Decreased camptothecin sensitivity of the stem-cell-like fraction of Caco2 cells correlates with an altered phosphorylation pattern of topoisomerase I.

The CD44+ and CD44- subpopulations of the colorectal cancer cell line Caco2 were analyzed separately for their sensitivities to the antitumor drug camptothecin. CD44+ cells were less sensitive to camptothecin than CD44- cells. The relative resistance of CD44+ cells was correlated with (i) reduced activity of the nuclear enzyme topoisomerase I and (ii) insensitivity of this enzyme to camptothecin when analyzed in extracts. In contrast, topoisomerase I activity was higher in extracts from CD44- cells and the enzyme was camptothecin sensitive. Topoisomerase I from the two subpopulations were differentially phosphorylated in a manner that appeared to determine the drug sensitivity and activity of the enzyme. This finding was further supported by the fact that phosphorylation of topoisomerase I in CD44+ cell extract by protein kinase CK2 converted the enzyme to a camptothecin sensitive, more active form mimicking topoisomerase I in extracts from CD44- cells. Conversely, dephosphorylation of topoisomerase I in extracts from CD44- cells rendered the enzyme less active and camptothecin resistant. These findings add to our understanding of chemotherapy resistance in the Caco2 CD44+ cancer stem cell model

The effect of phosphorylation or dephosphorylation on the CPT sensitivity of TopI activity in extracts from FACS separated CD44+ cells or CD44− Caco2 cells.

<p>(<b>A</b>) The TopI activity in CD44+ cell extracts after phosphorylation or dephosphorylation. Whole cell extract was either left untreated (indicated by Control) or incubated with phosphatase (indicated by Dephos) or CK2 (indicated by Phos) before TopI activity was measured by REEAD in the presence of 60 µM CPT or 5% DMSO as indicated in the figure. One example randomly picked out of 12 individual microscopic images of each reaction sample is shown in the top panel. A quantitative depiction of three independent experiments, obtained as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099628#pone-0099628-g001" target="_blank">figure 1</a> is shown in the lower panel. (<b>B</b>) Same as (<b>A</b>), except that the analyses were performed on extracts from CD44− cells.</p