ku-55933 and Ataxia-Telangiectasia

Ataxia-telangiectasia mutated (ATM) kinase is a serine/threonine protein kinase and plays a key role in DNA double-strand breaks repair. Thus, ATM is considered a promising target for radiotherapy and chemotherapy sensitizing. Herein, we report the discovery of ATM agonist A22 and inhibitor A41 by computational methods and further biological evaluation. Among them, A22 exhibited low cytotoxicity in vitro and might serve as a useful tool for ATM research. Moreover, we firstly proved that ATM inhibitors could sensitize Irinotecan and Etoposide in a time-dependent manner on MCF-7 and SW480 cells, antagonism in a short period treatment while synergy at a long-term treatment and ATM agonist worked in an opposite way of ATM inhibitors. Further mechanism study demonstrated that the antagonism effect of ATM inhibitors with chemotherapeutic agents in a short period was resulting from inhibiting the p53/p21 axis to accelerate G1/S phase cell-cycle transition and promote cell survival. Additionally, A41 displayed antitumor effects combined with a chemotherapeutic drug in the SW480 xenograft model, indicating that A41 is a promising ATM inhibitor, which could increase the antitumor effect of chemotherapeutic drugs in vivo. All in all, these findings will guide the combination of ATM inhibitors with chemotherapeutic agents in further preclinical and clinical studies.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line, Tumor; DNA-Binding Proteins; Humans; Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases

Inhibitors of ataxia-telangiectasia mutated (ATM), such as KU-55933 (Ku), represent a promising class of novel anticancer drugs. In addition, the biguanide derivative phenformin exhibits antitumor activity superior to that of the AMPK activator metformin. Herein, we assessed the potential combinatorial therapeutic efficacy of phenformin and Ku when used to inhibit the growth of liver cancer cells, and we assessed the mechanisms underlying such efficacy. The Hep-G2 and SMMC-7721 liver cancer cell lines were treated with phenformin and Ku either alone or in combination, after which the impact of these drugs on cellular proliferation was assessed via 3-(4,5-dimethylthiazol) 2, 5-diphenyltetrazolium and colony formation assays, whereas Transwell assays were used to gauge cell migratory activity. The potential synergy between these two drugs was assessed using the CompuSyn software, while flow cytometry was employed to evaluate cellular apoptosis. In addition, western blotting was utilized to measure p-ATM, p-AMPK, p-mTOR, and p-p70s6k expression, while mitochondrial functionality was monitored via morphological analyses, JC-1 staining, and measurements of ATP levels. Phenformin and Ku synergistically impacted the proliferation, migration, and apoptotic death of liver cancer cells. Together, these compounds were able to enhance AMPK phosphorylation while inhibiting the phosphorylation of mTOR and p70s6k. These data also revealed that phenformin and Ku induced mitochondrial dysfunction as evidenced by impaired ATP synthesis, mitochondrial membrane potential, and abnormal mitochondrial morphology. These findings suggest that combination treatment with phenformin and Ku may be an effective approach to treating liver cancer via damaging mitochondria within these tumor cells.

Topics: AMP-Activated Protein Kinases; Apoptosis; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; China; Drug Synergism; Drug Therapy, Combination; Humans; Liver Neoplasms; Mitochondria; Morpholines; Phenformin; Phosphorylation; Pyrones; Ribosomal Protein S6 Kinases, 70-kDa; TOR Serine-Threonine Kinases

To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines.. Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis.. The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells.. Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.

Topics: Antineoplastic Agents; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Bile Duct Neoplasms; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; DNA Damage; Humans; Morpholines; Protein Kinase Inhibitors; Pyrazines; Pyrones; Sulfones; Tumor Suppressor Protein p53

The nucleosomal histone protein H2AX is specifically phosphorylated (γ-H2AX) adjacent to DNA double-strand breaks (DSBs) and is used for quantifying DSBs. Many chemotherapies and ionizing radiation (IR) used in cancer treatment result in DSBs. Therefore, γ-H2AX has a significant potential as a biomarker in evaluating patient sensitivity and responsiveness to IR and chemotherapy.. Here, we report a flow cytometry-based quantification of γ-H2AX (FCM-γ-H2AX assay) customized for clinical practice.. We validated that our method is able to detect DNA damage in peripheral blood mononuclear cells (PBMCs) treated with DSB inducing agents. The method also detected the DNA repair deficiency in PBMCs treated with DNA repair inhibitors, as well as the deficiency in DNA repair signaling in PBMCs from two ataxia telangiectasia patients.. The FCM-γ-H2AX assay has sufficient analytical sensitivity and precision to measure levels of DNA damage and DNA repair for clinical purposes. © 2016 International Clinical Cytometry Society.

Topics: Aminoglycosides; Ataxia Telangiectasia; Chromones; DNA; DNA Breaks, Double-Stranded; DNA Repair; Dose-Response Relationship, Radiation; Enediynes; Flow Cytometry; Gamma Rays; Histones; Humans; Leukocytes, Mononuclear; Morpholines; Phosphorylation; Primary Cell Culture; Pyrones

The clonogenic assay examines cell sensitivity to toxic agents and has been shown to correlate with normal tissue sensitivity to radiotherapy in cancer patients. The clonogenic assay is not clinically applicable due to its intra-individual variability and the time frame of the protocol. We aimed to develop a clinically applicable assay that correlated with the clonogenic assay.. We have developed a faster and less labor-intensive cell division assay (CD assay) using flow cytometry and incorporation of a fluorescent thymidine analogue. The CD assay was calibrated to the clonogenic assay and optimized for peripheral blood lymphocytes.. Following ionizing radiation of primary human skin fibroblasts, the four-day CD assay gave similar results as the 14-day clonogenic survival assay. In lymphocytes isolated from patient blood samples, the CD assay was able to detect increased radiosensitivity in ataxia telangiectasia patients and increased radiosensitivity after in vitro treatment with DNA-PK and ATM inhibitors. The CD assay found a variation in the intrinsic radiosensitivity of lymphocytes isolated from healthy control samples. The CD assay was able to measure the anti-proliferation effect of different chemotherapeutic drugs in lymphocytes.. Our results indicate that the CD assay is a fast and reliable method to measure the anti-proliferation effect of DNA-damaging agents with a potential to find the most sensitive patients in the work-up before cancer treatment.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Proliferation; Cells, Cultured; Chromones; Clinical Laboratory Techniques; Colony-Forming Units Assay; DNA Damage; DNA-Activated Protein Kinase; Fanconi Anemia; Fibroblasts; Flow Cytometry; Humans; Lymphocytes; Morpholines; Pyrones; Radiation Tolerance; Radiation, Ionizing; Skin

To study the effects of Jaridonin, a novel diterpenoid from isodon rubescens, on the cell cycle of human gastric cancer cells and its molecular mechanism of action.. Flow cytometry was used to analyze the cell cycle distribution and expression of ataxia telangiectasia mutated kinase (ATM) after Jaridonin treatment. Western blot was performed to detect the expression of cell cycle-related proteins.. The results of flow cytometry showed that the percentages of MGC-803 cells in G(2)/M phase at 6 hours after 0, 10, 20 μmol/L Jaridonin-treatment were (10.8±2.2)%, (18.2±2.5)%, (27.3±3.2)%, respectively; those at 12 hours after Jaridonin-treatment were (12.0±1.5)%, (24.1±2.0)% and (39.7±5.2)%, respectively, indicating a G2/M phase arrest of MGC-803 cells was resulted in a time- and dose-dependent manner. The expressions of ATM, Chk1, Chk2, phosphorylated Cdc2 and CDK2 were up-regulated in the MGC-803 cells after Jaridonin treatment, while the levels of Cdc2 and CDK2 were decreased. KU-55933, an inhibitor of ATM, reversed the expression of relevant proteins and G(2)/M phase arrest induced by Jaridonin.. Jaridonin can significantly induce G(2)/M arrest in gastric cancer MGC-803 cells. Its mechanism may be related to the activation of ATM and Chk1/2, and inactivation of Cdc2 and CDK2 phosphorylation.

Topics: Antineoplastic Agents, Phytogenic; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Diterpenes, Kaurane; Humans; Isodon; Morpholines; Neoplasm Proteins; Phosphorylation; Pyrones; Stomach Neoplasms

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, ATM (A-T, mutated), encodes a 370-kDa Ser/Thr protein kinase. ATM not only mediates cellular response to DNA damage but also acts as an activator of Akt in response to insulin. However, despite intensive studies, the mechanism underlying the neuronal degeneration symptoms of human A-T is still poorly understood. We found that the topoisomerase inhibitors etoposide and camptothecin readily induced apoptosis in undifferentiated proliferating SH-SY5Y cells but could not induce apoptosis in neuronally differentiated SH-SY5Y cells. In addition, etoposide induced p53 phosphorylation and H2AX foci formation in proliferating SH-SY5Y cells but failed to do so in differentiated SH-SY5Y cells. Moreover, while inhibition of ATM in undifferentiated SH-SY5Y cells partially protected them from etoposide-induced apoptosis, the same treatment had no effect on cell viability in differentiated SH-SY5Y cells. These results suggest that DNA damage or defective response to DNA damage is not the cause of neuronal cell death in human A-T. In contrast, we discovered that Akt phosphorylation was inhibited when ATM activity was suppressed in differentiated SH-SY5Y cells. Furthermore, inhibition of ATM induced apoptosis following serum starvation in neuronally differentiated SH-SY5Y cells but could not trigger apoptosis under the same conditions in undifferentiated proliferating SH-SY5Y cells. These results demonstrate that ATM mediates the Akt signaling and promotes cell survival in neuron-like human SH-SY5Y cells, suggesting that impaired activation of Akt is the reason for neuronal degeneration in human A-T.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Camptothecin; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; DNA Damage; DNA-Binding Proteins; Etoposide; Histones; Humans; Morpholines; Neuroblastoma; Neurons; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrones; Signal Transduction; Topoisomerase Inhibitors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G(2) checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G(2) to M phase transition, impaired G(2) checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G(2) checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G(2) checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G(2) checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G(2) checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G(2) checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Case-Control Studies; Cell Cycle Proteins; Cell Division; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Centromere; Chromosomal Instability; Cyclin B1; DNA-Binding Proteins; Esophageal Neoplasms; G2 Phase; Gamma Rays; Gene Knockdown Techniques; HeLa Cells; Humans; Mitotic Index; Morpholines; Nasopharyngeal Neoplasms; Protein Serine-Threonine Kinases; Pyrones; Telomerase; Translocation, Genetic; Tumor Suppressor Proteins

Cells deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome) show increased yields of both simple and complex chromosomal aberrations after high doses (>0.5Gy) of ionizing radiation (X-rays or γ-rays), however less is known on how these cells respond at low dose. Previously we had shown that the increased chromosome aberrations in ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex exchanges. The linear dose-response term for simple exchanges was significantly higher in NBS cells compared to wild type cells, but not for AT cells. However, AT cells have a high background level of exchanges compared to wild type or NBS cells that confounds the understanding of low dose responses. To understand the sensitivity differences for high to low doses, chromosomal aberration analysis was first performed at low dose-rates (0.5Gy/d), and results provided further evidence for the lack of sensitivity for exchanges in AT cells below doses of 1Gy. Normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, showed increased numbers of exchanges at a dose of 1Gy and higher, but were similar to wild type cells at 0.5Gy or below. These results were confirmed using siRNA knockdown of ATM. The present study provides evidence that the increased radiation sensitivity of AT cells for chromosomal exchanges found at high dose does not occur at low dose.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line; Chromosome Aberrations; DNA Damage; DNA-Binding Proteins; Dose-Response Relationship, Radiation; Fibroblasts; Gamma Rays; Gene Knockdown Techniques; Humans; Morpholines; Nuclear Proteins; Protein Serine-Threonine Kinases; Pyrones; Radiation Tolerance; Tumor Suppressor Proteins

Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with K(i) and IC(50) values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Movement; Cell Survival; DNA-Binding Proteins; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gamma Rays; Glioma; Humans; Insulin; MAP Kinase Signaling System; Morpholines; Neoplasm Invasiveness; Phosphoserine; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrones; Radiation-Sensitizing Agents; Thioxanthenes; Tumor Suppressor Proteins

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.

Topics: Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; DNA Damage; DNA Repair Enzymes; DNA-Binding Proteins; Fibroblasts; Fluorescent Antibody Technique; G1 Phase; Genes, cdc; Histones; Humans; Infrared Rays; Intracellular Signaling Peptides and Proteins; Morpholines; Nijmegen Breakage Syndrome; Nuclear Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Pyrones; Tumor Suppressor p53-Binding Protein 1; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; X-Rays