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Exp Cell Res. 2007 Mar 10;313(5):867-77. Epub 2006 Dec 21.

Confluence-dependent resistance to doxorubicin in human MDA-MB-231 breast carcinoma cells requires hypoxia-inducible factor-1 activity.

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


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-1alpha subunit and HIF transcriptional activity as determined by immunocytochemistry, Western blot, and luciferase reporter assays. Importantly, inhibition of HIF-1alpha expression with siRNA significantly attenuated CDR. Similarly, shaking of cultures attenuated CDR, pimonidazole immunostaining, HIF-1alpha accumulation, and HIF-1 transcriptional activity. Having established a link between HIF-1 and CDR, we used HIF-1alpha and HIF-1 transcriptional activity as markers to investigate the role of additional factors in the regulation of CDR. Confluence-dependent increases in HIF-1alpha 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-1alpha 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.

[Indexed for MEDLINE]

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