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

Abstract

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.

Figure 1. CPT response of CSC or non-CSC cell subpopulations of Caco2.

(A) FACS analysis of Caco2 cells stained with PE-conjugated CD44 (top panel) or CD133 (lower panel) specific antibodies. (B) FACS analysis of Caco2 cells before or after FACS sorting based on CD44. (I) Unstained cells, (II) Caco2 cells stained for CD44 before sorting, (III) Caco2 cells after sorting for CD44+ cells, (IV) Caco2 cells after sorting for CD44− cells. The cells were analyzed after sorting by staining for CD44 (C) Survival assay of the FACS sorted CD44+ or CD44− cells. The cells were incubated with DMSO (the solvent of CPT) or 0.1, 0.2, or 0.4, µM of CPT for 60 hours before the percentage of viable cells was measured by the MTT method. (D) Graphical depiction of the REEAD assay. S(hTopI) folds into a dumbbell shaped structure, which supports cleavage and ligation activity mediated by TopI. Hereby the substrate is converted to a closed circle, which is hybridized to a surface-attached RCA-primer matching the specific primer annealing sequence (p) on the substrate. Subsequently, phi-polymerase is added to support RCA. The resulting RCA products are visualized by hybridization of fluorescently labeled probes matching the complementary identifier sequence (i) of the circles and the products analyzed using a fluorescence microscope. TopI is illustrated as a gray pacman with a “T”, the phi-polymerase as a dark gray circle. The RCA-primers and detection probes are indicated on the figure. (E) Measurement of TopI activity by REEAD in the CD44+ and CD44− FACS sorted cell populations, respectively. Representative microscopic images obtained by analyzing the TopI activity present in whole cell extracts from 10-10⁴ cells are shown in the top panel. Signals obtained from circularized S(hTopI) are shown in red, while signals obtained from control circles are shown in green. The lower panel shows a quantitative depiction of three independent experiments. (F) CPT sensitivity of TopI in the sorted CD44+ and CD44− cells, respectively. S(hTopI) was incubated with whole cell extract from 10⁴ cells in presence of 15 µM, 30 µM, 60 µM CPT or DMSO as indicated in the figure. One example out of 12 individual microscopic images of each reaction sample is shown in the top panel, where red signals correspond to circularized S(hTopI) while green signals were generated from control circles. The lower panel shows a quantitative depiction of three independent experiments. For all REEAD experiments, to avoid misinterpretation due to potential uneven distribution of RCA primers printed on the surface, the number of red and green signals were counted on 12 microscopic images and the results depicted as the ratio between the number of red (R) TopI specific- and green (G) control circle generated signals (R/G) as described .

Figure 2. CPT sensitivity of TopI purified from nuclear extract of FACS sorted CD44+ or CD44− cell subpopulations.

(A) Graphical illustration of column purification. (B) Shows the results of SDS phage followed by silver stain visualization of the purified enzyme fractions. Lane 1, is a size marker, lanes 2 and 3 show the enzyme fractions purified from CD44− and CD44+ cells, respectively. (C) Shows the CPT sensitivity of the purified enzyme as measured by REEAD. S(hTopI) was incubated with TopI purified from the CD44+ or CD44− cells in the presence of DMSO or 15 µM, 30 µM or 60 µM CPT as indicated. The results of three independent experiments were quantified and depicted as a bar chart as described in the legend of figure 1.

Figure 3. Phoshorylation pattern of TopI in extracts from CD44+ or CD44− cell subpopulations.

(A) Two-dimensional silver staining (left panel) and immunoblot analysis (right panel) of TopI expression pattern in the Caco2 cell line. The positions of TopI are underlined. The TopI identity of two major isoforms recognized by TopI antibodies was ensured by the MS/MS analysis of several bands excised from the silver stained gel (data not shown). (B) One-dimentional immunoblot analysis of TopI expression in CD44− and CD44+ FACS sorted cell fractions by immunoblotting with TopI antibodies. β-actin was used as a loading control. (C) Two-dimentional immunoblot analysis of TopI expression and post-translational modification pattern in CD44− and CD44+ cell fractions (left-hand panels) by immunoblotting with TopI antibodies. Equal protein amounts were loaded on the gels. The time of blot exposure is identical. The resulting pattern of TopI after cell treatment with λ-PPase prior the 2D PAGE analysis is presented (right-hand panels). Arrowheads indicate multiple forms of TopI (black arrowheads – the position of the non-phosphorylated TopI, blue arrowheads – the positions of phosphorylated TopI isoforms; red arrowheads – the positions of non-phosphorylated otherwise modified isoforms). The positions of parental and modified TopI isoforms are verified by the superimposing of all analyzed blots.

Figure 4. The effect of phosphorylation or dephosphorylation on the CPT sensitivity of TopI activity in extracts from FACS separated CD44+ cells or CD44− Caco2 cells.

(A) 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 figure 1 is shown in the lower panel. (B) Same as (A), except that the analyses were performed on extracts from CD44− cells.