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Title: Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

Abstract

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.

Authors:
 [1];  [2];  [3];  [3];  [3];  [3];  [4];  [1];  [2];  [5]
  1. Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan)
  2. (Japan)
  3. Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555 (Japan)
  4. Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan)
  5. Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555 (Japan). E-mail: rokayasu@nirs.go.jp
Publication Date:
OSTI Identifier:
20857926
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 351; Journal Issue: 3; Other Information: DOI: 10.1016/j.bbrc.2006.10.094; PII: S0006-291X(06)02331-X; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; APOPTOSIS; DNA REPAIR; GENETIC RADIATION EFFECTS; INHIBITION; IRRADIATION; PROTEINS; RECOMBINATION; STRAND BREAKS; TUMOR CELLS

Citation Formats

Noguchi, Miho, Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Yu, Dong, Hirayama, Ryoichi, Ninomiya, Yasuharu, Sekine, Emiko, Kubota, Nobuo, Ando, Koichi, Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, and Okayasu, Ryuichi. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2006.10.094.
Noguchi, Miho, Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Yu, Dong, Hirayama, Ryoichi, Ninomiya, Yasuharu, Sekine, Emiko, Kubota, Nobuo, Ando, Koichi, Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, & Okayasu, Ryuichi. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin. United States. doi:10.1016/j.bbrc.2006.10.094.
Noguchi, Miho, Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Yu, Dong, Hirayama, Ryoichi, Ninomiya, Yasuharu, Sekine, Emiko, Kubota, Nobuo, Ando, Koichi, Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, and Okayasu, Ryuichi. Fri . "Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin". United States. doi:10.1016/j.bbrc.2006.10.094.
@article{osti_20857926,
title = {Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin},
author = {Noguchi, Miho and Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555 and Yu, Dong and Hirayama, Ryoichi and Ninomiya, Yasuharu and Sekine, Emiko and Kubota, Nobuo and Ando, Koichi and Heavy-Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555 and Okayasu, Ryuichi},
abstractNote = {In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.},
doi = {10.1016/j.bbrc.2006.10.094},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 351,
place = {United States},
year = {Fri Dec 22 00:00:00 EST 2006},
month = {Fri Dec 22 00:00:00 EST 2006}
}
  • Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ andmore » aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation in RIC1 cells. It is known that 53BP1 is involved in both DNA-PK dependent and independent NHEJ. Therefore we suggest that DNA-PK independent NHEJ repair DSBs under the condition of decreased DNA-PK activity, which causes reduction of HR efficiency.« less
  • Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials:more » Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.« less
  • Purpose: In addition to invasive tumor cells, endothelial cells (ECs) of the tumor vasculature are an important target for anticancer radiotherapy. The purpose of the present work is to investigate how 17-N-allilamino-17-demethoxygeldanamycin (17AAG), known as an anticancer drug inhibiting heat shock protein 90 (Hsp90), modifies radiation responses of human vascular ECs. Methods and Materials: The ECs cultured from human umbilical veins were exposed to {gamma}-irradiation, whereas some EC samples were pretreated with growth factors and/or 17AAG. Postirradiation cell death/survival and morphogenesis were assessed by means of terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick end labeling or annexin V staining and clonogenicmore » and tube-formation assays. The 17AAG-affected expression and phosphorylation of radioresistance-related proteins were probed by means of immunoblotting. Dominant negative or constitutively activated Akt was transiently expressed in ECs to manipulate Akt activity. Results: It was found that nanomolar concentrations of 17AAG sensitize ECs to relatively low doses (2-6 Gy) of {gamma}-irradiation and abolish the radioprotective effects of vascular endothelial growth factor and basic fibroblast growth factor. The drug-induced radiosensitization of ECs seems to be caused by prevention of Hsp90-dependent phosphorylation (activation) of Akt that results in blocking the radioprotective phosphatidylinositol 3-kinase/Akt pathway. Conclusions: Clinically achievable concentrations of 17AAG can decrease the radioresistance intrinsic to vascular ECs and minimize the radioprotection conferred upon them by tumor-derived growth factors. These findings characterize 17AAG as a promising radiosensitizer for the tumor vasculature.« less
  • Gefitinib (Iressa{sup R}, ZD1839) is a selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT signaling activation. It has been shown that inhibition of Hsp90 function can enhance antitumor activity of EGFR-TKI. XRCC1 is an important scaffold protein in base excision repair, which could be regulated by ERK1/2 and AKT pathways. However, the role of ERK1/2 and AKT-mediated XRCC1 expression in gefitinib alone or combination with an Hsp90 inhibitor-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. In this study, gefitinib treatment decreasedmore » XRCC1 mRNA and protein expression through ERK1/2 and AKT inactivation in two NSCLC cells, A549 and H1975. Knocking down XRCC1 expression by transfection with small interfering RNA of XRCC1 enhanced the cytotoxicity and cell growth inhibition of gefitinib. Combining treatment of gefitinib with an Hsp90 inhibitor resulted in enhancing the reduction of XRCC1 protein and mRNA levels in gefitinib-exposed A549 and H1975 cells. Compared to a single agent alone, gefitinib combined with an Hsp90 inhibitor resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells. Furthermore, transfection with constitutive active MKK1 or AKT vectors rescued the XRCC1 protein level as well as the cell survival suppressed by an Hsp90 inhibitor and gefitinib. These findings suggested that down-regulation of XRCC1 can enhance the sensitivity of gefitinib for NSCLC cells. - Highlights: • Gefitinib treatment decreased XRCC1 mRNA and protein expression in NSCLC cells. • Knocking down XRCC1 expression enhanced the cytotoxic effect of gefitinib. • Gefitinib combined with an Hsp90 inhibitor resulted in synergistically cytotoxicity.« less
  • To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombinationmore » substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination.« less