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

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

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

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

Targeting of CRMP-2 to the Primary Cilium Is Modulated by GSK-3β

<div><p>CRMP-2 plays a pivotal role in promoting axon formation, neurite outgrowth and elongation in neuronal cells. CRMP-2′s role in other cells is unknown. Our preliminary results showed CRMP-2 expression in cilia of fibroblasts. To localize CRMP-2, define its role and study the regulation of CRMP-2′s expression in cilia we carried out the following experiments. We find that in fibroblasts CRMP-2 localizes to the centrosome and is associated with the basal body and -at a low level- is present in primary cilia. Phosphorylated pCRMP-2 can only be detected at the basal body. RNAi knockdown of CRMP-2 interfered with primary cilium assembly demonstrating a critical requirement for CRMP-2. Deletion analysis of CRMP-2 identified a 51 amino acid sequence in the C-terminus that is required for targeting to the basal body and primary cilium. This domain contains GSK-3β phosphorylation sites as well as two repeats of the VxPx motif, previously identified as a cilium targeting signal in other primary cilium proteins. To our surprise, mutation of the CRMP-2 VxPx motifs did not eliminate primary cilium targeting. Instead, mutation of the GSK-3β phosphorylation sites abolished CRMP-2 targeting to the primary cilium without affecting basal body localization. Treatment of cells with lithium, a potent GSK-3β inhibitor, or with two specific GSK-3β inhibitors (the L803-mts peptide inhibitor and CHIR99021) resulted in cilium elongation and decreased basal body levels of pCRMP-2 as well as increased levels of total CRMP-2 at the primary cilium. In summary, we identified CRMP-2 as a protein critically involved in primary cilia formation. To our knowledge this is the first demonstration of modulation of primary cilium targeting by GSK-3β.</p> </div

CRMP-2 mutation analysis delineates the primary cilium targeting sequence.

<p>A) Domain structure of wild type CRMP-2 (wt) and corresponding regions present in the mutants (mut1 - mut8) are indicated. DHC, dynein heavy chain interacting domain; Nb, Numb interacting domain; Mt, microtubule-binding domain; KLC, kinesin light chain interacting domain. CTS, cilium targeting capability, based on observations of mutant protein expression patterns in transfected fibroblasts. B) Immunofluorescence analysis of human foreskin fibroblasts expressing mut7-GFP or mut8-GFP as indicated. Human foreskin fibroblasts were transfected with mut7-GFP or mut8-GFP (green signal) and analyzed using anti-acetylated tubulin antibodies (red signal). Mut7-GFP localizes to the basal body (top panels: arrow) in cells lacking a primary cilium, and to the cilium in cells that express the organelle (middle panels). Mut8-GFP does not localize to basal body or cilium. Bars indicate 10 μm. Magnification: 100X. C) Nucleotide sequence and deduced amino acid sequence of the CRMP-2 51 residue C-terminal region present in mut7. Val and Pro residues in the VxPx sequences are indicated in blue and Thr and Ser residues in known GSK-3β phosphorylation sites are indicated in red (T509, T514 and S518). D) Point mutations were introduced to mutate the VxPx sites to AxAx (mut9: purple lettering) and GSK-3β phosphorylation sites to Ala (mut-10: red lettering). E) Analysis of the localization of mut9-GFP (top panels) and mut10-GFP (bottom panels) (green signals) was done by transfection of human foreskin fibroblasts and staining for acetylated tubulin (red signals). Although the intensity of mut9-GFP was lower than wild type, mut9-GFP was observed to localize to both the basal body (arrow) and the cilium. The bottom panels show that mut10-GFP only localizes to the basal body (arrow), not the cilium. Bars indicate 10 μm. Magnification: 100X.</p

Multi-potent differentiation of CD90+/− sfMPCs.

<p>Normal and OA CD90+ or CD90− sfMPCs were induced to differentiate into osteoblasts or adipocytes and stained with Akaline phosphatase (A) to indentify osteoblasts, or Oil Red-O (B) to identify lipid-containing cells (adipocytes). qRT-PCR was used to determine the relative level of adipocyte specific genes (Adiponectin, PPAR-gamma, [C]) and osteoblastic specific genes (Osteonectin, Sp7/Osterix [D]). * = p>0.05.</p

CRMP-2 Mutants.

<p>Notes:</p><p>1) indicated are the CRMP-2 amino acid residues <u>retained</u> in the indicated mutant.</p><p>2) indicated are the CRMP-2 amino acid residues <u>deleted</u> in the indicated mutant.</p><p>3) Mut9-GFP contains <u>mutated</u> VxPx motifs.</p><p>4) Mut10-GFP contains <u>mutated</u> GSK-3β phosphorylation sites.</p

CRMP-2-GFP expression pattern in fibroblasts.

<p>Human foreskin fibroblasts were transfected with CRMP-2-GFP (green signal), fixed and stained with anti-acetylated tubulin antibodies (red signal). The last column shows merged images. A) GFP signal was observed at the basal body (arrow) and –at a low level- at the primary cilium (arrowhead). The inset shows an offset of an enlarged part of the image to compare staining patterns for acetylated tubulin and CRMP-2-GFP. B) and C) show examples of transfected cells with CRMP-2-GFP in concentrations in the primary cilium (arrows). D) Shows an example of the unequal distribution of CRMP-2-GFP occasionally observed: the inset shows an offset for comparison of the signals. Bars indicate 10 μm. Magnification: 100X.</p