Jäger Heike, Dreker Tobias, Buck Anita, Giehl Klaudia, Gress Thomas, Grissmer Stephan
Department of Applied Physiology, University Ulm, Ulm, Germany.
Mol Pharmacol. 2004 Mar;65(3):630-8. doi: 10.1124/mol.65.3.630.
Ion channels are important in controlling cell cycle progression and proliferation in a variety of cell types. Using the whole-cell recording mode of the patch-clamp technique, functional ion channels were electrophysiologically characterized in PANC-1 (K-ras G12D (+/-), p53 R273C, Deltap16), BxPC-3 (smad4-, p53 Y220C, Deltap16), and MiaPaCa-2 [transforming growth factor-beta receptor type II defect, K-ras G12C(-/-), p53 R248W, Deltap16] human pancreatic cancer cell lines. In BxPC-3 and the MiaPaCa-2 cells, we could identify approximately 600 or approximately 1200 functional Ca2+-activated K+ channels (IK) per cell, respectively, whereas PANC-1 cells expressed approximately 200 functional IK channels per cell. These channels were observed by using pipette solutions buffering [Ca2+]i to 1 microM. The channels were voltage-independent, blocked by charybdotoxin, clotrimazole, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), and blocked by Ba2+ in a voltage-dependent manner. In the presence of 10 microM clotrimazole or TRAM-34, proliferation of the BxPC-3 as well as the MiaPaCa-2 cells was completely stopped. In contrast, proliferation of PANC-1 cells was hardly affected by clotrimazole or TRAM-34. Proliferation in all three cell lines could be inhibited in the presence of the Ca2+ channel antagonists verapamil, diltiazem, and nifedipine. By quantitative RT-PCR, we could show that MiaPaCa-2 cells exhibit a 2.8-fold and BxPC3 cells a more than 8-fold elevated level of IK mRNA level compared with PANC-1 cells. Interestingly, in primary pancreatic tumors we found a tremendous up-regulation of IK mRNA. In eight of nine (or 89%) primary pancreatic tumor tissues, we found a 6- to 66-fold increase in IK mRNA. Our findings suggest that a certain amount of functional IK channels is crucial for the proliferation of some pancreatic cancer types. The blockade of IK channels may ultimately prove useful as a therapeutic option for some patients with ductal adenocarcinoma of the pancreas with an up-regulated IK channel expression.
离子通道在控制多种细胞类型的细胞周期进程和增殖中起着重要作用。采用膜片钳技术的全细胞记录模式，对PANC-1（K-ras G12D（+/-），p53 R273C，Delta p16）、BxPC-3（smad4-，p53 Y220C，Delta p16）和MiaPaCa-2 [转化生长因子-β II型受体缺陷，K-ras G12C（-/-），p53 R248W，Delta p16] 人胰腺癌细胞系中的功能性离子通道进行了电生理学特征分析。在BxPC-3和MiaPaCa-2细胞中，我们分别可以识别出每个细胞约600个或约1200个功能性钙激活钾通道（IK），而PANC-1细胞每个细胞表达约200个功能性IK通道。通过使用将细胞内钙浓度（[Ca2+]i）缓冲至1微摩尔的移液管溶液观察到了这些通道。这些通道不依赖电压，可被蝎毒素、克霉唑、1-[(2-氯苯基)二苯基甲基]-1H-吡唑（TRAM-34）阻断，并以电压依赖性方式被钡离子阻断。在存在10微摩尔克霉唑或TRAM-34的情况下，BxPC-3以及MiaPaCa-2细胞的增殖完全停止。相比之下，克霉唑或TRAM-34对PANC-1细胞的增殖几乎没有影响。在存在钙离子通道拮抗剂维拉帕米、地尔硫卓和硝苯地平的情况下，所有三种细胞系的增殖都可受到抑制。通过定量逆转录聚合酶链反应（RT-PCR），我们可以表明，与PANC-1细胞相比，MiaPaCa-2细胞的IK mRNA水平升高了2.8倍，BxPC3细胞的IK mRNA水平升高了8倍以上。有趣的是，在原发性胰腺肿瘤中，我们发现IK mRNA有巨大的上调。在9个原发性胰腺肿瘤组织中的8个（或89%）中，我们发现IK mRNA增加了6至66倍。我们的研究结果表明，一定数量的功能性IK通道对于某些类型的胰腺癌增殖至关重要。IK通道的阻断最终可能被证明是一些IK通道表达上调的胰腺导管腺癌患者的一种治疗选择。
