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ABSTRACT: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT.
The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used.
Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G(2)-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7.
This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.
International journal of radiation oncology, biology, physics 07/2012; 84(4):e507-14. · 4.59 Impact Factor
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ABSTRACT: Altered expression of the INT6 gene, encoding the e subunit of the translational initiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in the DNA damage response. INT6 was required for cell survival following γ-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with γ-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, this result could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.
Cancer Research 04/2012; 72(8):2006-16. · 7.86 Impact Factor
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ABSTRACT: The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.
DNA repair 02/2012; 11(4):441-8. · 4.20 Impact Factor
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ABSTRACT: H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.
PLoS ONE 01/2012; 7(11):e49245. · 4.09 Impact Factor
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ABSTRACT: Inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy owing to the frequent mutational activation of the PI3K-Akt-mTORC1 pathway in many cancers, including glioblastomas (GBMs). NVP-BEZ235 is a novel and potent dual PI3K/mTOR inhibitor that is currently in phase 1/2 clinical trials for advanced solid tumors. Here, we show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the two major kinases responding to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Consequently, NVP-BEZ235 blocks both nonhomologous end joining and homologous recombination DNA repair pathways resulting in significant attenuation of DSB repair. In addition, phosphorylation of ATMtargets and implementation of the G(2)/M cell cycle checkpoint are also attenuated by this drug. As a result, NVP-BEZ235 confers an extreme degree of radiosensitization and impairs DSB repair in a panel of GBM cell lines irrespective of their Akt activation status. NVP-BEZ235 also significantly impairs DSB repair in a mouse tumor model thereby validating the efficacy of this drug as a DNA repair inhibitor in vivo. Our results, showing that NVP-BEZ235 is a potent and novel inhibitor of ATM and DNA-PKcs, have important implications for the informed and rational design of clinical trials involving this drug and also reveal the potential utility of NVP-BEZ235 as an effective radiosensitizer for GBMs in the clinic.
Neoplasia (New York, N.Y.) 01/2012; 14(1):34-43. · 5.48 Impact Factor
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Cell cycle (Georgetown, Tex.) 10/2011; 10(19):3230. · 5.36 Impact Factor
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ABSTRACT: In metastatic prostate cancer, DOC-2/DAB2 interactive protein (DAB2IP) is often downregulated and has been reported as a possible prognostic marker to predict the risk of aggressive prostate cancer (PCa). Our preliminary results show that DAB2IP-deficient PCa cells are radioresistant. In this study, we investigated the anticancer drug Epothilone B (EpoB) for the modulation of radiosensitivity in DAB2IP-deficient human PCa cells.
We used a stable DAB2IP-knock down human PCa cell line, PC3 shDAB2IP, treated with EpoB, ionizing radiation (IR), or the combined treatment of EpoB and IR. The modulation of radiosensitivity was determined by surviving fraction, cell cycle distribution, apoptosis, and DNA double-strand break (DSB) repair. For in vivo studies, the PC3shDAB2IP xenograft model was used in athymic nude mice.
Treatment with EpoB at IC(50) dose (33.3 nM) increased cellular radiosensitivity in the DAB2IP-deficient cell line with a dose enhancement ratio of 2.36. EpoB delayed the DSB repair kinetics after IR and augmented the induction of apoptosis in irradiated cells after G(2)/M arrest. Combined treatment of EpoB and radiation enhanced tumor growth delay with an enhancement factor of 1.2.
We have demonstrated a significant radiation dose enhancement using EpoB in DAB2IP-deficient prostate cancer cells. This radiosensitization can be attributed to delayed DSB repair, prolonged G(2) block, and increased apoptosis in cells entering the cell cycle after G(2)/M arrest.
International journal of radiation oncology, biology, physics 11/2010; 78(4):1210-8. · 4.59 Impact Factor
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Cristel V Camacho,
Bipasha Mukherjee,
Brian McEllin,
Liang-Hao Ding,
Burong Hu,
Amyn A Habib,
Xian-Jin Xie,
Chaitanya S Nirodi,
Debabrata Saha,
Michael D Story,
Adayabalam S Balajee,
Robert M Bachoo,
David A Boothman, Sandeep Burma
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ABSTRACT: DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.
Carcinogenesis 10/2010; 31(10):1889-96. · 5.70 Impact Factor
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ABSTRACT: DNA double-strand breaks (DSBs) are the most lethal type of DNA damage induced by ionizing radiation or chemotherapeutic drugs used to eradicate cancer cells. The ability of cancer cells to effectively repair DSBs significantly influences the outcome of therapeutic regimens. Therefore, a new and important area of clinical cancer research is the development of DNA repair inhibitors that can be used as radio- or chemosensitizers. Nonhomologous end joining (NHEJ) is the predominant pathway for the repair of radiation-induced DSBs. A series of recent reports indicates that the epidermal growth factor receptor (EGFR) or its downstream components may modulate NHEJ through direct interaction with the DNA repair enzyme, DNA-dependent protein kinase. Because EGFR is overexpressed or activated in many cancers, these findings provide a compelling rationale for combining radiotherapy with therapies that block EGFR or its downstream signaling components. In this review, we delineate how these novel connections between a cell-surface receptor (EGFR) and a predominantly nuclear event (NHEJ) provide vulnerable nodes that can be selectively targeted to improve cancer therapy.
Seminars in radiation oncology 10/2010; 20(4):250-7. · 4.32 Impact Factor
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ABSTRACT: Aberrant epidermal growth factor receptor (EGFR) signaling is common in cancer. Increased expression of wild type and mutant EGFR is a widespread feature of diverse types of cancer. EGFR signaling in cancer has been the focus of intense investigation for decades primarily for two reasons. First, aberrant EGFR signaling is likely to play an important role in the pathogenesis of cancer, and therefore, the mechanisms of EGFR-mediated oncogenic signaling are of interest. Second, the EGFR signaling system is an attractive target for therapeutic intervention. EGFR gene amplification and overexpression are a particularly striking feature of glioblastoma (GBM), observed in approximately 40% of tumors. GBM is the most common primary malignant tumor of the central nervous system in adults. In approximately 50% of tumors with EGFR amplification, a specific EGFR mutant (EGFRvIII, also known as EGFR type III, de2-7, Delta EGFR) can be detected. This mutant is highly oncogenic and is generated from a deletion of exons 2 to 7 of the EGFR gene, which results in an in-frame deletion of 267 amino acids from the extracellular domain of the receptor. EGFRvIII is unable to bind ligand, and it signals constitutively. Although EGFRvIII has the same signaling domain as the wild type receptor, it seems to generate a distinct set of downstream signals that may contribute to an increased tumorigenicity. In this review, we discuss recent progress in key aspects of EGFR signaling in GBM, focusing on neuropathology, signal transduction, imaging of the EGFR, and the role of the EGFR in mediating resistance to radiation therapy in GBM.
Neoplasia (New York, N.Y.) 09/2010; 12(9):675-84. · 5.48 Impact Factor
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ABSTRACT: Glioblastomas (GBM) are lethal brain tumors that are highly resistant to therapy. The only meaningful improvement in therapeutic response came from use of the S(N)1-type alkylating agent temozolomide in combination with ionizing radiation. However, no genetic markers that might predict a better response to DNA alkylating agents have been identified in GBMs, except for loss of O(6-)methylguanine-DNA methyltransferase via promoter methylation. In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we show that loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) confers sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a functional analogue of temozolomide. We find that MNNG induces replication-associated DNA double-strand breaks (DSB), which are inefficiently repaired in PTEN-deficient astrocytes and trigger apoptosis. Mechanistically, this is because PTEN-null astrocytes are compromised in homologous recombination (HR), which is important for the repair of replication-associated DSBs. Our results suggest that reduced levels of Rad51 paralogs in PTEN-null astrocytes might underlie the HR deficiency of these cells. Importantly, the HR deficiency of PTEN-null cells renders them sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 due to synthetic lethality. In sum, our results tentatively suggest that patients with PTEN-null GBMs (about 36%) may especially benefit from treatment with DNA alkylating agents such as temozolomide. Significantly, our results also provide a rational basis for treating the subgroup of patients who are PTEN deficient with PARP inhibitors in addition to the current treatment regimen of radiation and temozolomide.
Cancer Research 07/2010; 70(13):5457-64. · 7.86 Impact Factor
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ABSTRACT: Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of poleta and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner.
Mechanisms of ageing and development 06/2010; 131(6):436-44. · 4.18 Impact Factor
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ABSTRACT: DAB2IP (DOC-2/DAB2 interactive protein) is a member of the RAS-GTPase-activating protein family. It is often downregulated in metastatic prostate cancer and has been reported as a possible prognostic marker to predict the risk of aggressive prostate cancer. In this study, we furnish several lines of evidence indicating that metastatic human prostate cancer PC3 cells deficient in DAB2IP (shDAB2IP) exhibit increased clonogenic survival in response to ionizing radiation (IR) compared with control cells expressing an endogenous level of DAB2IP (shVector). Radioresistance was also observed in normal prostate cells that are deficient in DAB2IP. This enhanced resistance to IR in DAB2IP-deficient prostate cancer cells is primarily due to faster DNA double-strand break (DSB) repair kinetics. More than 90% of DSBs were repaired in shDAB2IP cells by 8 hours after 2 Gy radiation, whereas only 60% of DSB repair were completed in shVector cells at the same time. Second, upon irradiation, DAB2IP-deficient cells enforced a robust G(2)-M cell cycle checkpoint compared with control cells. Finally, shDAB2IP cells showed resistance to IR-induced apoptosis that could result from a striking decrease in the expression levels of proapoptotic proteins caspase-3, caspase-8, and caspase-9, and significantly higher levels of antiapoptotic proteins Bcl-2 and STAT3 than those in shVector cells. In summary, DAB2IP plays a significant role in prostate cell survival following IR exposure due to enhanced DSB repair, robust G(2)-M checkpoint control, and resistance to IR-induced apoptosis. Therefore, it is important to identify patients with dysregulated DAB2IP for (a) assessing prostate cancer risk and (b) alternative treatment regimens.
Cancer Research 03/2010; 70(7):2829-39. · 7.86 Impact Factor
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ABSTRACT: DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.
Nucleic Acids Research 12/2009; 38(6):1821-31. · 8.03 Impact Factor
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ABSTRACT: The cellular response to DNA double-strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell-cycle-dependent manner. Here, we report that the crucial checkpoint signalling proteins-p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2-are phosphorylated rapidly by ATR in an ATM/Mre11/cell-cycle-independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM-deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM-deficient cells enforce an early G2/M checkpoint that is ATR-dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM-deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP-1), a protein involved in chromatin remodelling, is mediated by DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in a spatio-temporal manner in addition to ATM. We posit that ATM substrates involved in cell-cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA-PKcs in addition to ATM.
EMBO Reports 06/2009; 10(6):629-35. · 7.36 Impact Factor
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Bipasha Mukherjee,
Brian McEllin,
Cristel V Camacho,
Nozomi Tomimatsu,
Shyam Sirasanagandala,
Suraj Nannepaga,
Kimmo J Hatanpaa,
Bruce Mickey,
Christopher Madden,
Elizabeth Maher,
David A Boothman,
Frank Furnari,
Webster K Cavenee,
Robert M Bachoo, Sandeep Burma
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ABSTRACT: Glioblastoma multiforme (GBM) is the most lethal of brain tumors and is highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key glioma-specific mutation, epidermal growth factor receptor variant III (EGFRvIII), confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSB), the most lethal lesion inflicted by IR. The EGFR inhibitor gefitinib (Iressa) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated Akt-1 accelerates repair, implicating the PI3K/Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevated activation of a key DSB repair enzyme, DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Enhanced radioresistance is abrogated by the DNA-PKcs-specific inhibitor NU7026, and EGFRvIII fails to confer radioresistance in DNA-PKcs-deficient cells. In vivo, orthotopic U87-EGFRvIII-derived tumors display faster rates of DSB repair following whole-brain radiotherapy compared with U87-derived tumors. Consequently, EGFRvIII-expressing tumors are radioresistant and continue to grow following whole-brain radiotherapy with little effect on overall survival. These in vitro and in vivo data support our hypothesis that EGFRvIII expression promotes DNA-PKcs activation and DSB repair, perhaps as a consequence of hyperactivated PI3K/Akt-1 signaling. Taken together, our results raise the possibility that EGFR and/or DNA-PKcs inhibition concurrent with radiation may be an effective therapeutic strategy for radiosensitizing high-grade gliomas.
Cancer Research 06/2009; 69(10):4252-9. · 7.86 Impact Factor
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Seongmi Park,
Dawen Zhao,
Kimmo J Hatanpaa,
Bruce E Mickey,
Debabrata Saha,
David A Boothman,
Michael D Story,
Eric T Wong, Sandeep Burma,
Maria-Magdalena Georgescu,
Vivek M Rangnekar,
Sandili S Chauncey,
Amyn A Habib
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ABSTRACT: Therapeutic inhibition of mammalian target of rapamycin (mTOR) in cancer is complicated by the existence of a negative feedback loop linking mTOR to the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Thus, mTOR inhibition by rapamycin or TSC1/2 results in increased PI3K-Akt activation. The death domain kinase receptor interacting protein 1 (RIP1) plays a key role in nuclear factor-kappaB (NF-kappaB) activation and also activates the PI3K-Akt pathway through unknown mechanisms. RIP1 has recently been found to be overexpressed in glioblastoma multiforme, the most common adult primary malignant brain tumor, but not in grade II to III glioma. Our data suggest that RIP1 activates PI3K-Akt using dual mechanisms by removing the two major brakes on PI3K-Akt activity. First, increased expression of RIP1 activates PI3K-Akt by interrupting the mTOR negative feedback loop. However, unlike other signals that regulate mTOR activity without affecting its level, RIP1 negatively regulates mTOR transcription via a NF-kappaB-dependent mechanism. The second mechanism used by RIP1 to activate PI3K-Akt is down-regulation of cellular PTEN levels, which appears to be independent of NF-kappaB activation. The clinical relevance of these findings is highlighted by the demonstration that RIP1 levels correlate with activation of Akt in glioblastoma multiforme. Thus, our study shows that RIP1 regulates key components of the PTEN-PI3K-Akt-mTOR pathway and elucidates a novel negative regulation of mTOR signaling at the transcriptional level by the NF-kappaB pathway. Our data suggest that the RIP1-NF-kappaB status of tumors may influence response to treatments targeting the PTEN-PI3K-mTOR signaling axis.
Cancer Research 06/2009; 69(10):4107-11. · 7.86 Impact Factor
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ABSTRACT: Phosphorylated histone H2AX (gamma-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with gamma-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether gamma-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a gamma-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-gamma-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, gamma-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.
Biochemical and Biophysical Research Communications 04/2009; 380(4):752-7. · 2.48 Impact Factor
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ABSTRACT: DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.
Radiation Research 05/2008; 169(4):437-46. · 2.68 Impact Factor
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ABSTRACT: Cells from patients with genomic instability syndromes have high predisposition to cancer. However, little is known about whether these mutant cells have high susceptibility to oncogenic transformation. We have tested the hypothesis that a defect in maintaining genome integrity is necessary but not sufficient alone for oncogenic transformation and needs to collaborate with other signals in order to produce full oncogenic transformation. Using genetically matched primary cells deficient for the Fanconi complementation group C gene (Fancc) and the ataxia telangiectasia mutated gene (Atm), we found that certain forms of oncogenic activation and cooperation require a combination of genomic instability with increased expression of nucleophosmin (NPM) to prevent oncogenic stress-induced apoptosis or senescence. Intriguingly, co-expression of c-Myc and NPM leads to a synergistic increase in the proliferation rate in Fancc-/- or Atm-/- cells. Analysis of p53 stabilization and activation by c-Myc demonstrates that over-expression of NPM significantly reduces the accumulation of the activated p53 but not the stability of p53. Moreover, NPM is shown to enhance transforming activity of co-expressed Myc and Ras in wild-type and, to a greater degree, in Fancc-/- or Atm-/- cells, suggesting a role in oncogenic cooperation. Finally, a partial knockdown of NPM is sufficient to cause massive apoptosis in Fancc-/- or Atm-/- cells co-expressing c-Myc and Ras while sparing untransformed cells. Our study demonstrates a novel mechanism of NPM tumorigenesis by establishing NPM as a crucial inhibitor of oncogene-induced apoptosis and senescence.
Carcinogenesis 07/2007; 28(6):1163-70. · 5.70 Impact Factor
