RESUMEN
The presence of hypoxic cells in human solid tumors is an important factor leading to resistance to radiation therapy and chemotherapy. However, differences in the oxygen tension between normal tissues and tumors also provide the potential for designing tumor-specific gene therapy. The strategy is to selectively induce the expression of suicide genes under hypoxia and thereby preferentially kill hypoxic cells. The hypoxia-responsive vector regulates gene expression via the hypoxia-responsive element, which can be activated through the transcriptional complex hypoxia-inducible factor 1. A gene therapy that is based on hypoxia-regulated gene expression needs to consider the suicide gene, the genetic vector, the delivery method and the bystander effect. These factors pose considerable challenges for the development of a successful hypoxia-directed gene therapy, but once this has been achieved, this type of therapy in combination with traditional radiation and chemotherapy should provide an improved clinical outcome for patients with these diseases.
Asunto(s)
Regulación de la Expresión Génica/genética , Terapia Genética , Hipoxia/genética , Neoplasias/terapia , Animales , Humanos , Neoplasias/metabolismo , Transducción de Señal/genéticaRESUMEN
Human malignant gliomas are thought to develop as the result of stepwise accumulations of multiple genetic alterations. Recently, we showed that E6/E7-mediated inactivation of p53/pRb, ras pathway activation (initiated by expression of mutant H-Ras), and expression of human telomerase reverse transcriptase (hTERT) in combination converted normal human astrocytes into cells that formed intracranial tumors resembling human anaplastic astrocytoma (AA). In this study, we created human astrocytes that, in addition to expressing E6/E7, hTERT, and Ras, also expressed a constitutive activated form of Akt intended to mimic the Akt activation noted in grade IV glioblastoma multiforme (GBM). Although these cells grew no differently than astrocytes expressing E6, E7, and H-Ras in vitro or in the first 28 days following s.c. implantation, they ultimately formed tumors four to six times larger than those formed by the E6/E7/hTERT/Ras cells. Unlike the poorly vascularized, necrosis-free AA formed by E6/E7/hTERT/Ras cells, the tumors formed by s.c. or intracranial injection of Akt-expressing cells had large areas of necrosis surrounded by neovascularization and were consistent in appearance with grade IV human GBM. These results show that activation of the Akt pathway is sufficient to allow conversion of human AA to human GBM.
Asunto(s)
Astrocitoma/enzimología , Astrocitoma/patología , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Glioblastoma/enzimología , Glioblastoma/patología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Animales , Astrocitos/enzimología , Astrocitos/patología , Astrocitos/fisiología , Astrocitoma/genética , Neoplasias Encefálicas/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Activación Enzimática , Glioblastoma/genética , Humanos , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Ratas , Transducción de Señal/fisiología , TransfecciónRESUMEN
The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen) produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE) from the erythropoietin gene (Epo), which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1). Under anoxic conditions, nine copies of HRE (9XHRE) yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV) in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Terapia Genética , Glioblastoma/metabolismo , Hipoxia Encefálica/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Factores de Transcripción , Adenoviridae/genética , Western Blotting , Neoplasias Encefálicas/terapia , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Glioblastoma/terapia , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Operón Lac/genética , Oxígeno/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , beta-Galactosidasa/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is an important mediator of the intense angiogenesis which is characteristic of glioblastoma. While genetic manipulation of VEGF/VEGF receptor expression has previously been shown to inhibit glioblastoma growth, to date, no study has examined the efficacy of pharmacologic blockade of VEGF activity as a means to inhibit intracranial growth of human glioblastoma. Using intraperitoneal administration of a neutralizing anti-VEGF antibody, we demonstrate that inhibition of VEGF significantly prolongs survival in athymic rats inoculated in the basal ganglia with G55 human glioblastoma cells. Systemic anti-VEGF inhibition causes decreased tumor vascularity as well as a marked increase in tumor cell apoptosis in intracranial tumors. Although intracranial glioblastoma tumors grow more slowly as a consequence of anti-VEGF treatment, the histologic pattern of growth suggests that these tumors adapt to inhibition of angiogenesis by increased infiltration and cooption of the host vasculature.
Asunto(s)
Anticuerpos/uso terapéutico , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/terapia , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Glioblastoma/irrigación sanguínea , Glioblastoma/terapia , Linfocinas/antagonistas & inhibidores , Neovascularización Patológica/prevención & control , Animales , Neoplasias Encefálicas/patología , Factores de Crecimiento Endotelial/inmunología , Femenino , Glioblastoma/patología , Humanos , Etiquetado Corte-Fin in Situ , Linfocinas/inmunología , Ratas , Ratas Desnudas , Tasa de Supervivencia , Factores de Tiempo , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The status of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP) in seven human glioma cell lines was evaluated to extend our knowledge of human glioblastoma multiforme tumor metabolism for future drug design. Cell lines SF-767, SF-763, A-172, U-87 MG, U-251 MG, U-343 MG, and SF-539 were used. Binding of 125I-labeled LDL to these cells at 4 degrees C was carried out to determine the number of LDL receptors on cells and the affinity of LDL for these receptors. The content of LRP was measured by immunoblotting. The presence of specific saturable LDL receptors was proven in six of the cell lines investigated. SF-767 cells revealed high-affinity LDL binding (equilibrium dissociation constant, Kd = 7 nM) and maximum binding capacity approximating 300,000 receptors/cell. Most of the remaining cell lines had relatively lower affinity (Kd = 38-62 nM) but also had very high numbers of receptors (128,000-950,000/cell). All cell lines exhibited LRP, but the expression was variable. The cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this protein. The data suggest that glioblastoma cells have high numbers of LDL receptors; however, there is considerable variation in binding affinity. Overall, this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents. LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target.
Asunto(s)
Glioblastoma/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de LDL/metabolismo , Sitios de Unión , Western Blotting , Diseño de Fármacos , Expresión Génica , Glioblastoma/patología , Humanos , Lipoproteínas LDL/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Termodinámica , Células Tumorales CultivadasRESUMEN
PURPOSE: Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. METHODS AND MATERIALS: The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 microg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). RESULTS: BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 microg/ml, NS), the amount of boron retained was near 10(9) atoms per cell (15 microg B/g cells). Lysosomes containing high concentrations of BOPP were randomly distributed throughout the cytoplasm; however, larger lysosomes containing BOPP were concentrated around the cell nucleus. Little or no BOPP accumulated in the cell nucleus. At incubation concentrations of 20 and 40 microg/ml (24-h time point), BOPP uptake in SF-767 cells was reduced in LPDS compared with NS (66% reduction). A similar result was observed for normal human fibroblasts (AG-1522 cells, 40 microg/ml, 24 h). At 40 microg/ml, in both NS and LPDS at 24 h, BOPP accumulation in LDL receptor-deficient human fibroblasts (GM019-15C cells) was reduced relative to AG-1522 cells. BOPP accumulation in GM019-15C cells (40 microg/ml, 24 h) was not affected by serum lipoprotein levels. CONCLUSION: In cell culture, BOPP is taken up by human glioma cells via the LDL pathway and is compartmentalized into cellular lysosomes. Knowledge of this mechanism of BOPP uptake and retention will be important in attempts to modify toxicity and efficacy of this drug.
Asunto(s)
Antineoplásicos/farmacocinética , Compuestos de Boro/farmacocinética , Glioblastoma/metabolismo , Lisosomas/metabolismo , Protoporfirinas/farmacocinética , Receptores de LDL/metabolismo , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lipoproteínas/metabolismo , Microscopía Fluorescente , Células Tumorales Cultivadas/metabolismoRESUMEN
Phenylacetate (PA) inhibits the growth of tumor cells in vitro and in vivo and shows promise as a relatively nontoxic agent for cancer treatment. A recent report shows that prolonged exposure of cells to low concentrations of PA can enhance the radiation response of brain tumor cells in vitro, opening up the possibility of using this drug to improve the radiation therapy of brain tumor patients. We investigated the cytotoxicity produced by sodium phenylacetate (NaPA) alone and in combination with X-rays in SF-767 human glioblastoma cells and in two medulloblastoma cell lines, Masden and Daoy. Exposure of all three cell lines to relatively low concentrations of NaPA for up to 5 days did not enhance the subsequent cell killing produced by X-irradiation. However, enhanced cell killing was achieved by exposing either oxic or hypoxic cells to relatively high drug concentrations ( > 50-70 mM) for 1 h immediately before X-irradiation. Because central nervous system toxicity can occur in humans at serum concentrations of approximately 6 mM PA, translation of these results into clinical trials will likely require local drug-delivery strategies to achieve drug concentrations that can enhance the radiation response. The safety of such an approach with this drug has not been demonstrated.
Asunto(s)
Fenilacetatos/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Neoplasias Encefálicas , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Hipoxia de la Célula , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Glioblastoma , Humanos , Meduloblastoma , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
PURPOSE: Glioblastoma multiforme brain tumors (GM) are treated with a spectrum of fractionation regimens based on the clinical and anatomical characteristics of the tumor but rarely based on the molecular characteristics of the individual neoplasm. This study tests the hypothesis that the response of cell lines derived from GM to fractionated radiotherapy depends on the function of wild-type p53 (wt p53), a tumor suppressor gene frequently mutated in GM tumors. METHODS & MATERIALS: Isogenic derivatives of glioblastoma cells differing only in p53 function were prepared using a retroviral vector expressing a dominant negative mutant of p53 (mt p53). Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Apoptosis was assayed by a terminal deoxynucleotide transferase-labeling technique and chromatin morphology. RESULTS: We have previously reported the generation of isogenic GM cell lines differing only in p53 function. U87-175.4, lacking wt p53 function, had a significantly lower alpha/beta value than U87-LUX.8, expressing functional wt p53, leading us to hypothesize that fractionated irradiation would preferentially spare GM cells harboring mt p53 compared with those expressing functional, wt p53. Survival curves following either 2.0 Gy or 3.5 Gy/fraction demonstrated that lack of functional wt p53 was associated with resistance to fractionated irradiation. Radiation-induced apoptosis could not account for the observed differences in clonogenic survival. Rather, our data suggested that a deficit in the G1-checkpoint contributed to increased resistance to fractionated irradiation of cells expressing mutant p53. CONCLUSIONS: The effect of fractionated radiotherapy in GM may depend on the function of the tumor suppressor gene p53. A potential clinical consequence of these findings is that hyperfractionation regimens may provide a therapeutic advantage specifically for tumors expressing wt p53 whereas a radiotherapy course of fewer, larger fractions may be appropriate for the treatment of tumors carrying p53 mutations. Further studies are needed to confirm our proposal that the p53 status of GM tumors can be used to guide our choice of fractionation schemes.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Ciclo Celular/efectos de la radiación , Genes p53/fisiología , Glioblastoma/genética , Glioblastoma/radioterapia , Apoptosis , Neoplasias Encefálicas/fisiopatología , Ciclo Celular/fisiología , Supervivencia Celular , Fraccionamiento de la Dosis de Radiación , Vectores Genéticos/administración & dosificación , Glioblastoma/fisiopatología , Humanos , Tolerancia a Radiación , Retroviridae , Células Tumorales CultivadasRESUMEN
PURPOSE: This study was performed to standardize experimental conditions for the quantification by pulsed-field gel electrophoresis (PFGE) of radiation-induced DNA double-strand breaks (dsb) and rejoining in a human malignant brain tumour xenograft model. MATERIALS AND METHODS: Because no correlation was found between DNA dsb induction or rejoining and clinical radiation response for six fresh glioblastoma (GBM) specimens, assay conditions were examined in detail. SF-767 human GBM cells were implanted into the flanks of athymic mice. Resulting tumours were irradiated in vivo, dissociated mechanically or using an enzyme cocktail, and assayed for DNA dsb induction and repair. In other experiments, excised tumour portions were irradiated and allowed to repair either as chunks (>50 mm3 pieces), as minced tumour (approximately 1 mm3 pieces), or as single-cell suspensions. Finally, the effect of holding excised tumours in vitro for times of up to 72 h before irradiation and assay for DNA dsb and cell survival was studied. RESULTS AND CONCLUSIONS: The method of tumour dissociation had no effect on results; however, both the configuration of specimens during irradiation and the in vitro maintenance time markedly affected the experimental outcome. Chunks irradiated in vitro had DNA dsb results that were very similar to those obtained when tumours were irradiated in situ, while minced pieces or single-cell suspensions resulted in steeper dose-response curves. When tumour chunks were maintained at 4 degrees C in medium, DNA dsb induction was not affected for 24 h and DNA dsb rejoining remained constant for 48 h but then decreased. Cell survival, however, decreased continually during the 72 h in vitro maintenance time.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN/efectos de la radiación , Glioma/radioterapia , Animales , Femenino , Glioma/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Factores de Tiempo , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
To facilitate investigation of the molecular mechanisms of tumor cell radiosensitivities, we have generated a set of clones with different radiosensitivities from a human glioma cell line U-251 MG-Ho. Forty-four colonies were isolated by subjecting parent cells to the mutagen N-methylnitrosourea and then irradiating these cells with increasing doses of x-rays. About half of the clones displayed different radiosensitivities than the parent cells. We selected one of the most sensitive clones (X3i) and one of the most resistant clones (Y6) for further study. Isoeffective doses for these two clones differed by about a factor of 1.7; the relative radiosensitivities of both clones were stable for at least 30 cell culture passages. These two clones do not differ significantly in either the induction or repair of radiation-induced DNA double-strand breaks as measured by pulsed field gel electrophoresis. Radiation-induced apoptosis measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay and micronucleus formation were similar in both clones. However, potentially lethal damage repair was greater in the radioresistant Y6 clone than in the radiosensitive X3i clone as determined by colony-forming efficiency assay.
Asunto(s)
Glioma/radioterapia , Alquilantes , Apoptosis/efectos de la radiación , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular/efectos de la radiación , Cromosomas/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Glioma/inducido químicamente , Humanos , Etiquetado Corte-Fin in Situ , Metilnitrosourea , Pruebas de Micronúcleos , Tolerancia a Radiación , Factores de Tiempo , Células Tumorales Cultivadas , Rayos XRESUMEN
The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE), which can be activated through hypoxia-inducible factor-1 (HIF-1). We transfected plasmids containing multiple copies of HRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HRE copy number, and the degree of hypoxia.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Glioma/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/biosíntesis , Factores de Transcripción , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Etiquetado Corte-Fin in Situ , Operón Lac/genética , Luciferasas/metabolismo , Oxígeno/metabolismo , Plásmidos , Transfección , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2 , beta-Galactosidasa/metabolismoRESUMEN
UNLABELLED: BACKGROUND-MATERIALS-METHODS: We initiated the studies reported in this paper to establish baseline growth data for tumors produced by several human brain tumor cell lines. Female athymic mice were injected with five established human glioblastoma cell lines subcutaneously. We optimized implantation conditions in SF-767 cells by evaluating tumor take and growth characteristics, and resulting growth data were compared to 2 other cell lines. RESULTS-CONCLUSIONS: Three (SF-767, U-251 MG-NCI, and U-87 MG) of the 5 cell lines produced solid, ellipsoid tumors. For SF-767 cells, the best tumor growth parameters were achieved when 3.0 x 10(6) cells in 0.1 ml medium containing fetal calf serum were injected unilaterally. These conditions produced a high percentage of usable tumors (77.6%) that were detectable approximately 3 days after implantation and reached a size of 100 mm3 in 23 days. Comparison of several growth characteristics of the tumors produced by the 3 cell lines revealed that SF-767 tumors displayed the most uniform growth rates, the fastest doubling times, and the most uniform usable group of tumors (> 100 mm3). U-87 MG and U-251 MG-NCI had a similar histopathologic appearance while SF-767 had a different histology. Our results indicate that these 3 human glioblastoma cells produce flank tumors that exhibit decidedly different growth parameters. We are currently using all 3 of these human brain tumor xenograft models in other in vivo studies.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Animales , Pruebas de Carcinogenicidad , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The polyamine inhibitor DL-alpha-difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase which is a rate-limiting enzyme in the polyamine bio-synthesis pathway. The present study describes the effects of DFMO on glioma cell proliferation, migration and invasion using multicellular spheroids from three glioma cell lines (GaMg, U-251 Mg and U-87 Mg). 10 mM DFMO reduced cell migration in the three cell lines by about 30-50%. 1 mM putrescine, added together with DFMO inhibited the DFMO effect. A stronger effect was observed in the growth assay where 10 mM DFMO reduced the spheroid growth, for all cell lines, by 90%. This effect was also reversed by adding 1 mM of putrescine. In vitro tumor cell invasion experiments indicated after 3 days of confrontation, an extensive invasion also after 10 mM DFMO treatment. The brain aggregate volumes were reduced to about the same extent as in the absence of drug, suggesting essentially no effects of DFMO on the invasive process. It is concluded that the tumor spheroids retained their ability to invade normal brain tissue even after DFMO exposure. However, DFMO inhibited spheroid growth and cell migration which supports the notion that cell growth, migration and invasion are biological properties that are not necessarily related to each other.
Asunto(s)
Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Eflornitina/farmacología , Glioma/patología , Adulto , Animales , División Celular/efectos de los fármacos , Femenino , Glioma/tratamiento farmacológico , Humanos , Invasividad Neoplásica , Ratas , Ratas Wistar , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
Matrigel, an extracellular matrix material, has been used to promote growth of experimental tumors. SF-767, a human glioblastoma cell line, is used in brain tumor research. We investigated Matrigel induced changes in tumor latency, growth rate, cell yield, plating efficiency, and histology of SF-767 tumors in athymic mice. Low volume (0.1 ml) Matrigel did not increase growth rate in comparison with control tumors but appeared to promote uniformity in growth. High volume (0.5 ml) Matrigel increased the initial rate of tumor growth, increased cell yield and produced tumors with a centralized area of residual Matrigel and necrotic cells, with viable cells on the periphery of the mass. On average, tumors grown with Matrigel had shorter latency periods and lower plating efficiencies. We conclude that important characteristics of the SF-767 tumor model, which may be important when evaluating efficacy of anticancer agents, are altered by Matrigel.
Asunto(s)
Colágeno/farmacología , Glioblastoma/patología , Laminina/farmacología , Proteoglicanos/farmacología , Animales , División Celular , Combinación de Medicamentos , Matriz Extracelular/fisiología , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Trasplante HeterólogoRESUMEN
PURPOSE: This study aimed to determine the extent of paclitaxel-induced cytotoxicity and cell-cycle perturbations when used alone and in combination with radiation in human glioma cells. METHODS AND MATERIALS: The effect of paclitaxel alone on three human glioma cells lines--SF-126, U-87 MG, and U-251 MG--was assessed after 24, 48, 72, or 96 h treatment. For experiments in combination with radiation, cells were exposed to either a long (48-h) or short (8-h) duration of paclitaxel treatment prior to irradiation. Cell survival was determined by clonogenic assay. Cell cycle perturbations were assessed by using flow cytometry to measure the proportion of cells in G1, S, and G2/M phases. RESULTS: When cells were treated with paclitaxel alone for > or = 24 h, cytotoxicity increased up to a threshold dose, after which it plateaued. When treatment duration was < or = 24 h, cytotoxicity was appreciably greater in U-251 MG cells than in SF-126 and U-87 MG cells. After 24 h of paclitaxel treatment, cells in plateau phase growth had increased survival compared to cells in log phase growth. In contrast, after 8 h paclitaxel treatment, mitotic cells had reduced survival compared to cells from an asynchronous population. Cell-cycle perturbations were consistent with the presence of a mitotic block after paclitaxel treatment, although changes in other cell-cycle phase fractions varied among cell lines. For experiments in combination with radiation, cytotoxicity was increased when cells were irradiated after 48 h of paclitaxel treatment but not after 8 h of treatment. CONCLUSION: The duration of paclitaxel treatment and the location of cells in the cell cycle modify the degree of radiation cytotoxicity. The mechanisms of paclitaxel cytotoxicity are likely to be multifactorial because varying effects are seen in different cell lines. Furthermore, it is clear that simply increasing the number of cells in G2/M is insufficient in itself to increase the response of cells to radiation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Ciclo Celular/efectos de los fármacos , Paclitaxel/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Terapia Combinada , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Fase G1/efectos de los fármacos , Fase G1/efectos de la radiación , Fase G2/efectos de los fármacos , Fase G2/efectos de la radiación , Glioma , Humanos , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Factores de Tiempo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
We measured radiation-induced damage and repair in MO59J and MO59K human glioma cell lines using the nucleoid halo assay. Although these two cell lines have different radiosensitivities when assayed for colony forming efficiency, our results indicated that there was no significant difference between the two in terms of the unwinding and rewinding of DNA supercoils, radiation-induced changes in nucleoid halo size or the kinetics of nucleoid halo lysis. The only differences noted were in the kinetics of recovery of radiation-induced changes in nucleoid halo size, with the more sensitive cell line (MO59J) showing a slightly faster recovery than the more resistant cell line (MO59K). However, this difference was not statistically different. Our data indicate that the different cellular radiosensitivities of MO59J and MO59K cells are probably not due to any differences in their supercoiled DNA structure as measured by the nucleoid halo assay.
Asunto(s)
Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN de Neoplasias/efectos de la radiación , ADN Superhelicoidal/efectos de la radiación , Núcleo Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Glioma , Humanos , Cinética , Células Tumorales Cultivadas , Rayos XRESUMEN
PURPOSE: Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS: Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION: We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.
Asunto(s)
Apoptosis/efectos de la radiación , Neoplasias Encefálicas/radioterapia , Ciclo Celular/efectos de la radiación , Glioblastoma/radioterapia , Proteína p53 Supresora de Tumor/fisiología , Neoplasias Encefálicas/patología , Citometría de Flujo , Fase G1/efectos de la radiación , Glioblastoma/patología , Humanos , Células Tumorales CultivadasRESUMEN
The assay of colony-forming efficiency is a mainstay in the measurement of cell response in vitro to many physical and chemical agents. Currently, data on colony-forming efficiency can be calculated in a variety of ways. Authors rarely describe in detail the methods used to determine the extent of biological variation within experiments. The use of standard methods of data analysis and presentation would improve interpretation of data and facilitate comparison between laboratories. Here we propose such a method. Binomial and Poisson probability theory were used to increase the accuracy of the estimate of the surviving fraction and to create an objective criterion for determining whether data obtained from serial dilutions of cell numbers used in the assay of colony-forming efficiency should be excluded or included for further analysis. The variability inherent in the calculation of surviving fraction was determined by using Fieller's theorem, a special statistical application for assessing ratios of estimates, to determine the 95% confidence interval. All calculations were done on a simple and commercially available spreadsheet program.
Asunto(s)
Supervivencia Celular , Modelos Estadísticos , Ensayo de Tumor de Célula Madre , Línea Celular , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Glioma , Humanos , Matemática , Distribución de Poisson , Probabilidad , Células Tumorales Cultivadas , Rayos XRESUMEN
PURPOSE: Radiation therapy, though routinely used in the treatment of patients with glioblastoma multiforme, is of limited efficacy in extending patients' lives. In this study we investigated the mechanism by which ionizing radiation causes death of glioblastoma cells in the hope of ultimately altering the intrinsic radioresistance of glioblastoma tumors. METHODS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Radiation-induced apoptosis was assayed by fluorescence-activated cell sorter analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cellular distribution within the cell cycle was quantitated by dual labeling with propidium iodide and bromodeoxyuridine. RESULTS: We examined whether in vitro clonogenic radioresistance of glioblastoma would reflect their susceptibility to radiation-induced apoptosis and their ability to undergo a G1 arrest--two cellular functions associated with wild-type p53 expression. We demonstrated that apoptosis contributed to the cytocidal effect of ionizing radiation on glioblastoma cells. The apoptosis observed in glioblastoma cell lines occurred in the absence of wild-type p53 expression. We identified a glioblastoma cell line expressing wild-type p53 and found that it did not exhibit radiation-induced apoptosis but rather underwent a prolonged G1 arrest not observed in any glioblastoma cell line lacking wild-type p53 expression. CONCLUSION: Apoptosis is an important component of the lethal effect of ionizing radiation on glioblastoma cells and does not require wild-type p53 expression. Glioblastoma expressing wild-type p53 exhibited no apoptosis, even after high radiation doses, but rather underwent a prolonged G1 arrest. The observation of p53-independent apoptosis and p53-dependent Gi arrest in glioblastoma cells have important radiobiologic and clinical implications.
Asunto(s)
Apoptosis/efectos de la radiación , Ciclo Celular/efectos de la radiación , Glioblastoma/radioterapia , Tolerancia a Radiación/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral , Citometría de Flujo , Humanos , Técnicas In Vitro , Proteína p53 Supresora de Tumor/efectos de la radiaciónRESUMEN
It is proposed that genomic integrity is preserved after DNA damage in a variety of ways. X irradiation induces a p53-dependent G1-phase cell cycle checkpoint which putatively allows time for repair of DNA damage. The p53 protein is also involved in the initiation of apoptosis after radiation-induced DNA damage, presumably leading to the elimination of lethally damaged cells from the irradiated population. To test the hypothesis that repair occurs in the additional time provided by the activation of the G1-phase checkpoint, we investigated whether the presence of a G1-phase arrest modified the frequency and type of chromosomal rearrangements at the first mitosis after irradiation. Isogenic cell lines derived from the same human glioma cell line, but differing in p53 status, were used. Purified G1-phase cells, isolated by centrifugal elutriation and X-irradiated, were studied. The wild-type p53 cell line demonstrated a dose-dependent arrest during G1 phase, as determined by flow cytometry. These cells remained in G1-phase as long as 48 h after irradiation. Cells expressing a dominant-negative p53 mutation accumulated to a much lesser extent in G1 phase after irradiation. Cells lacking the G1-phase checkpoint showed increased survival at all radiation doses. There were no significant differences in the type or frequency of total chromosomal aberrations in mitotic cells from either cell line after 1,2,4 or 6 Gy X rays, as measured by conventional cytogenetic analysis. There was an increase, however, in the number of reciprocal translocations in mitotic cells with mutant p53 (lacking a G1-phase checkpoint), as measured by fluorescence in situ hybridization with a chromosome 4-specific DNA library, but only after 6 Gy. The results suggest that the presence of a well-defined p53-dependent G1-phase arrest does not reduce chromosomal aberrations caused by low doses of ionizing radiation markedly, but may reduce the overall degree of survival by triggering other G1-phase events.