Ghosh A K, Shankar D B, Shackleford G M, Wu K, T'Ang A, Miller G J, Zheng J, Roy-Burman P
Department of Pathology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Cell Growth Differ. 1996 Oct;7(10):1425-34.
Three alternatively spliced mRNA isoforms of the human fibroblast growth factor-8 (FGF8) gene, expressed in a prostatic carcinoma cell line, have been isolated as cDNA clones and characterized by DNA sequencing. The clones, designated FGF8a, FGF8b, and FGF8e, differ from each other at the NH2-terminal region of the mature proteins and share extensive nucleotide sequence homology in the protein coding region to the corresponding mouse cDNA isoforms that were previously reported. FGF8a and FGF8b exhibit identical amino acid sequences to those of their murine counterparts. FGF8e displays partial sequence variation from the corresponding mouse clone only in the extra exon sequence found in this isoform in both species. There is extensive sequence diversity between FGF8 (human) and Fgf8 (murine) genes in the 3'-untranslated region of the mRNAs. Northern blot analyses revealed FGF8 mRNA expression only in fetal kidney tissue among the various fetal and adult human tissues tested. The reverse transcription-PCR amplification method, however, detected FGF8 mRNA expression in adult prostate, kidney, and testes (the tissues that were tested) and in all normal and tumor prostatic epithelial cell lines examined; although expression of both FGF8a and FGF8b was seen in kidney and testes, FGF8b appeared to be the predominantly expressed species in the prostatic tissue and cell lines analyzed by reverse transcription-PCR. To address the biological effect of specific isoform expression, NIH3T3 cells were transfected with a eukaryotic expression vector containing cDNA for FGF8a, FGF8b, or FGF8e. Consistent with previous reports on differences in the transforming potential of mouse FGF8 isoforms, human FGF8b was found to induce marked morphological transformation to NIH3T3 cells and strong tumorigenicity of the transfected cells in nude mice. Human FGF8a and FGF8e were moderately transforming in NIH3T3 cells, and the transfected cells were moderately tumorigenic in vivo. These results document the production of three alternatively spliced FGF8 mRNAs in human tissues and the transforming and tumorigenic potential of their protein products. Moreover, these data, combined with the tissue-specific expression of these isoforms, suggest that they may have different biological functions.
在前列腺癌细胞系中表达的人成纤维细胞生长因子8（FGF8）基因的三种可变剪接mRNA亚型，已作为cDNA克隆分离出来，并通过DNA测序进行了表征。这些克隆分别命名为FGF8a、FGF8b和FGF8e，它们在成熟蛋白的NH2末端区域彼此不同，并且在蛋白质编码区域与先前报道的相应小鼠cDNA亚型具有广泛的核苷酸序列同源性。FGF8a和FGF8b与其小鼠对应物的氨基酸序列相同。FGF8e仅在两种物种中该亚型特有的外显子序列中与相应的小鼠克隆存在部分序列差异。在mRNA的3'-非翻译区，FGF8（人）和Fgf8（小鼠）基因之间存在广泛的序列多样性。Northern印迹分析显示，在所测试的各种胎儿和成人人类组织中，FGF8 mRNA仅在胎儿肾脏组织中表达。然而，逆转录-PCR扩增方法检测到FGF8 mRNA在成人前列腺、肾脏和睾丸（所测试的组织）以及所有检测的正常和肿瘤前列腺上皮细胞系中均有表达；尽管在肾脏和睾丸中同时检测到FGF8a和FGF8b的表达，但通过逆转录-PCR分析，FGF8b似乎是前列腺组织和细胞系中主要表达的亚型。为了研究特定亚型表达的生物学效应，将含有FGF8a、FGF8b或FGF8e cDNA的真核表达载体转染到NIH3T3细胞中。与先前关于小鼠FGF8亚型转化潜能差异的报道一致，发现人FGF8b可诱导NIH3T3细胞发生明显的形态转化，并使转染细胞在裸鼠中具有很强的致瘤性。人FGF8a和FGF8e在NIH3T3细胞中具有中等转化能力，转染细胞在体内具有中等致瘤性。这些结果证明了在人类组织中产生了三种可变剪接的FGF8 mRNA及其蛋白质产物的转化和致瘤潜能。此外，这些数据与这些亚型的组织特异性表达相结合，表明它们可能具有不同的生物学功能。
