Cumulative per cent of heterogeneous populations transformed vs. time.

<p>Cumulative per cent of heterogeneous populations transformed vs. time.</p

Comparison of Approximate and Exact Values of Transformation Time T*.

<p>Comparison of Approximate and Exact Values of Transformation Time T*.</p

SCID mice treated with ABT-263 and rituximab independently and in combination (Reprinted from [6] under a CC BY license, with permission from Cancer Research, original copyright 2008).

<p>SCID mice treated with ABT-263 and rituximab independently and in combination (Reprinted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130590#pone.0130590.ref006" target="_blank">6</a>] under a CC BY license, with permission from Cancer Research, original copyright 2008).</p

Model calculations compared to data, with combination therapy prediction using parameters derived from monotherapy data.

<p>Model calculations compared to data, with combination therapy prediction using parameters derived from monotherapy data.</p

Cumulative per cent of heterogeneous populations transformed vs. time.

<p>Cumulative per cent of heterogeneous populations transformed vs. time.</p

Chk1 inhibition accelerates bendamustine-induced cell death.

<p><b>A.</b> Clonogenic assays were performed to assess cell survival following BDM and Chk1 inhibition. HeLa cells were treated with BDM (50 or 200 µM) for 24 h. Cells were grown for ∼10 days before being fixed and stained. The data presented are the mean absorbance value (O. D. 595 nm) relative to untreated cells, which is set to 100%. Each bar graph represents the average of 3 individual experiments performed in triplicate ± SD. †P<0.05 or *P<0.0001 or vs. untreated cells. <b>B.</b> Cell viability assessed by MTS assay was performed. HeLa cells were treated with BDM (3.125–200 µM) for 24 h. After this time, appropriate wells were co-treated with UCN-01 (100 nM) or Chk2 inhibitor (100 nM) for an additional 24 h. Data presented is the mean of 3 individual experiments performed in triplicate. Cell viability is expressed as a percentage of untreated cells ± SD. *P<0.0001 vs. 200 µM BDM alone. <b>C.</b> The percentage of apoptotic cells following indicated drug treatments was determined using Guava Nexin Reagent™. Data presented is the average of 3 individual experiments ± SD. *P<0.01 vs. untreated viable cells; †P<0.01 vs. untreated apoptotic cells.</p

Bendamustine induces both repairable and irreparable DNA damage.

<p>HeLa cells were treated for 24 or 48 h continuous treatment with either 50 or 200 µM BDM or 24 h followed by 24 h in the absence of drugs. <b>A.</b> Immunofluorescence analysis was performed to identify γ-H2AX, 53BP1 or RPA foci. Quantification of the average fluorescence per nucleus (nucleus outlined) is shown on the right. For γ-H2AX: *P = 8.9×10⁻²⁸ vs. untreated 24 h; ** P = 2.1×10⁻³¹ vs. untreated 24 h; †P = 5.2×10⁻⁴⁵ vs. untreated 48 h. For 53BP1: *P = 4.3×10⁻²⁰ vs. untreated 24 h; ** P = 4.2×10⁻⁴¹ vs. untreated 24 h; †P = 2.0×10⁻⁵⁷ vs. untreated 48 h. For RPA: *P = 2.5×10⁻¹⁶ vs. untreated 24 h; ** P = 2.8×10⁻⁵² vs. untreated 24 h; †P = 3.3×10⁻⁵⁸ vs. untreated 48 h. <b>B.</b> Lysates were probed to determine p-Chk1 (Ser345). Total Chk1 and alpha tubulin were used to determine loading.</p

Overcoming bendamustine-induced checkpoint arrest via Chk1 inhibition forces cells into premature mitosis.

<p>HeLa cells stably expressing GFP∶histone H2B were used for live cell video-microscopy. <b>A.</b> Representative montage of cells progressing through mitosis after mock treatment (upper panel), BDM at 50 µM (middle) or 200 µM (lower) followed by UCN-01 addition. <b>B.</b> Mitotic cells were fixed for metaphase spreads and dispersed onto glass slides, allowed to dry and then stained with DAPI. Metaphases were visualized using fluorescence microscopy. Images shown are representative of metaphases observed under each experimental condition. <b>C.</b> Representative electron micrographs of mitotic cells generated from untreated, 50 µM or 200 µM BDM+UCN-01 treatments.</p

Cell cycle perturbations induced by bendamustine are a widespread phenomenon in cancer cell lines.

<p>HeLa, BXPC3, MCF7, OVCAR 5 and U2932 cells were treated with bendamustine at the indicated concentrations for 24 h. Cell cycle profiles were determined using FACS analysis.</p

Involvement of base excision repair in bendamustine-induced DNA damage.

<p><b>A.</b> Immunofluorescence analyses were performed to detect remaining γ-H2AX foci after 48 h continuous treatment with 50 µM BDM in the presence or absence of either methoxyamine (MX) (6 mM) or the DNA PK inhibitor NU7441 (10 µM). Representative images are shown (left) along with quantitative analysis (right). Average values ± SD are shown. *P<0.005 vs. BDM alone. <b>B.</b> DNA damage induction and repair was conducted in assessed MEFs (<i>Tdg</i>^+/+ and <i>Tdg</i>^−/−) after treatment with BDM at 50 or 200 µM BDM for 24 h or 48 h. Representative images are shown, with nuclei outlined (circles) based on DAPI staining. Average γ-H2AX signal per nucleus ± SD is quantified (right). 24 h: *p = 0.009, **p = 2.0×10⁻⁸, ***p = 4.7×10⁻¹⁸ vs. untreated WT; †p = 0.00015, **p = 2.8×10⁻¹¹, ***p = 5.2×10⁻¹⁰ vs. untreated TDG −/−. 48 h: ***p = 4.1×10⁻¹⁰ vs. untreated WT; †p = 0.009, **p = 1.4×10⁻¹⁶, ***p = 1.9×10⁻¹⁶ vs. untreated TDG −/−.</p