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题名:Confluence-dependent resistance to doxorubicin in human MDA-MB-231 breast carcinoma cells requires hypoxia-inducible factor-1 activity
作者:Yu Fang, Richard Sullivan and Charles H. Graham
来源:Experimental Cell Research[IF=4.148], Volume 313, Issue 5, 10 March 2007, Pages 867-877
URL :http://dx.doi.org/10.1016/j.yexcr.2006.12.004
日期:070415
摘要: Yu Fanga, Richard Sullivana and Charles H. Graham, a,

aDepartment of Anatomy and Cell Biology, Botterell Hall Room 859, Queen s University, Kingston, ON, Canada K7L 3N6

Received 27 October 2006; revised 6 December 2006; accepted 11 December 2006. Available online 21 December 2006.

Summary

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 pimonidazole (a marker for hypoxia), as well as with increased levels of the HIF-1 subunit and HIF transcriptional activity as determined by immunocytochemistry, Western blot, and luciferase reporter assays. Importantly, inhibition of HIF-1 expression with siRNA significantly attenuated CDR. Similarly, shaking of cultures attenuated CDR, pimonidazole immunostaining, HIF-1 accumulation, and HIF-1 transcriptional activity. Having established a link between HIF-1 and CDR, we used HIF-1 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% O2 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 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. 14.

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