Schmitz F, Otte J M, Stechele H U, Reimann B, Banasiewicz T, Fölsch U R, Schmidt W E, Herzig K H
Department of Medicine, Christian-Albrechts-University Kiel, Germany.
Eur J Clin Invest. 2001 Sep;31(9):812-20. doi: 10.1046/j.1365-2362.2001.00870.x.
Mature amidated gastrin (G17 amide) mediates its effects in the gastrointestinal tract by activating G protein-coupled CCK-B/gastrin receptors. Although trophic actions of gastrin on the gastric mucosa have been well-established, the effect of G17 amide, progastrin and intermediates to colon neoplasia in humans is controversial. While epidemiological evidence from patients with elevated serum gastrin levels related to pernicious anaemia does not support an increased risk for colon cancer, a recent study suggests that prolonged hypergastrinaemia is associated with an increased risk for colon cancer. The extent to which trophic actions of gastrin in colorectal cancer are mediated by functional gastrin receptors remains to be defined. The aim of the present study was to determine CCK-B/gastrin receptor expression, structure, and function in 79 patients with colon cancer.
CCK-B/gastrin receptor cDNAs were isolated from 79 human colorectal cancer specimens and 15 control tissues, subcloned into the eukaryotic expression vector pCR3.1 and subjected to DNA sequence analysis. Wild-type and mutant cDNAs were transiently expressed in COS-7 cells to determine ligand affinities by 125I-labelled CCK-8S competition binding. Activation of the MAP kinase signalling cascade by G17 amide was determined in transfected Colo 320 cells expressing the wild-type or mutant CCK-B/gastrin receptors. Clonal expansion of single cells was quantified in transfected Colo 320 cells.
Gastrin mRNA is expressed in 44% of colorectal cancers and in 13% of control tissues. CCK-B/gastrin receptor mRNA is expressed in 38% of colorectal cancers and 13% of normal colonic tissue. Co-expression of gastrin and CCK-B/gastrin receptor message is significantly increased in colorectal cancer specimens (32% vs. 0%). There is no correlation between CCK-B/gastrin receptor expression and disease stage or histological grading. DNA sequence analysis revealed one spontaneous CCK-B/gastrin receptor mutation within the third intracellular loop with an exchange of valine-287 for phenylalanine. Pharmacological characterisation of the 287V --> F CCK-B/gastrin receptor reveals wild-type affinities for G17 amide, glycine-extended gastrin, CCK-8S and L-365,260. Mutation 287V --> F is associated with a loss of gastrin-induced MAPK p44/p42 signalling in Colo 320 cells while clonal expansion from single cells is increased by 53.1 +/- 15.9% when compared to Colo 320 cells expressing wild-type CCK-B/gastrin receptors.
Structural alterations of CCK-B/gastrin receptors may account for increased growth-promoting effects of amidated gastrins in colorectal cancer.
成熟的酰胺化胃泌素（G17酰胺）通过激活G蛋白偶联的CCK - B/胃泌素受体在胃肠道介导其效应。尽管胃泌素对胃黏膜的营养作用已得到充分证实，但G17酰胺、前胃泌素及其中间体对人类结肠肿瘤形成的影响仍存在争议。虽然来自与恶性贫血相关的血清胃泌素水平升高患者的流行病学证据不支持结肠癌风险增加，但最近的一项研究表明，长期高胃泌素血症与结肠癌风险增加有关。胃泌素在结直肠癌中的营养作用通过功能性胃泌素受体介导的程度仍有待确定。本研究的目的是确定79例结肠癌患者中CCK - B/胃泌素受体的表达、结构和功能。
从79例人类结直肠癌标本和15例对照组织中分离CCK - B/胃泌素受体cDNA，亚克隆到真核表达载体pCR3.1中并进行DNA序列分析。野生型和突变型cDNA在COS - 7细胞中瞬时表达，通过125I标记的CCK - 8S竞争结合测定配体亲和力。在表达野生型或突变型CCK - B/胃泌素受体的转染Colo 320细胞中测定G17酰胺对MAP激酶信号级联的激活。在转染的Colo 320细胞中对单细胞的克隆扩增进行定量。
胃泌素mRNA在44%的结直肠癌和13%的对照组织中表达。CCK - B/胃泌素受体mRNA在38%的结直肠癌和13%的正常结肠组织中表达。胃泌素和CCK - B/胃泌素受体信息的共表达在结直肠癌标本中显著增加（32%对0%）。CCK - B/胃泌素受体表达与疾病分期或组织学分级之间无相关性。DNA序列分析显示在第三个细胞内环内有一个自发的CCK - B/胃泌素受体突变，缬氨酸-287被苯丙氨酸取代。287V→F CCK - B/胃泌素受体的药理学特征显示对G17酰胺、甘氨酸延伸型胃泌素、CCK - 8S和L - 365,260具有野生型亲和力。287V→F突变与Colo 320细胞中胃泌素诱导的MAPK p44/p42信号传导丧失相关，而与表达野生型CCK - B/胃泌素受体的Colo 320细胞相比，单细胞的克隆扩增增加了53.1±15.9%。
CCK - B/胃泌素受体的结构改变可能解释了酰胺化胃泌素在结直肠癌中促进生长作用的增强。
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