FIGURE 2.

Breast cancer cells maintain osteoblasts in an immature state and induce differentiation of functional osteoclasts. A, bone marrow cells were grown for 12 days with AA (50 μg/ml) and β-glycerophosphate (10 mm) in the absence (left) or presence of MDA-MB-231 CM (10%, right). The cultures were fixed and stained for ALP (red) and mineralized deposits (black). Scale bar is 100 μm. B, bone marrow cells were grown for 3–9 days with AA (50 μg/ml) in the absence or presence of MDA-MB-231 CM (10%, left) or 4T1 CM (10%, right). Expression of Collagen-1 (Coll-1), osterix (Osx), and Runx2 was analyzed on day 3 (gray) or 9 (black). Expression of Cyclin A (CA), Cyclin D1 (CD1), and p53 was analyzed on day 9. Data are means ± S.E. (error bars), normalized to expression of β-actin (left) or GAPDH (right), and presented relative to levels observed in AA only samples (dashed line), n = 3–5 independent experiments, p < 0.05. C, bone marrow cells were grown for 9 days on dentin slices with AA (50 μg/ml) in the absence (left) or presence of MDA-MB-231 CM (10%, right), then the cells were removed, and dentin was stained with toluidine blue to reveal resorption pits. Scale bars represent 100 μm. D, expression of Cathepsin K (Cat K), TRAP, and MMP-9 was analyzed on day 9. Data are means ± S.E., normalized to expression of β-actin, and presented relative to levels observed in AA only samples (dashed line), n = 4–6 independent experiments, p < 0.05. E, bone marrow cells were grown for 9 days with AA (50 μg/ml) in the absence or presence of MDA-MB-231 CM (10%). The parallel samples were fixed, stained with DAPI nuclear stain, and the cell density was estimated (left). The rate of apoptosis was estimated as a proportion of cells demonstrating nuclear fragmentation from the total number of cells analyzed (center). Cell proliferation was measured by BrdU incorporation (right). Data are means ± S.E., n = 3–5 independent experiments, p < 0.05.