Individual based stochastic simulation as two type Moran process.

<p>We assume two possible cell types, wild type leukemic cancer cells sensitive to Imatinib (yellow) and resistant cancer cells (blue). In total, cells were included in the experiment and simulations. At time there are resistant cells and wild type cells. During the next time step three cases are possible: (i) the number of resistant cells increases by one with probability , (ii) stays the same with probability (iii) or decreases by one with probability , (see equation (2a) and (2b)).</p

Average fitness observed for samples A) with and B) without detected mutations.

<p>The black dots are due to data and the green dots due to simulations. The evaluated parameters differ (see the inset of the plots), the fitness of the mutated cells is on average higher but their transformation rate is much smaller.</p

Dynamics of resistance development in the experiment and the mathematical model.

<p><b>A</b>) Average fitness of the total population due to experiments (black dots), stochastic simulations (green dots) and analytical results (black line, due to equation (7)). The parameters of simulation and calculation were chosen to , , days and . The dashed red line shows the linear decrease of the wild type fitness, the slope is determined by the critical time . <b>B</b>) The average frequency of wild type cells due to simulation (yellow dots) and calculation (black line), as well as the frequency of resistant cancer cells (blue dots) over time. We always start with wild types only, but since the system selects for resistant cells, they fixate in the long run. This is why the average fitness of the total population exhibits a minimum. At the start of the experiments, the fitness of wild types decreases, but after some time resistant cells take over and the fitness increases until it saturates.</p

Stochastic simulations with three subpopulations.

<p><b>A</b>) Transitions between the three types considered, wild types (W), resistant types without observed mutations (T) and resistant types with observed mutations (M). The switching probabilities are according to the arrows. <b>B</b>) Average fitness of the system described in a) due to simulations (green dots) and experimental data (black dots, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028955#pone-0028955-g003" target="_blank">figure 3</a>). The parameters are those observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028955#pone-0028955-g004" target="_blank">figure 4</a>. <b>C</b>) Average frequency of the three types. Initially, only wild types (yellow line) are present, at day 37 of the experiment resistant types without observed mutations (T, dashed blue line) reach almost fixation, but in the long run mutated types (M, blue dots) take over due to their fitness advantage. At day 120 of the experiment we have 0.57 T types and 0.43 M types (6 T and 4 M types were observed in the experiment at day 120).</p

eIF-5A, DHS and DOHH are overexpressed in the glioblastoma cell lines G55T2 and U87-MG.

<p>(A) Expression levels of eIF-5A, -A2, DHS and DOHH were analysed by qPCR (mean ±SD, n = 3; *:P<0.05; **:P<0.001 ) in primary normal human astrocytes (NHA) compared to G55T2 and U87-MG cell lines. SYBR green Ct values were normalized against GAPDH expression and calculated using the 2^−ΔΔCT method. (B) Overexpression of eIF-5A, DHS and DOHH was confirmed by immunoblotting with whole cell lysates (35 µg protein per sample).</p

Effects of a combination of GC7 with TMZ on induction of apoptosis.

<p>(A) G55T2 or (B) U87-MG cells were treated with TMZ (150 µM) and/or GC7 (50 µM) for 72 hours.No significant cooperative effects of both drug in induction of apoptosis have been detected. Fractions of viable, early apoptotic and dead cells were assayed by an Annexin-V/PI double staining and FACS analysis (no significant differences; p>0.05 for all experiments).</p

Effect of GC7 on proliferation, hypusine status and viability in G55T2 and U87-MG cells.

<p>(A) Cells were incubated with 50 µM GC7 or vehicle for 48 hours. The effect of GC7 on eIF-5A hypusination was determined by a change of the pI of eIF-5A, visualized by 2D-Western blot using an antibody against eIF-5A (25 µg total protein, pH 4–7 first dimension strips). (B) To verify reduced hypusination, cells were co-incubated with GC7 and 25 µCi ³H-spermidine. 200 µg of total cellular proteins were precipitated with 10% TCA and excessive radioactivity was removed by washing. The protein pellet was resuspended in 1 N NaOH and the activity of ³H, incorporated into hypusine, was measured. (C) Normal human astrocytes (NHA), G55T2 and U87-MG cells were treated with the indicated concentrations of GC7 for 72 hours. Viable cells were counted by trypan blue exclusion and data are expressed as relative cell numbers compared to mock treated cells (mean ±SD, <i>n = </i>6). Significantly different cell numbers compared to controls are indicated by asterisks (*:P<0.05; **:P<0.001). (D) U87-MG and G55T2 cells were treated with 50 µM GC7 (black area) or water (dotted area) for 72 hours, fixed in 70% ethanol and stained with PI. Apoptotic/necrotic (sub-G₁) populations were determined by FACS analysis after PI staining. (E) U87-MG and G55T2 cells were treated with 50 µM GC7 (gray area) or water (dashed area) and stained with antibodies against cleaved caspase-3 or isotype control (dotted area) to determine the rate of ongoing apoptosis upon treatment. (F) Microphotograph of mock and GC7 treated U87-MG and G55T2 cells after five days of drug treatment. The photos were taken at x100 magnification. Arrows mark examples of morphologic changes upon treatment. Each bar represents 10 µm.</p

Induction of cell cycle arrest and cellular senescence by GC7.

<p>GC7-treatment of U87-MG and G55T2 cells leads to a higher proportion of cells in G₁-cell cycle phase, upregulation of p21<i>^Waf1/Cip1</i>, downregulation of phospho-AKT and activity of senescence associate β-galactosidase. (A) After incubation cells with 100 µM GC7, the cell cycle profile was compare to untreated cells by PI staining and FACS analysis. (B and C) Total protein lysates (35 µg) of G55T2 and U87-MG cells after treatment with 100 µM GC7 (duplicates) or water were immunoblotted for p53, p21<i>^Waf1/Cip1</i>, phospho-AKT, AKT and β-tubulin. Signals were quantified after normalization against β-tubulin. (D and E) Microphotograph at x100 magnification of mock and GC7 treated U87-MG cells after SA-β-galactosidase assay. Scale bars represents 100 µm. Quantification of SA-β-gal⁺ cells (triplicates, 200 cells per sample were counted; n.d. = no SA-β-gal⁺ cells detected).</p

Restauration of p53 in G55T2 cells leads to increased premature senescence by DHS inhibition.

<p>(A) Empty vector- and p53-transduced G55T2 populations were monitored by FACS, 4 and 8 days post transduction. (B and C) GC7 and mock treated G55T2 cells +/− p53^wt were stained for SA-β-galactosidase activity. Microphotographs were taken at x100 magnification. Scale bars represents 100 µm. SA-β-gal⁺ cells were determined in triplicates, 200 cells per sample were counted. (D) Expression of p21<i>^Waf1/Cip1</i>, phospho-AKT, AKT and β-tubulin in GC7 treated G55T2 cells +/− p53^wt was analysed by immunoblotting.</p

Knockdown of DHS mRNA in U87-MG and G55T2 cell lines by gene specific shRNAs.

<p>(A) Relative DHS-mRNA expression in G55T2 and U87-MG cells after lentiviral transduction with a scrambled shRNA (G55T2-ctrl and U87 ctrl) or DHS-specific shRNA pools normalized against GAPDH (mean±SD, <i>n = </i>3). (B) Immunoblot (30 µg total protein per sample) against DHS and β-Tubulin in G55T2 and U87-MG cells after lentiviral transduction with a scrambled shRNA (G55T2-ctrl and U87-ctrl) or DHS-specific shRNAs (G55T2 DHS-sh and U87 DHS-sh) after two days of puromycin selection. (C) Glioblastoma cell lines after transduction with a scrambled control shRNA or an shRNA-pool against DHS (100× magnification) with 100 µm scale bars. (D) Proliferation of transduced cells measured by trypan blue exclusion over five days. Significantly different cell numbers or DHS expression compared to controls are indicated (*:P<0.05; **:P<0.001). (E) Immunoblot results of U87-MG and G55T2 cells after transduction with a scrambled control shRNA (ctrl) or four different shRNAs against eIF-5A (30 µg total protein). Transduced cells were selected by incubation with puromycin for two days, cell lysates were obtained seven days post-selection. (F) Proliferative capacity of transduced cells with strong (A1-sh2 for U87-MG and A1-sh1 for G55T2) or moderate (A1-sh3 for U87-MG and A1-sh2 for G55T2) eIF-5A knockdown was determined by trypan blue exclusion cell counting over five days. Significantly different cell numbers compared to controls are marked by an asterisk (*:p<0.05).</p