The active metabolite of irinotecan is toxic to primary human pheochromocytoma cells.

<p>XTT assay results showing killing of human PCC cells by camptothecin and SN-38. Cultures were treated with the indicated drug concentrations for one week. Data represent mean +/− SEM of triplicate wells. (**, p<.01).</p

Cytotoxicity of camptothecin against MPC cells is increased in the presence of 5-azacytidine.

<p>Cytotoxicity of camptothecin against MPC 4/30/PRR cells was tested in the presence or absence of 5-azacytidine (1 uM) by XTT assay. Absorbance is proportional to cell survival. Captions under each bar indicate whether 5-aza was present during the first week/second week of a 2-week experiment. Data are from a representative experiment that was repeated on 3 independent occasions. Bars indicate mean +/− SEM of quadruplicate wells.</p

MPC cells treated with camptothecin show morphological changes of apoptosis.

<p>Representative fluorescence micrographs showing nuclear morphology of DAPI-stained MPC cultures. Panel A shows nuclei of cells maintained in control medium for 7 days. Nuclei are round to oval with finely stippled chromatin. One mitosis is evident (m). Panels B–D show typical apoptotic changes seen at day 7 in cultures with camptothecin or camptothecin +5-aza. (B, early peripheral margination of chromatin; C, nuclear shrinkage and marked chromatin margination; d, nuclear fragmentation). In addition, B–D contain fewer cells, consistent with ongoing attrition. Bar = 20 um. Original magnification 100 x.</p

Killing of cells from <i>SDHB</i>-mutated and apparently sporadic human PCC/PGL s by camptothecin.

<p>Dissociated human tumor cells in primary cultures maintained with 0 (control), 1 or 10 uM camptothecin (Cmpt) for 2 wks, then fixed and stained for TH (red cytoplasm) to discriminate the tumor cells from other cell types. At 10 uM, camptothecin eliminated almost all background cells (faintly visible as hematoxylin-counterstained blue nuclei in top row control and 1 uM panels) and was therefore considered too toxic for the purposes of this investigation.</p

Clinically utilized TOP1 inhibitors show variable toxicities to MPC cells.

<p>Concurrent tests of camptothecin versus clinically utilized TOP1 inhibitors on MPC cells in monolayer cultures at one week. Equivalency of camptothecin and SN-38 is seen at 10-fold lower concentrations of SN38. Data are from three independent experiments, each with triplicate wells. Bars indicate mean +/− SEM. (**, p<.01; *, p<.05).</p

Camptothecin paradoxically increases bioluminescence and luciferase expression.

<p>(A) Effects of camptothecin and 5-azacytidine on bioluminescence of MPC GL-9 cells compared to survival measured by Absorbance in XTT assay at 1 week. (B) corresponding immunoblot from the same experiment showing increased levels of firefly luciferase protein and copepod GFP (copGFP) in camptothecin-treated cultures. Expression of CgA is not increased, indicating that the effect is specific for the luciferase construct. The paradoxically increased bioluminescence of cells treated with camptothecin obscured obvious actual toxicity that was quantifiable by XTT assay.</p

Comparative cytotoxicity of camptothecin against MPC and MTT cells.

<p>Parallel tests of camptothecin toxicity on MPC and MTT cell lines at two time points, demonstrate greater sensitivity of the more aggressive MTT line to low camptothecin concentrations. Data are from a representative experiment that was repeated on 2 independent occasions. Bars indicate mean +/− SEM of quadruplicate wells.</p

Camptothecin and 5-azacytidine cooperatively increase MPC cell apoptosis.

<p>Immunoblots show the cooperative effects of camptothecin and 5-azacytidine on MPC cell apoptosis, which is indicated by the presence of a 25 kDa fragment of PARP. A marked increase in intensity of the PARP25 band is seen at 24 hrs with the combination of camptothecin and 5-aza, with little effect of 5-aza alone. This pattern is still evident, but diminished, after 4 days.</p

Cytotoxicity of camptothecin against human PCC/PGL cells in primary cultures.

<p>Dissociated primary tumor cells from PCCs or PGLs representing different genotypes were cultured in the presence of 1 uM or 10 uM camptothecin compared to control medium. Counts were derived by counting all stained cells defined by a randomly placed square coverslip in a 35 mm culture dish (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087807#pone-0087807-g001" target="_blank">Figure 1</a>). All counts were done at 2 weeks except for tumor 7 (**), which was counted at 1 week because of extensive cell death caused by particular sensitivity to camptothecin. The two tumors listed as sporadic negative were tested negative for MEN2 <i>RET</i> mutation and for <i>SDHB</i>, <i>SDHC</i> and <i>SDHD</i> mutations and deletions.</p

Clinicopathological characteristics.

<p>M, male; F, female; U, unknown; PCC, pheochromocytoma; PGL, paraganglioma; Meta, metastasis; B, benign; M, malignant; FU, follow-up; D, died.</p><p>Clinicopathological characteristics.</p