RESUMO
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.
Assuntos
Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Hipertermia Induzida/efeitos adversos , Animais , Cricetinae , Humanos , Hipertermia Induzida/métodosRESUMO
In recent years, genes associated with cancer have been studied to assess their possible use as predictive indicators for cancer therapies. Among these, the gene product of the tumor suppressor gene p53 was found to play an important role in cancer therapy. The p53 molecule induces cell-cycle arrest, apoptosis and DNA repair after cells are subjected to cancer therapies involving radiation, heat and various anti-cancer agents. Mutations in p53 are observed at a high frequency in human tumors, and are present in about half of all malignant tumors in humans. Sensitization to radiation, heat and anti-cancer agents was observed in cells containing wild type p53, but not in cells containing mutated p53. This review discusses p53 activation of signaling pathways after exposure to cancer therapies which target p53; such therapies include chemical chaperones, the p53 gene, p53-C terminal peptides, and p53-targeting agents which enhance p53-central signal transduction pathways.
Assuntos
Antineoplásicos/uso terapêutico , Hipertermia Induzida , Luz , Neoplasias/terapia , Radiossensibilizantes/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Terapia Genética , Humanos , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13-100 KeV/microm), or Fe-ion beams (200 KeV/microm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/microm, and then decreased at values over 100 KeV/microm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53.
Assuntos
Apoptose/efeitos da radiação , Caspase 9/fisiologia , Genes p53/fisiologia , Mutação , Neoplasias/radioterapia , Caspase 3/fisiologia , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Ativação Enzimática , Íons Pesados , Humanos , Transferência Linear de Energia , Neoplasias/enzimologia , Neoplasias/patologia , Raios XRESUMO
The aim of this study was to ascertain whether LY294002, an inhibitor of PI-3K, enhances heat sensitivity in human cancer cells regardless of their p53 status. Colony formation assays showed that LY294002 enhanced heat sensitivity in two human lung cancer cell lines; H1299/wild-type p53 (wtp53) and H1299/mutated p53 (mp53) cells. These cell lines have identical genetic backgrounds except for their p53 status. LY294002 suppressed the heat-induced accumulation of heat shock protein 27 (hsp27) and heat shock protein 72 (hsp72) in these cell lines. Heat-induced apoptosis was observed more frequently in H1299/wtp53 cells than in H1299/mp53 cells, and was enhanced by LY294002 in both cell lines. In addition, both the heat-induced phosphorylation of Akt and the accumulation of survivin were suppressed by LY294002. These results suggest that LY294002 inhibits anti-apoptosis signaling through hsp27 and hsp72 as well as cell survival signaling through Akt and survivin. LY294002 appears to be an attractive candidate for a p53-independent heat sensitizer in hyperthermic cancer therapy.
Assuntos
Cromonas/farmacologia , Temperatura Alta , Hipertermia Induzida , Neoplasias Pulmonares/enzimologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares , Mutação , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Elementos de Resposta/genética , Survivina , Fatores de Transcrição/metabolismo , Transfecção , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: To determine the effects of mild temperature hyperthermia (MTH) and p53 status of tumor cells on the size of hypoxic fractions (HFs) in solid tumors, with reference to the effect on intratumor quiescent (Q) cell populations. METHODS AND MATERIALS: Human head-and-neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into left hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received nicotinamide injection or carbogen gas (95% O(2), 5% CO(2)) inhalation combined with or without MTH. Nicotinamide prevents intermittent blood flow that could induce perfusion-limited acute hypoxia. Chronically hypoxic cells in regions beyond the limitation of oxygen diffusion in tumors are oxygenated by increasing the oxygen transport capacity of circulating blood with carbogen gas inhalation. After each treatment, the mice received a series of test doses of gamma-rays while alive or after tumor clamping to obtain HFs in the tumors. Immediately after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B) to inhibit cytoplasmic division while allowing nuclear division. Tumor cells not labeled with BrdU were detected with immunofluorescence staining of BrdU for P cells, and the micronucleus frequency in cells without BrdU labeling [ = Q cells] was determined. The micronucleus frequency in total (P + Q) tumor cells was determined from the tumors that were not pretreated with BrdU. RESULTS: SAS/mp53 tumors showed larger values for the size of not only the HF but also the diffusion-limited chronically HF than SAS/neo tumors. Q cell populations included a larger HF, particularly the chronically HF, than total cell populations in both tumors, especially in SAS/neo tumors. MTH could efficiently oxygenate the chronically HF, irrespective of p53 status. CONCLUSION: MTH is a useful combined treatment with a radioenhancement effect on intratumor Q cells, irrespective of the p53 status of tumor cells. The p53 status has the potential to affect microenvironmental conditions within solid tumors.
Assuntos
Carcinoma de Células Escamosas/fisiopatologia , Hipóxia Celular , Genes p53/genética , Neoplasias de Cabeça e Pescoço/fisiopatologia , Hipertermia Induzida/métodos , Mutação , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/radioterapia , Hipóxia Celular/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Nus , Testes para Micronúcleos , TransfecçãoRESUMO
Hyperthermia is useful for the treatment of human head and neck cancer, as it is relatively easy to perform thermoregulation when compared with deep organs. In this study, we focused attention on the p53 as a predictive indicator of hyperthermic cancer therapy. We used two kinds of cell lines of a human squamous cell carcinoma (SAS) with identical backgrounds of function except for the p53 protein. We assayed the heat sensitivity, frequency of apoptosis, and apoptosis-related gene expression after heat treatment using DNA array. The SAS/neo (wild-type p53; wtp53) cells were sensitive to heat, and the induction of Caspase-3 activation and apoptosis in the wtp53 cells was clearly high compared with the SAS/mp53 (mutated p53; mp53) cells. The gene expression of apoptosis suppressive-genes such as IL-12 p35 decreased in the wtp53 cells, and IL-12 R beta1 increased in the mp53 cells, though apoptosis-promotive genes of Caspase-9, CD30 and CD40 were induced p53-independently by hyperthermia. It is suggested that heat-induced apoptosis was suppressed by IL-12-related genes in the mp53 cells. These findings strongly imply that p53 status is a useful candidate for a predictive indicator of the effectiveness in hyperthermic therapy.
Assuntos
Apoptose/genética , Carcinoma de Células Escamosas/terapia , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Hipertermia Induzida , Neoplasias da Língua/terapia , Antígenos CD40/metabolismo , Carcinoma de Células Escamosas/genética , Caspase 3 , Caspase 9 , Caspases/metabolismo , Ativação Enzimática , Perfilação da Expressão Gênica , Humanos , Interleucina-12/metabolismo , Antígeno Ki-1/metabolismo , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Língua/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received tirapazamine (TPZ) with or without mild temperature hyperthermia (40 degrees C, 60 min) (MTH), gamma-ray irradiation with or without MTH and/or TPZ, cisplatin (CDDP) with or without MTH and/or TPZ, or paclitaxel (TXL) with or without MTH and/or TPZ. After each treatment, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling (i.e., quiescent (Q) cells) was determined by using immunofluorescence staining for BrdU. Meanwhile, 6 h after gamma-ray irradiation or 24 h after other cytotoxic treatments, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN frequency and apoptosis frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. On the whole, gamma-ray irradiation and CDDP injection induced a higher frequency of apoptosis and lower frequency of MN in SAS/neo cells than SAS/mp53 cells. There were no apparent differences in the induced frequency of apoptosis and MN between SAS/neo and SAS/mp53 cells after TPZ or TXL treatment. MTH sensitized cells to TPZ-inducing cytotoxicity more markedly in SAS/mp53 and Q cells than in SAS/neo cells and total cells, respectively. In gamma-ray irradiation and CDDP treatment, the enhancement in combination with MTH and/or TPZ was more remarkable in SAS/mp53 cells and Q cells than in SAS/neo and total tumor cells, respectively. Also in the case of TXL treatment, the combination with MTH and/or TPZ induced a slightly greater enhancement effect in SAS/mp53 cells and Q cells. In view of the difficulty in controlling mutated p53 status tumors and intratumor Q cells, combination treatment with MTH and/or TPZ as a cooperative modality in cancer therapy is considered to have potential for controlling solid tumors as a whole.