High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts.

Hematopoietic stem cells (HSCs) and their progeny sustain lifetime hematopoiesis. Aging alters HSC function, number, and composition and increases risk of hematological malignancies, but how these changes occur in HSCs remains unclear. Signaling via p38 mitogen-activated kinase (p38MAPK) has been proposed as a candidate mechanism underlying induction of HSC aging. Here, using genetic models of both chronological and premature aging, we describe a multimodal role for p38α, the major p38MAPK isozyme in hematopoiesis, in HSC aging. We report that p38α regulates differentiation bias and sustains transplantation capacity of HSCs in the early phase of chronological aging. However, p38α decreased HSC transplantation capacity in the late progression phase of chronological aging. Furthermore, codeletion of p38α in mice deficient in ataxia-telangiectasia mutated, a model of premature aging, exacerbated aging-related HSC phenotypes seen in ataxia-telangiectasia mutated single-mutant mice. Overall, these studies provide new insight into multiple functions of p38MAPK, which both promotes and suppresses HSC aging context dependently.

ATR activation is regulated by dimerization of ATR activating proteins.

The checkpoint kinase ATR regulates DNA repair, cell cycle progression, and other DNA damage and replication stress responses. ATR signaling is stimulated by an ATR activating protein, and in metazoan cells, there are at least two ATR activators: TOPBP1 and ETAA1. Current evidence indicates TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism, but several aspects of this mechanism remain undefined. For example, ATR and its obligate binding partner ATR interacting protein (ATRIP) form a tetrameric complex consisting of two ATR and two ATRIP molecules, but whether TOPBP1 or ETAA1 dimerization is similarly required for ATR function is unclear. Here, we show that fusion of the TOPBP1 and ETAA1 ATR activation domains (AADs) to dimeric tags makes them more potent activators of ATR in vitro. Furthermore, induced dimerization of both AADs using chemical dimerization of a modified FKBP tag enhances ATR kinase activation and signaling in cells. ETAA1 forms oligomeric complexes mediated by regions of the protein that are predicted to be intrinsically disordered. Induced dimerization of a "mini-ETAA1" protein that contains the AAD and Replication Protein A (RPA) interaction motifs enhances ATR signaling, rescues cellular hypersensitivity to DNA damaging agents, and suppresses micronuclei formation in ETAA1-deficient cells. Together, our results indicate that TOPBP1 and ETAA1 dimerization is important for optimal ATR signaling and genome stability.

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.

First radiobiological characterization of the McCune-Albright syndrome: influence of the ATM protein and effect of statins + bisphosphonates treatment.

PURPOSE: MacCune-Albright syndrome (MAS) is a rare autosomal dominant osteo-hormonal disorder. MAS is characterized by a severe form of polyostotic fibrous dysplasia, 'café-au-lait' pigmentation of the skin and multiple endocrinopathies. MAS was shown to be caused by mosaic missense somatic mutations in the GNAS gene coding for the alpha-subunit of the stimulatory G-protein. MAS is also associated with radiation-induced malignant tumors, like osteosarcoma, fibrosarcoma and chondrosarcoma but their origin remains misunderstood. In parallel, bisphosphonates treatment was shown to improve the MAS patients' outcome, notably by increasing bone density but, again, the molecular mechanisms supporting these observations remain misunderstood. MATERIALS AND METHODS: Here, by using fibroblast and osteoblast cell lines derived from 2 MAS patients, the major radiobiological features of MAS were investigated. Notably, the clonogenic cell survival, the micronuclei and the γH2AX, pATM and MRE11 immunofluorescence assays were applied to MAS cells. RESULTS: It appears that cells from the 2 MAS patients are associated with a moderate but significant radiosensitivity, a delayed radiation-induced nucleoshuttling of the ATM kinase likely caused by its sequestration in cytoplasm, suggesting impaired DNA double-strand breaks (DSB) repair and signaling in both fibroblasts and osteoblasts. Such delay may be partially corrected by using bisphosphonates combined with statins, which renders cells more radioresistant. CONCLUSIONS: Our findings represent the first radiobiological characterization of fibroblasts and osteoblasts providing from MAS patients. Although the number of studied cases is reduced, our findings suggest that the MAS cells tested belong to the group of syndromes associated with moderate but significant radiosensitivity. Further investigations are however required to secure the clinical transfer of the combination of bisphosphonates and statins, to reduce the disease progression and to better evaluate the potential risks linked to radiation exposure.

Phosphorylation of seryl-tRNA synthetase by ATM/ATR is essential for hypoxia-induced angiogenesis.

Hypoxia-induced angiogenesis maintains tissue oxygen supply and protects against ischemia but also enhances tumor progression and malignancy. This is mediated through activation of transcription factors like hypoxia-inducible factor 1 (HIF-1) and c-Myc, yet the impact of hypoxia on negative regulators of angiogenesis is unknown. During vascular development, seryl-tRNA synthetase (SerRS) regulates angiogenesis through a novel mechanism by counteracting c-Myc and transcriptionally repressing vascular endothelial growth factor A (VEGFA) expression. Here, we reveal that the transcriptional repressor role of SerRS is inactivated under hypoxia through phosphorylation by ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and RAD3-related (ATR) at Ser101 and Ser241 to attenuate its DNA binding capacity. In zebrafish, SerRSS101D/S241D, a phosphorylation-mimicry mutant, cannot suppress VEGFA expression to support normal vascular development. Moreover, expression of SerRSS101A/S241A, a phosphorylation-deficient and constitutively active mutant, prevents hypoxia-induced binding of c-Myc and HIF-1 to the VEGFA promoter, and activation of VEGFA expression. Consistently, SerRSS101A/S241A strongly inhibits normal and tumor-derived angiogenesis in mice. Therefore, we reveal a key step regulating hypoxic angiogenesis and highlight the importance of nuclear SerRS in post-developmental angiogenesis regulation in addition to vascular development. The role of nuclear SerRS in inhibiting both c-Myc and HIF-1 may provide therapeutic opportunities to correct dysregulation of angiogenesis in pathological settings.

Complete loss of ATM function augments replication catastrophe induced by ATR inhibition and gemcitabine in pancreatic cancer models.

BACKGROUND: Personalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC. METHODS: Through kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo. RESULTS: Complete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts. CONCLUSIONS: ATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

Hepatitis C Virus Mediated Inhibition of miR-181c Activates ATM Signaling and Promotes Hepatocyte Growth.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection promotes hepatocyte growth and progress to hepatocellular carcinoma. We previously observed that HCV infection of hepatocytes transcriptionally down-regulates miR-181c expression through CCAAT/enhancer binding protein ß (C/EBP-ß). Here, we examined the role of miR-181c in the regulation of cell cycle progression in relation to HCV infection. In silico analysis suggested that ataxia-telangiectasia mutated (ATM) protein, a protein kinase, is a direct target of miR-181c. ATM is a central mediator of response for cellular DNA double-strand break. APPROACH AND RESULTS: Our results demonstrated that ATM expression is higher in HCV-infected hepatocytes and chronic HCV-infected liver biopsy specimens. We have shown a direct interaction of miR-181c with the 3' untranslated region of ATM, and the presence of ATM in miR-181c-associated RNA-induced silencing complex. Exogenous expression of miR-181c inhibited ATM expression and activation of its downstream molecules, Chk2 and Akt. On the other hand, introduction of anti-miR-181c restored ATM and phosphorylated Akt. Furthermore, introduction of miR-181c significantly inhibited phospho-cyclin-dependent kinase 2 (CDK2) and cyclin-A expression, arresting cell cycle progression, whereas overexpression of miR-181c promoted apoptosis of HCV-infected hepatocytes and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. In addition, miR-181c significantly regressed tumor growth in the xenograft human hepatocellular carcinoma mouse model. CONCLUSIONS: Together, our results suggest that HCV infection suppresses miR-181c in hepatocytes, resulting in ATM activation and apoptosis inhibition for promotion of cell cycle progression. The results provide mechanistic insight into understanding the role of miR-181c in HCV-associated hepatocyte growth promotion, and may have the potential for therapeutic intervention.

Accumulation of Cytoplasmic DNA Due to ATM Deficiency Activates the Microglial Viral Response System with Neurotoxic Consequences.

ATM (ataxia-telangiectasia mutated) is a PI3K-like kinase best known for its role in the DNA damage response (DDR), especially after double-strand breaks. Mutations in the ATM gene result in a condition known as ataxia-telangiectasia (A-T) that is characterized by cancer predisposition, radiosensitivity, neurodegeneration, sterility, and acquired immune deficiency. We show here that the innate immune system is not spared in A-T. ATM-deficient microglia adopt an active phenotype that includes the overproduction of proinflammatory cytokines that are toxic to cultured neurons and likely contribute to A-T neurodegeneration. Causatively, ATM dysfunction results in the accumulation of DNA in the cytoplasm of microglia as well as a variety of other cell types. In microglia, cytoplasmic DNA primes an antiviral response via the DNA sensor, STING (stimulator of interferon genes). The importance of this response pathway is supported by our finding that inhibition of STING blocks the overproduction of neurotoxic cytokines. Cytosolic DNA also activates the AIM2 (absent in melanoma 2) containing inflammasome and induces proteolytic processing of cytokine precursors such as pro-IL-1ß. Our study furthers our understanding of neurodegeneration in A-T and highlights the role of cytosolic DNA in the innate immune response.SIGNIFICANCE STATEMENT Conventionally, the immune deficiencies found in ataxia-telangiectasia (A-T) patients are viewed as defects of the B and T cells of the acquired immune system. In this study, we demonstrate the microglia of the innate immune system are also affected and uncover the mechanism by which this occurs. Loss of ATM (ataxia-telangiectasia mutated) activity leads to a slowing of DNA repair and an accumulation of cytoplasmic fragments of genomic DNA. This ectopic DNA induces the antivirus response, which triggers the production of neurotoxic cytokines. This expands our understanding of the neurodegeneration found in A-T and offers potentially new therapeutic options.

Inhibition of CHK 1 (Checkpoint Kinase 1) Elicits Therapeutic Effects in Pulmonary Arterial Hypertension.

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a debilitating disease associated with progressive vascular remodeling of distal pulmonary arteries leading to elevation of pulmonary artery pressure, right ventricular hypertrophy, and death. Although presenting high levels of DNA damage that normally jeopardize their viability, pulmonary artery smooth muscle cells (PASMCs) from patients with PAH exhibit a cancer-like proproliferative and apoptosis-resistant phenotype accounting for vascular lumen obliteration. In cancer cells, overexpression of the serine/threonine-protein kinase CHK1 (checkpoint kinase 1) is exploited to counteract the excess of DNA damage insults they are exposed to. This study aimed to determine whether PAH-PASMCs have developed an orchestrated response mediated by CHK1 to overcome DNA damage, allowing cell survival and proliferation. Approach and Results: We demonstrated that CHK1 expression is markedly increased in isolated PASMCs and distal PAs from patients with PAH compared with controls, as well as in multiple complementary animal models recapitulating the disease, including monocrotaline rats and the simian immunodeficiency virus-infected macaques. Using a pharmacological and molecular loss of function approach, we showed that CHK1 promotes PAH-PASMCs proliferation and resistance to apoptosis. In addition, we found that inhibition of CHK1 induces downregulation of the DNA repair protein RAD 51 and severe DNA damage. In vivo, we provided evidence that pharmacological inhibition of CHK1 significantly reduces vascular remodeling and improves hemodynamic parameters in 2 experimental rat models of PAH. CONCLUSIONS: Our results show that CHK1 exerts a proproliferative function in PAH-PASMCs by mitigating DNA damage and suggest that CHK1 inhibition may, therefore, represent an attractive therapeutic option for patients with PAH.

Pellino1 regulates reversible ATM activation via NBS1 ubiquitination at DNA double-strand breaks.

DNA double-strand break (DSB) signaling and repair are critical for genome integrity. They rely on highly coordinated processes including posttranslational modifications of proteins. Here we show that Pellino1 (Peli1) is a DSB-responsive ubiquitin ligase required for the accumulation of DNA damage response proteins and efficient homologous recombination (HR) repair. Peli1 is activated by ATM-mediated phosphorylation. It is recruited to DSB sites in ATM- and γH2AX-dependent manners. Interaction of Peli1 with phosphorylated histone H2AX enables it to bind to and mediate the formation of K63-linked ubiquitination of NBS1, which subsequently results in feedback activation of ATM and promotes HR repair. Collectively, these results provide a DSB-responsive factor underlying the connection between ATM kinase and DSB-induced ubiquitination.

SMAD7 in keratinocytes promotes skin carcinogenesis by activating ATM-dependent DNA repair and an EGFR-mediated cell proliferation pathway.

SMA- and MAD-related protein 7 (SMAD7) is a general inhibitor of transforming growth factor-ß (TGF-ß) signaling that acts through interaction and degradation of TGF-ß receptors. SMAD7 has been demonstrated to be transcriptionally upregulated in chemical-induced skin tumors and TGF-ß-treated normal keratinocytes. To evaluate the function of SMAD7 in skin carcinogenesis in vivo, Smad7 transgenic mice that specifically express either wild-type (WT) SMAD7 (TG-Smad7-WT) or mutant SMAD7 (TG-Smad7-MT) in keratinocytes, as well as Smad7 keratinocyte-specific knockout (Smad72f/2f-K14Cre) mice, were subjected to chemical-induced skin carcinogenesis. WT-SMAD7-expressing transgenic mice showed significantly greater papilloma formation than did non-TG control and Smad7-MT mice. The expression of WT-SMAD7 attenuated DNA damage-induced apoptosis in epidermal keratinocytes by stimulating the ATM-dependent DNA repair pathway. Nonetheless, overexpression of WT-SMAD7 caused a susceptibility to 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation through activation of epidermal growth factor (EGF) signaling. In agreement with the transgenic mouse data, keratinocyte-specific deletion of SMAD7 markedly suppressed the tumor formation by inhibiting ATM and epidermal growth factor receptor (EGFR) signaling. Moreover, specific inhibition of EGFR signaling attenuated the hyperproliferation and tumor formation in TG-Smad7-WT mice. Taken together, these data support a novel role for SMAD7 as a tumor promoter in skin carcinogenesis where SMAD7 stimulates the DNA repair pathway and EGFR signaling activation.

Deletion of Atm in Tumor but not Endothelial Cells Improves Radiation Response in a Primary Mouse Model of Lung Adenocarcinoma.

Stereotactic body radiotherapy is utilized to treat lung cancer. The mechanism of tumor response to high-dose radiotherapy (HDRT) is controversial, with competing hypotheses of increased direct tumor cell killing versus indirect effects on stroma including endothelial cells. Here we used dual recombinase technology in a primary murine lung cancer model to test whether tumor cells or endothelial cells are critical HDRT targets. Lenti-Cre deleted one or two copies of ataxia-telangiectasia mutated gene (Atm; KPAFL/+ or KPAFL/FL), whereas adeno-FlpO-infected mice expressed Cre in endothelial cells to delete one or both copies of Atm (KPVAFL/+ or KPVAFL/FL) to modify tumor cell or endothelial cell radiosensitivity, respectively. Deletion of Atm in either tumor cells or endothelial cells had no impact on tumor growth in the absence of radiation. Despite increased endothelial cell death in KPVAFL/FL mice following irradiation, tumor growth delay was not significantly increased. In contrast, a prolonged tumor growth delay was apparent in KPAFL/FL mice. Primary tumor cell lines lacking Atm expression also demonstrated enhanced radiosensitivity as determined via a clonogenic survival assay. These findings indicate that tumor cells, rather than endothelial cells, are critical targets of HDRT in primary murine lung cancer. SIGNIFICANCE: These findings establish radiosensitizing tumor cells rather than endothelial cells as the primary mechanism of tumor response to high-dose radiotherapy, supporting efforts to maximize local control by radiosensitizing tumors cells.See related commentary by Hallahan, p. 704.

Regulation of ATM and ATR by SMARCAL1 and BRG1.

The G2/M checkpoint is activated on DNA damage by the ATM and ATR kinases that are regulated by post-translational modifications. In this paper, the transcriptional co-regulation of ATM and ATR by SMARCAL1 and BRG1, both members of the ATP-dependent chromatin remodeling protein family, is described. SMARCAL1 and BRG1 co-localize on the promoters of ATM and ATR; downregulation of SMARCAL1 and BRG1 results in transcriptional repression of ATM/ATR and overriding of the G2/M checkpoint leading to mitotic abnormalities. On doxorubicin-induced DNA damage, SMARCAL1 and BRG1 are upregulated and these two proteins in turn, upregulate the expression of ATM/ATR. The transcriptional response to DNA damage is feedback regulated by phospho-ATM as it binds to the promoters of SMARCAL1, BRG1, ATM and ATR on DNA damage. The regulation of ATM/ATR is rendered non-functional in Schimke Immuno-Osseous Dysplasia where SMARCAL1 is mutated and in Coffin-Siris Syndrome where BRG1 is mutated. Thus, an intricate transcriptional regulation of DNA damage response genes mediated by SMARCAL1 and BRG1 is present in mammalian cells.

ATR-mediated proteome remodeling is a major determinant of homologous recombination capacity in cancer cells.

The ATR kinase is crucial for genome maintenance, but the mechanisms by which ATR controls the DNA repair machinery are not fully understood. Here, we find that long-term chronic inhibition of ATR signaling severely impairs the ability of cells to utilize homologous recombination (HR)-mediated DNA repair. Proteomic analysis shows that chronic ATR inhibition depletes the abundance of key HR factors, suggesting that spontaneous ATR signaling enhances the capacity of cells to use HR-mediated repair by controlling the abundance of the HR machinery. Notably, ATR controls the abundance of HR factors largely via CHK1-dependent transcription, and can also promote stabilization of specific HR proteins. Cancer cells exhibit a strong dependency on ATR signaling for maintaining elevated levels of HR factors, and we propose that increased constitutive ATR signaling caused by augmented replication stress in cancer cells drives the enhanced HR capacity observed in certain tumor types. Overall, these findings define a major pro-HR function for ATR and have important implications for therapy by providing rationale for sensitizing HR-proficient cancer cells to PARP inhibitors.

Artesunate promotes G2/M cell cycle arrest in MCF7 breast cancer cells through ATM activation.

BACKGROUND: Recent studies have revealed that artesunate (ART) has clear anti-tumor activity, suggesting that it could be a good candidate chemotherapeutic agent. In this study, we researched the inhibitory effect of ART on MCF7 cells and explored the possible mechanisms. METHODS: MTT assay was used to detect the effect of ART on the proliferation of MCF7 cells. Crystal violet staining was used to observe morphological and quantitative changes. Flow cytometry was used to detect the cell cycle of the drug-acting MCF7 cells. In addition, western blotting was used to detect the drug influence on expression of the ATM, phospho-ATM(S1981), H2AX, γH2AX(S139), CHK2 and phospho-CHK2(T68), cdc25C, and phospho-cdc25C(S216). RESULTS: In the experimental groups, the proliferation of MCF7 cells was inhibited in a dose-dependent manner and the original cell morphology was lost. The number of G2/M phase cells in the experimental groups increased significantly, and the expression of DNA damage response-associated proteins was significantly increased, such as phospho-ATM(S1981), γH2AX(S139), phospho-CHK2(T68), and phospho-cdc25C(S216). CONCLUSIONS: ART can inhibit cell proliferation and promote G2/M arrest in MCF7 cells through ATM activation and the ensuing "ATM-Chk2-Cdc25C" pathway, thus implicating ART as a novel candidate for breast cancer chemotherapy.

[Topoisomerase inhibitor upregulates MICA/B expression in breast cancer cells through ATM/ATR and NF-κB pathway].

OBJECTIVE: To investigate the effects of chemotherapeutic agents widely used in clinical practice on major histocompatibility complex class I-related chain A and B (MICA/B) expression in breast cancer cells, and to explore the molecular mechanisms involved. METHODS: We examined MICA/B mRNA and surface protein expressions in breast cancer cells treated with chemotherapeutic agents by real-time RT-PCR and flow cytometry respectively. The blocking effects of ataxia telangiectasia mutated and Rad3-related kinase (ATM/ATR) inhibitor caffeine and nuclear factor κB (NF-κB) inhibitor pynolidine dithiocarbamate (PDTC) on etoposide-upregulated MICA/B mRNA and surface protein expressions were investigated. Electrophoretic mobility shift assay (EMSA) was taken to investigate whether etoposide enhanced the binding of NF-κB to MICA/B gene promoter. RESULTS: Three topoisomerase inhibitors etoposide, camptothecin and doxorubicine upregulated MICA and MICB mRNA expressions in breast cancer cell MCF-7. Comparing to no-drug-treated cells, MICA mRNA levels increased to (1.68±0.17), (2.54±0.25) and (3.42±0.15) fold, and levels of MICB mRNA increased to (1.82±0.24), (1.56±0.05) and (5.84±0.57) fold respectively in cancer cells treated by etoposide at the concentrations of 5, 20 and 100 µmol/L (P<0.05). MICA and MICB mRNA levels also increased significantly when MCF-7 cells were incubated with camptothecin or doxorubicine at the specific concentrations (P<0.05). MICB mRNA expression also increased slightly in another breast cancer cell SK-BR-3 treated by topoisomerase II inhibitors etoposide and camptothecin (P<0.05). Furthermore, etoposide and camptothecin upregulated MICA/B surface protein expression in MCF-7 cells (P<0.05), and the upregulation was found in both living and apoptotic cells. Our study showed that etoposide induced-MICA/B expression in MCF-7 was inhibited by caffeine at different concentrations. When cancer cells were treated by caffeine with 1, 5 and 10 mmol/L, MICA mRNA levels decreased from (3.75±0.25) to (0.89±0.05), (0.81±0.02) and (0.48±0.04) fold respectively (P<0.001), and MICB mRNA levels decreased from (6.85±0.35) to (1.36±0.13), (0.76±0.06) and (0.56±0.03) fold (P<0.05), while MICA/B protein levels decreased from (3.42±0.05) to (1.32±0.03), (1.21±0.06) and (1.14±0.03) fold (P<0.001), indicating that etoposide-induced MICA/B expression was inhibited by ATM/ATR inhibitor. Similarly, NF-κB inhibitor PDTC also inhibited MICA/B mRNA and protein expressions induced by etoposide significantly when MCF-7 cells were incubated with PDTC at the concentrations of 10, 50 and 100 µmol/L (P<0.05), indicating that NF-κB was also involved in this process. EMSA showed that the binding of NF-κB to MICA/B promoter enhanced in MCF-7 cells after etoposide treatment. CONCLUSION: Topoisomerase inhibitor increased MICA/B mRNA and protein expressions in breast cancer cells, indicating that chemotherapeutic agents might increase the recognizing and killing ability of immunocytes to breast cancer cells. ATM/ATR and NF-κB pathways might be involved in it.

53BP1 loss suppresses the radiosensitizing effect of icotinib hydrochloride in colorectal cancer cells.

BACKGROUND: This study aimed to investigate the influence of the expression of P53-binding protein 1 (53BP1), a key component in DNA damage repair pathways, on the radiosensitizing effect of icotinib hydrochloride in colorectal cancer and to elucidate the mechanisms underlying this influence. MATERIALS AND METHODS: Real-time RT-PCR and Western blotting were performed to verify the gene-knockout effect of 53BP1 small hairpin RNA (ShRNA), and colony formation assay was employed to investigate the influence of 53BP1 downregulation on the radiosensitizing effect of icotinib hydrochloride in HCT116 cells. Cell apoptosis, cell cycle distributions, and histone H2AX (γ-H2AX) fluorescence foci after 53BP1 knockdown were evaluated. Relative protein expression in the ataxia telangiectasia mutated kinase (ATM)-checkpoint kinase-2 (CHK2)-P53 pathway was measured by Western blot analysis to unravel the molecular mechanisms linking the pathway to the above phenomena. RESULTS: Icotinib hydrochloride increased the radiosensitivity of HCT116 cells; however, this effect was suppressed by the downregulation of 53BP1 expression, a change that inhibited cell apoptosis, increased the percentage of HCT116 cells arrested in S-phase and inhibited the protein expression of key molecules in the ATM-CHK2-P53 apoptotic pathway. CONCLUSION: Our studies confirmed that the loss of 53BP1 serves as a negative regulator of the radiosensitizing effect of icotinib in part by suppressing the ATM-CHK2-P53 apoptotic pathway.

Radiation-induced G2/M arrest rarely occurred in glioblastoma stem-like cells.

PURPOSE: The purpose of this study is to systematically study the cell-cycle alterations of glioblastoma stem-like cells (GSLCs) after irradiation, possibly enriching the mechanisms of radioresistance of GSLCs. MATERIALS AND METHODS: GSLCs were enriched and identified, and then the radioresistance of GSLCs was validated by analyzing cell survival, cell proliferation, and radiation-induced apoptosis. The discrepancy of the cell-cycle distribution and expression of cell-cycle-related proteins between GSLCs and glioblastoma differentiated cells (GDCs) after irradiation was completely analyzed. RESULTS: The survival fractions and the cell viabilities of GSLCs were significantly higher than those of GDCs after irradiation. Radiation-induced apoptosis was less prominent in GSLCs than in GDCs. After irradiation with high-dose X-rays, the percentages of GDCs in G2/M phase was evidently increased. However, radiation-induced G2/M arrest occurred less frequently in GSLCs, but S-phase arrest occurred in GSLCs after irradiation with 8 Gy. Further mechanistic studies showed that the expressions levels of Cdc25c, Cdc2, and CyclinB1 in GSLCs were not apparently changed after irradiation, while those of p-ATM and p-Chk1 were sharply increased after irradiation in GSLCs. The basal level of Cdc25c expression in GSLCs was much higher than that in GDCs. CONCLUSIONS: We explored the cell-cycle alterations and cell-cycle-related proteins expression levels in GSLCs after irradiation, providing a novel mechanism of radioresistance of GSLCs.

Ionizing radiation does not impair the mechanisms controlling genetic stability during T cell receptor gene rearrangement in mice.

PURPOSE: To determine whether low dose/low dose rate radiation-induced genetic instability may result from radiation-induced inactivation of mechanisms induced by the ATM-dependent DNA damage response checkpoint. To this end, we analysed the faithfulness of T cell receptor (TR) gene rearrangement by V(D)J recombination in DNA from mice exposed to a single dose of X-ray or chronically exposed to low dose rate γ radiation. MATERIALS AND METHODS: Genomic DNA obtained from the blood or the thymus of wild type or Ogg1-deficient mice exposed to low (0.1) or intermediate/high (0.2-1 Gy) doses of radiation either by acute X-rays exposure or protracted exposure to low dose-rate γ-radiation was used to analyse by PCR the presence of illegitimate TR gene rearrangements. RESULTS: Radiation exposure does not increase the onset of TR gene trans-rearrangements in irradiated mice. In mice where it happens, trans-rearrangements remain sporadic events in developing T lymphocytes. CONCLUSION: We concluded that low dose/low dose rate ionizing radiation (IR) exposure does not lead to widespread inactivation of ATM-dependent mechanisms, and therefore that the mechanisms enforcing genetic stability are not impaired by IR in developing lymphocytes and lymphocyte progenitors, including BM-derived hematopoietic stem cells, in low dose/low dose rate exposed mice.