Shattuck-Brandt R L, Richmond A
Department of Cell Biology, Vanderbilt University School of Medicine, and Veterans Affairs Medical Center, Nashville, Tennessee 37232-2175, USA.
Cancer Res. 1997 Jul 15;57(14):3032-9.
The expression of the CXC chemokine MGSA is often deregulated during viral infection, chronic inflammation, and melanoma tumor progression. In Hs294T melanoma cells, the increased constitutive expression of MGSA is due to increased gene transcription. Moreover, nuclear extracts from unstimulated Hs294T cells contain 19-fold more immunoreactive NF-kappaB p65 than that observed in normal retinal pigment epithelial (ARPE) cells. This increase in NF-kappaB p65 correlates with increased NF-kappaB DNA binding activity in Hs294T nuclear extracts. After stimulation with interleukin 1, Western and electrophoretic mobility shift assay analysis indicate that in both cell types, additional activated NF-kappaB p65 is translocated to the nucleus. However, the rate of postinduction repression of NF-kappaB DNA binding is delayed in Hs294T melanoma cells compared to ARPE cells. Western analysis of whole-cell lysates from both Hs294T and ARPE cells indicates that protein levels of the inhibitor of NF-kappaB, I-kappaB alpha, are 3-fold lower in Hs294T cells. The decrease in I-kappaB alpha cannot be attributed to alterations in the transcription or translation of I-kappaB alpha. Rather, the posttranslational processing has been altered. In Hs294T cells, the half-life of the I-kappaB alpha protein is 45 min, compared to 120 min in ARPE cells. These results indicate that in Hs294T melanoma cells the equilibrium between I-kappaB alpha degradation and resynthesis has been altered, leading to constitutive nuclear translocation and activation of NF-kappaB. Similar mechanisms could also operate in other tumorigenic processes, as well as in viral and chronic inflammatory disorders, to produce high constitutive and unregulated chemokine expression.
CXC趋化因子MGSA的表达在病毒感染、慢性炎症和黑色素瘤肿瘤进展过程中常常失调。在Hs294T黑色素瘤细胞中，MGSA组成型表达增加是由于基因转录增加所致。此外，未受刺激的Hs294T细胞的核提取物中免疫反应性NF-κB p65比正常视网膜色素上皮（ARPE）细胞中多19倍。Hs294T细胞核提取物中NF-κB p65的这种增加与NF-κB DNA结合活性的增加相关。用白细胞介素1刺激后，蛋白质免疫印迹法和电泳迁移率变动分析表明，在两种细胞类型中，额外激活的NF-κB p65都易位至细胞核。然而，与ARPE细胞相比，Hs294T黑色素瘤细胞中NF-κB DNA结合的诱导后抑制速率延迟。对Hs294T和ARPE细胞全细胞裂解物的蛋白质免疫印迹分析表明，NF-κB抑制剂I-κBα的蛋白水平在Hs294T细胞中低3倍。I-κBα的减少不能归因于I-κBα转录或翻译的改变。相反，其翻译后加工发生了改变。在Hs294T细胞中，I-κBα蛋白的半衰期为45分钟，而在ARPE细胞中为120分钟。这些结果表明，在Hs294T黑色素瘤细胞中，I-κBα降解与再合成之间的平衡发生了改变，导致NF-κB的组成型核易位和激活。类似的机制也可能在其他致瘤过程以及病毒和慢性炎症性疾病中起作用，从而产生高水平的组成型和不受调控的趋化因子表达。
