Brennan L, Jongstra J
Arthritis Centre-Research Unit, Toronto Hospital Research Institute, Ontario, Canada.
Carcinogenesis. 1996 Apr;17(4):771-7. doi: 10.1093/carcin/17.4.771.
The mouse Lsp1 gene encodes a 330 amino acid intracellular F-actin binding protein. Previously we showed that the mouse and human Lsp1 genes are expressed in normal B-cells and T-cells, including Thy1+ thymocytes and in normal macrophages and neutrophils. No or little LSP1 RNA and protein was found in a series of transformed mouse and human T-lymphoma cell lines, although normal antigen and lymphokine dependent T-cell lines expressed the Lsp1 gene. Here we show by Northern analysis that three mature antigen independent T-cell lines (CTLL-2, HT-2 and 532.10) which grow in the presence of lymphokine only, do not express LSP1 RNA, while mature resting and activated lymph node T-cells express high levels of LSP1 RNA. To determine whether the down-regulation of LSP1 expression is an early event which occurs in vivo in the tumor, rather than as a consequence of in vitro propagation of T-lymphoma cells, we analyzed LSP1 expression in primary T-lymphomas induced in AKR/J or AKR/J x BALB/cJ mice by a single intraperitoneal (i.p.) injection with 75 mg N-methyl-N-nitrosourea (MNU) per kg body-weight. Two- color Fluorescence Activated Cell Sorter (FACS) analysis showed that all tumors had an immature CD4+/CD8+ double positive (DP) phenotype. Many tumors contained a substantial population of CD4+ single positive (SP) cells. Since these cells may be infiltrating lymphocytes which can be expected to express the Lsp1 gene at a high level these tumors were not included in our analysis. Nineteen tumors were analyzed for Lsp1 gene expression and 13 contained reduced levels of LSP1 RNA, ranging from 4% to 44% of those found in age-matched normal thymus. Six tumors showed either no or only a small reduction in LSP1 RNA. These tumors had developed later than those expressing low levels of LSP1. The level of LSP1 RNA in tumors developing <110 days after injection of MNU was 19.1% +/- 5.2 (mean +/- SEM), while the level of LSP1 in later tumors was 78.4% +/- 13.0 (P = 0.004). Similar data were obtained when the expression of LSP1 protein was analyzed. These findings extend our previous data in several ways. First, they suggest a correlation between the down-regulation of LSP1 expression and abnormal regulation of growth or survival of immature and mature T-lymphocytes. Second, they show that down-regulation of the Lsp1 gene in transformed T-cells is not the result of prolonged in vitro culture, but occurs in the majority of primary tumors. Third, they show that there are two classes of T-lymphoma, which differ in their expression of LSP1 RNA and that the down-regulation of LSP1 is specifically associated with the early appearance of T-lymphoma after injection with MNU. This strongly suggests that the absence or reduced expression of LSP1 contributed to the transformation process and argues against the possibility that loss of LSP1 expression is a mere inconsequential epigenetic event.
小鼠Lsp1基因编码一种含330个氨基酸的细胞内F - 肌动蛋白结合蛋白。此前我们发现，小鼠和人类的Lsp1基因在正常B细胞和T细胞中表达，包括Thy1 + 胸腺细胞，以及正常巨噬细胞和中性粒细胞。在一系列转化的小鼠和人类T淋巴瘤细胞系中未发现或仅发现少量LSP1 RNA和蛋白，尽管正常的抗原和淋巴因子依赖性T细胞系表达Lsp1基因。在此我们通过Northern分析表明，仅在淋巴因子存在下生长的三种成熟的抗原非依赖性T细胞系（CTLL - 2、HT - 2和532.10）不表达LSP1 RNA，而成熟的静止和活化淋巴结T细胞则表达高水平的LSP1 RNA。为了确定LSP1表达下调是否是肿瘤在体内发生的早期事件，而非T淋巴瘤细胞体外增殖的结果，我们分析了通过腹腔单次注射每千克体重75 mg N - 甲基 - N - 亚硝基脲（MNU）诱导的AKR/J或AKR/J×BALB/cJ小鼠原发性T淋巴瘤中LSP1的表达。双色荧光激活细胞分选仪（FACS）分析表明所有肿瘤均具有不成熟的CD4 + /CD8 + 双阳性（DP）表型。许多肿瘤含有大量CD4 + 单阳性（SP）细胞。由于这些细胞可能是浸润性淋巴细胞，预计会高水平表达Lsp1基因，因此这些肿瘤未纳入我们的分析。对19个肿瘤进行Lsp1基因表达分析，其中13个肿瘤的LSP1 RNA水平降低，为年龄匹配的正常胸腺中发现水平的4%至44%。6个肿瘤的LSP1 RNA要么未降低，要么仅略有降低。这些肿瘤比那些LSP1表达水平低的肿瘤出现得晚。注射MNU后<110天出现的肿瘤中LSP1 RNA水平为19.1%±5.2（平均值±标准误），而后期肿瘤中LSP1水平为78.4%±13.0（P = 0.004）。分析LSP1蛋白表达时获得了类似数据。这些发现从几个方面扩展了我们之前的数据。首先，它们表明LSP1表达下调与未成熟和成熟T淋巴细胞生长或存活的异常调节之间存在相关性。其次，它们表明转化T细胞中Lsp1基因的下调不是长期体外培养的结果，而是发生在大多数原发性肿瘤中。第三，它们表明有两类T淋巴瘤，其LSP1 RNA表达不同，且LSP1的下调与注射MNU后T淋巴瘤的早期出现特异性相关。这强烈表明LSP1的缺失或表达降低促成了转化过程，并且反对LSP1表达缺失仅仅是一个无关紧要的表观遗传事件的可能性。
