Induction of pro-apoptotic and cell cycle-inhibiting genes in chromium (VI)-treated human lung fibroblasts: Lack of effect of ERK
Induction of pro-apoptotic and cell cycle-inhibiting genes in chromium (VI)-treated human lung fibroblasts: Lack of effect of ERK
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DOI：
10.1023/b:mcbi.0000007270.82431.3e
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发表时间：
2004-01-01
影响因子：
4.3
通讯作者：
Patierno, SR
中科院分区：
文献类型：
--
作者：
Ceryak, S;Zingariello, C;Patierno, SR
细胞增殖和凋亡是由紧密协调的信号通路控制的，其最终导致多种蛋白质的转录激活/抑制。细胞周期和/或凋亡控制的失调可能导致基因组不稳定、肿瘤转化和肿瘤进展。在某些条件下，一些六价铬[Cr（VI）]化合物在人体呼吸道中是有毒和致癌的，我们已经表明，它们诱导细胞凋亡和/或细胞周期阻滞在p53依赖的方式。越来越多的证据表明，细胞外信号调节激酶（ERK）的激活与DNA损伤反应，p53依赖性和非依赖性机制。在这里，目的是研究Cr（VI）的转录调控的关键细胞周期抑制剂和促凋亡和抗凋亡蛋白的影响，以及ERK激活的Cr（VI）的遗传毒性反应中的作用。将二倍体人肺成纤维细胞与3-9 μ M Na 2CrO 4孵育，并在4、8和24 h以及Cr（VI）暴露终止（恢复）后24 h分离RNA。使用P-32标记的多转录物探针通过RNA酶保护试验定量mRNA表达，所述探针含有cdk抑制剂p21（waf 1/cip 1）、p27（kip 1）、p16（INK 4a）、p15（INK 4 b）;促凋亡蛋白bcl-X-S和bax;抗凋亡蛋白bcl-W、bcl-X-L和bc 12、GADD 45和细胞周期蛋白A的基因序列。在一般情况下，bcl-W和bcl-X-L的表达都下调铬暴露后，约50%，在24小时，这是更明显的恢复期后。当Cr（VI）浓度小于或等于6 μ M时，bc 12表达上调。特别令人感兴趣的是，bax的表达减少，在剂量和时间依赖性的方式，然而，bcl-X-S的8小时后升高了近3倍，并下降到控制水平的恢复期结束时。GADD 45和p21的表达在8 h时均上调2倍，但在恢复期间下降至对照水平。p27和p16的表达均未受到Cr（VI）暴露的明显影响，但p15的表达在暴露于所有浓度的Cr（VI）后均显著增加。Cr（VI）暴露24 h后，细胞周期蛋白A的表达下降。Cr（VI）在暴露后约0.5-3 h诱导ERK活性的瞬时爆发（超过对照的2-6倍）。然而，抑制ERK激活与PD 98059铬诱导的基因表达的改变没有影响。此外，Cr（VI）诱导的克隆形成性致死率，暴露于1和2 μ M Cr（VI）24小时后评估，也不受ERK抑制。这些数据表明，p53依赖性和非依赖性细胞凋亡和生长抑制途径显着影响Cr（VI）曝光。然而，Cr（VI）影响关键凋亡和生长停滞基因的能力，从而克隆致死，似乎是独立的ERK。继续研究铬（VI）诱导的细胞周期和细胞凋亡控制的细胞和分子机制，将进一步了解铬（VI）相关的致癌作用。
Cell proliferation and apoptosis are controlled by tightly orchestrated signaling pathways that culminate in transcriptional activation/repression of multiple proteins. Dysregulation of cell cycle and/or apoptosis control may lead to genomic instability, neoplastic transformation and tumor progression. Under certain conditions, some hexavalent chromium [Cr(VI)] compounds are toxic and carcinogenic in the human respiratory tract, and we have shown that they induce apoptosis and/or cell cycle arrest in a p53-dependent fashion. There is increasing evidence linking extracellular signal-regulated kinase (ERK) activation with the DNA damage response, by both p53-dependent and -independent mechanisms. Here, the aim was to study the effect of Cr(VI) transcriptional regulation of key cell cycle inhibitors and pro- and anti-apoptotic proteins, as well as the role of ERK activation in the Cr(VI) genotoxic response. Diploid human lung fibroblasts were incubated with 3-9 uM Na2CrO4, and RNA was isolated at 4, 8, and 24 h, as well as 24 h after Cr(VI) exposure was terminated (recovery). mRNA expression was quantitated by RNase protection assay with a P-32-labeled multi-transcript probe containing gene sequences for the cdk inhibitors, p21(waf1/cip1), p27(kip1), p16(INK4a), p15(INK4b); the pro-apoptotic proteins bcl-X-S and bax; the anti-apoptotic proteins bcl-W, bcl-X-L, and bc12, GADD45, and cyclin A. In general, bcl-W and bcl-X-L expression were both downregulated after Cr exposure, to around 50% at 24 h, which was more pronounced after the recovery period. At Cr(VI) concentrations less than or equal to6 uM, bc12 expression was upregulated. Of particular interest is that bax expression was reduced, in a dose and time-dependent fashion, however that of bcl-X-S was elevated by nearly 3-fold after 8 h, and declined to control levels at the end of the recovery period. Expression of GADD45 and p21 were both upregulated by 2-fold at 8 h, but declined to control levels during recovery. Neither the expression of p27 nor that of p16 were apparently affected by Cr(VI) exposure, however the expression of p15 was markedly increased after exposure to all concentrations of Cr(VI). Finally, the expression of cyclin A was decreased after 24 h Cr(VI) exposure. Cr(VI) induced a transient burst of ERK activity (2-6-fold over control) around 0.5-3 h after exposure. However, inhibition of ERK activation with PD98059 had no effect on the Cr-induced alterations in gene expression. Moreover, Cr(VI)-induced clonogenic lethality, as assessed after 24 h exposure to 1 and 2 uM Cr(VI), was also not affected by ERK inhibition. These data suggest that both p53-dependent and -independent apoptotic and growth-inhibitory pathways are markedly affected by Cr(VI) exposure. However, the ability of Cr(VI) to affect key apoptotic and growth arresting genes, and thus clonogenic lethality, appears to be independent of ERK. Continued investigation into the cellular and molecular mechanisms of Cr(VI)-induced cell cycle and apoptosis control should further the understanding of Cr(VI)-associated carcinogenesis.