Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Toxicol Appl Pharmacol. 2011 Jan 15;250(2):213-20. doi: 10.1016/j.taap.2010.10.026. Epub 2010 Nov 8.
Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in the spleen.
苯胺暴露与脾脏毒性有关，导致脾脏肿大、增生、纤维化和各种脾脏肉瘤，这是在慢性暴露的情况下发生的。在早期的研究中，我们已经表明，苯胺暴露会导致铁过载、氧化应激和激活氧化还原敏感的转录因子，这些因子可以调节各种基因，导致脾脏发生肿瘤性反应。然而，导致苯胺诱导脾脏细胞增殖的分子机制在很大程度上仍然未知。因此，本研究旨在研究 G1 期细胞周期蛋白（细胞周期蛋白）的调节、细胞周期蛋白依赖性激酶（CDK）的表达、视网膜母细胞瘤蛋白（pRB）的磷酸化和脾脏细胞增殖，在肿瘤发生反应之前的实验条件下。雄性 SD 大鼠用苯胺（通过饮用水每天 0.5mmol/kg）处理 30 天（对照组仅接受饮用水），并测量脾细胞增殖、G1 期细胞周期蛋白、CDK 和 pRB 的蛋白表达。苯胺处理导致脾细胞增殖显著增加，基于细胞计数、细胞增殖标志物包括增殖细胞核抗原（PCNA）、核 Ki67 蛋白（Ki67）和微染色体维持（MCM）、MTT 测定和流式细胞术分析。来自苯胺处理大鼠的脾细胞蛋白的 Western blot 分析显示，与对照组相比，细胞周期蛋白 D1、D2、D3 和 E 的表达显著增加。同样，实时 PCR 分析显示，苯胺处理大鼠脾脏中细胞周期蛋白 D1、D2、D3 和 E 的 mRNA 表达显著增加。这些细胞周期蛋白的过表达与 CDK4、CDK6、CDK2 的表达增加以及 pRB 蛋白的磷酸化有关。我们的数据表明，细胞周期蛋白、CDK 和 pRB 蛋白的磷酸化表达增加可能是细胞增殖的关键，可能有助于苯胺诱导的脾脏肿瘤发生反应。
