Adelmant G, Gilbert J D, Freytag S O
Departments of Molecular Biology and Radiation Oncology, Henry Ford Health System, Detroit, Michigan 48202-3450, USA.
J Biol Chem. 1998 Jun 19;273(25):15574-81. doi: 10.1074/jbc.273.25.15574.
Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process.
人易位性脂肪肉瘤（TLS）-CCAAT/增强子结合蛋白（C/EBP）同源蛋白（CHOP）是一种在脂肪组织恶性肿瘤中特异性发现的融合癌蛋白，由t(12;16)易位产生，该易位将TLS的氨基末端部分与CHOP的整个编码区域融合。鉴于CHOP是C/EBP转录因子家族的成员，而该家族蛋白是脂肪细胞分化机制的一部分，我们研究了TLS-CHOP是否通过直接干扰C/EBP功能来阻断脂肪细胞分化。采用单步逆转录病毒感染方案，将野生型或突变型TLS-CHOP与C/EBPβ在未处理的NIH3T3细胞中共同表达，并测定它们抑制C/EBPβ驱动的脂肪生成的能力。当TLS-CHOP以与人类黏液样脂肪肉瘤中观察到的水平相当的水平表达时，它在阻断脂肪细胞分化方面极其有效。TLS-CHOP的这种作用需要一个功能性的亮氨酸拉链结构域，并且与其与C/EBPβ异源二聚化以及原位抑制C/EBPβ DNA结合和反式激活活性的能力相关。相比之下，TLS-CHOP的碱性区域是可有可无的，这使得TLS-CHOP的抑制作用不太可能归因于由其假定的反式激活活性导致的意外基因表达。另一种脂肪生成转录因子PPARγ2能够挽救受TLS-CHOP抑制的细胞，这表明TLS-CHOP主要干扰C/EBPβ驱动的脂肪生成，而不干扰脂肪细胞分化程序的其他必要事件。总之，这些结果表明TLS-CHOP通过直接阻止C/EBPβ与其靶基因结合并进行反式激活来阻断脂肪细胞分化。此外，它们为正常分化的阻断是癌症过程的一个重要方面这一论点提供了有力支持。
