RESUMO
PURPOSE: High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. METHODS AND MATERIALS: In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. RESULTS: We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. CONCLUSIONS: Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.
Assuntos
Antineoplásicos , Neoplasias , Radiossensibilizantes , Feminino , Humanos , Ensaios de Triagem em Larga Escala/métodos , Detecção Precoce de Câncer , Radiossensibilizantes/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular TumoralRESUMO
Bladder cancer is a common cancer; it is the tenth most common cancer in the world. Around one fourth of all diagnosed patients have muscle-invasive bladder cancer (MIBC), characterized by advanced tumors and which remains a lethal disease. The standard treatment for MIBC is the bladder removal by surgery. However, bladder-preserving alternatives are emerging by combining chemotherapy, radiotherapy and minimal surgery, aiming to increase the patient's quality of life. The aim of the study was to improve these treatments by investigating a novel approach where in addition to radiotherapy, a receptor, TYRO3, a member of TAM receptor tyrosine kinase family known to be highly expressed on the bladder cancer cells and involved in the control of cell survival is targeted. For this, we evaluated the influence of TYRO3 expression levels on a colony or cell survival assays, DNA damage, γH2AX foci formation, gene expression profiling and cell cycle regulation, after radiation on different bladder cell models. We found that TYRO3 expression impacts the radiation response via the cell cycle dysregulation with noeffets on the DNA repair. Therefore, targeting TYRO3 is a promising sensitization marker that could be clinically employed in future treatments.
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Neoplasias da Bexiga Urinária , Ciclo Celular/genética , Cistectomia , Humanos , Qualidade de Vida , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/radioterapiaRESUMO
Proton therapy allows to avoid excess radiation dose on normal tissues. However, there are some limitations. Indeed, passive delivery of proton beams results in an increase in the lateral dose upstream of the tumor and active scanning leads to strong differences in dose delivery. This study aims to assess possible differences in the transcriptomic response of skin in C57BL/6 mice after TBI irradiation by active or passive proton beams at the dose of 6 Gy compared to unirradiated mice. In that purpose, total RNA was extracted from skin samples 3 months after irradiation and RNA-Seq was performed. Results showed that active and passive delivery lead to completely different transcription profiles. Indeed, 140 and 167 genes were differentially expressed after active and passive scanning compared to unirradiated, respectively, with only one common gene corresponding to RIKEN cDNA 9930021J03. Moreover, protein-protein interactions performed by STRING analysis showed that 31 and 25 genes are functionally related after active and passive delivery, respectively, with no common gene between both types of proton delivery. Analysis showed that active scanning led to the regulation of genes involved in skin development which was not the case with passive delivery. Moreover, 14 ncRNA were differentially regulated after active scanning against none for passive delivery. Active scanning led to 49 potential mRNA-ncRNA pairs with one ncRNA mainly involved, Gm44383 which is a miRNA. The 43 genes potentially regulated by the miRNA Gm44393 confirmed an important role of active scanning on skin keratin pathway. Our results demonstrated that there are differences in skin gene expression still 3 months after proton irradiation versus unirradiated mouse skin. And strong differences do exist in late skin gene expression between scattered or scanned proton beams. Further investigations are strongly needed to understand this discrepancy and to improve treatments by proton therapy.
Assuntos
Prótons , Pele/metabolismo , Pele/efeitos da radiação , Transcriptoma/genética , Irradiação Corporal Total , Animais , Peso Corporal/efeitos da radiação , Relação Dose-Resposta à Radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Ontologia Genética , Queratinas/metabolismo , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Bladder cancer is among the top ten most common cancer types in the world. Around 25% of all cases are muscle-invasive bladder cancer, for which the gold standard treatment in the absence of metastasis is the cystectomy. In recent years, trimodality treatment associating maximal transurethral resection and radiotherapy combined with concurrent chemotherapy is increasingly used as an organ-preserving alternative. However, the use of this treatment is still limited by the lack of biomarkers predicting tumour response and by a lack of targeted radiosensitising drugs that can improve the therapeutic index, especially by limiting side effects such as bladder fibrosis. In order to improve the bladder-preserving treatment, experimental studies addressing these main issues ought to be considered (both in vitro and in vivo studies). Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we conducted a literature search in PubMed on experimental studies investigating how to improve bladder cancer radiotherapy with different radiosensitising agents using a comprehensive search string. We made comments on experimental model selection, experimental design and results, formulating the gaps of knowledge still existing: such as the lack of reliable predictive biomarkers of tumour response to chemoradiation according to the molecular tumour subtype and lack of efficient radiosensitising agents specifically targeting bladder tumour cells. We provided guidance to improve forthcoming studies, such as taking into account molecular characteristics of the preclinical models and highlighted the value of using patient-derived xenografts as well as syngeneic models. Finally, this review could be a useful tool to set up new radiation-based combined treatments with an improved therapeutic index that is needed for bladder preservation.
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BACKGROUND: Over the past few years, anti-HER2 targeted therapies have proven to be a key treatment in the management of human epidermal growth receptor 2 (HER2)-positive breast cancers, as well as gastrointestinal tract tumors and head and neck tumors. Anti-HER2 therapies administered alone or in combination with chemotherapy have been extensively studied, but only limited robust data are available concerning the safety and efficacy of anti-HER2 molecules in combination with radiotherapy. METHODS: We searched on Medline, Embase and Cochrane databases the articles providing data on the concomitant association between the antiHER2 therapies used in clinical practice (trastuzumab, pertuzumab, lapatinib and T-DM1) with radiotherapy. The articles were selected according to their pre-clinical and clinical relevance. RESULTS: The trastuzumab-irradiation combination is the most studied, with a focus on the cardiac toxicity. The combination of lapatinib-irradiation was particularly studied in the context of cerebral metastases of HER2-positive breast cancer. The data on pertuzumab and T-DM1 were poor and are mainly case reports. CONCLUSION: To date, reliable conclusions about the toxicity and/or efficacy of concomitant irradiation with anti-HER2 therapies are difficult to make due to the heterogeneity of the data in the literature and need to be confirmed on a larger scale and long term follow-up. Nevertheless, no serious adverse events are reported and the toxicity profile seems to be manageable.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Quimiorradioterapia , Feminino , Humanos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells' poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.
Assuntos
Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Radiação Ionizante , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
PURPOSE: Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). METHODS AND MATERIALS: The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells. RESULTS: We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role. CONCLUSIONS: Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.
Assuntos
Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA/fisiologia , Proteínas Nucleares/fisiologia , Prótons , Eficiência Biológica Relativa , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Institutos de Câncer , Sobrevivência Celular/fisiologia , DNA Glicosilases/fisiologia , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endonucleases , França , Células HeLa , Histonas/análise , Humanos , Método de Monte Carlo , Proteínas Nucleares/deficiência , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/fisiologia , Terapia com Prótons , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
The goal of the present study was to evaluate the role of the tyrosine kinase receptor fibroblast growth factor-1 (FGFR1) and its ligand, the fibroblast growth factor 2 (FGF2) in determining the response to chemoradiotherapy of breast cancers. S14 was a phase II neoadjuvant study carried out at the Institut Curie that recruited 59 patients between November 2001 and September 2003. This prospective study aimed to assess the pathological response after preoperative radiochemotherapy (5FU-Navelbine-radiotherapy) for large breast cancers. The expression of FGFR1 and FGF2 in tumor cells were assessed by immunohistochemistry. Tumors in which no staining was seen, were considered as negative for that protein. We used the Khi-2 test or the Fisher test to compare the qualitative variables and the Student t test or the non-parametric Wilcoxon test for the quantitative variables. We included in the present study all the 32 patients from the S14 cohort for whom the tissue blocks from the biopsy specimens were available with sufficient tumoral tissue. FGFR1 and FGF2 staining were observed respectively in 17 (56%) and 22 (68%) of the 32 tumoral biopsies. The expression of FGFR1 was associated with the hormone receptor positive status (p=0.0191). Only 11% (1/9) of the high grade tumors failed to respond to chemoradiotherapy compared to 68 % resistant tumors (15/22) among the low/intermediate grade tumors (p=0.0199). Among the low/intermediate grade tumors, FGFR1 negative tumors did not respond to chemoradiotherapy (0/9), compared with tumors expressing FGFR1 among which, almost one half had a good response (6/13) (p=0.0167). Among the low and intermediate grade breast cancers, the FGFR1 negative tumors were resistant to chemoradiotherapy. The expression of FGFR1 in patients' biopsies may serve as a marker of response to chemoradiotherapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Carcinoma Ductal de Mama/patologia , Carcinoma Ductal de Mama/terapia , Carcinoma Lobular/patologia , Carcinoma Lobular/terapia , Quimiorradioterapia , Ensaios Clínicos Fase II como Assunto , Resistencia a Medicamentos Antineoplásicos , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fluoruracila/administração & dosagem , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Gradação de Tumores , Tolerância a Radiação , Resultado do Tratamento , Vimblastina/administração & dosagem , Vimblastina/análogos & derivados , VinorelbinaRESUMO
Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5(KD) cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5(KD) cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5(KD) cells is due to a role of Cdk5 in other pathways or the altered polymer levels.
Assuntos
Quinase 5 Dependente de Ciclina/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Tolerância a Radiação , Sequência de Bases , Dano ao DNA , Reparo do DNA , Células HeLa , Humanos , Dados de Sequência Molecular , Inibidores de Poli(ADP-Ribose) PolimerasesRESUMO
PURPOSE: Treatment planning in proton therapy uses a generic value for the relative biological efficiency (RBE) of 1.1 throughout the spread-out Bragg peak (SOBP) generated. In this article, we report on the variation of the RBE with depth in the SOBP of the 76- and 201-MeV proton beams used for treatment at the Institut Curie Proton Therapy Center in Orsay. METHODS AND MATERIALS: The RBE (relative to (137)Cs γ-rays) of the two modulated proton beams at three positions in the SOBP was determined in two human tumor cells using as endpoints clonogenic cell survival and the incidence of DNA double-strand breaks (DSBs) as measured by pulse-field gel electrophoresis without and with enzymatic treatment to reveal clustered lesions. RESULTS: The RBE for induced cell killing by the 76-MeV beam increased with depth in the SOBP. However for the 201-MeV protons, it was close to that for (137)Cs γ-rays and did not vary significantly. The incidence of DSBs and clustered lesions was higher for protons than for (137)Cs γ-rays, but did not depend on the proton energy or the position in the SOBP. CONCLUSIONS: Until now, little attention has been paid to the variation of RBE with depth in the SOBP as a function of the nominal energy of the primary proton beam and the molecular nature of the DNA damage. The RBE increase in the 76-MeV SOBP implies that the tumor tissues at the distal end receives a higher biologically equivalent dose than at the proximal end, despite a homogeneous physical dose. This is not the case for the 201-MeV energy beam. The precise determination of the effects of incident beam energy, modulation, and depth in tissues on the linear energy transfer-RBE relationship is essential for treatment planning.
Assuntos
Dano ao DNA , DNA de Neoplasias/efeitos da radiação , Transferência Linear de Energia , Terapia com Prótons , Eficiência Biológica Relativa , Institutos de Câncer , Linhagem Celular Tumoral , Sobrevivência Celular , Radioisótopos de Césio , Relação Dose-Resposta à Radiação , França , Raios gama/uso terapêutico , Células HeLa , Humanos , Radiobiologia , Ensaio Tumoral de Célula-Tronco/métodosRESUMO
Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD(+). The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD(+) at the enzyme's activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , HumanosRESUMO
Two molecularly distinct G2/M cell cycle arrests are induced after exposure to ionising radiation (IR) depending on the cell cycle compartment in which the cells are irradiated. The aims of this study were to determine whether there are threshold doses for their activation and investigate the molecular pathways and possible links between the G2 to M transition and hyper-radiosensitivity (HRS). Two human glioblastoma cell lines (T98G-HRS(+) and U373-HRS(-)) unsynchronized or enriched in G2 were irradiated and flow cytometry with BrdU or histone H3 phosphorylation analysis used to assess cell cycle progression and a clonogenic assay to measure radiation survival. The involvement of ATM, Wee1 and PARP was studied using chemical inhibitors. We found that cells irradiated in either the G1 or S phase of the cell cycle transiently accumulate in G2 in a dose-dependent manner after exposure to doses as low as 0.2Gy. Only Wee1 inhibition reduced this G2 accumulation. A block of the G2 to M transition was found after irradiation in G2 but occurs only above a threshold dose, which is cell line dependent, and requires ATM activity after exposure to doses above 0.5Gy. A failure to activate this early G2/M checkpoint correlates with low dose radiosensitization. These results provide evidence that after exposure to low doses of IR two distinct G2/M checkpoints are activated, each in a dose-dependent manner, with distinct threshold doses and involving different damage signalling pathways and confirm links between the early G2/M checkpoint and hyper-radiosensitivity.
Assuntos
Divisão Celular/efeitos da radiação , Fase G2/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , Radiação IonizanteRESUMO
The consequences of PARP-1 disruption or inhibition on DNA single-strand break repair (SSBR) and radio-induced lethality were determined in synchronized, isogenic HeLa cells stably silenced or not for poly(ADP-ribose) polymerase-1 (PARP-1) (PARP-1(KD)) or XRCC1 (XRCC1(KD)). PARP-1 inhibition prevented XRCC1-YFP recruitment at sites of 405 nm laser micro irradiation, slowed SSBR 10-fold and triggered the accumulation of large persistent foci of GFP-PARP-1 and GFP-PCNA at photo damaged sites. These aggregates are presumed to hinder the recruitment of other effectors of the base excision repair (BER) pathway. PARP-1 silencing also prevented XRCC1-YFP recruitment but did not lengthen the lifetime of GFP-PCNA foci. Moreover, PARP-1(KD) and XRCC1(KD) cells in S phase completed SSBR as rapidly as controls, while SSBR was delayed in G1. Taken together, the data demonstrate that a PARP-1- and XRCC1-independent SSBR pathway operates when the short patch repair branch of the BER is deficient. Long patch repair is the likely mechanism, as GFP-PCNA recruitment at photo-damaged sites was normal in PARP-1(KD) cells. PARP-1 silencing elicited hyper-radiosensitivity, while radiosensitization by a PARP inhibitor reportedly occurs only in those cells treated in S phase. PARP-1 inhibition and deletion thus have different outcomes in terms of SSBR and radiosensitivity.
Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , Inibidores de Poli(ADP-Ribose) Polimerases , Tolerância a Radiação , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacologia , DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fase G1 , Raios gama , Células HeLa , Humanos , Lasers , Naftalimidas/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Quinolonas/farmacologia , Interferência de RNA , Fase S , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Radiosensitization caused by the poly(ADP-ribose) polymerase (PARP) inhibitor 4-amino-1,8-naphthalimide (ANI) was investigated in 10 asynchronously growing rodent (V79, CHO-Xrs6, CHO-K1, PARP-1+/+ 3T3, and PARP-1-/- 3T3) or human (HeLa, MRC5VI, IMR90, M059J, and M059K) cell lines, either repair proficient or defective in DNA-PK (CHO-Xrs6 and M059J) or PARP-1 (PARP-1-/- 3T3). Pulse exposure to ANI (1-hour contact) potentiated radiation response in rodent cells except in PARP-1(-/-) 3T3 fibroblasts. In contrast, ANI did not significantly enhance radiation susceptibility in asynchronously dividing human cells; yet, single-strand break rejoining was lengthened by ca. 7-fold in all but mouse PARP-1-/- 3T3s. Circumstantial evidence suggested that radiosensitization by ANI occurs in rapidly dividing cells only. Experiments using synchronized HeLa cells consistently showed that ANI-induced radiosensitization is specific of the S phase of the cell cycle and involves stalled replication forks. Under these conditions, prolonged contact with ANI ended in the formation of de novo DNA double-strand breaks hours after irradiation, evoking collision with uncontrolled replication forks of DNA lesions whose repair was impaired by inhibition of the PARP catalytic activity. The data suggest that increased response to radiotherapy by PARP inhibitors may be achieved only in rapidly growing tumors with a high S-phase content.
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1-Naftilamina/análogos & derivados , DNA/biossíntese , Neoplasias/radioterapia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Quinolonas/farmacologia , Radiossensibilizantes/farmacologia , 1-Naftilamina/farmacologia , Animais , Cricetinae , Replicação do DNA/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Naftalimidas , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/genética , Tolerância a Radiação/efeitos dos fármacos , Fase S/efeitos dos fármacos , Fase S/efeitos da radiação , Células Tumorais CultivadasRESUMO
Enhanced cytotoxicity of etoposide by wortmannin, an inhibitor of enzymes holding a phosphatidylinositol 3-kinase domain, was investigated in eight cell lines proficient or deficient for DNA double-strand break repair. Wortmannin stimulated the decatenating activity of topoisomerase II, promoted etoposide-induced accumulation of DNA double-strand breaks, shifted the specificity for cell killing by etoposide from the S to G1 phase of the cell cycle, and potentiated the cytotoxicity of etoposide through two mechanisms. (a) Sensitization to high, micromolar amounts of etoposide required integrity of the nonhomologous end-joining repair pathway. (b) Wortmannin dramatically increased the susceptibility to low, submicromolar amounts of etoposide in a large fraction of the cell population irrespective of the status of ATM, Ku86, and DNA-PKCS. It is shown that this process correlates depression of phosphatidylinositol 3-kinase-dependent phosphorylation of the atypical, zeta isoform of protein kinase C (PKCzeta). Stable expression of a dominant-negative, kinase-dead mutant of PKCzeta in a tumor cell line reproduced the hypersensitivity pattern induced by wortmannin. The results are consistent with up-regulation of the topoisomerase II activity in relation to inactivation of PKCzeta and indicate that PKCzeta may be a useful target to improve the efficiency of topoisomerase II poisons at low concentration.
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Androstadienos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Etoposídeo/farmacologia , Proteína Quinase C/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Sinergismo Farmacológico , Células HeLa , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C/genética , Inibidores de Proteínas Quinases/farmacologia , Coelhos , Inibidores da Topoisomerase II , Transfecção , Células U937 , WortmaninaRESUMO
Ligands that stabilize the telomeric G-rich single-stranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication. Ligand 12459, a potent G-quadruplex ligand that belongs to the triazine series, has been previously shown to induce both telomere shortening and apoptosis in the human A549 cell line as a function of its concentration and time exposure. We show here that A549 clones obtained after mutagenesis and selected for resistance to the short term effect of ligand 12459 frequently displayed hTERT transcript overexpression (2-6-fold). Overexpression of hTERT was also characterized in two resistant clones (JFD10 and JFD18) as an increase in telomerase activity, leading to an increase in telomere length. An increased frequency of anaphase bridges was also detected in JFD10 and JFD18, suggesting an alteration of telomere capping functions. Transfection of either hTERT or DN-hTERT cDNAs into A549 cells did not confer resistance or hypersensitivity to the short term effect of ligand 12459, indicating that telomerase expression is not the main determinant of the antiproliferative effect of ligand 12459. In contrast, transfection of DN-hTERT cDNA into resistant JFD18 cells restored sensitivity to apoptotic concentrations of ligand 12459, suggesting that telomerase does participate in the resistance to this G-quadruplex ligand. This work provides evidence that telomerase activity is not the main target for the 12459 G-quadruplex ligand but that hTERT functions contribute to the resistance phenotype to this class of agents.
Assuntos
Compostos de Quinolínio/química , Telomerase/biossíntese , Telômero/ultraestrutura , Triazinas/química , Divisão Celular , Linhagem Celular Tumoral , DNA/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Resistência a Múltiplos Medicamentos , Humanos , Hibridização in Situ Fluorescente , Concentração Inibidora 50 , Ligantes , Mutação , Oligonucleotídeos/metabolismo , Fenótipo , Ligação Proteica , Compostos de Quinolínio/farmacologia , RNA/metabolismo , Radiação Ionizante , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Telômero/metabolismo , Transfecção , Triazinas/farmacologiaRESUMO
BACKGROUND: Growth inhibition by RPR-130401, a non-peptidomimetic farnesyltransferase inhibitor, was investigated without or with combined exposure to ionizing radiation in three human tumor cell lines (HCT-116, MiAPaCa-2 and A-549) bearing a point mutation in the K-Ras gene. RESULTS: RPR-130401 inhibited cell growth with an IC50 of 50 nM (HCT-116), 120 nM (MiAPaCa-2) and 710 nM (A-549), with a poor incidence of apoptosis. The drug brought about G1 and S phase depletion together with arrest of cells in G2 phase and induced a significant accumulation of hyperploid cells showing active S phase DNA synthesis, with HCT-116 and A-549 cells being the most and least responsive, respectively. The drug also produced dramatic changes of the nuclear lamin B pattern, without lamin B cleavage and perturbation of the actin cytoskeleton. On the other hand, RPR-130401 elicited strictly additive interaction in combined treatment with ionizing radiation with regard to cell kill, altered cell cycle progression and induced hyperploidy. CONCLUSIONS: The data suggest that disruption of orderly progression through mitosis and cytokinesis, is a major outcome of drug action and that this effect proceeds from inhibition of lamin B farnesylation. It is anticipated from the strict additivity of RPR-130401 and radiation that neither induced radiation resistance nor acute or late complications of radiotherapy, should occur in combined treatment with RPR-130401.