Intratumoral heterogeneity for and epigenetic modulation of mdr-1 expression in murine melanoma

We determined whether tumour size in vivo and cell density in vitro modulate the expression of the mdr-1 gene in B16 melanoma cells. Cells were injected subcutaneously into syngeneic mice. Small (5 mm in diameter) and large (1520 mm in diameter) tumours were harvested. Tumour cells from small subcutaneous turnouts exhibited higher levels of mdr-1 mRNA (measured using Northern blot and in situ hybridization) and P-glycoprotein (P-gp) (measured using immunohistochemistry and fluorescent activated cell sorter analysis), as well as greater in vitro resistance to doxorubicin (DXR) than cells from large subcutaneous tumours. Immunohistochemical studies using an antibody against proliferating cell nuclear antigen revealed that the small subcutaneous tumours contained a larger fraction of proliferating cells than the large tumours. To determine whether cell proliferation correlated with expression of mdr-1, we plated B16-F10 cells to yield sparse and confluent monolayer cultures. The levels of mdr-1 mRNA and P-gp and resistance to DXR and phosphotyrosine activity were higher in the sparse cultures than in the confluent cultures. These results demonstrate an intratumoral heterogeneity for the expression of mdr-1 that directly correlates with intratumoral heterogeneity for cell division.