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首页> 外文学位 >The role of Akt1 in G1/S cell cycle checkpoint bypass and cell migration after genotoxin stress.
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The role of Akt1 in G1/S cell cycle checkpoint bypass and cell migration after genotoxin stress.

机译：Akt1在基因毒素应激后在G1 / S细胞周期检查点旁路和细胞迁移中的作用。

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摘要

A growing body of evidence underscores the central role of the Akt pathway in the pathogenesis of lung cancer. Akt has been documented to play a role not only in cell survival but also in cell migration and invasion. Certain forms of hexavalent chromium [Cr(VI)] are human respiratory carcinogens and genotoxins. Our studies identified Akt as crucial to the death/survival balance after Cr(VI) exposure in both lung fibroblasts and epithelial cells. The objective of this dissertation was to elucidate the role of Akt1 in G1/S checkpoint bypass and migration after genotoxin exposure. Our studies indicate that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV) partially abrogated Cr(VI)-induced growth arrest and induced G1/S cell cycle checkpoint override in human lung fibroblasts (HLF). This checkpoint override was dependent on Akt activation, resulting in the modulation of subcellular localization and expression of the G1/S transition effectors, pRb and p27, following genotoxin exposure. Furthermore, biological upregulation of Akt1 indicated that Akt1 was sufficient to bypass a Cr(VI)-induced G1/S checkpoint arrest via the regulation of G1/S checkpoint effector expression and localization. Recent evidence suggests that p27 localization and phosphorylation status is responsible for cytoskeletal reorganization via its interaction with RhoA. Our data indicates that Akt upregulation, either biologically or in the presence of SOV, causes an increase in the interaction between p27 and RhoA. Furthermore, PTP inhibition significantly decreased the GTPase activity of RhoA after Cr(VI) treatment, whereas Akt1 activation significantly increased GPTase activity of RhoA after Cr(VI) treatment. Moreover, PTP inhibition and Akt1 upregulation both significantly increased HLF migration after Cr(VI) exposure, as assessed by a wound healing assay. Similarly, Cr(VI)-induced decrease in cell adhesion was abrogated by PTP inhibition and Akt1 upregulation. Consistent with its effect on migration, the Cr(VI)-induced disruption of F-actin stress fibers was abrogated by PTP inhibition. Notably, Akt1 activation was sufficient to overcome Cr(VI)-induced cytoskeletal alterations in our preliminary studies, as transient MyrAkt1 transfection abrogated the Cr(VI)-induced disruption in F-actin stress fibers. Given the evidence for the purported role of fibroblasts in epithelial mesenchymal transition (EMT), we investigated the effect of PTP inhibition and Akt1 upregulation in the presence of Cr(VI) treatment in fibroblasts, on the migratory capacity of epithelial cells. Our data indicate that PTP inhibition in fibroblasts, in the context of genotoxic stress, abrogates the Cr(VI)-induced decrease in migration of virally transformed BEAS2B lung epithelial cells. Furthermore, PTP inhibition in the presence of genotoxic stress in fibroblasts further enhances the Cr(VI)-induced increase in migration of metastatic Calu3 cells. Akt1 upregulation in the context of genotoxic stress in fibroblasts increases the migration of BEAS2B cells, while having no effect on the migration of Calu3 lung adenocarcinoma cells. In conclusion, the results of our studies provide new insights to the understanding of Cr(VI)-induced lung carcinogenesis. The ability of Akt activation to enhance cell migration in conjunction with checkpoint override in the face of genotoxic exposure could play a role in neoplastic progression. Since fibroblasts are involved in accelerating the EMT, our data also highlight a potential role by which survival pathway activation may affect the cellular microenvironment after initial exposure to a genotoxin.
机译：越来越多的证据强调了Akt途径在肺癌发病机理中的核心作用。 Akt已被证明不仅在细胞存活中而且在细胞迁移和侵袭中都起作用。某些形式的六价铬[Cr（VI）]是人类呼吸道致癌物和遗传毒素。我们的研究表明，Akt对暴露于肺成纤维细胞和上皮细胞中的六价铬后的死亡/生存平衡至关重要。本文的目的是阐明Akt1在暴露基因毒素后在G1 / S检查点旁路和迁移中的作用。我们的研究表明，广泛的蛋白质酪氨酸磷酸酶（PTP）抑制剂，原钒酸钠（SOV）可以部分消除Cr（VI）引起的生长停滞并在人肺成纤维细胞（HLF）中引起G1 / S细胞周期检查点超控。此检查点替代取决于Akt激活，导致基因毒素暴露后，亚细胞定位的调节和G1 / S过渡效应子pRb和p27的表达。此外，Akt1的生物学上调表明，Akt1足以通过调节G1 / S检查点效应子的表达和定位来绕开Cr（VI）诱导的G1 / S检查点停滞。最近的证据表明，p27的定位和磷酸化状态是通过与RhoA相互作用而引起细胞骨架重组的原因。我们的数据表明，无论从生物学上还是在SOV存在下，Akt的上调都会导致p27与RhoA之间的相互作用增加。此外，PTP抑制显着降低Cr（VI）处理后RhoA的GTPase活性，而Akt1激活显着增加Cr（VI）处理后RhoA的GPTase活性。此外，PTP抑制和Akt1上调都显着增加了Cr（VI）暴露后的HLF迁移，如伤口愈合试验所评估。同样，PTP抑制和Akt1上调消除了Cr（VI）诱导的细胞粘附减少。与其对迁移的影响一致，Cr（VI）诱导的F-肌动蛋白应激纤维破坏被PTP抑制所废除。值得注意的是，在我们的初步研究中，Akt1激活足以克服Cr（VI）诱导的细胞骨架改变，因为瞬时MyrAkt1转染消除了Cr（VI）诱导的F-肌动蛋白应激纤维的破坏。给定据称成纤维细胞在上皮间充质转化（EMT）中发挥作用的证据，我们研究了在成纤维细胞中Cr（VI）处理下PTP抑制和Akt1上调对上皮细胞迁移能力的影响。我们的数据表明，在遗传毒性胁迫的背景下，成纤维细胞中的PTP抑制作用消除了Cr（VI）诱导的病毒转化BEAS2B肺上皮细胞迁移的减少。此外，在成纤维细胞中存在遗传毒性应激的情况下，PTP抑制作用进一步增强了Cr（VI）诱导的转移性Calu3细胞迁移的增加。在成纤维细胞遗传毒性应激的情况下，Akt1上调增加了BEAS2B细胞的迁移，而对Calu3肺腺癌细胞的迁移没有影响。总之，我们的研究结果为理解Cr（VI）诱导的肺癌发生提供了新的见识。面对遗传毒性暴露，Akt激活增强细胞迁移的能力与检查点覆盖相结合，可能在肿瘤进展中起作用。由于成纤维细胞参与了EMT的加速，因此我们的数据也突显了潜在的作用，即在最初暴露于基因毒素后，生存途径的激活可能会影响细胞的微环境。

著录项

  • 作者

    Lal, Madhu.;

  • 作者单位

    The George Washington University.;

  • 授予单位 The George Washington University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 227 p.
  • 总页数 227
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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