Zhong Q, Chen C F, Chen Y, Chen P L, Lee W H
Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center, San Antonio 78245, USA.
Cancer Res. 1997 Oct 1;57(19):4225-8.
tsg101 was identified as a tumor susceptibility gene by homozygous functional inactivation of allelic loci in mouse 3T3 fibroblasts. The human homologue was mapped at chromosome 11p15.1-2 and found to have intragenic deletion in 7 of 15 breast cancer specimens. To further confirm the relevance of defects in this gene to breast cancer, antibodies specific for the putative gene product were prepared and used to identify cellular TSG101 protein. The antibodies recognized a 46-kDa protein in human retinoblastoma WERI-27 cells labeled with [35S]methionine. This protein was not detected with preimmune sera. In cell fractionation studies, the 46-kDa protein cofractionating with glutathione S-transferase was found mainly in the cytoplasm. Similarly, when cells were immunostained with anti-TSG101 antibodies, fluorescence was localized in the cytoplasm of most of the cells. A full-size 46-kDa TSG101 protein was detected in a panel of 10 breast cancer cell lines and 2 normal breast epithelial cell lines with the same antibodies. Consistently, the full-length TSG101 mRNA was also detected in these breast cells using reverse transcription-PCR. These results indicate that homozygous intragenic deletion of TSG101 is rare in breast cancer cells.
通过对小鼠3T3成纤维细胞中等位基因座的纯合功能失活，tsg101被鉴定为肿瘤易感基因。人类同源基因定位于染色体11p15.1 - 2，并且在15例乳腺癌标本中有7例发现该基因存在基因内缺失。为了进一步证实该基因缺陷与乳腺癌的相关性，制备了针对假定基因产物的特异性抗体，并用于鉴定细胞中的TSG101蛋白。这些抗体在经[35S]甲硫氨酸标记的人视网膜母细胞瘤WERI - 27细胞中识别出一种46 kDa的蛋白质。用免疫前血清未检测到这种蛋白质。在细胞分级分离研究中，发现与谷胱甘肽S - 转移酶共分级的46 kDa蛋白质主要存在于细胞质中。同样，当用抗TSG101抗体对细胞进行免疫染色时，荧光定位于大多数细胞的细胞质中。用相同抗体在一组10个乳腺癌细胞系和2个正常乳腺上皮细胞系中检测到了全长46 kDa的TSG101蛋白。一致地，使用逆转录 - PCR在这些乳腺细胞中也检测到了全长TSG101 mRNA。这些结果表明TSG101的纯合基因内缺失在乳腺癌细胞中很少见。
