Autocrine action of amphiregulin in a colon carcinoma cell line and immunocytochemical localization of amphiregulin in human colon.
Autocrine action of amphiregulin in a colon carcinoma cell line and immunocytochemical localization of amphiregulin in human colon.
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DOI：
10.1083/jcb.118.3.741
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发表时间：
1992-08
期刊：
影响因子：
--
通讯作者：
Stromberg K
中科院分区：
文献类型：
--
作者：
Johnson GR;Saeki T;Gordon AW;Shoyab M;Salomon DS;Stromberg K
双调蛋白（Amphiregulin，AR）是一种新发现的糖基化的84个氨基酸残基的多肽生长调节因子，与EGF家族蛋白具有序列同源性。为了获得研究AR生物学作用的免疫学试剂，使用两种含有AR不同区域对应序列的合成肽在兔中制备多克隆抗体。一种针对AR残基26-44的抗肽抗体制剂（AR-Ab 2）在检测天然AR中最有效，而另一种针对残基8-26的抗体制剂（AR-Ab 1）在检测福尔马林固定和石蜡包埋组织中的AR中最有效。结肠癌细胞系Geo在无血清条件下自主增殖，其生长受到外源性AR或EGF的刺激。在40和200 pM的EGF和AR，分别观察到这种增长的半最大刺激。EGF受体胞外结构域的mAb阻断了由外源性添加AR诱导的细胞增殖刺激，表明这种刺激是通过EGF受体介导的。发现Geo细胞组成型表达显著水平的AR mRNA转录物，如通过聚合酶链反应扩增的cDNA产物的分析所确定的，并且使用这些细胞中的AR-Ab 1抗体免疫细胞化学检测AR蛋白。使用来自Geo细胞条件培养基的AR-Ab 2抗体特异性地免疫沉淀AR，所述Geo细胞条件培养基已经用[35 S]半胱氨酸代谢标记。在SDS-PAGE中，分泌的AR以宽带（18.5-22.5 kD）迁移，中位分子量约为20.7 kD。从Geo细胞条件化的无血清培养基中免疫特异性去除AR，并将AR耗尽的培养基重新加入Geo细胞，导致相对于对照组约40%的细胞生长抑制。此外，通过单独添加抗EGF受体mAb，Geo细胞的生长也被抑制约50%。这些结果表明，AR和EGF受体参与这些细胞的自分泌生长，并表明AR可能通过EGF受体通过细胞外自分泌回路起作用。为了研究AR在体内人结肠中的表达，使用AR-Ab 1抗体将AR免疫细胞化学定位在来自正常和恶性人结肠的福尔马林固定的石蜡包埋切片中。（400字处删节）
Amphiregulin (AR) is a newly discovered glycosylated, 84-amino acid residue polypeptide growth regulator which has sequence homology to the EGF family of proteins. To obtain immunological reagents to study the biological role of AR, two synthetic peptides containing sequences corresponding to distinct regions of AR were used to generate polyclonal antibodies in rabbits. One preparation of antipeptide antibodies directed against residues 26-44 of AR (AR-Ab2) was most effective in the detection of native AR, whereas another preparation of antibodies against residues 8-26 (AR-Ab1) was found to be most efficacious in the detection of AR in formalin-fixed and paraffin- embedded tissues. The growth of a colon carcinoma cell line, Geo, which proliferates autonomously under serum-free conditions, was stimulated by the exogenous addition of AR or EGF. Half-maximal stimulation of this growth was observed at 40 and 200 pM of EGF and AR, respectively. A mAb to the extracellular domain of the EGF receptor blocked the stimulation of cell proliferation induced by the exogenous addition of AR, suggesting that this stimulation was mediated via the EGF receptor. Geo cells were found to constitutively express significant levels of the AR mRNA transcript as determined by analysis of the polymerase chain reaction-amplified cDNA product and AR protein was detected immunocytochemically using the AR-Ab1 antibodies in these cells. AR was immunoprecipitated specifically using the AR-Ab2 antibodies from the conditioned medium of Geo cells, which had been metabolically labeled with [35S]cysteine. The secreted AR migrated as a broad band (18.5-22.5 kD) with a median molecular weight of approximately 20.7 kD in SDS- PAGE. Immunospecific removal of AR from serum-free medium conditioned by the Geo cells and readdition of the AR-depleted medium to Geo cells resulted in an approximately 40% inhibition of cell growth relative to controls. Furthermore, the growth of the Geo cells was also inhibited by approximately 50% by the addition of the anti-EGF receptor mAb alone. These results indicate that AR and the EGF receptor are involved in the autocrine growth of these cells and suggests that AR may act through the EGF receptor via an extracellular autocrine loop. To study the expression of AR in human colon in vivo, AR was localized immunocytochemically in formalin-fixed, paraffin-embedded sections from normal and malignant human colon using the AR-Ab1 antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)