RESUMO
Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.
Assuntos
Actinas/efeitos da radiação , Divisão Celular/efeitos da radiação , Radiação Terahertz , Actinas/metabolismo , Citocinese/efeitos da radiação , Células HeLa/efeitos da radiação , Humanos , Interfase/efeitos da radiação , Microscopia de Fluorescência , Análise de Célula ÚnicaRESUMO
In this study, we determined the potential of polyethylene glycol-encapsulated iron oxide nanoparticles (IONPCO) for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to enhance the cytotoxic effects of ionizing radiation. The biological effects of IONP and X-ray irradiation (50 kV and 6 MV) were determined in HeLa cells using the colony formation assay (CFA) and detection of γH2AX foci. Data are presented as mean ± SEM. IONP were efficiently internalized by HeLa cells. IONPCO radiomodulating effect was dependent on nanoparticle concentration and photon energy. IONPCO did not radiosensitize HeLa cells with 6 MV X-rays, yet moderately enhanced cellular radiosensitivity to 50 kV X-rays (DMFSF0.1 = 1.13 ± 0.05 (p = 0.01)). IONPDOX did enhance the cytotoxicity of 6 MV X-rays (DMFSF0.1 = 1.3 ± 0.1; p = 0.0005). IONP treatment significantly increased γH2AX foci induction without irradiation. Treatment of HeLa cells with IONPCO resulted in a radiosensitizing effect for low-energy X-rays, while exposure to IONPDOX induced radiosensitization compared to IONPCO in cells irradiated with 6 MV X-rays. The effect did not correlate with the induction of γH2AX foci. Given these results, IONP are promising candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos , Compostos Férricos , Nanopartículas Metálicas , Tolerância a Radiação/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Portadores de Fármacos/química , Compostos Férricos/química , Células HeLa/efeitos dos fármacos , Células HeLa/patologia , Células HeLa/efeitos da radiação , Células HeLa/ultraestrutura , Humanos , Nanopartículas Metálicas/química , Radiação IonizanteRESUMO
The effect of terahertz (THz) radiation on deep tissues of human body has been considered negligible due to strong absorption by water molecules. However, we observed that the energy of THz pulses transmits a millimeter thick in the aqueous solution, possibly as a shockwave, and demolishes actin filaments. Collapse of actin filament induced by THz irradiation was also observed in the living cells under an aqueous medium. We also confirmed that the viability of the cell was not affected under the exposure of THz pulses. The potential of THz waves as an invasive method to alter protein structure in the living cells is demonstrated.
Assuntos
Citoesqueleto de Actina/efeitos da radiação , Radiação Terahertz , Citoesqueleto de Actina/metabolismo , Transferência de Energia , Células HeLa/efeitos da radiação , Humanos , Polimerização/efeitos da radiação , Soluções/efeitos da radiação , Radiação Terahertz/efeitos adversos , ÁguaRESUMO
This paper investigated an ultrasound vibration cell patterning technique. The ultrasound cell culture dish consisted of a culture dish with a glass bottom and a glass disc with a piezoelectric ring that generated a resonance flexural vibration mode on the bottom of the dish. The growth of HeLa cells on the dish was observed under ultrasound excitation for 24â¯h. Large ultrasound vibrations on the dish inhibited the cell growth. The acoustic field was predicted with finite element analysis and it was found that the cell growth depended strongly on both the acoustic field in the culture medium and the vibration distribution of the dish. The ultrasound vibrations did not affect the viability of the cells, and the cell growth could be controlled by the flexural vibration of the cultured dish.
Assuntos
Técnicas de Cultura de Células/instrumentação , Células HeLa/fisiologia , Células HeLa/efeitos da radiação , Ultrassom/instrumentação , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Análise de Elementos Finitos , Vidro/química , Humanos , VibraçãoRESUMO
BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) has been widely used to treat malignant tumors. Our previous studies indicated that connexin (Cx) 32- and Cx26-composed gap junctional intercellular communication (GJIC) could improve the phototoxicity of PDT. However, the role of heterotypic Cx32/Cx26-formed GJIC in PDT phototoxicity is still unknown. Thus, the present study was aimed to investigate the effect of Cx32/Cx26-formed GJIC on PDT efficacy. METHODS: CCK8 assay was used to detect cell survival after PDT. Western blot assay was utilized to detect Cx32/Cx26 expression. "Parachute" dye-coupling assay was performed to measure the function of GJ channels. The intracellular Ca2+ concentrations were determined using flow cytometer. ELISA assay was performed to detect the intracellular levels of PGE2 and cAMP. RESULTS: The present study demonstrates there is a Cx32/Cx26-formed GJIC-dependent reduction of phototoxicity when cells were exposure to low concentration of Photofrin. Such a protective action is missing at low cell density due to the lack of GJ coupling. Under high-cell density condition, where there is opportunity for the cells to contact each other and form GJ, suppressing Cx32/Cx26-formed GJIC by either inhibiting the expression of Cx32/Cx26 or pretreating with GJ channel inhibitor augments PDT phototoxicity after cells were treated with at 2.5 µg/ml Photofrin. The above results suggest that at low Photofrin concentration, the presence of Cx32/Cx26-formed GJIC may decrease the phototoxicity of PDT, leading to the insensitivity of malignant cells to PDT treatment. The GJIC-mediated PDT insensitivity was associated with Ca2+ and prostaglandin E2 (PGE2 ) signaling pathways. CONCLUSION: The present study provides a cautionary note that for tumors expressing Cx32/Cx26, the presence of Cx32/Cx26-composed GJIC may cause the resistance of tumor cells to PDT. Oppositely, treatment strategies designed to downregulate the expression of Cx32/Cx26 or restrain the function of Cx32/Cx26-mediated GJIC may increase the sensitivity of malignant cell to PDT. Lasers Surg. Med. 51:301-308, 2019. © 2019 Wiley Periodicals, Inc.
Assuntos
Comunicação Celular/efeitos da radiação , Conexina 26/fisiologia , Conexinas/fisiologia , Junções Comunicantes/efeitos da radiação , Células HeLa/efeitos da radiação , Fotoquimioterapia/efeitos adversos , Técnicas de Cultura de Células , Sobrevivência Celular , Éter de Diematoporfirina/farmacologia , Células HeLa/patologia , Humanos , Fármacos Fotossensibilizantes/farmacologia , Proteína beta-1 de Junções ComunicantesRESUMO
Ultraviolet (UV) irradiation is a common form of DNA damage that can cause pyrimidine dimers between DNA, which can cause gene mutations, even double-strand breaks and threaten genome stability. If DNA repair systems default their roles at this stage, the organism can be damaged and result in disease, especially cancer. To better understand the cellular response to this form of damage, we applied highly sensitive mass spectrometry to perform comparative proteomics of phosphorylation in HeLa cells. A total of 4367 phosphorylation sites in 2100 proteins were identified, many of which had not been reported previously. Comprehensive bioinformatics analysis revealed that these proteins were involved in many important biological processes, including signaling, localization and cell cycle regulation. The nuclear pore complex, which is very important for RNA transport, was changed significantly at phosphorylation level, indicating its important role in response to UV-induced cellular stress. Protein-protein interaction network analysis and DNA repair pathways crosstalk were also examined in this study. Proteins involved in base excision repair, nucleotide repair and mismatch repair changed their phosphorylation pattern in response to UV treatment, indicating the complexity of cellular events and the coordination of these pathways. These systematic analyses provided new clues of protein phosphorylation in response to specific DNA damage, which is very important for further investigation. And give macroscopic view on an overall phosphorylation situation under UV radiation.
Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Dímeros de Pirimidina/efeitos da radiação , Células HeLa/efeitos da radiação , Humanos , Espectrometria de Massas , Fosforilação/efeitos da radiação , Mapas de Interação de Proteínas/efeitos da radiação , Raios UltravioletaRESUMO
We report on a highly efficient magneto-actuated cancer cell apoptosis method using a biaxial pulsed magnetic field configuration, which maximizes the induced magnetic torque. The light transmissivity dynamics show that the biaxial magnetic field configuration can actuate the magnetic nanoparticles with higher responsiveness over a wide range of frequencies as compared to uniaxial field configurations. Its efficacy was demonstrated in in vitro cell destruction experiments with a greater reduction in cell viability. Magnetic nanoparticles with high aspect ratios were also found to form a triple vortex magnetization at remanence which increases its low field susceptibility. This translates to a larger magneto-mechanical actuated force at low fields and 12% higher efficacy in cell death as compared to low aspect ratio nanoparticles.
Assuntos
Apoptose/efeitos da radiação , Células HeLa/fisiologia , Células HeLa/efeitos da radiação , Campos Magnéticos , Nanopartículas Metálicas/efeitos da radiação , HumanosRESUMO
Objective: To investigate the killing effect of low-temperature plasma (LTP) on HepG2, A549 and HeLa cell lines and explore its possible mechanism. Methods: The inhibitory effect of LTP on the proliferation of HepG2, A549 and HeLa cells was determined by MTT assay. Transmission electron microscopy was used to observe the ultrastructural changes of HepG2, A549 and HeLa cells treated with LTP. Cell apoptosis was detected by Muse cytometry. Western blot was used to detect the expression of apoptosis-related proteins. Results: The survival rates of LTP-irradiated HepG2 cells (irradiated for 107 s), HeLa cells (irradiated for 121 s) and A549 cells (irradiated for 127 s) were 50%. LTP destroyed the ultrastructure of HepG2, A549 and HeLa cells to different degrees, showing nuclear fragmentation and organelle damages. The apoptosis rates of the three cell lines were increased at 24 h after exposure to LTP for 1/6 IC50 irradiation time. Furthermore, LTP irradiation also suppressed the protein expression of Bcl-2 and XRCC1 and increased that of Bax. Conclusions: LTP has an obvious killing effect on HepG2, A549 and HeLa cancer cell lines. This effect may be related to the induction of cell apoptosis and inhibition of DNA repair.
Assuntos
Células A549/fisiologia , Apoptose , Proliferação de Células , Crioterapia/métodos , Células HeLa/fisiologia , Células Hep G2/fisiologia , Células A549/efeitos da radiação , Células A549/ultraestrutura , Proteínas Reguladoras de Apoptose/metabolismo , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células HeLa/efeitos da radiação , Células HeLa/ultraestrutura , Células Hep G2/efeitos da radiação , Células Hep G2/ultraestrutura , HumanosRESUMO
Ultraviolet (UV) radiation is a convenient stimulus for triggering cell death that is available in most laboratories. We use a Stratalinker UV cross-linker because it is a safe, cheap, reliable, consistent, and easily controlled source of UV irradiation. This protocol describes using a Stratalinker to trigger UV-induced death of HeLa cells.
Assuntos
Morte Celular , Células HeLa/efeitos da radiação , Raios Ultravioleta , HumanosRESUMO
The tumor suppressor, cylindromatosis (CYLD), is a negative regulator of NF-κB signaling by removing lysine 63-linked ubiquitin chains from multiple NF-κB signaling components, including TRAF2, TRAF6, and NEMO. How CYLD itself is regulated, however, remains yet to be characterized. In this study, we present the first evidence that UV irradiation is able to induce CYLD translocation from the cytoplasm to microtubules and that the cytoskeleton-associated CYLD is subject to posttranslational modification and degradation in a proteasome-independent manner. By immunostaining, we found that CYLD displayed microtubule-like filament localization under ultraviolet (UV) irradiation. Further studies revealed that the cytoskeleton-associated CYLD underwent posttranslational modification, which in turn contributed to CYLD degradation in an unknown manner, distinct from proteasome-mediated degradation under normal conditions. Collectively, our data suggest that UV-induced CYLD degradation might serve as an underlying mechanism for UV-induced NF-κB pathway activation.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Raios Ultravioleta/efeitos adversos , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Citoesqueleto/metabolismo , Enzima Desubiquitinante CYLD , Células HeLa/efeitos da radiação , Humanos , Células MCF-7/efeitos da radiação , Microtúbulos/metabolismo , Microtúbulos/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Transporte Proteico/efeitos da radiaçãoRESUMO
Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell-based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.
Assuntos
Ciclo Celular/efeitos da radiação , Raios X/efeitos adversos , Divisão Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Fase G1/efeitos da radiação , Fase G2/efeitos da radiação , Células HeLa/efeitos da radiação , Humanos , Cinética , Microscopia de Fluorescência , Fase S/efeitos da radiaçãoRESUMO
Histone modifications are involved in the DNA damage response (DDR). Here, by utilizing an ELISA immunoassay we assessed the methylation at H3K9 (H3K9me2 and H3K9me3) in two cell lines with differential sensitivity to radiation-induced apoptosis, HeLa (sensitive) and MCF-7 (resistant). We found that DNA damage induction by γ-irradiation leads to considerable accumulation (up to 5-fold) of H3K9me2 and H3K9me3, but not of H4K20me3 (control modification) in MCF-7 cells (p<0.05). Interestingly, a lower dose (2 Gy) was more effective than 5 Gy. In HeLa cells a smaller effect (approx. 1.5-1.8-fold) was evident only at 5 Gy. In conclusion, our findings reveal that DNA damage leads to specific accumulation of H3K9me2 and H3K9me3 in a cell-type specific manner.
Assuntos
Heterocromatina/metabolismo , Histonas/metabolismo , Radiação Ionizante , Dano ao DNA/fisiologia , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Heterocromatina/efeitos da radiação , Humanos , Lisina/metabolismo , Células MCF-7/metabolismo , Células MCF-7/efeitos da radiação , Metilação , Tolerância a Radiação , Proteína Supressora de Tumor p53/metabolismoRESUMO
In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , Fatores de Transcrição/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa/efeitos da radiação , Humanos , Imidazóis/farmacologia , Células MCF-7/efeitos dos fármacos , Células MCF-7/efeitos da radiação , Complexo Mediador/genética , Piperazinas/farmacologia , Ligação Proteica , Transporte Proteico/efeitos da radiação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Polimerase II/metabolismo , Ativação Transcricional , Raios UltravioletaRESUMO
Crosstalk between cancer cells and the surrounding cancer associated fibroblasts (CAFs) plays an illusive role in cancer radiotherapy. This study investigated the effect of cancer cell-cancer associated fibroblasts crosstalk on the proliferation and survival of irradiated cervical cancer cells. A pretreatment with conditioned medium from a mixed culture of CAF and HeLa cells (mixCAF) had a stronger effect on enhancing the proliferation and survival of irradiated HeLa cells compared to pretreatment with CAF conditioned medium alone. In addition, pretreatment with a mixed culture of CAF and HeLa cells conditioned medium reduced the levels of two major radiation-induced genes, GADD45 and BTG2, and phosphorylation of p38. Profiling of the growth and survival factors in the conditioned medium revealed PDGF and VEGF, and IGF2, EGF, FGF-4, IGFBPs and GM-CSF to be specifically secreted from HeLa cells and CAFs, respectively. This study demonstrated radiation protective effects of CAF-cancer cell crosstalk, and identified multiple growth factors and radiation response genes that might be involved in these effects.
Assuntos
Carcinoma de Células Escamosas/patologia , Comunicação Celular , Células Epiteliais/patologia , Fibroblastos/patologia , Células Estromais/patologia , Microambiente Tumoral , Neoplasias do Colo do Útero/patologia , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos da radiação , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Células HeLa/transplante , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fosforilação/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Tolerância a Radiação , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/metabolismo , Ensaio Tumoral de Célula-Tronco , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation.
Assuntos
Ficusina/química , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Complexo de Inativação Induzido por RNA/genética , Adenosina , Reagentes de Ligações Cruzadas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células HeLa/efeitos da radiação , Humanos , Oligonucleotídeos Antissenso/farmacologia , Fotoquímica/métodos , Complexo de Inativação Induzido por RNA/química , Termodinâmica , Thermus thermophilus/genética , Raios UltravioletaRESUMO
OBJECTIVE: To compare the efficacies and side effects of Iodine-125 (125I) seeds implantation with afterloading radiotherapy on tumor xenografts derived from Hela cells. METHODS: Mice bearing Hela cells were randomly divided into three groups: two therapeutic groups receiving four 125I seeds implantation and afterloading therapy, respectively, and the one control group without any intervention. Comparisons were evaluated in aspects of curative efficacies (tumor volume, tumor inhibition rate and metastasis) and side effects (body weight, ovarian endocrine functions, skin lesion surrounding the tumor). RESULTS: The xenografts tumor volume of therapeutic groups were significantly smaller than that of controls(p < 0.05),both of the 125I seeds implantation and afterloading therapy showed excellent tumor inhibition rate. Furthermore, 125I seeds implantation had milder side effects on skin, weight loss, ovarian endocrine functions. CONCLUSIONS: (125)I seed implantation may be an alternative minimally invasive therapy for cervical cancer.
Assuntos
Braquiterapia/métodos , Radioisótopos do Iodo/uso terapêutico , Neoplasias do Colo do Útero/radioterapia , Animais , Feminino , Células HeLa/efeitos da radiação , Xenoenxertos/efeitos da radiação , Humanos , Iodo , Camundongos , Camundongos Endogâmicos BALB C , Dosagem Radioterapêutica , Distribuição Aleatória , Resultado do TratamentoRESUMO
Radioresistance of cancer stem cells (CSCs) is regarded as one of the possible causes of cancer recurrence after radiotherapy. Since the regularities and mechanisms of radiation effects on this population of cells have not been sufficiently studied, the aim of this work is to elucidate the changes in the CSC number after γ-irradiation in stable cultures of tumor cells in vitro and tumor tissue in vivo (in the course of radiation therapy of patients with cancers of the upper respiratory tract). CSCs were identified in the cell lines B16, MCF-7, HeLa by the ability to exclude the fluorescent dye Hoechst 33342 (SP method) 48-72 h after irradiation at the doses of 1-20 Gy and in biopsy material by immunophenotype CD44+CD24(-/low) before and 24 h after irradiation at the total dose of 10 Gy. The essential differences in the response of CSCs and other cancer cells were found after exposure to low-LET radiation. The absolute number of CSCs increased after a single exposure at the doses ranging from 1 to 5-10 Gy in different cell cultures, but a further dose increase maintained the current number of CSCs or decreased it. At the same time, the number of non CSCs significantly decreased with increasing doses of radiation exposure, as expected. Fractionated irradiation in vivo at a total dose of 10 Gy increased the relative amount of CSCs in most patients. The registered changes are an integral indicator of cell death, cell division delay immediately after irradiation, proliferation at a later time, possible dedifferentiation of non CSCs, etc. The exact contribution of each of them to the radiation-induced increase of the CSCs number is of considerable interest and requires further research.
Assuntos
Raios gama , Neoplasias Laríngeas/radioterapia , Células-Tronco Neoplásicas/efeitos da radiação , Tolerância a Radiação/genética , Adulto , Idoso , Animais , Feminino , Células HeLa/efeitos da radiação , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Imunofenotipagem , Neoplasias Laríngeas/patologia , Células MCF-7/efeitos da radiação , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/ultraestruturaRESUMO
The response of HeLa cells to subcellular spatially localized two-photon irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. Upon irradiation under these conditions, a localized population of PpIX excited states can be produced with meaningful intracellular spatial resolution; the dimensions of the domain where the incident light flux is high enough for PpIX two-photon absorption are defined by the microscope optics and by the diffraction of light (spot diameter at beam waist of Ë0.5-1.0 µm). In turn, the dimensions of the intracellular domain containing cytotoxic PpIX-sensitized singlet oxygen will likewise be confined. Most importantly, cell response (e.g., morphological signs of cell death) correlates with the light dose delivered and the intracellular domain irradiated. Thus, controlling light delivery can complement other techniques used to impart intracellular spatial localization in mechanistic studies of photoinitiated reactive oxygen species. Such controlled light delivery is also expected to be a particularly useful tool to study the so-called bystander effect in which a selectively-perturbed cell can influence a neighboring cell through intercellular signaling mechanisms.
Assuntos
Fótons , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/metabolismo , Oxigênio Singlete/metabolismo , Apoptose/efeitos da radiação , Efeito Espectador , Células HeLa/efeitos da radiação , Humanos , Lasers , Luz , Transdução de Sinais/efeitos da radiaçãoRESUMO
FANCJ mutations are linked to Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double-strand breaks (DSBs) and interstrand cross-links (ICLs), but its mechanism of action is not well understood. Here we show with live-cell imaging that FANCJ recruitment to laser-induced DSBs but not psoralen-induced ICLs is dependent on nuclease-active MRE11. FANCJ interacts directly with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ regulates the MRE11 nuclease to facilitate DSB processing and appropriate end resection. Cells deficient in FANCJ and MRE11 show increased ionizing radiation (IR) resistance, reduced numbers of γH2AX and RAD51 foci, and elevated numbers of DNA-dependent protein kinase catalytic subunit foci, suggesting that HR is compromised and the nonhomologous end-joining (NHEJ) pathway is elicited to help cells cope with IR-induced strand breaks. Interplay between FANCJ and MRE11 ensures a normal response to IR-induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and the FA pathway. Our findings are discussed in light of the current model for HR repair.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Dano ao DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Hidrolases Anidrido Ácido , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Ficusina/farmacologia , Células HeLa/efeitos dos fármacos , Células HeLa/efeitos da radiação , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Radiação Ionizante , Reparo de DNA por RecombinaçãoRESUMO
The aim of this in vitro study was to evaluate the effects of low level laser irradiation on the proliferation of HeLa cells using 405 nm diode laser, 514 nm argon laser, 633 nm He-Ne laser, or 785 nm diode laser, The cells were seeded on 96-well microplates for 24 h in 5% fetal bovine serum containing medium, then irradiated with the laser at dose of 100 and 1 000 J x m(-2), respectively. At the time point of 24, 48, 72 h after irradiation, cell viability was assessed by MTT assay. The results show that 405, 633 and 785 nm laser irradiation induces wavelength-dependent and time-dependent proliferation. 633 nm laser irradiation results in a stimulatory proliferation effect that is most significant, whereas 514 nm laser irradiation produces little increase in cell proliferation. Low level laser irradiation increases cell proliferation in a dose-dependent manner. 1 000 J x m(-2) laser irradiation is more effective in increasing cell proliferation than 100 J x m(-2) laser irradiation using 405 nm diode laser, 633 nm He-Ne laser, or 785 nm diode laser, but not as effective as using 514 nm argon laser.