Fig 2. Selection of MCF10A cells by intermittent hypoxia (IH) induces drug resistance and reduced expression of p53 and E-cadherin.

(A) Multiple intermittent hypoxia regimens were tested at 1%O₂ and 0.2% O₂ over 6 days. Factor survival is expressed as compared to cells grown in parallel in normoxia (21%O₂). MCF10A cells undergoing repetitive cycles of 16 hours hypoxia followed by 8 hrs reoxygenation exhibited the most cell death. (B) MCF10A cells were cultured in 50 cycles of IH in the selective regimen described in (A), and individual cells were isolated to be raised as clones, and then passaged for 2 months in normoxia. These clonal populations were tested for etoposide resistance. Heterogeneity in the IH-selected clones was detected, while a significant overall increase in etoposide resistance was measured. (C) MCF10A IH-selected clones 4 and 9 were further tested for resistance to multiple cytotoxic conditions, including treatment to the microtubule stabilizing docetaxel, the folate metabolism inhibitor methotrexate, as well as hypoxia and reduction of growth factors. Intermittent hypoxia exhibited increased survival to all of these conditions except for survival to growth factor removal. (D,E) Intermittent hypoxia exhibits changes in the expression of p53 and E-cadherin. Quantitative Real-Time PCR was performed on each IH-selected and passage control clone. Reduced p53 and E-cadherin mRNA expression levels were detected in IH-selected control clones. Factor expression is relative to the average of control clones.