Angelloz-Nicoud P, Binoux M
Institut National de la Santé et de la Recherche Médicale, Hôpital Saint Antoine, Paris, France.
Endocrinology. 1995 Dec;136(12):5485-92. doi: 10.1210/endo.136.12.7588299.
PC-3 cells, whose growth is androgen-independent, were shown to be capable of slow proliferation in serum-free medium and in the absence of added growth factor for 7 days. They secreted insulin-like growth factor (IGF)-II but no detectable IGF-I. This IGF-II, although produced in small amounts, plays a role in their proliferation because growth could be inhibited dose dependently by up to 80% in the presence of monoclonal antibodies directed against IGFs or the type 1 IGF receptor. PC-3 cells also secreted IGF binding proteins (IGFBPs) -2, -3, -4, and -6. Immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of the same molecular size as those generated from IGFBP-3 in vivo. With the addition to the culture medium of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc-SC), at concentrations < 0.2 mM that were nontoxic to the cells, cell proliferation was dose dependently inhibited up to 80% and, at the same time, proteolysis of the IGFBP-3 secreted by the cells was depressed. Urokinase activity detected in the conditioned media was depressed by Pefabloc, suggesting that the urokinase-type plasminogen activator was involved in the proteolysis of IGFBP-3. In addition, 0.01-5 micrograms/ml plasminogen induced a dose-dependent increase in both proliferation and the proportions of proteolysed IGFBP-3 in the media. The stimulation of proliferation was totally blocked in the presence of anti-type 1 IGF receptor antibody. Recombinant human IGF-II (5-200 ng/ml) added to cell-free medium conditioned by 48 h of culture dose dependently stimulated PC-3 cell proliferation. At concentrations < or = 100 ng/ml, its mitogenic action was potentiated when medium had been conditioned by cells cultured in the presence of plasminogen but inhibited when medium had been conditioned by cells cultured in the presence of Pefabloc. We conclude from these results 1) that IGF-II is involved in the autocrine control of PC-3 cell proliferation via the type 1 IGF receptor; and 2) that this proliferation is directly dependent on IGF-II bioavailability that itself is modulated by the limited IGFBP-3 proteolysis induced, at least in part, by urokinase-type plasminogen activator and plasmin.
PC-3细胞的生长不依赖雄激素，已证明其能够在无血清培养基中，且在不添加生长因子的情况下缓慢增殖7天。它们分泌胰岛素样生长因子（IGF）-II，但未检测到IGF-I。这种IGF-II虽然分泌量少，但在其增殖过程中发挥作用，因为在存在针对IGF或1型IGF受体的单克隆抗体时，细胞生长可被剂量依赖性抑制达80%。PC-3细胞还分泌IGF结合蛋白（IGFBP）-2、-3、-4和-6。免疫印迹分析显示IGFBP-3发生选择性蛋白水解，产生的片段与体内IGFBP-3产生的片段分子大小相同。向培养基中添加浓度<0.2 mM且对细胞无毒的丝氨酸蛋白酶抑制剂4-（2-氨基乙基）-苯磺酰氟（Pefabloc-SC），细胞增殖被剂量依赖性抑制达80%，同时，细胞分泌的IGFBP-3的蛋白水解受到抑制。在条件培养基中检测到的尿激酶活性被Pefabloc抑制，提示尿激酶型纤溶酶原激活剂参与了IGFBP-3的蛋白水解。此外，0.01 - 5微克/毫升的纤溶酶原可诱导培养基中细胞增殖和IGFBP-3蛋白水解比例呈剂量依赖性增加。在存在抗1型IGF受体抗体时，增殖刺激被完全阻断。添加到经48小时培养的无细胞培养基中的重组人IGF-II（5 - 200纳克/毫升）剂量依赖性刺激PC-3细胞增殖。在浓度≤100纳克/毫升时，当培养基由在纤溶酶原存在下培养的细胞预处理时，其促有丝分裂作用增强，而当培养基由在Pefabloc存在下培养的细胞预处理时，其促有丝分裂作用受到抑制。我们从这些结果得出结论：1）IGF-II通过1型IGF受体参与PC-3细胞增殖的自分泌控制；2）这种增殖直接依赖于IGF-II的生物利用度，而IGF-II的生物利用度本身至少部分受尿激酶型纤溶酶原激活剂和纤溶酶诱导的有限IGFBP-3蛋白水解调节。
