RESUMEN
BACKGROUND: Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) have been shown to be of merit as biomarkers for a variety of cancers. Prostate tissue resections from patients with prostate cancer (PCa) and benign prostatic hyperplasia were analysed to determine the influence of freeze-drying on the recovery of uPA and PAI-1 and their predictive performance. METHODS: Prostate tissue was frozen in liquid nitrogen and homogenised into a fine powder in a precooled stainless steel punch homogeniser. One aliquot of the powder was extracted directly, and a second aliquot was freeze-dried overnight and then extracted. The extracts were analysed by FEMTELLE assay to determine the concentrations of uPA and PAI-1. uPA/PAI-1 ratios were calculated for each sample, and the mean ratios for the frozen and the lyophilised tissue were compared. RESULTS: The concentrations of uPA measured for the frozen and lyophilised samples are strongly correlated (R = 0.90 ± 0.05). The same applies to the PAI-1 measured (R = 0.89 ± 0.03). The uPA/PAI-1 ratios for the lyophilised and frozen samples were strongly correlated. The uPA/PAI-1 ratios for frozen and lyophilised samples were found to be essentially the same with values of 0.0344 ± 0.0066 and 0.0340 ± 0.0068, respectively (P = 0.9633). CONCLUSION: The recovery of uPA and PAI-1 from a deep frozen prostate tissue homogenate followed by freeze-drying proceeds with a loss of 10 and 11%, respectively, with no influence on the uPA/PAI-1 ratio. Lyophilisation is a safe procedure for the preservation of frozen prostate tissue samples as it permits recovery of the markers without compromising their use for diagnostic purposes.
Asunto(s)
Biomarcadores de Tumor/análisis , Liofilización/métodos , Inhibidor 1 de Activador Plasminogénico/análisis , Neoplasias de la Próstata , Activador de Plasminógeno de Tipo Uroquinasa/análisis , Anciano , Anciano de 80 o más Años , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Neoplasias de la Próstata/patologíaRESUMEN
Normal tissue toxicity resulting from chemoradiotherapy is of significant clinical concern. This study used normal Chinese hamster fibroblasts from lung (V79) and ovary (CHO-K1) to assess the modulation of cellular response to photons and neutrons by cisplatin, vinblastine, and bleomycin. Based on the colony formation assay, the drug concentration corresponding to 50% cell survival (EC50) of V79 cells was 1.50 +/- 0.21 micromol/L for cisplatin, 0.97 +/- 0.06 nmol/L for vinblastine, and 1.68 +/- 0.11 micromol/L for bleomycin. The corresponding values for CHO-K1 cells were significantly lower for vinblastine (0.54 +/- 0.02 nmol/L) and bleomycin (0.49 +/- 0.13 micromol/L), but not for cisplatin (1.57 +/- 0.20 micromol/L). No radiosensitivity enhancement was apparent when cells were exposed to p(66)/Be neutrons or photons (60Co gamma-rays) in the presence of these drugs at EC50 concentrations. These data suggest that concurrent use of these drugs with radiation for the treatment of lung and ovarian diseases radiation does not exacerbate radiation-induced normal tissue toxicity, regardless of the quality of radiation. The relatively higher sensitivity of the ovarian cells to vinblastine and bleomycin might constitute a limitation in the use of these drugs for the treatment of lung lesions.
Asunto(s)
Antineoplásicos/farmacología , Bleomicina/farmacología , Cisplatino/farmacología , Neutrones Rápidos , Fotones , Tolerancia a Radiación/efectos de los fármacos , Vinblastina/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a DrogaRESUMEN
The effects of cisplatin exposure time, concentration, and irradiation sequence on the sensitivity of Chinese hamster lung fibroblasts (V79) to gamma-ray exposure were examined. Based on clonogenic cell survival, the cisplatin concentrations corresponding to 50% cell survival (EC(50)) for exposure times of 1 h to 7 days followed a 2-phase exponential decay and ranged from 28.26 +/- 3.32 to 1.53 +/- 0.24 micromol/L, respectively. When cells were treated at EC(50) for exposures of less than 4 h and irradiated immediately, cisplatin inhibited the effect of radiation. Exposures of 4-6 h did not affect radiosensitivity. For exposures of 8-12 h, radiosensitization was observed, which disappeared at 14 h and reappeared for much longer cisplatin treatments. At the lowest achievable EC(50) (1.53 micromol/L), radiosensitization was observed if irradiation was delayed for 1-8 h. This enhancement in radiosensitivity disappeared for irradiation delays of 10-12 h, but reappeared when irradiation was delayed for 14-18 h. These data demonstrate that the mode of interaction between cisplatin and gamma-irradiation depends on the concentration and exposure time of cisplatin, as well as on the timing of irradiation after cisplatin administration. Consideration of changes in cell cycle kinetics may contribute to the improvement of treatment outcomes in adjuvant chemoradiotherapy involving cisplatin.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Fibroblastos/efectos de los fármacos , Pulmón/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de la radiación , Concentración 50 Inhibidora , Pulmón/efectos de la radiación , Factores de TiempoRESUMEN
PURPOSE: To examine the role of DNA double-strand break (DSB) rejoining in cell survival and micronucleus yield after 60Co gamma-irradiation. MATERIALS AND METHOD: Thirteen human cell lines (six glioblastoma, five prostate, one melanoma, one squamous cell carcinoma) were irradiated with 60Co gamma-rays to doses of 0-10Gy for cell survival and micronucleus measurements and 0-100Gy for DSB rejoining. Measurements were performed using standard clonogenic, micronucleus and constant-field gel electrophoresis assays. RESULTS: Radioresistance and micronucleus yield were positively correlated (r=0.74, p=0.004). A significant cell type-dependent correlation was demonstrated between total (0-20 h) DSB rejoining and cell survival (r=0.86, p=0.03 for glioblastomas; r=0.79, p=0.04 for other cell lines), with more resistant cell lines showing higher levels of DSB rejoining. No relationship was apparent between fast (0-2 h) or slow (2-20 h) DSB rejoining and clonogenic survival. While there was no relationship between total or slow DSB rejoining and micronucleus yield, a significant and cell type-specific correlation emerged between fast rejoining and micronucleus yield for the glioblastomas (r=0.89, p=0.04) and other cell lines (r=0.76, p=0.04). Cell lines with higher levels of DSB rejoining within 2 h of irradiation showed higher yields of micronuclei. CONCLUSION: Fast DSB rejoining, possibly through interaction with slow DSB rejoining, appears to play an important role in the formation of micronuclei. However, total DSB rejoining reflects intrinsic radiosensitivity. Consideration of differences in DSB rejoining kinetics might contribute to a better understanding of the significance of cell survival and micronucleus data in the clinical and radiation protection setting.
Asunto(s)
Radioisótopos de Cobalto/uso terapéutico , Daño del ADN , Rayos gamma , Glioblastoma/radioterapia , Pruebas de Micronúcleos , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta en la Radiación , Humanos , Cinética , Micronúcleos con Defecto Cromosómico/genética , Factores de TiempoRESUMEN
BACKGROUND: Determination of the drug concentration required to kill 50% of the tumour cells (EC50) does not take into account the propensity of cells to undergo apoptosis and necrosis. These 2 parameters and the viable cells are here assessed by a flow cytometric (FC) approach using propidium iodide (PI) and FITC-Annexin V staining. MATERIALS AND METHODS: A number of carcinoma cell lines of defined p53 status were exposed to cis-PtII for 24 hours, stained with PI and FITC-Annexin V and analyzed by FC. Unstained viable cells, early apoptotic cells and necrotic cells were scored separately in dual parameter plots of green fluorescence (FITC) against red fluorescence (PI) to generate dose response curves. RESULTS: EC50 values for cell viability were found to be 1-4 times higher than survival data from colony assays resembling data obtained by MTT or Crystal Violet vital dye staining. Percentage apoptosis measured by Annexin V binding was in agreement with microscopic scoring of apoptotic cells after Acridine Orange staining. CONCLUSION: The FC assay described gives a good estimate of cell viability resembling data from vital dye staining assays and provides additional information on apoptosis and necrosis. FC data from Annexin V binding and microscopic scoring after Acridine Orange staining were in excellent agreement.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Citometría de Flujo/métodos , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Supervivencia Celular/efectos de los fármacos , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
At present micronucleus data cannot predict cellular radiosensitivity. The inclusion of data from apoptosis and abnormal morphology has not entirely resolved this problem. Here, we assess the probability of cell death arising from events other than micronucleation, apoptosis and abnormal morphology (i.e. lesions not detected by these damage assays) P(oe), for its ability to reflect intrinsic cellular radiosensitivity. Analysis of data from 17 cell lines used in two separate studies, spanning a wide range of radiosensitivity (0.09
Asunto(s)
Apoptosis , Pruebas de Micronúcleos/métodos , Línea Celular , Supervivencia Celular , Relación Dosis-Respuesta en la Radiación , Humanos , Micronúcleos con Defecto Cromosómico/patología , Modelos Teóricos , Tolerancia a Radiación , Factores de TiempoRESUMEN
The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.
Asunto(s)
Glioblastoma/patología , Insecticidas/toxicidad , Limoninas , Triterpenos/toxicidad , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Humanos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
It is now well established that micronuclei frequency does not always rank cell lines according to radiosensitivity. There is, however, a growing interest in reconstructing cellular radiosensitivity (measured by colony assay) from concurrent micronucleus and apoptosis data. Using a variety of radiosensitive and radioresistant cell lines, we have derived a missing parameter--Poe, the probability of cell death by other events such as small deletions, chromosome aberrations, late apoptosis and necrosis which are undetectable by micronucleus and apoptosis assays performed at a single time point. In the radioresistant glioma cell lines G120, G60, G28, G44 and G62 (SF2 > or =0.59), a characteristic threshold dose exists above which cell loss due to undetectable lesions occurs. In the radiosensitive SK-N-SH and KELLY cell lines (SF2 < or =0.43), the Poe parameter is positive at very low doses, reaches a maximum and declines at higher doses. In the radiation resistant G28 cells, Poe was found to be below zero for doses up to 6 Gy. In the G62, G44 and G120 cell lines, the threshold doses to induce Poe events were 0.87, 3.04 and 3.85 Gy, respectively. Cell death by undetectable lesions is a cell-specific and time-dependent variable. Micronucleus and apoptosis assays performed concurrently and at a specific time point miss cell death due to other events and this may be the reason why reconstruction of cellular radiosensitivity from micronucleus and apoptosis data fails.
Asunto(s)
Glioma/metabolismo , Micronúcleos con Defecto Cromosómico/metabolismo , Neuroblastoma/metabolismo , Apoptosis/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Pruebas de Micronúcleos/métodos , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2