Stanwell C, Burke T R, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 1995 Nov 1;55(21):4950-6.
Differentiation therapy is an attractive option for the treatment of superficial, localized neoplastic lesions of the skin. Topical application of agents that induce differentiation could selectively inhibit tumor cell growth, inducing a program of cell death with the production of cross-linked protein envelopes as the terminal event of this process at the skin surface, effectively eliminating the neoplastic phenotype. The nonspecific kinase inhibitor staurosporine induces cornified envelope assembly in neoplastic keratinocytes and causes tumor regression (A. A. Dlugosz and S. H. Yuspa, Cancer Res., 51: 4677-4684, 1991). In pursuit of less toxic agents, specific tyrosine kinase inhibitors were tested for the ability to induce differentiation in keratinocyte-derived cells. Of a range of inhibitors tested, only MC was able to induce cross-linked protein and consequent cell death in mouse and human primary normal keratinocytes, 308 neoplastic mouse keratinocytes, HPV-18-infected immortalized human keratinocytes, and human lines SQCC-Y1 (squamous carcinoma) and A431 (epidermoid carcinoma). MC increased cross-linked protein in a dose-dependent manner (0.05-1 mM). To confirm differentiation, MC-treated mouse primary normal keratinocytes were tested for activation of the endogenous cross-linking enzyme transglutaminase, but no association was found between transglutaminase activity and MC-induced protein cross-linking. MC also induced protein cross-linking in the fibroblast cell line NIH3T3 and in B16 melanoma cells, in which cornified envelope assembly is not part of the differentiation process. This cross-linking occurred at 4 degrees C, suggesting a nonphysiological process. Western blot analysis of an in vitro assay with purified EGF receptor showed that MC was able to cross-link the receptor. As in NIH3T3 cells, DTT inhibited cross-linking, suggesting that oxidation of MC or an acceptor group may be required for this effect. Thus, MC does not induce differentiation by a physiological mechanism in epithelial cells but causes chemical protein cross-linking into cornified envelope-like structures at high concentration.
分化疗法是治疗皮肤浅表性、局限性肿瘤病变的一个有吸引力的选择。局部应用诱导分化的药物可以选择性地抑制肿瘤细胞生长，诱导细胞死亡程序，在皮肤表面产生交联蛋白包膜作为该过程的终末事件，有效消除肿瘤表型。非特异性激酶抑制剂星形孢菌素可诱导肿瘤角质形成细胞中角质包膜组装并导致肿瘤消退（A. A. 德鲁戈什和S. H. 尤斯帕，《癌症研究》，51: 4677 - 4684，1991）。为了寻找毒性较小的药物，对特异性酪氨酸激酶抑制剂诱导角质形成细胞衍生细胞分化的能力进行了测试。在所测试的一系列抑制剂中，只有MC能够在小鼠和人原代正常角质形成细胞、308个肿瘤性小鼠角质形成细胞、HPV - 18感染的永生化人角质形成细胞以及人细胞系SQCC - Y1（鳞状细胞癌）和A431（表皮样癌）中诱导交联蛋白并导致细胞死亡。MC以剂量依赖性方式（0.05 - 1 mM）增加交联蛋白。为了确认分化，对经MC处理的小鼠原代正常角质形成细胞进行内源性交联酶转谷氨酰胺酶激活测试，但未发现转谷氨酰胺酶活性与MC诱导的蛋白交联之间存在关联。MC还能在成纤维细胞系NIH3T3和B16黑色素瘤细胞中诱导蛋白交联，而角质包膜组装并非这些细胞分化过程的一部分。这种交联在4℃时发生，提示这是一个非生理性过程。对纯化的表皮生长因子受体进行体外试验的蛋白质印迹分析表明，MC能够使该受体交联。与在NIH3T3细胞中一样，二硫苏糖醇抑制交联，提示这种作用可能需要MC或受体基团氧化。因此，MC并非通过上皮细胞中的生理机制诱导分化，而是在高浓度下导致化学性蛋白交联形成角质包膜样结构。
