CLONAL ANALYSIS OF SUCRASE ISOMALTASE EXPRESSION IN THE HUMAN COLON ADENOCARCINOMA CACO-2 CELLS
CLONAL ANALYSIS OF SUCRASE ISOMALTASE EXPRESSION IN THE HUMAN COLON ADENOCARCINOMA CACO-2 CELLS
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DOI：
10.1042/bj2800599
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发表时间：
1991-12-15
影响因子：
4.1
通讯作者：
QUARONI, A
中科院分区：
文献类型：
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作者：
BEAULIEU, JF;QUARONI, A
为了研究汇合的 Caco-2 细胞（一种表现出成人小肠肠上皮细胞特征的人结肠癌细胞系）中刷状缘酶嵌合表达的生物合成基础，我们获得了一系列在生长速率、释放到培养基中的转化生长因子-α/表皮生长因子样活性量以及蔗糖酶异麦芽酶（SI）活性方面显着不同的克隆。其他肠道标志物（氨肽酶 N、二肽基肽酶 IV、乳糖酶、碱性磷酸酶和“隐窝细胞抗原”）的表达变异性更为有限，表明我们获得的 Caco-2 细胞克隆的总体分化能力没有差异。采用免疫荧光染色、放射性蛋氨酸代谢标记和 SI mRNA 丰度杂交分析来研究具有低、中或高蔗糖酶活性的克隆中的 SI 合成及其调控。获得的结果表明，即使在克隆细胞系中，SI 表达也存在异质性，并且 SI 表达与培养基中生长因子浓度之间呈负相关，表明汇合 Caco-2 细胞中细胞增殖和分化的自分泌调节。使用具有最低和最高水平 SI 活性的两个克隆进行脉冲追踪实验，然后用表位特异性抗体对标记的 SI 进行免疫沉淀和 SDS/PAGE 分析，表明转录和翻译后机制在肠细胞中 SI 表达的调节中发挥作用。
To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.