胞骨架蛋白之一是否会从存活的肿瘤细胞中完整释放蛋白，以及该特性是否与乳腺癌患者的转移进展相关。
通过各种来源的癌细胞进行 EPISPOT（上皮免疫斑点）检测，以分析全长 CK19 的释放，并通过质谱分析 CK19 的序列。用环己酰亚胺、布雷菲德菌素 A 或长春新碱进行额外的功能实验，以分析 CK19 释放的生物学特性。CK19-EPISPOT 用于检测 45 例乳腺癌患者骨髓中的播散性肿瘤细胞，然后对这些患者进行中位随访 6 年。
CK19 由结直肠（HT-29、HCT116、Caco-2）和乳腺（MCF-7、SKBR3 和 MDA-MB-231）癌细胞系表达和释放。CK19-EPISPOT 比 CK19-ELISA 更敏感。针对不同 CK19 表位的双荧光 EPISPOT 显示全长 CK19 的释放，该释放通过质谱得到证实。功能实验表明 CK19 释放是一个主动过程，而不仅仅是细胞死亡的结果。在 44%至 70%的乳腺癌患者的骨髓中可检测到 CK19 释放细胞（RC）。这种发生率和 CK19-RC 的数量与明显转移的存在相关，并且具有 CK19-RC 的患者的生存时间比没有这些细胞的患者短（P=0.025，对数秩检验；P=0.0019，风险比，4.7；多变量分析）。
全长 CK19 由存活的上皮肿瘤细胞释放，CK19-RC 可能构成具有高转移特性的乳腺癌细胞的生物学活性亚群。
