Hartley J A, Gibson N W, Zwelling L A, Yuspa S H
Cancer Res. 1985 Oct;45(10):4864-70.
We have studied the appearance of single strand breaks (SSB) in DNA of mouse keratinocytes exposed in vitro to various tumor promoters. Mouse basal keratinocytes were selectively cultured in low calcium medium, prelabeled with [14C]thymidine, exposed to test agents, and SSB quantified by alkaline elution. 12-O-Tetradecanoylphorbol-13-acetate (TPA) caused a dose-dependent (10(-9)-10(-7) M) increase in SSB after 24 h but not after shorter exposures. DNA containing TPA-induced SSB was found only in cells which had detached from the culture plate as a consequence of TPA-induced terminal differentiation. Attached cells, resistant to the differentiation-inducing effects of TPA, had the low level of SSB found in DNA from vehicle-treated control cells. Attached cells were resistant to the formation of SSB and to induced differentiation when reexposed to TPA. Other tumor-promoting phorbol esters, mezerein and retinyl phorbol acetate, also produced SSB in detached cells, whereas phorbol or resiniferatoxin caused neither SSB or cell detachment. Retinoic acid, which blocks the induction of differentiation by TPA, inhibited the production of SSB by TPA; however, fluocinolone acetonide, chymostatin, catalase, or superoxide dismutase blocked neither TPA-induced SSB nor terminal differentiation. Epidermal cell lines resistant to TPA-induced differentiation were also resistant to SSB production by TPA. Benzoyl peroxide (BP) (10(-4) M) induced SSB in basal keratinocytes within 1 h, and attached cells showed extensive SSB by 12 h. Retinoic acid had only a slight effect on BP-induced SSB, and 1 of 3 TPA-resistant cell lines developed SSB when exposed to BP. These results suggest that TPA-induced SSB in epidermal cells are an indirect consequence of the induction of terminal differentiation, whereas BP produces SSB by a more direct mechanism of DNA damage.
我们研究了体外暴露于各种肿瘤启动剂的小鼠角质形成细胞DNA中单链断裂（SSB）的出现情况。将小鼠基底角质形成细胞在低钙培养基中进行选择性培养，用[14C]胸腺嘧啶核苷进行预标记，暴露于测试剂中，然后通过碱性洗脱对SSB进行定量。12 - 十四酰佛波醇 - 13 - 乙酸酯（TPA）在24小时后导致SSB呈剂量依赖性（10(-9)-10(-7) M）增加，但较短暴露时间后则无此现象。含有TPA诱导的SSB的DNA仅在因TPA诱导的终末分化而从培养板上脱离的细胞中发现。对TPA诱导的分化有抗性的贴壁细胞，其DNA中的SSB水平与载体处理的对照细胞中的水平一样低。当再次暴露于TPA时，贴壁细胞对SSB的形成和诱导分化具有抗性。其他促肿瘤佛波酯，如大戟二萜醇酯和视黄基佛波醇乙酸酯，也在脱离的细胞中产生SSB，而佛波醇或树脂毒素既不引起SSB也不导致细胞脱离。维甲酸可阻断TPA诱导的分化，也抑制TPA诱导的SSB产生；然而，氟轻松丙酮、抑肽酶、过氧化氢酶或超氧化物歧化酶既不阻断TPA诱导的SSB也不阻断终末分化。对TPA诱导的分化有抗性的表皮细胞系对TPA产生SSB也具有抗性。过氧化苯甲酰（BP）（10(-4) M）在1小时内诱导基底角质形成细胞产生SSB，到12小时时贴壁细胞显示出广泛的SSB。维甲酸对BP诱导的SSB只有轻微影响，并且3个对TPA有抗性的细胞系中有1个在暴露于BP时产生了SSB。这些结果表明，表皮细胞中TPA诱导的SSB是终末分化诱导的间接结果，而BP通过更直接的DNA损伤机制产生SSB。
