Dovhey S E, Ghosh N S, Wright K L
H. Lee Moffitt Cancer Center and Research Institute, Department of Medical Microbiology and Immunology, University of South Florida, Tampa 33612, USA.
Cancer Res. 2000 Oct 15;60(20):5789-96.
The inadequate ability of cancer cells to present antigen on the cell surface via MHC class I molecules is one mechanism by which tumor cells evade antitumor-associated antigen immunity. In many cases, such as in renal cell carcinoma (RCC), the lack of MHC class I antigen presentation can be attributed to the down-regulation of genes needed for antigen processing, such as the transporters associated with antigen processing (TAP)1 and TAP2, and the proteasomal components low molecular weight proteins (LMP)2 and LMP7. The TAP1 and LMP2 genes are transcribed from a shared bidirectional promoter containing an IFN response factor element that confers IFN-gamma inducibility. Here, we investigate the differential responsiveness to IFN-gamma of RCC cell lines, Caki-1 and Caki-2, which have been reported to have abnormally low expressions of TAP1 and LMP2. We now demonstrate that the Caki-2 cell line is defective in the IFN-gamma signaling pathway. The effects of IFN-gamma on TAP1 and LMP2 expression revealed a loss of up-regulation in Caki-2 cells, but not in Caki-1 cells. In vivo DNA footprinting shows a specific loss of occupancy at the IFN response factor element site in Caki-2 cells, whereas Caki-1 cells show full promoter occupancy. Furthermore, in vitro DNA-binding studies indicated that Caki-2 cells do not have IFN-regulatory factor 1- or signal transducer and activator of transcription 1 (Stat1)-binding activity after IFN-gamma stimulation. Examination of Stat1, Jak1, and Jak2 proteins demonstrated that the proteins were expressed, however, not phosphorylated, upon IFN-gamma treatment in Caki-2 cells. Also, this cell line expressed both IFN-gamma receptor chains. IFN-gamma inducibility could not be rescued by introduction of normal Jak1 and/or Jak2 proteins. However, overexpression of Jak1 did increase TAP1 and LMP2 expression independent of IFN-gamma, indicating that the Stat1 and IFN-regulatory factor 1 proteins present in Caki-2 can be activated. These findings suggest that the loss of TAP1 and LMP2 induction is a defect in the earliest steps of the IFN-gamma signaling pathway resulting in the inability of Caki-2 cells to up-regulate the MHC class I antigen-processing pathway. Because immunotherapy may be one of the most promising approaches for treating RCC, understanding the mechanisms by which these tumors circumvent cytokine signaling, thereby evading antitumor-specific-antigen immunity, would greatly aid the efficacy of such therapy.
癌细胞通过主要组织相容性复合体I类分子在细胞表面呈递抗原的能力不足，是肿瘤细胞逃避抗肿瘤相关抗原免疫的一种机制。在许多情况下，如在肾细胞癌（RCC）中，MHC I类抗原呈递的缺乏可归因于抗原加工所需基因的下调，如与抗原加工相关的转运体（TAP）1和TAP2，以及蛋白酶体成分低分子量蛋白（LMP）2和LMP7。TAP1和LMP2基因从一个共享的双向启动子转录而来，该启动子含有一个赋予γ干扰素诱导性的干扰素反应因子元件。在此，我们研究了肾癌细胞系Caki-1和Caki-2对γ干扰素的不同反应性，据报道这两种细胞系中TAP1和LMP2的表达异常低。我们现在证明Caki-2细胞系在γ干扰素信号通路中存在缺陷。γ干扰素对TAP1和LMP2表达的影响显示，Caki-2细胞中上调作用丧失，但Caki-1细胞中没有。体内DNA足迹分析显示Caki-2细胞中干扰素反应因子元件位点的占有率特异性丧失，而Caki-1细胞显示启动子完全被占据。此外，体外DNA结合研究表明，γ干扰素刺激后，Caki-2细胞没有干扰素调节因子1或信号转导和转录激活因子1（Stat1）的结合活性。对Stat1、Jak1和Jak2蛋白的检测表明，这些蛋白在Caki-2细胞中经γ干扰素处理后表达，但未磷酸化。此外，该细胞系表达两种γ干扰素受体链。引入正常的Jak1和/或Jak2蛋白无法挽救γ干扰素诱导性。然而，Jak1的过表达确实增加了TAP1和LMP2的表达，且与γ干扰素无关，这表明Caki-2细胞中存在的Stat1和干扰素调节因子1蛋白可以被激活。这些发现表明，TAP1和LMP2诱导的丧失是γ干扰素信号通路早期步骤中的缺陷，导致Caki-2细胞无法上调MHC I类抗原加工途径。由于免疫疗法可能是治疗肾细胞癌最有前景的方法之一，了解这些肿瘤规避细胞因子信号传导从而逃避抗肿瘤特异性抗原免疫的机制，将极大地有助于提高此类疗法的疗效。
