Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission.

Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA-resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission

Dose Dependent Effects on Cell Cycle Checkpoints and DNA Repair by Bendamustine

Bendamustine (BDM) is an active chemotherapeutic agent approved in the U. S. for treating chronic lymphocytic leukemia and non-Hodgkin lymphoma. Its chemical structure suggests it may have alkylator and anti-metabolite activities; however the precise mechanism of action is not well understood. Here we report the concentration-dependent effects of BDM on cell cycle, DNA damage, checkpoint response and cell death in HeLa cells. Low concentrations of BDM transiently arrested cells in G2, while a 4-fold higher concentration arrested cells in S phase. DNA damage at 50, but not 200 µM, was efficiently repaired after 48 h treatment, suggesting a difference in DNA repair efficiency at the two concentrations. Indeed, perturbing base-excision repair sensitized cells to lower concentrations of BDM. Timelapse studies of the checkpoint response to BDM showed that inhibiting Chk1 caused both the S- and G2-arrested cells to prematurely enter mitosis. However, whereas the cells arrested in G2 (low dose BDM) entered mitosis, segregated their chromosomes and divided normally, the S-phase arrested cells (high dose BDM) exhibited a highly aberrant mitosis, whereby EM images showed highly fragmented chromosomes. The vast majority of these cells died without ever exiting mitosis. Inhibiting the Chk1-dependent DNA damage checkpoint accelerated the time of killing by BDM. Our studies suggest that BDM may affect different biological processes depending on drug concentration. Sensitizing cells to killing by BDM can be achieved by inhibiting base-excision repair or disrupting the DNA damage checkpoint pathway

Cell death in insulin-containing cells : induction and prevention

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Pixantrone induces cell death through mitotic perturbations and subsequent aberrant cell divisions

Pixantrone is a novel aza-anthracenedione active against aggressive lymphoma and is being evaluated for use against various hematologic and solid tumors. The drug is an analog of mitoxantrone, but displays less cardiotoxicity than mitoxantrone or the more commonly used doxorubicin. Although pixantrone is purported to inhibit topoisomerase II activity and intercalate with DNA, exact mechanisms of how it induces cell death remain obscure. Here we evaluated the effect of pixantrone on a panel of solid tumor cell lines to understand its mechanism of cell killing. Initial experiments with pixantrone showed an apparent discrepancy between its anti-proliferative effects in MTS assays (short-term) compared with clonogenic assays (long-term). Using live cell videomicroscopy to track the fates of cells, we found that cells treated with pixantrone underwent multiple rounds of aberrant cell division before eventually dying after approximately 5 d post-treatment. Cells underwent abnormal mitosis in which chromosome segregation was impaired, generating chromatin bridges between cells or within cells containing micronuclei. While pixantrone-treated cells did not display γH2AX foci, a marker of DNA damage, in the main nuclei, such foci were often detected in the micronuclei. Using DNA content analysis, we found that pixantrone concentrations that induced cell death in a clonogenic assay did not impede cell cycle progression, further supporting the lack of canonical DNA damage signaling. These findings suggest pixantrone induces a latent type of DNA damage that impairs the fidelity of mitosis, without triggering DNA damage response or mitotic checkpoint activation, but is lethal after successive rounds of aberrant division

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

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

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