Gniadecki R, Gajkowska B, Bartosik J, Hansen M, Wulf H C
Department of Dermatology, University of Copenhagen, Bispebjerg Hospital, Denmark.
Acta Derm Venereol. 1998 Jul;78(4):248-57. doi: 10.1080/000155598441800.
Apoptosis is a form of cellular suicide and is activated in various cells, including keratinocytes, in response to physiological and pathological stimuli. A current hypothesis holds that apoptotic cells in a concerted manner express characteristic phenotypic features comprising cytoplasmic budding, pyknosis, chromatin condensation, karyorhexis and internucleosomal DNA fragmentation. In this study we investigated the effects of different potential inducers of apoptosis on cultured human keratinocytes. Viability was determined with vital dyes (ethidium bromide, trypan blue), cell morphology was investigated with electron microscopy, and nuclear and DNA integrity was assessed with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) or DNA gel electrophoresis. Irradiation with 50 mJ/cm2 ultraviolet B (UVB), treatment with C8 ceramide, or suspension in a semisolid medium caused apoptosis which was ultrastructurally different from necrotic induced by very high (300 mJ/cm2) doses of UVB. However, the phenotype of dying cells did not exhibit all typical features of apoptosis and cell morphology depended on the method used to induce apoptosis. Cells irradiated with ultraviolet B or treated with C8 ceramide developed large and small budding and DNA nicks but not chromatin condensation or classical karyorhexis. In UVB-irradiated cells a novel form of karyorhexis was observed manifested by formation of a few very small chromatin fragments in nuclear periphery. Cells suspended in methylcellulose developed DNA nicks and pyknosis, but not budding or karyorhexis. In neither case could the typical internucleosomal DNA fragments be detected by gel electrophoresis. These morphological results indicate that in keratinocytes induction of effect or death mechanisms is not concerted but depends on the stimulus inducing apoptosis.
细胞凋亡是一种细胞自杀形式，在包括角质形成细胞在内的各种细胞中，对生理和病理刺激作出反应时被激活。目前的一种假说认为，凋亡细胞以协同的方式表达特征性表型特征，包括细胞质出芽、核固缩、染色质凝聚、核碎裂和核小体间DNA片段化。在本研究中，我们调查了不同潜在凋亡诱导剂对培养的人角质形成细胞的影响。用活性染料（溴化乙锭、台盼蓝）测定细胞活力，用电子显微镜研究细胞形态，并用TUNEL（末端脱氧核苷酸转移酶介导的dUTP缺口末端标记）或DNA凝胶电泳评估核和DNA完整性。用50 mJ/cm2的紫外线B（UVB）照射、用C8神经酰胺处理或悬浮在半固体培养基中均可导致细胞凋亡，其超微结构与极高剂量（300 mJ/cm2）UVB诱导的坏死不同。然而，死亡细胞的表型并未表现出凋亡的所有典型特征，细胞形态取决于诱导凋亡的方法。用紫外线B照射或用C8神经酰胺处理的细胞会出现大小不等的出芽和DNA缺口，但没有染色质凝聚或典型的核碎裂。在UVB照射的细胞中，观察到一种新的核碎裂形式，表现为核周边形成一些非常小的染色质片段。悬浮在甲基纤维素中的细胞出现DNA缺口和核固缩，但没有出芽或核碎裂。在这两种情况下，凝胶电泳均未检测到典型的核小体间DNA片段。这些形态学结果表明，在角质形成细胞中，效应或死亡机制的诱导并非协同一致，而是取决于诱导凋亡的刺激因素。
