Confluence-dependent resistance to doxorubicin in human MDA-MB-231 breast carcinoma cells requires hypoxia-inducible factor-1 activity
Confluence-dependent resistance to doxorubicin in human MDA-MB-231 breast carcinoma cells requires hypoxia-inducible factor-1 activity
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DOI：
10.1016/j.yexcr.2006.12.004
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发表时间：
2007-03-10
影响因子：
3.7
通讯作者：
Graham, Charles H.
中科院分区：
文献类型：
--
作者：
Fang, Yu;Sullivan, Richard;Graham, Charles H.
与作为稀疏单层培养的肿瘤细胞相比，作为汇合单层培养的肿瘤细胞表现出对抗癌剂的高水平抗性。这种现象被称为汇合依赖性抗性（CDR）。我们确定了缺氧诱导因子-1（HIF-1）对MDA-MB-231乳腺癌细胞中CDR发展的贡献，HIF-1是细胞适应缺氧的关键转录调节因子。克隆形成试验表明，密度依赖性增加耐阿霉素。细胞密度也与还原性激活的pimoniclazole（缺氧的标志物）染色增加，以及与HIF-1 α亚基和HIF转录活性的增加水平相关，如通过免疫细胞化学，Western印迹和荧光素酶报告基因测定所确定的。重要的是，用siRNA抑制HIF-1 α表达显著减弱了CDR。类似地，振荡培养物减弱CDR、pimoniclazole免疫染色、HIF-1 α。积累和HIF-1转录活性。在建立了HIF-1和CDR之间的联系后，我们使用HIF-1 α和HIF-1转录活性作为标记来研究其他因子在CDR调节中的作用。HIF-1的融合依赖性增加。即使在0.1%O-2培养的细胞中以及在用来自汇合单层的条件培养基孵育的稀疏培养物中也观察到HIF-1的积累和HIF-1转录活性。在稀疏和汇合培养物中的血清剥夺也导致HIF-1 α积累。这些结果表明，虽然细胞周围缺氧可能是HIF-1活性的主要贡献者，可溶性因子水平的变化也可能发挥作用。这项研究表明，HIF-1是CDR所必需的。(c)2006年爱思唯尔公司All rights reserved.
Compared with tumour cells cultured as sparse monolayers, tumour cells cultured as confluent monolayers exhibit high levels of resistance to anti-cancer agents. This phenomenon is called confluence-dependent resistance (CDR). We determined the contribution of hypoxia-inducible factor-1 (HIF-1), a key transcriptional regulator of cellular adaptations to hypoxia, to the development of CDR in MDA-MB-231 breast cancer cells. Clonogenic assays revealed a density-dependent increase in resistance to doxorubicin. Cell density also correlated with increased staining for reductively activated pimoniclazole (a marker for hypoxia), as well as with increased levels of the HIF-1 alpha subunit and HIF transcriptional activity as determined by immunocytochemistry, Western blot, and luciferase reporter assays. Importantly, inhibition of HIF-1 alpha expression with siRNA significantly attenuated CDR. Similarly, shaking of cultures attenuated CDR, pimoniclazole immunostaining, HIF-1 alpha. accumulation, and HIF-1 transcriptional activity. Having established a link between HIF-1 and CDR, we used HIF-1 alpha and HIF-1 transcriptional activity as markers to investigate the role of additional factors in the regulation of CDR. Confluence-dependent increases in HIF-1. accumulation and HIF-1 transcriptional activity were observed even in cells cultured at 0.1% O-2 as well as in sparse cultures incubated with conditioned medium from confluent monolayers. Serum deprivation in both sparse and confluent cultures also resulted in HIF-1 alpha accumulation. These results reveal that, although pericellular hypoxia may be a major contributor to HIF-1 activity, changes in the levels of soluble factors may also play a role. This study demonstrates that HIF-1 is required for CDR. (c) 2006 Elsevier Inc. All rights reserved.