Mitotic Death Research Articles

An essential role for p73 in regulating mitotic cell death

The molecular mechanisms regulating cell death during mitosis are poorly understood. We show here a critical role for p73, but not p53, in regulating mitotic cell death induced by various means. Prolonged mitotic arrest and the activation of spindle checkpoint are required for mitotic death, which occurs before mitotic exit and which can be ameliorated by accelerated mitotic exit. Absence or silencing of p73 expression abrogated mitotic death without accelerating mitotic exit, and was independent of BubR1 and Mad2, the loss of which promotes mitotic exit. However, the absence of p73 reduced mitotic death by compromising the expression of the proapoptotic BH3-only protein Bim and thereby affecting cytochrome c release and caspase activation. p73 was found to induce bim expression through direct binding to regulatory elements in intron 1. Congruently, mitotic cell death was rescued to similar extents by silencing either bim or p73 expression. Taken together, the data show an important role for the p73-Bim axis in regulating cell death during mitosis that is independent of p53.

DNA damage-induced cell death is enhanced by progression through mitosis

Progression through the G2/M transition following DNA damage is linked to cytokinesis failure and mitotic death. In four different transformed cell lines and two human embryonic stem cell lines, we find that DNA damage triggers mitotic chromatin decondensation and global phosphorylation of histone H2AX, which has been associated with apoptosis. However, extended time-lapse studies in HCT116 colorectal cancer cells indicate that death does not take place during mitosis, but 72% of cells die within 3 days of mitotic exit. By contrast, only 11% of cells in the same cultures that remained in interphase died, suggesting that progression through mitosis enhances cell death following DNA damage. These time-lapse studies also confirmed that DNA damage leads to high rates of cytokinesis failure, but showed that cells that completed cytokinesis following damage died at higher rates than cells that failed to complete division. Therefore, post-mitotic cell death is not a response to cytokinesis failure or polyploidy. We also show that post-mitotic cell death is largely independent of p53 and is only partially suppressed by the apical caspase inhibitor Z-VAD-FMK. These findings suggest that progression through mitosis following DNA damage initiates a p53- and caspase-independent cell death response that prevents propagation of genetic lesions.

Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G(1), with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype.

Anticancer activity of RecQL1 helicase siRNA in mouse xenograft models

Small interfering RNAs (siRNAs) are expected to have a medical application in human therapy as drugs with a high specificity for their molecular target mRNAs. RecQL1 DNA helicase in the human RecQ helicase family participates in DNA repair and recombination pathways in the cell cycle of replication. Silencing the RecQL1 expression by RecQL1-siRNA induces mitotic death in vitro specifically in growing cancer cells. By contrast, the same RecQL1 silencing does not affect the growth of normal cells, emphasizing that RecQL1 helicase is an ideal molecular target for cancer therapy. In this study, we show that local and systemic administration of RecQL1-siRNA mixed with polyethyleneimine polymer or cationic liposomes prevented cancer cell proliferation in vivo in mouse models of cancer without noticeable adverse effects. The results indicate that RecQL1-siRNA in a complex with a cationic polymer is a very promising anticancer drug candidate, and that in particular, RecQL1-siRNA formulated with a cationic liposome has an enormous potential to be used by intravenous injection for therapy specific for liver cancers, including metastasized cancers from the colon and pancreas.

Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of α-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of α-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. γ-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.

Low-dose hyper-radiosensitivity of progressive and regressive cells isolated from a rat colon tumour: Impact of DNA repair

Purpose: To ask whether highly metastatic sublines show more marked low-dose hyper-radiosensitivity (HRS) response than poorly metastatic ones.Materials and methods: The progressive (PRO) subline showing tumourigenicity and metastatic potential and the regressive (REG) subline showing neither tumourigenicity nor metastatic potential were both isolated from a parental rat colon tumour. Clonogenic survival, micronuclei and apoptosis, cell cycle distribution, DNA single- (SSB) and double-strand breaks (DSB) induction and repair were examined.Results: HRS phenomenon was demonstrated in PRO subline. Before irradiation, PRO cells show more spontaneous damage than REG cells. After 0.1 Gy, PRO cells displayed: (i) More DNA SSB 15 min post-irradiation, (ii) more unrepaired DNA DSB processed by the non-homologous end-joining (NHEJ) and by the RAD51-dependent recombination pathways, (iii) more micronuclei, than REG cells while neither apoptosis nor p53 phosphorylation nor cell cycle arrest was observed in both sublines.Conclusions: HRS response of PRO subline may be induced by impairments in NHEJ repair that targets G1 cells and RAD51-dependent repair that targets S-G2/M cells. The cellular consequences of such impairments are a failure to arrest in cell cycle, the propagation of damage through cell cycle, mitotic death but not p53-dependent apoptosis. Tumourigenic cells with high metastatic potential may preferentially show HRS response.

Mechanisms underlying the pro-survival pathway of p53 in suppressing mitotic death induced by adriamycin

The p53 tumor suppressor responds to chemotherapeutic stress by triggering apoptosis or eliciting pro-survival pathway through arresting cell cycle progression for DNA damage repair. Here we examined the pro-survival activity of p53 on the adriamycin-induced stress using H1299 cells stably expressing tsp53 V143A, a temperature-sensitive mutant activating only the subset of p53 target genes related to growth arrest and DNA repair, but not apoptosis. At 38 °C, cells evaded from adriamycin-induced G2 arrest and died of apoptosis and mitotic catastrophe, which could be inhibited by Cdk inhibitors. Activation of functional tsp53 V143A at 32 °C led to suppression of Cdk1/2 activities and Cyclin B1/Cdk1 expression, cells exhibited prolonged G2 arrest, regained reproductive potential and were protected from mitotic catastrophe induced by adriamycin. Inhibition of mitotic catastrophe and Cyclin B1/Cdk1 expression was ablated upon silencing p21 Waf1 expression in tsp53 V143A-H1299 cells or in HCT116 cells. Together we show that p21 Waf1 is a key component of G2 checkpoint necessary and sufficient for protecting tumor cells against adriamycin-induced mitotic catastrophe.

BUB1 mediation of caspase-independent mitotic death determines cell fate

The spindle checkpoint that monitors kinetochore–microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.

Future Strategies for Mitigation and Treatment of Chronic Radiation-Induced Normal Tissue Injury

Until the mid-1990s, radiation-induced normal-tissue injury was generally assumed to be solely caused by the delayed mitotic death of parenchymal or vascular cells, and these injuries were held to be progressive and untreatable. From this assumption, it followed that postirradiation interventions would be unlikely to reduce either the incidence or the severity of radiation-induced normal tissue injury. It is now clear that parenchymal and vascular cells are active participants in the response to radiation injury, an observation that allows for the possibility of pharmacologic mitigation and/or treatment of these injuries. Mitigation or treatment of chronic radiation injuries has now been experimentally shown in multiple organ systems (eg, lung, kidney, and brain), with different pharmacologic agents (eg, angiotensin-converting enzyme inhibitors, pentoxifylline, and superoxide dismutase mimetics) and with seemingly different mechanisms (eg, suppression of the renin-angiotensin system and suppression of chronic oxidative stress). Unfortunately, the mechanistic basis for most of the experimental successes has not been established, and assessment of the utility of these agents for clinical use has been slow. Clinical development of pharmacologic approaches to mitigation or treatment of chronic radiation injuries could lead to significant improvement in survival and quality of life for radiotherapy patients and for victims of radiation accidents or nuclear terrorism.

Sphingosine-1-Phosphate Protects Proliferating Endothelial Cells from Ceramide-Induced Apoptosis but not from DNA Damage–Induced Mitotic Death

Because of the central role of the endothelium in tissue homeostasis, protecting the vasculature from radiation-induced death is a major concern in tissue radioprotection. Premitotic apoptosis and mitotic death are two prevalent cell death pathways induced by ionizing radiation. Endothelial cells undergo apoptosis after radiation through generation of the sphingolipid ceramide. However, if mitotic death is known as the established radiation-induced death pathway for cycling eukaryotic cells, direct involvement of mitotic death in proliferating endothelial radiosensitivity has not been clearly shown. In this study, we proved that proliferating human microvascular endothelial cells (HMEC-1) undergo two waves of death after exposure to 15 Gy radiation: an early premitotic apoptosis dependent on ceramide generation and a delayed DNA damage-induced mitotic death. The fact that sphingosine-1-phosphate (S1P), a ceramide antagonist, protects HMEC-1 only from membrane-dependent apoptosis but not from DNA damage-induced mitotic death proves the independence of the two pathways. Furthermore, adding nocodazole, a mitotic inhibitor, to S1P affected both cell death mechanisms and fully prevented radiation-induced death. If our results fit with the standard model in which S1P signaling inhibits ceramide-mediated apoptosis induced by antitumor treatments, such as radiotherapy, they exclude, for the first time, a significant role of S1P-induced molecular survival pathway against mitotic death. Discrimination between ceramide-mediated apoptosis and DNA damage-induced mitotic death may give the opportunity to define a new class of radioprotectors for normal tissues in which quiescent endothelium represents the most sensitive target, while excluding malignant tumor containing pro-proliferating angiogenic endothelial cells that are sensitive to mitotic death.

Arsenite‐induced mitotic death is distinct from both nocodazole and Taxol

Arsenite causes mitotic arrest and death at anaphase, but the mechanism is highly disputed. We compared arsenite-induced mitotic arrest to mitotic arrest induced by two spindle poisons, nocodazole and Taxol. Mitotic index peaked earlier during nocodazole and Taxol treatments vs. arsenite treatment in TR9-7 cells. Spindle formation occurred in arsenite- and Taxol-treated cells but not in nocodazole-treated cells. Nocodazole-treated mitotic cells always contained two centrosomes. In contrast, many arsenite-treated mitotic cells contained more than two centrosomes. Taxol-induced centrosome duplication was concentration dependent and only occurred at sub-therapeutic doses. We tested additional cell lines to examine if arsenite sensitivity correlated with Taxol and nocodazole. HeLa, A375, and A2780 cells were sensitive to all three drugs. CP70 cells, cisplatin-resistant derivative of A2780 cells, were as sensitive to mitotic arresting drugs as their parental line. A549 cells were sensitive to nocodazole and Taxol, but were resistant to arsenite. Co-treatment with BSO, a glutathione synthase inhibitor, and MK571, an MRP export pump inhibitor, sensitized A549 cells to arsenite but did not alter nocodazole or Taxol sensitivity. In conclusion, arsenite-induced mitotic arrest appears to be independent of direct effects on tubulin polymerization. (Supported by PHS grants R01 ES011314 and F30 ES013372.)

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Abdominal radiation therapy causes cell death of gastrointestinal (GI) epithelial and mesenchymal cells (endothelial cells and lymphocytes). Above a threshold dose of abdominal radiation, death can occur acutely from the GI syndrome. Below this threshold dose, acute radiation toxicity is rarely fatal, but late effects of radiation therapy can be severe and ultimately cause death. The cellular target (epithelial vs. mesenchymal cells) and the molecular mechanism of cell death (apoptosis vs. mitotic catastrophe), which mediate these toxicities, are unclear. To determine the cellular target and molecular mechanism which mediate the acute toxicity of the GI Syndrome and late effects of radiation therapy. The Cre-lox system was utilized to delete genes in a tissue specific manner in mice. To assess the role of apoptosis in mediating toxicity following abdominal irradiation, mice were generated that lacked the intrinsic pathway of apoptosis (deleted for BAX and BAK) in either GI epithelial cells or in endothelial cells and lymphocytes. GI epithelial cells or endothelial cells were isolated and deletion of BAK and BAX were confirmed by Western blot. To investigate the role of mitotic catastrophe in mediating toxicity following abdominal irradiation, p53 was deleted in either GI epithelial cells or in endothelial cells and lymphocytes. After abdominal irradiation, apoptosis and mitotic catastrophe were evaluated by histology and immunohistochemistry. Mice were treated with whole abdominal irradiation and followed for survival. Deletion of the intrinsic pathway of apoptosis in the GI epithelium dramatically decreased GI epithelial apoptosis after abdominal irradiation, but did not affect survival from the GI syndrome. Deletion of the intrinsic pathway of apoptosis in endothelial cells and lymphocytes decreased mesenchymal cell death after abdominal irradiation, but also did not affect survival from the GI syndrome. In contrast, deletion of p53 from the GI epithelium completely blocked GI epithelial apoptosis and increased mitotic catastrophe. Increased mitotic catastrophe sensitized mice to death from the GI syndrome. Deletion of p53 from endothelial cells did not affect acute toxicity after abdominal irradiation. However, deletion of p53 from endothelial cells significantly enhanced the late vascular complications of radiation therapy. Apoptosis of GI epithelial cells or mesenchymal cells (endothelial cells and lymphocytes) do not mediate the GI syndrome. Instead, mitotic catastrophe of GI epithelial cells appears to mediate the acute toxicity of the GI syndrome. Late vascular effects of abdominal irradiation are enhanced when p53 is deleted from endothelial cells. Therefore, mitotic death of endothelial cells may be an important mechanism regulating the late effects of radiation therapy.

Concise Review: Bone Morphogenetic Protein Pleiotropism in Neural Stem Cells and Their Derivatives—Alternative Pathways, Convergent Signals

Bone morphogenetic proteins (BMPs) are a class of morphogens that are critical regulators of the central nervous system (CNS), peripheral nervous system, and craniofacial development. Modulation of BMP signaling also appears to be an important component of the postnatal stem cell niche. However, describing a comprehensive model of BMP actions is complicated by their paradoxical effects in precursor cells, which include dorsal specification, promoting proliferation or mitotic arrest, cell survival or death, and neuronal or glial fate. In addition, in postmitotic neurons BMPs can promote dendritic growth, act as axonal chemorepellants, and stabilize synapses. Although many of these responses depend on interactions with other incoming signals, some reflect the recruitment of distinct BMP signal transduction pathways. In this review, we classify the diverse effects of BMPs on neural cells, focus on the known mechanisms that specify distinct responses, and discuss the remaining challenges in identifying the cellular basis of BMP pleiotropism. Addressing these issues may have importance for stem cell mobilization, differentiation, and cell integration/survival in reparative therapies.

The range of optimal concentration and mechanisms of paclitaxel in radio-enhancement in gastrointestinal cancer cell lines

This study was performed to investigate the range of optimal concentration and mechanisms of paclitaxel (PXL) radio-enhancement in gastrointestinal cancer cell lines HT29 and MKN45. Cell growth inhibition by PXL pretreatment at various concentrations (0-10 microM) followed by irradiation was investigated using a modified MTT assay. To investigate the mechanisms of the observed radio-enhancement, flow cytometry was conducted to define the cell cycle distributions. Furthermore, the alterations in expression of a DNA repair molecule [excision repair cross-complementation group1 (ERCC1)] and an angiogenesis factor [vascular endothelial growth factor (VEGF)] induced by PXL were investigated. Cytotoxic concentrations of PXL (0.1-10 microM) that cause accumulation of cells in the G2/M phase have strong radio-enhancing effects and inhibit total cell growth. The maximal non-cytotoxic concentration of PXL (0.01 microM) also had a radio-enhancing effect. The expression of the genes of ERCC1 and VEGF induced by radiation was suppressed by PXL pretreatment. The protein secretion of VEGF induced by radiation was suppressed at cytotoxic doses of PXL, and the induced protein secretion of ERCC1 was also suppressed even at maximal non-cytotoxic doses of PXL. The range of optimal concentration for PXL pretreatment was 0.01-0.1 microM in these cells. Two major mechanisms of radio-enhancement are suggested: (1) PXL induces G2/M arrest leading to increased DNA damage after radiation, which results in mitotic death, and (2) PXL suppresses the expression of radiation-induced DNA repair molecules and angiogenesis factors, resulting in inhibition of cell growth and cell death.

Differential mechanisms of radiosensitization by 2-deoxy-D-glucose in the monolayers and multicellular spheroids of a human glioma cell line

In vitro studies using monolayer cultures of human tumor cell lines have shown that 2-DG selectively inhibits energy-dependent DNA repair and cellular recovery processes in cancer cells. However, monolayer cultures differ greatly from the complex environmental conditions generated in solid tumors that develop inhomogeneous hypoxic and necrotic regions. In contrast, multicellular spheroids mimic heterogeneous cellular behavior and the consequent functional characteristics of in vivo solid tumors, and serve as important in vitro model to investigate tumor biology and responses to potential therapeutic agents. The present study compares the radiomodification by 2-DG in monolayer cultures and spheroids of a human glioma cell line (BMG-1) to gain insight into the effects in solid tumors. In spheroids, the glucose consumption (2.1 p mole/cell/h) and lactate production (3.67 p mole/cell/h) was nearly 2-3 fold higher than in monolayer cells (0.83 & 1.43 p mole/cell/h respectively). Presence of 2-DG (5 mM) for 2-4 h inhibited the glucose usage and lactate production by 70 % in spheroids, while a 35 % reduction was observed in monolayer cells. Under these conditions, 2-DG drastically enhanced the radiation-induced cell death of spheroids (by 2-3 folds); while a 40% increase was observed in monolayer cells. Radiosensitization by 2-DG in monolayer cells was primarily due to an increase in mitotic death (23%) linked to cytogenetic damage (micronuclei), whereas a profound induction of apoptosis (40%) accounted for the sensitization in spheroids. Although the Bcl-2 and Bax levels were significantly higher in spheroids, Bcl-2/Bax ratio was similar in monolayers and spheroids. Comet assay revealed a late onset of DNA breaks in the presence of 2- DG following irradiation only in spheroids, which corroborated well with the late onset of oxidative stress. 2- DG did not induce a significant cell cycle delay in monolayers, while a transient G2 delay was apparent in spheroids.

Induction of Centrosome Amplification during Arsenite-Induced Mitotic Arrest in CGL-2 Cells

Arsenite-induced mitotic abnormalities result in mitotic death in several cancer cell lines. However, how arsenite induces these effects is not known. We have previously shown that arsenite induces mitotic arrest, mitotic abnormalities, and mitotic death in CGL-2 cells. To further delineate the mechanism of action of arsenite, we examined its effect on centrosome duplication and the possible link between centrosome dysregulation and arsenite-induced mitotic death. Immunofluorescence staining of gamma-tubulin revealed that centrosome amplification was induced in arsenite-arrested mitotic cells but not in nocodazole-arrested cells. When S phase-enriched cells were treated with arsenite, they progressed into and arrested at mitosis and then formed supernumerary centrosomes. A further increase in arsenite-induced centrosome amplification was seen during the prolonged mitotic arrest. The arsenite-induced supernumerary centrosomes might result from uneven fragmentation of centrosome, overexpression of pericentriolar materials, and inhibition of centrosomal coalescence during mitosis. Furthermore, termination of mitotic arrest by treatment of arsenite-arrested mitotic cells with cyclin-dependent kinase 1 inhibitors or by suppression of spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 or Mad2 markedly reduced the induction of centrosome amplification and mitotic death in arsenite-treated cells. These results indicate that centrosome amplification is induced in arsenite-arrested mitotic CGL-2 cells in a spindle checkpoint-dependent manner and is involved in the induction of arsenite-induced mitotic death.

GL15 and U251 glioblastoma-derived human cell lines are peculiarly susceptible to induction of mitotic death by very low concentrations of okadaic acid

The challenging possibility of selectively inducing mitotic death in tumor cells by combining genotoxic agents with the inhibition of G2 checkpoints of the cell cycle is the subject of intensive investigation. We show that very low concentrations (3.5 and 5 nM) of okadaic acid induce mitotic death in two glioblastoma cell lines, in the absence of genotoxic agents. At the concentrations used, the main target of okadaic acid action is protein phosphatase 2A (PP2A), an enzyme deeply involved in the negative control of cell-cycle progression. The peculiar susceptibility of glioblastoma cells to induction of mitotic death by very low concentrations of okadaic acid must be related to an impairment of PP2A activity and to a specific deficiency in some cell-cycle checkpoints. In addition to its ability to induce abnormal mitoses in actively proliferating glioblastoma cells, okadaic acid possesses the ability to force semi-confluent glioblastoma cells to the M phase of the cell cycle, where they show the same abnormalities observed in actively proliferating glioblastoma cells. In semi-confluent cells the induction of mitotic death involves the activity of both the extracellular signal regulated kinases (ERKs) and the M-phase promoting factor: okadaic acid overstimulates ERK activity, and PD98059 (inhibitor of ERK activation) as well as roscovitine (S)-isomer (specific inhibitor of M-phase promoting factor activity) counteract the induction of mitotic death. Our results show that, without the use of genotoxic agents, it is possible to induce mitotic death in glioblastoma cells by activating the same uncontrolled pathways responsible for the uncontrolled proliferation.

Cell Death Mechanisms Associated with G2Radiosensitivity in Patients with Prostate Cancer and Benign Prostatic Hyperplasia

Cells respond to genotoxic insults such as ionizing radiation by halting in the G2 phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G2, and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G2 chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G2 chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G2 chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G2 assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G2 compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G2 checkpoint control in the prostate patients. There was no significant correlation of G2 radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G2 radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G2 aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

Heterogeneity in the radiosensitizing effects of the DNA ligand hoechst-33342 in human tumor cell lines.

The AT specific minor grove DNA binding ligands bisbenzimidazole derivatives like hoechst-33342 and hoechst-33258 which scavenge free radicals and stabilize macromolecular structure have been shown to afford radioprotection by reducing the induction of DNA damage. However, their ability to inhibit topoisomerases I & II, which play important roles in damage response pathways including DNA repair can enhance radiation damage under certain conditions. Since pool sizes of the topoisomerases differ not only between normal and tumor cells, but also among different tumors, it is anticipated that radiosensitization by hoechst-33342 can vary among tumors. The present studies were, therefore, undertaken to verify this proposition in human glioma (BMG-1 &U-87) and squamous carcinoma (4197 &4451) cell lines which differ in their biological behavior (ploidy, p53, cyclins, bcl, bax etc). Isotoxic concentrations of hoechst-33342 (IC50 i.e producing 50% cell kill) administered immediately following irradiation resulted in the radiosensitization of all cell lines, with a 4&7 fold increase in the cell death (loss of clonogenic cell survival) in U-87&BMG-1 and a 3 fold increase in 4197 &4451 cells. Growth inhibition and increase in cytogenetic damage (micronuclei formation) as well as delayed apoptosis observed under these conditions corroborated well with the enhanced cell death. The ligand induced a significant cell cycle delay, particularly in the late S and G2 phases of BMG-1, U-87 and 4197 cells, while no significant changes could be observed in 4451 cells. Higher endogenous levels of cyclin B1 found in both the glioma cell lines, was enhanced further by the ligand as compared to the squamous carcinoma cells. These results clearly demonstrate that the radiosensitizing effects of the ligand are indeed heterogeneous among different human tumor cell lines. The radiaosensitization is p53 independent and accompanied by enhanced mitotic death (linked to cytogenetic damage) as well as induction of cyclin B1 mediated apoptosis.

SDX-105 (Treanda™), Active in Non-Hodgkin's Lymphoma Cells, Induces the Mitotic Catastrophe Death Pathway.

SDX-105 (Treanda™) is an alkylating agent with a distinct mechanism of action that has been shown to be active in clinical trials in NHL and CLL patients refractory to traditional DNA-damaging agents. SDX-105 induces unique changes in gene expression in NHL cells and displays a lack of cross resistance with other 2-chloroethylamine alkylating agents. Quantitative PCR analysis confirmed that the G2/M checkpoint regulators Polo-like kinase 1 (PLK-1) and Aurora A kinase (AurkA) are down-regulated in the NHL cell line SU-DHL-1 after 8 hours of exposure to clinically relevant concentrations of SDX-105. No changes in these same genes were observed when cells were exposed to equi-toxic doses of chlorambucil or an active metabolite of cyclophosphamide. Because our previous studies demonstrated that SDX-105 treatment can activate apoptotic cell death pathways, we examined the ability of SDX-105 to induce cytotoxicity in cells unable to undergo ‘classical' caspase-mediated apoptosis. Multi-drug resistant MCF-7/ADR cells and p53 deficient RKO-E6 colon adenocarcinoma cells were exposed for two or three days to either 50 μM SDX-105 alone or 50 μM SDX-105 and 20 μM pan-caspase inhibitor zVAD-fmk. Although zVAD-fmk was able to inhibit SDX-105 induced increases in Annexin-V-positive cells, microscopic analysis of nuclear morphology using the DNA stain DAPI in cells treated with either SDX-105 alone or in combination with zVAD-fmk showed increased incidence of micronucleation. Multi/micro-nucleation and abnormal chromatin condensation are both hallmarks of mitotic catastrophe and have been observed in tumor cells exposed to microtubule-binding drugs such as the vinca alkaloids and taxanes. Activation of mitotic catastrophe may amplify the cytotoxicity of SDX-105 and its activity in tumor cells where classical apoptotic pathways are inhibited. This may explain, at least in part, the potent anti-tumor activity of SDX-105 in tumor cells refractory to conventional 2-chloroethylamine DNA-damaging agents. Additional studies are ongoing to further elaborate the role of mitotic catastrophe in SDX-105's mechanism of action. The capacity to induce mitotic catastrophe may explain the anti-tumor activity of SDX-105 in chemotherapy relapsed and resistant patients.