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Arese M, Mahmoudian M, Bussolino F. RNA aptamer-mediated gene therapy of prostate cancer: lessons from the past and future directions. Expert Opin Drug Deliv 2023; 20:1609-1621. [PMID: 38058168 DOI: 10.1080/17425247.2023.2292691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2023] [Accepted: 12/04/2023] [Indexed: 12/08/2023]
Abstract
INTRODUCTION Prostate cancer (PCa) is one of the most prevalent cancers in the world, and the fifth cause of death from cancer in men. Among the non-surgical treatments for PCa, gene therapy strategies are in the early stages of development and recent clinical trials have provided new insights suggesting promising future. AREAS COVERED Recently, the creation of targeted gene delivery systems, based on specific PCa cell surface markers, has been viewed as a viable therapeutic approach. Prostate-specific membrane antigen (PSMA) is vastly expressed in nearly all prostate malignancies, and the intensity of expression increases with tumor aggressiveness, androgen independence, and metastasis. RNA aptamers are short and single-stranded oligonucleotides, which selectively bind to a specific ligand on the surface of the cells, which makes them fascinating small molecules for target delivery of therapeutics. PSMA-selective RNA aptamers represent great potential for developing targeted-gene delivery tools for PCa. EXPERT OPINION This review provides a thorough horizon for the researchers interested in developing targeted gene delivery systems for PCa via PSMA RNA aptamers. In addition, we provided general information about different prospects of RNA aptamers including discovery approaches, stability, safety, and pharmacokinetics.
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Affiliation(s)
- Marco Arese
- Department of Oncology, University of Torino, Candiolo, Italy
- Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Mohammad Mahmoudian
- Department of Oncology, University of Torino, Candiolo, Italy
- Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
| | - Federico Bussolino
- Department of Oncology, University of Torino, Candiolo, Italy
- Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Italy
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Ihara M, Shichijo K, Kudo T, Ohtsuka K. Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73. Int J Hyperthermia 2019; 36:438-443. [PMID: 30922135 DOI: 10.1080/02656736.2019.1587009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
PURPOSE Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity. METHODS Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72. RESULTS The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37 °C after heat treatment at 44 °C for 15 min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3 h of incubation at 37 °C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3 h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment. CONCLUSIONS These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.
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Affiliation(s)
- Makoto Ihara
- a Department of Radioisotope Medicine, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute , Nagasaki University , Nagasaki , Japan
| | - Kazuko Shichijo
- b Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute , Nagasaki University , Nagasaki , Japan
| | - Takashi Kudo
- a Department of Radioisotope Medicine, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute , Nagasaki University , Nagasaki , Japan
| | - Kenzo Ohtsuka
- c Laboratory of Cell and Stress Biology, College of Bioscience and Biotechnology , Chubu University , Kasugai , Japan
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Nakagawa Y, Kajihara A, Takahashi A, Murata AS, Matsubayashi M, Ito SS, Ota I, Nakagawa T, Hasegawa M, Kirita T, Ohnishi T, Mori E. BRCA2 protects mammalian cells from heat shock. Int J Hyperthermia 2017; 34:795-801. [PMID: 28891354 DOI: 10.1080/02656736.2017.1370558] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
PURPOSE Heat shock induces DNA double-strand breaks (DSBs) in mammalian cells. Mammalian cells are capable of repairing DSBs by utilising the homologous recombination (HR) pathway. Breast cancer susceptibility gene 2 (BRCA2) is known to regulate the HR pathway. Here, we investigate the role of BRCA2 in repairing DNA damage induced by heat shock. MATERIALS AND METHODS Chinese hamster lung fibroblast cell lines and human tongue squamous cell carcinoma SAS cells were used. RAD51 foci formation assay was used as an HR indicator. Heat sensitivity was analysed with colony forming assays. Phosphorylated histone H2AX (γH2AX) intensity, which correlates with the number of DSBs, was analysed with flow cytometry. RESULTS RAD51 foci appeared with heat shock, and the number of cells with RAD51 foci was maximal at about 4 h after heat shock. Heat-induced RAD51 foci co-localised with γH2AX foci. BRCA2-deficient cells were sensitive to heat when compared to their parental wild-type cells. Heat-induced γH2AX was higher in BRCA2-deficient cells compared to parental cells. In SAS cells, cells transfected with BRCA2-siRNA were more sensitive to heat than cells transfected with negative control siRNA. Apoptotic bodies increased in number more rapidly in BRCA2-siRNA transfected cells than in cells transfected with negative control siRNA when cells were observed at 48 h after a heat treatment. In addition, cells deficient in BRCA2 were incapable of activating heat-induced G2/M arrest. CONCLUSION BRCA2 has a protecting role against heat-induced cell death. BRCA2 might be a potential molecular target for hyperthermic cancer therapy.
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Affiliation(s)
- Yosuke Nakagawa
- a Department of Oral and Maxillofacial Surgery , Nara Medical University , Nara , Japan
| | - Atsuhisa Kajihara
- a Department of Oral and Maxillofacial Surgery , Nara Medical University , Nara , Japan
| | | | - Akiho S Murata
- c Department of Future Basic Medicine , Nara Medical University , Nara , Japan
| | - Masaya Matsubayashi
- c Department of Future Basic Medicine , Nara Medical University , Nara , Japan
| | - Soichiro S Ito
- a Department of Oral and Maxillofacial Surgery , Nara Medical University , Nara , Japan
| | - Ichiro Ota
- d Department of Otolaryngology - Head and Neck Surgery , Nara Medical University , Nara , Japan
| | - Takahiko Nakagawa
- c Department of Future Basic Medicine , Nara Medical University , Nara , Japan
| | - Masatoshi Hasegawa
- e Department of Radiation Oncology , Nara Medical University , Nara , Japan
| | - Tadaaki Kirita
- a Department of Oral and Maxillofacial Surgery , Nara Medical University , Nara , Japan
| | - Takeo Ohnishi
- e Department of Radiation Oncology , Nara Medical University , Nara , Japan
| | - Eiichiro Mori
- c Department of Future Basic Medicine , Nara Medical University , Nara , Japan
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Howard M, Beltran C, Sarkaria J, Herman MG. Characterization of relative biological effectiveness for conventional radiation therapy: a comparison of clinical 6 MV X-rays and 137Cs. JOURNAL OF RADIATION RESEARCH 2017; 58:608-613. [PMID: 28444207 PMCID: PMC5737853 DOI: 10.1093/jrr/rrx018] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2016] [Revised: 12/23/2016] [Indexed: 05/28/2023]
Abstract
Various types of radiation are utilized in the treatment of cancer. Equal physical doses of different radiation types do not always result in the same amount of biological damage. In order to account for these differences, a scaling factor known as the relative biological effectiveness (RBE) can be used. 137Cesium (137Cs) has been used as a source of radiation in a significant body of radiation therapy research. However, high-energy X-rays, such as 6 MV X-rays, are currently used clinically to treat patients. To date, there is a gap in the literature regarding the RBE comparison of these two types of radiation. Therefore, the purpose of this study was to investigate the RBE of 137Cs relative to that of 6 MV X-rays. To determine the RBE, five cell lines were irradiated [Chinese hamster ovary (CHO); human lung adenocarcinoma (A549); human glioma (U251); human glioma (T98); and human osteosarcoma (U2OS)] by both types of radiation and assessed for cell survival using a clonogenic assay. Three of the five cell lines resulted in RBE values of ~1.00 to within 11% for all survival fractions, showing the physical and biological dose for these two types of radiation were equivalent. The other two cell lines gave RBE values differing from 1.00 by up to 36%. In conclusion, the results show the range in biological effect seen between cell lines, and therefore cell type must be considered when characterizing RBE.
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Affiliation(s)
- Michelle Howard
- Corresponding author. Department of Radiation Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Tel.: 1-507-293-2841; Fax: 1-507-293-2870;
| | - Chris Beltran
- Department of Radiation Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
| | - Jann Sarkaria
- Department of Radiation Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
| | - Michael G Herman
- Department of Radiation Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
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Oei AL, Ahire VR, van Leeuwen CM, Ten Cate R, Stalpers LJA, Crezee J, Kok HP, Franken NAP. Enhancing radiosensitisation of BRCA2-proficient and BRCA2-deficient cell lines with hyperthermia and PARP1-i. Int J Hyperthermia 2017; 34:39-48. [PMID: 28540821 DOI: 10.1080/02656736.2017.1324642] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Poly(ADP-ribose)polymerase1 (PARP1) is an important enzyme in regulating DNA replication. Inhibition of PARP1 can lead to collapsed DNA forks which subsequently causes genomic instability, making DNA more susceptible in developing fatal DNA double strand breaks. PARP1-induced DNA damage is generally repaired by homologous recombination (HR), in which BRCA2 proteins are essential. Therefore, BRCA2-deficient tumour cells are susceptible to treatment with PARP1-inhibitors (PARP1-i). Recently, BRCA2 was shown to be down-regulated by hyperthermia (HT) temporarily, and this consequently inactivated HR for several hours. In this study, we investigated whether HT exclusively interferes with HR by analysing thermal radiosensitisation of BRCA2-proficient and deficient cells. After elucidating the equitoxicity of PARP1-i on BRCA2-proficient and deficient cells, we studied the cell survival, apoptosis, DNA damage (γ-H2AX foci and comet assay) and cell cycle distribution after different treatments. PARP1-i sensitivity strongly depends on the BRCA2 status. BRCA2-proficient and deficient cells are radiosensitised by HT, indicating that HT does not exclusively act by inhibition of HR. In all cell lines, the addition of HT to radiotherapy and PARP1-i resulted in the lowest cell survival, the highest levels of DNA damage and apoptotic levels compared to duo-modality treatments. Concluding, HT not only inhibits HR, but also has the capability of radiosensitising BRCA2-deficient cells. Thus, in case of BRCA2-mutation carriers, combining HT with PARP1-i may boost the treatment efficacy. This combination therapy would be effective for all patients with PARP1-i regardless of their BRCA status.
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Affiliation(s)
- Arlene L Oei
- a Laboratory for Experimental Oncology and Radiobiology (LEXOR) , Center for Experimental and Molecular Medicine , Amsterdam , The Netherlands.,b Department of Radiotherapy , University of Amsterdam , Amsterdam , The Netherlands
| | - Vidhula R Ahire
- a Laboratory for Experimental Oncology and Radiobiology (LEXOR) , Center for Experimental and Molecular Medicine , Amsterdam , The Netherlands.,b Department of Radiotherapy , University of Amsterdam , Amsterdam , The Netherlands
| | - C M van Leeuwen
- c Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
| | - Rosemarie Ten Cate
- a Laboratory for Experimental Oncology and Radiobiology (LEXOR) , Center for Experimental and Molecular Medicine , Amsterdam , The Netherlands.,b Department of Radiotherapy , University of Amsterdam , Amsterdam , The Netherlands
| | - Lukas J A Stalpers
- a Laboratory for Experimental Oncology and Radiobiology (LEXOR) , Center for Experimental and Molecular Medicine , Amsterdam , The Netherlands.,b Department of Radiotherapy , University of Amsterdam , Amsterdam , The Netherlands
| | - Johannes Crezee
- c Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
| | - H Petra Kok
- c Academic Medical Center, University of Amsterdam , Amsterdam , The Netherlands
| | - Nicolaas A P Franken
- a Laboratory for Experimental Oncology and Radiobiology (LEXOR) , Center for Experimental and Molecular Medicine , Amsterdam , The Netherlands.,b Department of Radiotherapy , University of Amsterdam , Amsterdam , The Netherlands
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Maeda J, Fujii Y, Fujisawa H, Hirakawa H, Cartwright IM, Uesaka M, Kitamura H, Fujimori A, Kato TA. Hyperthermia-induced radiosensitization in CHO wild-type, NHEJ repair mutant and HR repair mutant following proton and carbon-ion exposure. Oncol Lett 2015; 10:2828-2834. [PMID: 26722249 PMCID: PMC4665357 DOI: 10.3892/ol.2015.3732] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2014] [Accepted: 08/17/2015] [Indexed: 12/19/2022] Open
Abstract
The DNA repair mechanisms involved in hyperthermia-induced radiosensitization with proton and carbon ion radiation exposure were investigated in the present study. In a previous study, Chinese hamster ovary (CHO) cells were exposed to low linear energy transfer (LET) photon radiation. These cells can be sensitized by hyperthermia as a result of inhibition of homologous recombination (HR) repair. The present study used wild-type, non-homologous end joining (NHEJ) and HR repair-deficient CHO cells to define the contributions of each repair pathway to cellular lethality following hyperthermia-induced hadron radiation sensitization. The cells were exposed to ionizing radiation, followed by hyperthermia treatment (42.5°C for 1 h). Hyperthermia-induced radiosensitization was determined by the colony formation assay and thermal enhancement ratio. HR repair-deficient cells exhibited no hyper-sensitization to X-rays, protons, or low and high LET carbon ions when combined with hyperthermia. Wild-type and NHEJ repair-deficient cells exhibited significant hyperthermia-induced sensitization to low LET photon and hadron radiation. Hyperthermia-induced sensitization to high LET carbon-ion radiation was less than at low LET radiation. Relative biological effectiveness (RBE) between radiation alone and radiation combined with hyperthermia cell groups was not significantly different in any of the cell lines, with the exception of wild-type cells exposed to high LET radiation, which exhibited a lower RBE in the combined group. The present study investigated additional cell lines to confirm the lower RBE observed in DNA repair-deficient cell lines. These findings suggested that hyperthermia-induced hyper-sensitization to hadron radiation is also dependent on inhibition of HR repair, as was observed with photon radiation in a previous study.
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Affiliation(s)
- Junko Maeda
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA
| | - Yoshihiro Fujii
- Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki, Ibaraki 300-0394, Japan
| | - Hiroshi Fujisawa
- School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
| | - Hirokazu Hirakawa
- Research Center for Charged Particle Therapy, International Open Laboratory, National Institute of Radiological Sciences, Chiba 263-8555, Japan
| | - Ian M Cartwright
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA
| | - Mitsuru Uesaka
- School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
| | - Hisashi Kitamura
- Research Development and Support Center, National Institute of Radiological Sciences, Chiba 263-8555, Japan
| | - Akira Fujimori
- Research Center for Charged Particle Therapy, International Open Laboratory, National Institute of Radiological Sciences, Chiba 263-8555, Japan
| | - Takamitsu A Kato
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA
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Oei AL, Vriend LEM, Crezee J, Franken NAP, Krawczyk PM. Effects of hyperthermia on DNA repair pathways: one treatment to inhibit them all. Radiat Oncol 2015; 10:165. [PMID: 26245485 PMCID: PMC4554295 DOI: 10.1186/s13014-015-0462-0] [Citation(s) in RCA: 193] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Accepted: 07/13/2015] [Indexed: 12/03/2022] Open
Abstract
The currently available arsenal of anticancer modalities includes many DNA damaging agents that can kill malignant cells. However, efficient DNA repair mechanisms protect both healthy and cancer cells against the effects of treatment and contribute to the development of drug resistance. Therefore, anti-cancer treatments based on inflicting DNA damage can benefit from inhibition of DNA repair. Hyperthermia – treatment at elevated temperature – considerably affects DNA repair, among other cellular processes, and can thus sensitize (cancer) cells to DNA damaging agents. This effect has been known and clinically applied for many decades, but how heat inhibits DNA repair and which pathways are targeted has not been fully elucidated. In this review we attempt to summarize the known effects of hyperthermia on DNA repair pathways relevant in clinical treatment of cancer. Furthermore, we outline the relationships between the effects of heat on DNA repair and sensitization of cells to various DNA damaging agents.
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Affiliation(s)
- Arlene L Oei
- Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands. .,Department of Radiotherapy, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands.
| | - Lianne E M Vriend
- Van Leeuwenhoek Centre for Advanced Microscopy (LCAM)-AMC, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.
| | - Johannes Crezee
- Department of Radiotherapy, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands.
| | - Nicolaas A P Franken
- Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands. .,Department of Radiotherapy, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands.
| | - Przemek M Krawczyk
- Van Leeuwenhoek Centre for Advanced Microscopy (LCAM)-AMC, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.
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Matsuya Y, Ohtsubo Y, Tsutsumi K, Sasaki K, Yamazaki R, Date H. Quantitative estimation of DNA damage by photon irradiation based on the microdosimetric-kinetic model. JOURNAL OF RADIATION RESEARCH 2014; 55:484-93. [PMID: 24515253 PMCID: PMC4014172 DOI: 10.1093/jrr/rrt222] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/14/2013] [Revised: 12/12/2013] [Accepted: 12/13/2013] [Indexed: 05/23/2023]
Abstract
The microdosimetric-kinetic (MK) model is one of the models that can describe the fraction of cells surviving after exposure to ionizing radiation. In the MK model, there are specific parameters, k and yD, where k is an inherent parameter to represent the number of potentially lethal lesions (PLLs) and yD indicates the dose-mean lineal energy in keV/μm. Assuming the PLLs to be DNA double-strand breaks (DSBs), the rate equations are derived for evaluating the DSB number in the cell nucleus. In this study, we estimated the ratio of DSBs for two types of photon irradiation (6 MV and 200 kVp X-rays) in Chinese hamster ovary (CHO-K1) cells and human non-small cell lung cancer (H1299) cells by observing the surviving fraction. The estimated ratio was then compared with the ratio of γ-H2AX foci using immunofluorescent staining. For making a comparison of the number of DSBs among a variety of radiation energy cases, we next utilized the survival data in the literature for both cells exposed to other photon types, such as (60)Co γ-rays, (137)Cs γ-rays and 100 kVp X-rays. The ratio of DSBs based on the MK model with conventional data was consistent with the ratio of γ-H2AX foci numbers, confirming that the γ-H2AX focus is indicative of DSBs. It was also shown that the larger yD is, the larger the DSB number is. These results suggest that k and yD represent the characteristics of the surviving fraction and the biological effects for photon irradiation.
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Affiliation(s)
- Yusuke Matsuya
- Graduate School of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060-0812, Japan
| | - Yosuke Ohtsubo
- Hokkaido PWFAC Sapporo-Kosei General Hospital, Kita-3 Higashi-8, Chuo-ku, Sapporo 060-0033, Japan
| | - Kaori Tsutsumi
- Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060-0812, Japan
| | - Kohei Sasaki
- Graduate School of Engineering, Kyoto University, Kyoto Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8530, Japan
| | - Rie Yamazaki
- Graduate School of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060-0812, Japan
| | - Hiroyuki Date
- Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060-0812, Japan
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Ihara M, Takeshita S, Okaichi K, Okumura Y, Ohnishi T. Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair. Int J Hyperthermia 2014; 30:102-9. [DOI: 10.3109/02656736.2014.887793] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
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Abstract
Combining long duration mild temperature hyperthermia (LDMH) and low dose-rate (LDR) brachytherapy to enhance therapeutic killing of cancer cells was proposed many years ago. The cellular and tumour research that supports this hypothesis is presented in this review. Research describing LDMH interaction with pulsed brachytherapy and high dose-rate brachytherapy using clinically relevant parameters are compared with LDMH/LDR brachytherapy. The mechanism by which LDMH sensitizes LDR has been established as the inhibition of sublethal damage repair. The molecular mechanisms have been shown to involve DNA repair enzymes, but the exact nature of these processes is still under investigation. The relative differences between LDMH interactions with human and rodent cells are presented to help in the understanding of possible roles of LDMH in clinical application. The role of LDMH in modifying tumour blood flow and its possible role in LDR sensitization of tumours is also presented. The positive aspects of LDMH-brachytherapy for clinical application are sixfold; (1) the thermal goals (temperature, time and volume) are achievable with currently available technology, (2) the hyperthermia by itself has no detectable toxic effects, (3) thermotolerance appears to play a minor if any role in radiation sensitization, (4) TER of around 2 can be expected, (5) hypoxic fraction may be decreased due to blood flow modification and (6) simultaneous chemotherapy may also be sensitized. Combined LDMH and brachytherapy is a cancer therapy that has established biological rationale and sufficient technical and clinical advancements to be appropriately applied. This modality is ripe for clinical testing.
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Affiliation(s)
- E P Armour
- Department of Radiation Oncology, William Beaumont Hospital, 3811 West Thirteen Mile Road, Royal Oak, MI 48073, USA.
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Yin HL, Suzuki Y, Matsumoto Y, Tomita M, Furusawa Y, Enomoto A, Morita A, Aoki M, Yatagai F, Suzuki T, Hosoi Y, Ohtomo K, Suzuki N. Radiosensitization by hyperthermia in the chicken B-lymphocyte cell line DT40 and its derivatives lacking nonhomologous end joining and/or homologous recombination pathways of DNA double-strand break repair. Radiat Res 2004; 162:433-41. [PMID: 15447039 DOI: 10.1667/rr3239] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70(-/-), DNA-PKcs(-/-/-), RAD54(-/-) and KU70(-/-)/RAD54(-/-). Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70(-/-)/RAD54(-/-), which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70(-/-) and DNA-PKcs(-/-/-), consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G(1) and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G(2), capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G(1) and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.
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Affiliation(s)
- Hong Lan Yin
- Department of Radiation Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
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Kampinga HH, Dynlacht JR, Dikomey E. Mechanism of radiosensitization by hyperthermia (> or = 43 degrees C) as derived from studies with DNA repair defective mutant cell lines. Int J Hyperthermia 2004; 20:131-9. [PMID: 15195507 DOI: 10.1080/02656730310001627713] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
All biochemical and cytogenetic data on radiosensitization by heat treatment at and above 43 degrees C indicate that inhibition of DNA repair plays a central role. There are several DNA repair pathways involved in restoration of damage after ionising irradiation and the kinetics of all of them are affected by heat shock. This, however, does not imply that the inhibition of each of these pathways is relevant to the effect of heat on cellular radiosensitivity. The current review evaluates the available data on heat radiosensitization in mutant or knockout cell lines defective in various DNA repair proteins and/or pathways. The data show that thermal inhibition of the non-homologous end-joining pathway (NHEJ) plays no role in heat radiosensitization. Furthermore, limited data suggest that the homologous recombination pathway may also not be a major heat target. By deduction, it is suggested that inhibition of base damage repair (BER) could be the crucial step in radiosensitization by heat. While a lack of mutant cell lines and redundancy of the BER pathway have hampered efforts toward a conclusive study, biochemical and correlative evidence support this hypothesis.
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Affiliation(s)
- H H Kampinga
- Department of Radiation and Stress Cell Biology, University of Groningen, A Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
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Kinuya S, Yokoyama K, Michigishi T, Tonami N. Optimization of radioimmunotherapy interactions with hyperthermia. Int J Hyperthermia 2004; 20:190-200. [PMID: 15195513 DOI: 10.1080/02656730310001611044] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Abstract
The efficacy of radioimmunotherapy (RIT) employing radiolabelled monoclonal antibodies (MAb) is currently limited in most solid tumours. The combination of local hyperthermia (HT) with RIT has the potential to enhance tumour targeting of MAb; moreover, this approach may add an antitumour effect to radioresistant hypoxic and S-phase cells and may inhibit the cells from repairing sublethal damage or potentially lethal damage caused by ionizing radiation. There are distinct types of protocols in this combination. Hyperthermic temperature and timing relative to RIT administration appear to affect the efficacy of the combination therapy. Responses to heating at any particular condition are not always the same among different tumour types. There are many papers describing influence of HT on the biodistribution of radiolabelled MAb, but only limited information is currently available on 'therapeutic' outcomes regarding the dependency of combination protocols. A previous study suggested that the best therapeutic improvement would be achieved when HT was combined immediately after the administration of MAb, which significantly increases the radiation absorbed dose to tumours and produces a uniform intratumoural dose distribution. Further therapeutic investigation should be required to reach the optimal protocol of combining these two modalities.
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Affiliation(s)
- S Kinuya
- Department of Biotracer Medicine, Kanazawa University Graduate School of Medical Sciences, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8640, Japan.
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Seno JD, Dynlacht JR. Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock. J Cell Physiol 2004; 199:157-70. [PMID: 15039997 DOI: 10.1002/jcp.10475] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.
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Affiliation(s)
- Joshua D Seno
- Department of Radiation Oncology, Indiana University School of Medicine, Indianapolis 46202, USA
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Dynlacht JR, Xu M, Pandita RK, Wetzel EA, Roti Roti JL. Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells. Int J Hyperthermia 2004; 20:144-56. [PMID: 15195509 DOI: 10.1080/02656730410001660634] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Abstract
The mechanism by which hyperthermia sensitizes mammalian cells to ionizing radiation remains to be elucidated, but an overwhelming amount of circumstantial evidence suggests that heat radiosensitization might be mediated by inhibition of double-strand break repair, particularly after exposure of irradiated cells to heat treatments in excess of about 43 degrees C. In mammalian cells, double-strand break repair usually occurs via two pathways, non-homologous end-joining and homologous recombination. Several reports suggest a role for non-homologous end-joining in heat radiosensitization, while others implicate homologous recombination as a target. However, cell lines that are compromised in either the non-homologous end-joining or homologous recombination pathway are still capable of being radiosensitized, suggesting that heat affects both pathways. Indeed, several of the proteins involved in one or both of these pathways have been observed to undergo alterations or translocation after unirradiated or irradiated cells are exposed to heat shock. The work summarized in this review implicates proteins of the Mre11/Rad50/Nbs1 complex as targets for heat radiosensitization.
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Affiliation(s)
- J R Dynlacht
- Department of Radiation Oncology, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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16
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Raaphorst GP, Maude-Leblanc J, Li L. Evaluation of Recombination Repair Pathways in Thermal Radiosensitization. Radiat Res 2004; 161:215-8. [PMID: 14731068 DOI: 10.1667/rr3103] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Thermal radiosensitization has been shown to cause inhibition of repair of sublethal and potentially lethal damage and DNA DSBs. In this study we assessed thermal radiosensitization in mutants deficient in homologous recombinational (HR) repair and nonhomologous end joining repair (NHEJ). Using cells of the mouse wild-type embryo fibroblast cell line MEF and its Ku80(-/-) derivative that is deficient in NHEJ, we showed that thermal radiosensitization is the same in both cell lines. Further studies with cells of the wild-type CHO-AA8 cell line and its derivative IRS(ISF), which is deficient in HR, also showed comparable thermal radiosensitization in both cell lines. Further experiments using cells of chicken DT40 cell lines also showed comparable thermal radiosensitization between the wild-type HR mutant Rad54, the NHEJ mutant Ku70, and the double mutant Rad 54-Ku70. These results indicate that the HR and NHEJ pathways may not be targets for thermal radiosensitization.
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Affiliation(s)
- G P Raaphorst
- Medical Physics Department, Ottawa Regional Cancer Centre, Ottawa, Ontario K1H 1C4, Canada.
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Dynlacht JR, Bittner ME, Bethel JA, Beck BD. The non-homologous end-joining pathway is not involved in the radiosensitization of mammalian cells by heat shock. J Cell Physiol 2003; 196:557-64. [PMID: 12891712 DOI: 10.1002/jcp.10334] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
A synergistic increase in cell killing is observed when a heat-shock is administered prior to, during, or immediately after exposure to ionizing radiation (IR). This phenomenon, known as heat-radiosensitization, is believed to be mediated by inhibition of repair of radiation-induced double strand breaks (DSB) when cells are exposed to temperatures above 42 degrees C. However, the mechanism by which heat inhibits DSB repair is unclear. The bulk of radiation-induced DSBs are repaired via the non-homologous end-joining pathway (NHEJ). Several reports indicate that the Ku70 and Ku80 subunits of the mammalian DNA-dependent protein kinase (DNA-PK), a complex involved in NHEJ, appear to be susceptible to a heat-induced loss of DNA-binding activity, with Ku80 representing the heat-sensitive component. Since the heat-induced loss and subsequent recovery of Ku-DNA binding activity correlates well with heat-radiosensitization, a role for Ku80 and NHEJ in heat-radiosensitization has been proposed. However, direct evidence implicating Ku80 (and NHEJ) in heat-radiosensitization has been indeterminate. In this study, we demonstrate that equitoxic heat treatments at 42.5-45.5 degrees C induce a similar amount of aggregation of Ku80 in human U-1 melanoma cells. These data suggest that the time-temperature-dependent relationship between heat lethality and Ku80 aggregation are similar. However, the aggregation/disaggregation of Ku80 and its transient or permanent inactivation is unrelated to heat-radiosensitization. When survival curves were obtained for irradiated or irradiated and heated Ku80(-/-) mouse embryo fibroblasts (MEFs) and compared with survival curves obtained for wild-type (WT) cells, we found that heat-radiosensitization was not reduced in the Ku80(-/-) cells, but actually increased. Thus, our findings indicate that Ku80 is not essential for heat-radiosensitization. Non-involvement of Ku-dependent or Ku-independent NHEJ pathways in heat-radiosensitization was confirmed by comparing clonogenic survival between DNA ligase IV-defective and WT human cells. Our data therefore implicate homologous recombination in inhibition of repair of radiation-induced DSBs and as a target for heat-radiosensitization.
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Affiliation(s)
- Joseph R Dynlacht
- Department of Radiation Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
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18
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Zeng ZC, Jiang GL, Wang GM, Tang ZY, Curran WJ, Iliakis G. DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma hepG2 cell line. World J Gastroenterol 2002; 8:797-803. [PMID: 12378618 PMCID: PMC4656564 DOI: 10.3748/wjg.v8.i5.797] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the role of DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma HepG2 cell lines.
METHODS: HepG2 cells were exposed to hyperthermia and irradiation. Hyperthermia was given at 45.5 °C. Cell survival was determined by an in vitro clonogenic assay for the cells treated with or without hyperthermia at various time points. DNA DSB rejoining was measured using asymmetric field inversion gel electrophoresis (AFIGE). The DNA-PKcs activities were measured using DNA-PKcs enzyme assay system.
RESULTS: Hyperthermia can significantly enhance irradiation-killing cells. Thermal enhancement ratio as calculated at 10% survival was 2.02. The difference in radiosensitivity between two treatment modes manifested as a difference in the α components and the almost same β components, which α value was considerably higher in the cells of combined radiation and hyperthermia as compared with irradiating cells (1.07 Gy-1vs 0.44 Gy-1). Survival fraction showed 1 logarithm increase after an 8-hour interval between heat and irradiation, whereas DNA-PKcs activity did not show any recovery. The cells were exposed to heat 5 min only, DNA-PKcs activity was inhibited at the nadir, even though the exposure time was lengthened. Whereas the ability of DNA DSB rejoining was inhibited with the increase of the length of hyperthermic time. The repair kinetics of DNA DSB rejoining after treatment with Wortmannin is different from the hyperthermic group due to the striking high slow rejoining component.
CONCLUSION: Determination with the cell extracts and the peptide phosphorylation assay, DNA-PKcs activity was inactivated by heat treatment at 45.5 °C, and could not restore. Cell survival is not associated with the DNA-PKcs inactivity after heat. DNA-PKcs is not a unique factor affecting the DNA DSB repair. This suggests that DNA-PKcs do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.
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Affiliation(s)
- Zhao-Chong Zeng
- Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
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Abstract
XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.
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Affiliation(s)
- B D Beck
- Department of Radiation Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA
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Giampuzzi M, Botti G, Di Duca M, Arata L, Ghiggeri G, Gusmano R, Ravazzolo R, Di Donato A. Lysyl oxidase activates the transcription activity of human collagene III promoter. Possible involvement of Ku antigen. J Biol Chem 2000; 275:36341-9. [PMID: 10942761 DOI: 10.1074/jbc.m003362200] [Citation(s) in RCA: 99] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis.
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Affiliation(s)
- M Giampuzzi
- Department of Nephrology, Gaslini Children's Hospital, Genova, Italy
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