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Cell-cell contact induced resistance to etoposide Oloumi, Arusha


Many tumour cell lines grown in close three dimensional cell-cell contact either as multicell spheroids or tumours in mice exhibit a form of multicellular drug and radiation resistance that has been called the "contact effect". This resistance is often associated with agents that produce DNA double-strand breaks such as ionizing radiation and the anticancer drug and topoisomerase II inhibitor, etoposide. The hypothesis was that growth in three dimensional contact results in changes in gene expression that act, directly or indirectly, to increase resistance to etoposide. The objectives were to 1) determine the mechanism for etoposide resistance of spheroids, 2) identify genes that are differentially expressed in monolayers and spheroids, and 3) based on these results, examine the importance of intracellular free calcium levels as mediators of contact resistance. Cycling cells from Chinese hamster V79 spheroids are about 10 times more resistant than monolayers to cell killing by etoposide. Previous results indicated that the outer cells of spheroids and monolayers contained the same total amount and activity of the target enzyme, topo lice, and grew at the same rate. Using immunoblotting and immunohistochemistry, topo Hoc was found to be localized primarily in the cytoplasm of the proliferating outer cells of V79, SiHa and C6 spheroids, while nuclear localization was observed in their corresponding monolayers. Conversely, only WiDr cells, which did not show an increase in resistance to etoposide when grown as spheroids, demonstrated a predominantly nuclear localization of topo Ila. This difference in localization pattern was subsequently explained by the 10-fold decrease in phosphorylation of topo Ila in spheroids relative to monolayers, since phosphorylation is apparently required for nuclear translocation of this enzyme. Cells sorted from xenograft tumours grown in immunodeficient mice resembled the spheroid pattern both in terms of sensitivity to etoposide and location of topo Hoc. When the outer cells of V79 spheroids were returned to monolayer growth, the rate of redistribution of topo Hot to the nucleus occurred with the same kinetics as the increase in sensitivity to the cytotoxic effects of etoposide. Thus, close 3-dimensional cell-cell contact can lead to a change in posttranslational modification of topo Hoc that results in resistance to etoposide. A more direct approach was taken to identify changes in gene expression that occur when cells are grown as spheroids. Using the technique of differential display, 8 reproducible genes were found to be differentially expressed in the outer layer of V79 spheroids compared to monolayers. Up-regulation of 3 genes (cytochrome oxidase c, mtsl and calretinin) was associated with an increase in calcium binding capacity in outer cycling cell of spheroids, suggesting a possible role for calcium for the development of a contact effect. Consistent with this hypothesis, a 2-fold lower concentration of intracellular calcium was found in spheroids compared to monolayers using fluo-3 as a calcium indicator dye. Exposure of monolayers and outer spheroid cells to non-cytotoxic concentrations of BAPTA-AM, a calcium chelating agent, eliminated the difference in etoposide sensitivity between V79 monolayers and spheroids. Calcium depletion has been previously shown to protect against etoposide-induced damage by affecting both the phosphoryaltion of topo Ila and by stablizing the cleavable complex. To determine whether over-expression of a calcium binding protein would increase resistance to etoposide, V79, SiHa and C6 monolayers were transduced with metastasin (mtsl). Expression of this transgene did not reduce killing by etoposide, however because the ultimate goal of reduction in intracellular free Ca²⁺ was also not achieved with this method, the importance of calcium regulation in etoposide resistance cannot be ruled out. In conclusion, growth of cells in 3-dimensional contact as spheroids or as solid tumours induces resistance to etoposide. The basis for this resistance could lie in a change in intracellular Ca²⁺ that alters cleavable complex formation and affects phosphorylation of topo Ila, both of which can cause resistance to the anti-cancer drug etoposide.

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