Alitalo R, Andersson L C, Tapiovaara H, Sistonen L, Vaheri A, Stephens R
Transplantation Laboratory, University of Helsinki, Finland.
J Cell Physiol. 1989 Jul;140(1):119-30. doi: 10.1002/jcp.1041400115.
The human chronic myeloid leukemia cell line K562 acquires several megakaryoblastoid features when cultured in the presence of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We observed strongly increased secretion of several proteins into the culture media of K562 cells within a few hours of TPA treatment. Two of the major secreted polypeptides were identified by immunoprecipitation from media of metabolically labeled cultures as the tissue inhibitor of metalloproteinases (TIMP) and the type 1 plasminogen activator inhibitor (PAI-1). Maximal amounts of PAI-1 mRNA and secretion of PAI-1 polypeptides were observed after 24 hr of TPA treatment and PAI-1 persisted at elevated levels for several days. The induction of PAI-1 mRNA was dependent on de novo protein synthesis. Uninduced and induced cells secreted urokinase plasminogen activator in its single-chain proenzyme form (pro-u-PA), which was cleaved extracellularly to the active two-chain form as shown by pulse-chase labeling experiments. Upon TPA induction, the secretion of u-PA polypeptides increased severalfold, and there was a transient accumulation of pro-u-PA in the culture medium. However, this did not lead to increased u-PA activity in the cultures, since active u-PA was removed by complex formation with the large excess of coinduced PAI-1. Induction of u-PA mRNA was biphasic: The first peak of about tenfold increase in steady-state u-PA mRNA at 3 hr was followed by a steep decline to the baseline level at 12 hr, and a second, slower accumulation of u-PA mRNA occurred over the next few days. The biphasic accumulation of u-PA mRNA was also reflected in u-PA protein synthesis. We conclude that concerted changes in favor of a nonproteolytic extracellular environment occur in TPA-induced K562 cultures undergoing megakaryoblastoid differentiation. These changes include excessive secretion of TIMP and inhibition of the induced u-PA by the simultaneous accumulation of PAI-1.
人类慢性髓系白血病细胞系K562在肿瘤启动子12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）存在的情况下培养时，会获得一些巨核母细胞样特征。我们观察到，在TPA处理后的几小时内，K562细胞向培养基中分泌的几种蛋白质显著增加。通过对代谢标记培养物的培养基进行免疫沉淀，鉴定出两种主要的分泌多肽为金属蛋白酶组织抑制剂（TIMP）和1型纤溶酶原激活物抑制剂（PAI - 1）。TPA处理24小时后观察到PAI - 1 mRNA的最大量以及PAI - 1多肽的分泌，并且PAI - 1在数天内持续保持在较高水平。PAI - 1 mRNA的诱导依赖于从头合成蛋白质。未诱导和诱导的细胞均以单链酶原形式（pro - u - PA）分泌尿激酶型纤溶酶原激活物，脉冲追踪标记实验表明，该酶原在细胞外被切割成活性双链形式。在TPA诱导下，u - PA多肽的分泌增加了数倍，并且培养基中出现了pro - u - PA的短暂积累。然而，这并没有导致培养物中u - PA活性增加，因为活性u - PA通过与大量共同诱导的PAI - 1形成复合物而被清除。u - PA mRNA的诱导是双相的：在3小时时，稳态u - PA mRNA约增加十倍，随后在12小时急剧下降至基线水平，在接下来的几天内出现了u - PA mRNA的第二次较慢积累。u - PA mRNA的双相积累也反映在u - PA蛋白质合成中。我们得出结论，在经历巨核母细胞样分化的TPA诱导的K562培养物中，发生了有利于非蛋白水解性细胞外环境的协同变化。这些变化包括TIMP的过度分泌以及PAI - 1的同时积累对诱导的u - PA的抑制。
