Phase Cell Cycle Checkpoint Research Articles

Synergistic lethality between auranofin-induced oxidative DNA damage and ATR inhibition in cancer cells

AimsStudies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments, however, clinical use of ATR inhibitors in combination with DNA damaging chemotherapy is limited due to toxicity in healthy tissues. In this study, we investigated the synergistic anticancer effect between ATR inhibition and oxidative DNA damage induced by the thioredoxin reductase inhibitor auranofin. Main methodsCytotoxicity was evaluated by cell viability assays. Western blot, comet assay, immunostaining and flow cytometry were performed to dissect the underlying mechanisms. In vivo efficacy was examined against tumor xenografts. Key findingsNontoxic doses of auranofin alone increased the levels of reactive oxygen species (ROS) in cancer but not noncancerous cells, resulting in oxidative DNA damage and activation of the ATR DNA damage response pathway selectively in cancer cells. Inhibition of ATR in auranofin-treated cancer cells resulted in unscheduled firing of dormant DNA replication origins, abrogation of the S phase cell cycle checkpoint and extensive DNA breakage, leading to replication catastrophe and potent synergistic lethality. Both the antioxidant NAC and the DNA polymerase inhibitor aphidicolin reduced replication stress and synergistic cytotoxicity, implicating replication stress-driven catastrophic cell death resulted from collision between oxidative DNA damage and dysregulated DNA replication. In vivo, auranofin and VE822 coadministration enabled marked regressions of tumor xenografts, while each drug alone had no effect. SignificanceAs increased generation of ROS is a universal feature of tumors, our findings may open new routes to broaden the therapeutic potential of ATR inhibitors.

Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours

Defective DNA repair is being demonstrated to be a useful target in cancer treatment. Currently, defective repair is identified by specific gene mutations, however defective repair is a common feature of cancers without these mutations. DNA damage triggers cell cycle checkpoints that are responsible for co-ordinating cell cycle arrest and DNA repair. Defects in checkpoint signalling components such as ataxia telangiectasia mutated (ATM) occur in a low proportion of cancers and are responsible for reduced DNA repair and increased genomic instability. Here we have investigated the AURKA-PLK1 cell cycle checkpoint recovery pathway that is responsible for exit from the G2 phase cell cycle checkpoint arrest. We demonstrate that dysregulation of PP6 and AURKA maintained elevated PLK1 activation to promote premature exit from only ATM, and not ATR-dependent checkpoint arrest. Surprisingly, depletion of the B55α subunit of PP2A that negatively regulates PLK1 was capable of overcoming ATM and ATR checkpoint arrests. Dysregulation of the checkpoint recovery pathway reduced S/G2 phase DNA repair efficiency and increased genomic instability. We found a strong correlation between dysregulation of the PP6-AURKA-PLK1-B55α checkpoint recovery pathway with signatures of defective homologous recombination and increased chromosomal instability in several cancer types. This work has identified an unrealised source of G2 phase DNA repair defects and chromosomal instability that are likely to be sensitive to treatments targeting defective repair.

STAT3 imparts BRCAnessby impairing homologous recombination repair in Epstein-Barr virus-transformed B lymphocytes.

Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV re-shapes cellular DNA repair, ii) provide the first genome-wide evidence for insertions resulting from MMEJ in human cells, and iii) expand the range of cancers (EBV-related and -unrelated) that are likely to respond to synthetic lethal inhibitors given the high prevalence of cancers with constitutively active STAT3.

CircHMCU Promotes Proliferation and Metastasis of Breast Cancer by Sponging the let-7 Family

Circular RNA (circRNA), as a kind of novel identified non-coding RNA, has become the focus of attention for its vital physiological and pathological roles. However, the function and mechanism of circRNAs in the regulation of cancer progression are largely unknown. In the present study we found a circRNA termed circHMCU whose expression was associated with poor prognosis. It was upregulated in cell lines with high metastatic potential compared with its parental cell line and in breast cancer tissues compared with normal tissues. In vitro results proved that circHMCU could significantly promote proliferation, migration, and invasion abilities of breast cancer cells via affecting the G1 phase cell cycle checkpoint and the epithelial-mesenchymal transition (EMT) pathway. Further in vivo studies showed that overexpression of circHMCU contributed to rapid proliferation and lung metastasis of breast cancer. For determination of the mechanisms, bioinformatics analysis revealed two complementary sequences within circHMCU for let-7 microRNAs, which was validated by a luciferase reporter assay. Finally, let-7 microRNAs could rescue the functions of circHMCU in breast cancer via suppressing the expression of MCY, HMGA2, and CCND1. Taken together, our findings demonstrated that circHMCU exerted oncogenic functions in breast cancer and could be a used as a novel biomarker in the diagnosis and prognosis of breast cancer.

Drosophila Caliban mediates G1-S transition and ionizing radiation induced S phase checkpoint

ABSTRACTCell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Understanding the underlying mechanisms of cell cycle regulation is essential to address how normal development and tissue homeostasis are achieved. Here, we present a novel cell cycle regulator Caliban (Clbn), the Drosophila ortholog of human Serologically defined colon cancer antigen 1 (SDCCAG1) gene. We show that ionizing radiation induces expression of clbn, and over-expression of clbn blocks G1-to-S cell cycle transition in Drosophila, while flies loss of clbn have defective S phase checkpoint in response to irradiation. Mechanistically, induced expression of clbn suppressed E2F1 activity and down-regulates the DNA replication and expression of its downstream target cyclin E, a key regulator of G1-to-S transition. Meanwhile, clbn over-expression leads to upregulation of the CDK inhibitor Dacapo (Dap), and upregulated Dap is decreased when e2f1 is over-expressed. Furthermore, expression of clbn is down-regulated in cells with e2f1 over-expression or rbf1 knockdown, indicating that Clbn and E2F1 act antagonistically in mediating G1-to-S transition. Thus we provide genetic evidence that Clbn works together with E2F1 in regulating cell cycle progression, and Clbn is required for S phase cell cycle checkpoint in response to DNA damage.

Abstract A069: Increasing replication stress by reducing nucleoside levels sensitizes melanomas and non-small cell lung cancers to CHK1 inhibitor in vitro and in vivo

Abstract CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and as combinations with subclinical doses of hydroxyurea in melanoma and non-small cell lung cancer (NSCLC). We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is associated with defective S phase cell cycle checkpoint response to low dNTP levels. Loss of checkpoint response by overexpressing components of the checkpoint or inhibition of Wee1, convert CHK1 inhibitor insensitive cells to sensitive, and similarly depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. The increased DNA damage found with CHK1 inhibitor treatment is not sufficient to induce cell death, and cell death was triggered without entry into mitosis. We have found that about 20% of melanomas are sensitive to CHK1 inhibitor as a single agent, but we show that >70% of melanomas and NSCLCs are sensitive to combination with subclinical doses of hydroxyurea. The mechanism of cell death in the combination appears identical to CHK1 inhibitor alone in inhibitor-sensitive melanomas, with cells dying without entering mitosis. In half the cell melanomas tested, the combination produced a complete loss of viability; in the other half, the viable population after treatment was incapable of further proliferation in the absence in drug. The sensitivity to the combination is independent of other known risk factors, suggesting a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination. Citation Format: Zay Yar Oo, Martina Proctor, Alexander Stevenson, Jill Larsen, Brian G. Gabrielli. Increasing replication stress by reducing nucleoside levels sensitizes melanomas and non-small cell lung cancers to CHK1 inhibitor in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A069.

Abstract A33: Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo

Abstract CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and lung cancer. We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is loss of the S phase cell cycle checkpoint response. This is through a number of mechanisms including the uncoupling of CHK1 activation with the destabilization of CDC25A. Loss of checkpoint by over-expressing components of the checkpoint or inhibition of Wee1, covert CHK1 inhibitor insensitive cells to sensitive, and similarly depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. Loss of the S phase checkpoint provides cells with an adaptive advantage through introduction of moderate levels of genomic instability. The increased DNA damage found with CHK1 inhibitor treatment is not sufficient to induce cell death, but also involves a mechanism that is dependent in part on DNA-PK activity. Loss of S phase checkpoint function is predicted for <25% of melanomas and non-small cell lung squamous cell cancers. This is independent of other known risk factors, suggesting a significant proportion of melanoma and lung cancer patients could benefit from treatment with these drugs. Note: This abstract was not presented at the conference. Citation Format: Zay Yar Oo, Alexander Stevenson, Catherine Lanagan, Loredana Spoerri, Jill Larsen, Brian Gabrielli. Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A33.

DNA Fiber Assay upon Treatment with Ultraviolet Radiations

Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV type C (UVC) exposure, the DNA fiber assay can be used to analyse the new origin firing and DNA synthesis rate ( Jackson et al., 1998 ; Merrick et al., 2004 ; Alfano et al., 2016 ). The DNA fiber assay technique was conceived in the 90s and then further developed through the use of thymidine analogues (such as CldU and IdU), which are incorporated into the nascent DNA strands. By treating the cells in sequential mode with these analogues, which can be visualized through specific antibodies carrying different fluorophores, it is possible to monitor the replication fork activity and assess how this is influenced by UV radiations or others agents.

Abstract C189: Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma

Abstract The S phase cell cycle checkpoint is critical for maintaining genomic integrity. This checkpoint is triggered in response to DNA damage and replication stresses to control cell cycle progression, halting DNA replication and allowing the time to repair to maintain genomic integrity. If this checkpoint is defective, cells lose their ability to ensure the fidelity of replication and repair any damage, resulting in replicative stress and genomic instability which will subsequently increase the risk of cancer. We have discovered that a large proportion of melanoma cell lines lose the ability to halt replication when challenged with high dose hydroxyurea-induced replicative stress, despite normal cell cycle checkpoint signalling. We found that failure in the crosstalk between ATR-CHK1 checkpoint signalling and the cell cycle mechanism in destabilization of CDC25A. Although CHK1-dependent phosphorylation of CDC25A which triggers its degradation was detected, the S phase checkpoint defective melanoma cell lines fail to destabilize CDC25A, maintaining normal activation of CDK2/Cyclin E. The melanoma cells with this S phase checkpoint defect are also hyper-sensitive to killing by CHK1 inhibitor. We have previously shown that melanoma with high levels of replicative stress are very sensitive to CHK1 inhibitors. I will report our studies investigating whether CHK1 inhibitor sensitivity is a direct outcome of the replicative stress arising from the S phase checkpoint defect, or whether the loss of the ATR-CHK1-CDC25A dependent S phase checkpoint signalling mechanism is directly responsible for the hyper-sensitivity to CHK1 inhibitors. Citation Format: Zay Y. Oo, Sheena Daignault, James Chen, Brian Gabrielli. Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C189.

Autocrine regulation of glioma cell proliferation via pHe-sensitive K+ channels

Since the seminal studies of Otto Warburg in the 1920s, it has been widely recognized that cancers grow glycolytically, even in the presence of oxygen. This generates an abundance of protons in a gradient across most solid tumors with an acidic core and an alkaline rim. Whether and how this proton gradient may also serve in an autocrine fashion in these tumors is unclear. We demonstrate that human glioma cells form spheroids that act as a viable three-dimensional tumor model, forming physiologically relevant extracellular pH (pHe) and cell proliferation gradients. Using fluorescent cell cycle trackers, we determined that the rate of cell proliferation is directly dependent on pHe and that cells adjust their growth rate according to their position within the pH gradient. We further show that glioma cells sense pH via H(+)-sensitive K(+) channels, which translate changes in pH into changes in membrane voltage. These channels are tonically active and blocked by acidic pHe, quinine, and ruthenium red. Blockade of this K(+) conductance by acidic pHe or drug inhibition depolarized glioma cells and tumor spheroids and prevented their passage through the hyperpolarization-dependent G1-to-S phase cell cycle checkpoint, thereby inhibiting cell division. In this way, pHe directly determines the proliferative state of glioma cells.

Upregulation of the ATR‐CHEK1 pathway in oral squamous cell carcinomas

The ATR-CHEK1 pathway is upregulated and overactivated in Ataxia Telangiectasia (AT) cells, which lack functional ATM protein. Loss of ATM in AT confers radiosensitivity, although ATR-CHEK1 pathway overactivation compensates, leads to prolonged G(2) arrest after treatment with ionizing radiation (IR), and partially reverses the radiosensitivity. We observed similar upregulation of the ATR-CHEK1 pathway in a subset of oral squamous cell carcinoma (OSCC) cell lines with ATM loss. In the present study, we report copy number gain, amplification, or translocation of the ATR gene in 8 of 20 OSCC cell lines by FISH; whereas the CHEK1 gene showed copy number loss in 12 of 20 cell lines by FISH. Quantitative PCR showed overexpression of both ATR and CHEK1 in 7 of 11 representative OSCC cell lines. Inhibition of ATR or CHEK1 with their respective siRNAs resulted in increased sensitivity of OSCC cell lines to IR by the colony survival assay. siRNA-mediated ATR or CHEK1 knockdown led to loss of G(2) cell cycle accumulation and an increased sub-G(0) apoptotic cell population by flow cytometric analysis. In conclusion, the ATR-CHEK1 pathway is upregulated in a subset of OSCC with distal 11q loss and loss of the G(1) phase cell cycle checkpoint. The upregulated ATR-CHEK1 pathway appears to protect OSCC cells from mitotic catastrophe by enhancing the G(2) checkpoint. Knockdown of ATR and/or CHEK1 increases the sensitivity of OSCC cells to IR. These findings suggest that inhibition of the upregulated ATR-CHEK1 pathway may enhance the efficacy of ionizing radiation treatment of OSCC.

Oxidative stress leads to increased mutation frequency in a murine model of myelodysplastic syndrome

The myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it has been suggested that additional mutations lead to progression of MDS to AML, the causative agent(s) for such mutations remains unclear. Oxidative stress is a potential cause, therefore, we evaluated levels of reactive oxygen species (ROS) in NUP98-HOXD13 (NHD13) transgenic mice, a murine model for MDS. Increased levels of ROS were detected in bone marrow nucleated cells (BMNC) that express CD71, a marker for cell proliferation, as well as immature, lineage negative bone marrow nucleated cells from NHD13 mice. In addition to the increase in ROS, increased DNA double strand breaks and activation of a G2/M phase cell cycle checkpoint were noted in NHD13 BMNC. Finally, using an in vivo assay for mutation frequency, we detected an increased mutation frequency in NHD13 BMNC. These results suggest that oxidative stress may contribute to disease progression of MDS to AML through ineffective repair of DNA damage and acquisition of oncogenic mutations.

Abstract 3113: Effects of an up-regulated ATR-CHEK1 pathway in head and neck squamous cell carcinomas

Abstract Background: The ATR-CHEK1 pathway is upregulated in a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with loss of the G1 cell cycle checkpoint. We hypothesized that the upregulated ATR-CHEK1 pathway protects HNSCC from mitotic catastrophe (cell death) by enhancing the G2M phase checkpoint and that inhibition of this pathway may sensitize HNSCC to DNA damaging agents. Methods and Results: In HNSCC, we observed gain, amplification or translocation of the ATR gene in 8 of 20 cell lines studied; whereas the CHEK1 gene is partially lost in 12 of 20 cell lines. ATR and CHEK1 FISH in paraffin sections of primary head and neck squamous cell carcinomas confirmed our findings in HNSCC cell lines. We observed that both ATR and CHEK1 are overexpressed by quantitative PCR in five of eight HNSCC cell lines with loss of the G1 cell cycle checkpoint. Inhibition of ATR and CHEK1 with caffeine or specific inhibition with the respective siRNAs results in increased susceptibility of HNSCC to ionizing radiation by clonogenic cell survival assay. siRNA-mediated ATR or CHEK1 knockdown leads to loss of G2M checkpoint accumulation and increased sub-G0 apoptotic population by flow cytometric analysis. The mechanism of cell death after ATR and CHEK1 knockdown is by mitotic catastrophe, as evidenced by increased premature chromatin condensation. Conclusions: The ATR-CHEK1 pathway is up-regulated in a subset of HNSCC with loss of G1 phase cell cycle checkpoint. The up-regulated ATR-CHEK1 pathway protects these HNSCC from mitotic catastrophe by enhancing the G2M phase cell cycle checkpoints and inhibition of ATR and/or CHEK1 increase the sensitivity of the HNSCC to ionizing radiation. We conclude that inhibition of the up-regulated ATR-CHEK1 pathway in HNSCC may potentiate or increase the efficacy of the current therapeutic modalities for HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3113. doi:1538-7445.AM2012-3113

Dedifferentiated peripheral chondrosarcoma: a clinicopathologic, immunohistochemical, and molecular analysis of four cases

Peripheral dedifferentiated chondrosarcoma (DCS) is an exceedingly rare aggressive surface bone neoplasm in which a high-grade sarcoma arises within an osteochondroma. Four such examples were identified in our files, representing 11.1% of all DCS treated at our hospital in the years 1995-2010, and were the object of the present study. The patients were two men and two women ranging in age between 30 and 64 years, with tumors located in the pelvis (n = 2), in the scapula (n = 1), and the tibia (n = 1). Radiologically, there was evidence of a preexisting osteochondroma associated with aggressive osteolytic areas at the base and periphery of the exostosis, extending to the bone segment of origin and to the soft tissues. Immunohistochemical analysis of cell cycle regulators showed consistent loss in the expression of p16 and overexpression of cyclin D1, and to a lesser extent of RB and p53, in the anaplastic compartments of peripheral DCS in comparison with the well-differentiated cartilaginous components, while no significant expression of MDM2 was observed in any of the cases studied. Moreover, PDGFRα was absent in both tumor components, and PDGF-Rβ was strongly and diffusely positive in all the cases. Finally, no mutations were identified in exons 4-9 of the TP53 gene in both cases, showing positivity for p53 in the anaplastic component. In conclusion, our study shows that alterations of genes implicated in the regulation of the G1 to the S phase cell cycle checkpoint contribute to the process of dedifferentiation in peripheral secondary chondrosarcoma (CS), although the molecular mechanisms seem at least in part to differ from those involved in the process of dedifferentiation of central CS. PDGFRβ could represent a potential target for treatments with receptor tyrosine kinase inhibitors in peripheral DCS.

Abstract 4197: A DNA damage checkpoint response to unrepaired ultraviolet radiation-induced lesions which is defective in melanoma

Abstract Ultraviolet radiation (UVR) is a major environmental risk factor in the development of skin cancer. Here we describe a G2 phase cell cycle checkpoint response to suberythemal doses of UVR that is defective in a majority of melanoma cell lines tested. The majority of cells in the basal layer of the epidermis are quiescent, and exposure to suberythemal doses of UVR produces not only DNA damage but also provides a mitogenic stimulus to drive cells into the cell cycle. The majority of UVR-induced DNA lesions are rapidly repaired by NER mechanisms, but a small number of DNA lesions fail to be repaired during G1 phase and are encountered by the replication fork during S phase. This triggers Werner helicase (Wrn) dependent lesion bypass, allowing replication to continue relatively unhindered, but producing gaps in the newly replicated DNA strand opposite the lesions. These single stranded DNA gaps recruit replication protein A (RPA) and components of the ATR-Chk1 checkpoint signaling responsible for the G2 phase arrest observed. The RPA foci also co-localise components of the error free RAD18-RAD51 post replication repair pathway which are essential for repair of the gaps and checkpoint signaling. This checkpoint mechanism ensures detection and repair of all UVR induced DNA damage before proceeding into mitosis. Loss of this checkpoint response increases the rate of UVR induced mutation. Thus this checkpoint response is critical for maintaining genomic stability in the epidermal melanocytes and keratinocytes, and loss of this checkpoint mechanism may account for the increased UVR signature DNA mutations found in melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4197. doi:10.1158/1538-7445.AM2011-4197

Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G(2)-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 microg/mL chloroquine, 15 microM genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eight cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G(2)-phase cells entering mitosis, using histone H3 phosphorylation analysis. The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G(2)-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Overcoming HRS is reliant on the function of the early G(2)-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.

Deficient G2-M and S Checkpoints are Associated with Increased Lung Cancer Risk: A Case-Control Analysis

Loss or attenuation of cell cycle checkpoint function can compromise the fidelity of DNA due to insufficient time to repair DNA damage. We evaluated cell cycle checkpoints in 747 patients with lung cancer and 745 controls by measuring the proportions of cultured peripheral blood lymphocytes in G2-M and S phases. As an indicator of G2-M phase or S phase cell cycle checkpoint function, the gamma-radiation-induced cell accumulation index at G2-M or S phase was defined as (percentage of cells in G2-M or S with ionizing radiation exposure - percentage of cells in G2-M or S without ionizing radiation exposure) / (percentage of cells in G2-M or S without ionizing radiation exposure). We found that the median cell accumulation index was significantly lower in patients than that in controls at both the G2-M phase (0.774 versus 0.882, P = 0.002) and the S phase (0.226 versus 0.243, P = 0.001). When the median value for the cell accumulation index at the G2-M or S phase in the controls was used as the cutoff point, the reduced indices at G2-M and S phases were associated with 1.28-fold (95% confidence interval, 1.04-1.58) and 1.30-fold (95% confidence interval, 1.06-1.61) increased lung cancer risks, respectively. Analyses stratified by histology showed some heterogeneity. Additionally, cell accumulation indices at both G2-M and S phases were not associated with clinical stages. We conclude that attenuated functions of G2-M and S cell cycle checkpoints might be susceptibility markers for lung cancer.

Low-Dose Hyper-radiosensitivity is not Caused by a Failure to Recognize DNA Double-Strand Breaks

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.

Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.

ATM-dependent and -independent gene expression changes in response to oxidative stress, gamma irradiation, and UV irradiation.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiencies, telangiectasias, sensitivity to ionizing radiation, and high predisposition for malignancies. The ataxia telangiectasia mutated (ATM) gene encodes a protein (ATM) with serine/threonine kinase activity. DNA-double strand breaks are known to increase its kinase activity. While cells from individuals with AT are attenuated in their G(1)-, S- and G(2)-phase cell cycle checkpoint functions in response to gamma irradiation and oxidative stress, their response to UV irradiation appears to be equivalent to that of wild-type cells. In this study, we investigated changes in gene expression in response to gamma irradiation, oxidative stress, and UV irradiation, focusing on the dependence on ATM. Doses for all three treatments were selected that resulted in roughly an equivalent induction of a G(1) checkpoint response and inhibition of progression through S phase. To investigate gene expression changes, logarithmically growing wild-type and AT dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 micro M t-butyl-hydroperoxide), or UV radiation (7.5 J/m(2)), and RNA was harvested 6 h after treatment. Gene expression analysis was performed using the NIEHS Human ToxChip 2.0 with approximately 1900 cDNA clones representing known genes and ESTs. All three treatments resulted in distinct patterns of gene expression changes, as shown previously. ATM-dependent and ATM-independent components were detected within these patterns, as were novel indications of involvement of ATM in regulation of transcription factors such as SP1, AP1 and MTF1.