Dilley Tarrah K, Bowden G Tim, Chen Qin M
Department of Pharmacology, College of Medicine, University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724, USA.
Exp Cell Res. 2003 Oct 15;290(1):38-48. doi: 10.1016/s0014-4827(03)00308-2.
Aging is the highest risk factor for cancer. Although oxidants are thought to contribute to both aging and cancer, the interplay between oxidative stress, aging, and cancer has not been well studied. Human diploid fibroblasts (HDFs) undergo premature senescence in response to sublethal doses of H(2)O(2). To test the hypothesis that senescent or senescent-like HDFs function as a tumor promoter, we have employed an in vitro skin tumor promotion model, in which colony formation is measured using initiated mouse keratinocyte 308 cells seeded at clonal density. 308 cells form colonies when co-cultured with normal HDFs only in the presence of the tumor promoter phorbol 12-myristate 13-acetate (TPA), which induces an average of 5.75 colonies. When co-cultured with H(2)O(2)-treated HDFs, 308 cells form an average of 30.3 colonies. To understand the mechanism behind this phenomenon, we tested whether conditioned medium of HDFs, HDF extracellular matrix (ECM), density of HDFs, or the contact between keratinocytes and HDFs plays a role in 308 cell colony formation. The conditioned medium from prematurely senescent cells resulted in an average of eightfold more 308 cell colonies formed than the conditioned medium from normal HDFs, and the growth-promoting effect of the conditioned medium was trypsin sensitive. The ECM alone was not able to induce 308 cell colony formation. Increasing the density of normal HDFs or contact with normal HDFs but not senescent-like HDFs was inhibitory to the growth of 308 cells. Measurement of Connexin 43 indicated a decreased expression of the protein, which suggests an impaired gap junction communication in senescent-like HDFs. We conclude that H(2)O(2)-treated fibroblasts not only lose contact inhibition of the growth of initiated keratinocytes perhaps related to reduced gap junction communication but also increase production of secreted protein factors to enhance the growth of 308 keratinocytes.
衰老为癌症的最高风险因素。尽管氧化剂被认为与衰老和癌症均有关联，但氧化应激、衰老和癌症之间的相互作用尚未得到充分研究。人二倍体成纤维细胞（HDFs）在受到亚致死剂量的H₂O₂刺激后会发生早衰。为验证衰老或类衰老HDFs发挥肿瘤促进剂作用这一假说，我们采用了一种体外皮肤肿瘤促进模型，其中使用以克隆密度接种的起始小鼠角质形成细胞308细胞来测量集落形成。仅在肿瘤促进剂佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（TPA）存在的情况下，308细胞与正常HDFs共培养时才会形成集落，TPA平均诱导形成5.75个集落。当与经H₂O₂处理的HDFs共培养时，308细胞平均形成30.3个集落。为了解这一现象背后的机制，我们测试了HDFs的条件培养基、HDF细胞外基质（ECM）、HDFs的密度或角质形成细胞与HDFs之间的接触是否在308细胞集落形成中起作用。早衰细胞的条件培养基导致形成的308细胞集落平均比正常HDFs的条件培养基多八倍，且条件培养基的促生长作用对胰蛋白酶敏感。单独的ECM无法诱导308细胞集落形成。增加正常HDFs的密度或与正常HDFs接触（而非类衰老HDFs）会抑制308细胞的生长。连接蛋白43的测量表明该蛋白表达降低，这提示类衰老HDFs中的间隙连接通讯受损。我们得出结论，经H₂O₂处理的成纤维细胞不仅可能因间隙连接通讯减少而失去对起始角质形成细胞生长的接触抑制，还会增加分泌蛋白因子的产生以促进308角质形成细胞的生长。
