PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells
PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells
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DOI：
10.1053/gast.2001.24843
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发表时间：
2001-06-01
期刊：
影响因子：
29.4
通讯作者：
Brasitus, TA
中科院分区：
文献类型：
--
作者：
Cerda, SR;Bissonnette, M;Brasitus, TA
（背景和目的）bar下：先前的研究表明，氧化偶氮甲烷诱导的大鼠和散发性人类结肠肿瘤中蛋白激酶C（PHC）-delta表达减少。为了阐明PKC-δ对人结肠癌细胞的肿瘤表型的作用，我们在CaCo-2细胞中建立了这种同工酶的稳定转染子。（方法）在bar下：将人PKC-δ互补DNA亚克隆到两个不同的金属硫蛋白调控的表达载体中。通过Western印迹法对PKC-δ转染子的多克隆群体进行表征，使用PKC-δ特异性底物原位测量PKC-δ活性，通过Coulter计数器测定增殖，并通过流式细胞术分析细胞周期分布，通过在软琼脂中的生长和通过碱性磷酸酶和蔗糖酶异麦芽糖酶的变化来评估体外转化。通过4 '，6-二脒基-2-苯基吲哚二盐酸盐和末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记染色来评估细胞凋亡。（结果）在条形图下：在Zn 2+存在下，PKC-δ转染子表达的蛋白质增加4倍，PKC-δ活性增加2倍。PKC-δ转染后细胞生长下降30%（P < 0.05），分化表型增强。PKC-δ表达增加导致G 0/G1期阻滞，抑制锚定非依赖性生长（50%，P < 0.05），并导致凋亡增加2倍（P < 0.05）。（结论）在酒吧之下：我们的研究表明，增加表达的PKC-δ抑制锚定依赖性和非依赖性的生长，同时诱导细胞分化和限制这种人结肠癌细胞系的生存。
(Background & Aims) under bar: Previous studies showed decreased protein kinase C (PHC)-delta expression in azoxymethane-induced rat and sporadic human colonic tumors. To elucidate the role of PKC-delta on the neoplastic phenotype of human colon cancer cells, we established stable transfectants of this isoenzyme in CaCo-2 cells. (Methods) under bar: Human PKC-delta complementary DNA was subcloned into 2 distinct metallothionein-regulated expression vectors. Polyclonal populations of PKC-delta transfectants were characterized by Western blotting PKC-delta activity was measured in situ using a PKC-delta -specific substrate, Proliferation was determined by Coulter counter, and cell cycle distribution was analyzed by flow cytometry, In vitro transformation was assessed by growth in soft agar and differentiation by changes in alkaline phosphatase and sucrase isomaltase. Apoptosis was evaluated by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, (Results) under bar: In the presence of Zn2+, PKC-delta transfectants expressed a 4-fold increase in the protein and a 2-fold increase in activity of PKC-delta. PKC-delta transfectants exhibited a 30% decrease (P < 0.05) in cell growth and an enhanced differentiation phenotype, Increased PKC- expression induced a significant G0/G1 arrest, inhibited anchorage-independent growth (50%, P < 0.05), and caused a 2-fold increase in apoptosis (P < 0.05). (Conclusions) under bar: Our studies show that increased expression of PKC-delta inhibits anchorage-dependent and -independent growth, while inducing cellular differentiation and limiting survival of this human colon cancer cell line.