Kopp R, Fichter M, Assert R, Pfeiffer A F, Classen S
Department of Surgery, Klinikum Grosshadern, University of Munich, D-81377 Munich, Germany.
Int J Mol Med. 2009 May;23(5):639-49. doi: 10.3892/ijmm_00000175.
Initiation of cell growth and neoplastic transformation frequently involves activation of growth factor receptor-coupled tyrosine kinases and stimulation of the phosphoinositide second messenger system. Altered expression of CD44 variants was reported in several malignant tumor types with possible implications for tumor progression and prognosis. CD44 variant expression was reported to be associated with second messenger activation and differentiation. We therefore investigated the effects of butyrate-induced short-term differentiation on phosphoinositide signaling, phospholipase C and protein kinase C activity and alteration of CD44 variant expression in human HT-29 colon carcinoma cells. HT-29 cells were cultured with sodium butyrate for 6 days. Phosphoinositide turnover was measured by [32P]orthophosphate incorporation and phospholipase C activity by determination of the release of [3H]inositolphosphates from [3H]myoinositol prelabeled cells. Protein kinase C activity was determined by histone III-S phosphorylation, PKC subtype expression by RNase protection analysis, and CD44 variant expression was determined by RT-PCR using variant-specific primers. Treatment of HT-29 human colon carcinoma cells with sodium butyrate caused a dose-dependent inhibition of cell proliferation (IC50, 2.5 mM) with morphologic signs of an enterocytic differentiation following 6 days of treatment. The phosphoinositide turnover as determined by 32P-incorporation under non-equilibrium conditions showed a 30-40% inhibition of labeled phosphoinositides and phosphatidic acid and a dose-dependent inhibition of cholinergically stimulated phospholipase C activity as a secondary event following butyrate-induced enterocytic differentiation. However, long-term incubation of HT-29 cells with phorbol ester or an inhibitor of classical and novel PKC subtypes did not affect cell proliferation. In butyrate-treated HT-29 cells activation of calcium-dependent protein kinase C by cholinergic stimulation or phorbolester treatment induced an increase in membrane-bound cPKC activity, while expression of distinct high- molecular CD44 variant transcripts v3 (670 bp), v5 (940 bp) and v8 (535 bp) were drastically reduced after butyrate pretreatment. Enterocytic differentiation of HT-29 colon carcinoma cells seems to be associated with alterations in phosphoinositide resynthesis, phospholipase C activity and ligand/receptor-induced PKC translocation. The observed reduction of distinct high-molecular CD44v3, v5 and v8 variants following butyrate-induced differentiation indicates an association of specific CD44 variant expression with the malignant phenotype of HT-29 colon cancer cells, thus being possible targets for new diagnostic and therapeutic strategies.
细胞生长的启动和肿瘤转化常常涉及生长因子受体偶联酪氨酸激酶的激活以及磷酸肌醇第二信使系统的刺激。据报道，在几种恶性肿瘤类型中CD44变体的表达发生改变，这可能对肿瘤进展和预后有影响。据报道，CD44变体表达与第二信使激活和分化有关。因此，我们研究了丁酸盐诱导的短期分化对人HT - 29结肠癌细胞中磷酸肌醇信号传导、磷脂酶C和蛋白激酶C活性以及CD44变体表达改变的影响。HT - 29细胞用丁酸钠培养6天。通过[32P]正磷酸盐掺入测量磷酸肌醇周转，通过测定[3H]肌醇预先标记细胞中[3H]肌醇磷酸的释放来测量磷脂酶C活性。通过组蛋白III - S磷酸化测定蛋白激酶C活性，通过核糖核酸酶保护分析测定PKC亚型表达，通过使用变体特异性引物的RT - PCR测定CD44变体表达。用丁酸钠处理HT - 29人结肠癌细胞导致细胞增殖的剂量依赖性抑制（IC50，2.5 mM），处理6天后出现肠上皮细胞分化的形态学迹象。在非平衡条件下通过32P掺入测定的磷酸肌醇周转显示标记的磷酸肌醇和磷脂酸受到30 - 40%的抑制，并且作为丁酸盐诱导的肠上皮细胞分化后的继发事件，胆碱能刺激的磷脂酶C活性受到剂量依赖性抑制。然而，用佛波酯或经典和新型PKC亚型抑制剂对HT - 29细胞进行长期孵育并不影响细胞增殖。在丁酸盐处理的HT - 29细胞中，胆碱能刺激或佛波酯处理激活钙依赖性蛋白激酶C会导致膜结合cPKC活性增加，而在丁酸盐预处理后，不同的高分子量CD44变体转录本v3（670 bp）、v5（940 bp）和v8（535 bp）的表达大幅降低。HT - 29结肠癌细胞的肠上皮细胞分化似乎与磷酸肌醇再合成、磷脂酶C活性以及配体/受体诱导的PKC易位的改变有关。丁酸盐诱导分化后观察到的特定高分子量CD44v3、v5和v8变体的减少表明特定CD44变体表达与HT - 29结肠癌细胞的恶性表型有关，因此可能成为新的诊断和治疗策略的靶点。
