Confluence-dependent resistance to doxorubicin in human MDA-MB-231 breast carcinoma cells requires hypoxia-inducible factor-1 activity.
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.
Fang
Yu
Y
Department of Anatomy and Cell Biology, Botterell Hall Room 859, Queen's University, Kingston, ON, Canada K7L 3N6.
Sullivan
Richard
R
Graham
Charles H
CH
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism*; D002277:Carcinoma / Q000378:metabolism*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D000903:Antibiotics, Antineoplastic; D051793:Hypoxia-Inducible Factor 1; D004317:Doxorubicin