RESUMEN
BACKGROUND: The measurement of the heterogeneity of radiolabeled monoclonal antibody uptake in tumor has an essential role in the calculation and interpretation of the absorbed dose of radiation. Large data arrays and long calculation times have been limiting factors in the calculation of three-dimensional dose-rate distributions used to study the relationship between uptake heterogeneity and dose. METHODS: Serial autoradiographs of tumor sections were digitized with approximately 100 microns resolution using a laser densitometer. The section images were aligned to form a registered tumor-image data set. The image data were corrected for film response versus activity density to create a three dimensional activity density distribution using features of a three-dimensional radiotherapy treatment planning system. Dose-rate distributions were formed by convolution with a beta dose kernel using fast Fourier transforms. RESULTS: Differential dose-rate-volume histograms (derived from the dose-rate distribution) were created to summarize the dose-rate nonuniformity throughout the tumor volume. Effects of section sampling interval, interpolation methods between section planes, and calculation resolution on the dose-rate-volume histograms were illustrated. CONCLUSIONS: The several orders of magnitude improvement in calculational speed provided by the fast Fourier transform technique allowed an investigation of the effects of the calculational parameters. This investigation enabled tuning of both data acquisition and dose computation. These studies can lead to further enhancements in the calculational efficiency of three-dimensional dose-rate distributions. These improvements will allow the study of summing techniques to yield average total dose distributions.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Neoplasias/radioterapia , Radioinmunoterapia , Animales , Autorradiografía , Neoplasias del Colon/radioterapia , Relación Dosis-Respuesta en la Radiación , Humanos , Linfoma/radioterapia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Dosificación Radioterapéutica , Trasplante HeterólogoRESUMEN
PURPOSE: To determine the potential advantage of combining halogenated pyrimidine radiosensitization and continuous low dose rate irradiation in human malignant glioma. METHODS AND MATERIALS: An established glioma line (U-251) was incubated with 5-bromo-2-doxyuridine (BrdUrd) at clinically achievable concentrations at three dose rates of interest--100 cGy/min (typical of external beam therapy), 43 cGy/hr (typical of temporary afterloaded implants), and 12 cGy/hr (typical of permanent implants). RESULTS: After exposure to 1 microM BrdUrd, the greatest enhancement ratio was seen at the 12 cGy/hr dose rate, implying a BrdUrd induced inverse dose rate effect independent of a G2M block. Under these conditions, the mean inactivation dose after 1 microM BrdUrd exposure was equivalent for 100 cGy/min and 12 cGy/hr. CONCLUSION: These results support the use of halopyrimidines as sensitizers of temporary afterloaded and permanent implants.
Asunto(s)
Bromodesoxiuridina/uso terapéutico , Glioma/radioterapia , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Relación Dosis-Respuesta en la Radiación , Humanos , Técnicas In Vitro , Dosificación Radioterapéutica , Tasa de Supervivencia , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
The choice of radionuclide remains an important question in clinical radioimmunotherapy. Therefore, a study was initiated, using an in vivo model system, to assess the relative merits of 131I- and 90Y-labeled 17-1A monoclonal antibody as therapeutic agents in the treatment of colon cancer. 131Iodine- and 90Y-labeled 17-1A were assessed in animal therapy trials using athymic nude mice bearing LS174T human colon cancer xenografts. 131Iodine-labeled 17-1A decreased tumor growth in a dose-dependent fashion without lethality. In contrast, the doses of 90Y-labeled 17-1A which were required to produce a significant increase in tumor doubling time also caused marked toxicity. Although similar tumor growth inhibition was produced by 250 microCi 90Y- and 150 microCi 131I-labeled 17-1A, Medical Internal Radiation Dose calculations based on biodistribution data estimated that the dose delivered by 90Y was greater than that delivered by 131I. To investigate this discrepancy, 3-dimensional dose distributions within LS174T tumors were assessed using autoradiography and 3-dimensional calculational techniques. It was found that a greater fraction of the dose was deposited in the tumor after treatment with 131I- compared to 90Y-labeled 17-1A. When the Medical Internal Radiation Dose calculations were adjusted using the 3-dimensional dose distributions, 250 microCi of 90Y- and 150 microCi of 131I-labeled 17-1A were found to deliver similar tumor doses. These studies suggest that 131I-labeled 17-1A is superior to 90Y-labeled 17-1A, since 131I-labeled antibody produced less hematological and animal toxicity and was more effective at inhibiting LS174T tumor growth than 90Y-labeled antibody across the range of radionuclide doses tested. Furthermore, they suggest that it will be necessary to perform 3-dimensional dose calculations in addition to Medical Internal Radiation Dose calculations in order to interpret tumor dosimetry.
Asunto(s)
Adenocarcinoma/radioterapia , Neoplasias del Colon/radioterapia , Radioisótopos de Yodo/uso terapéutico , Radioinmunoterapia , Radioisótopos de Itrio/uso terapéutico , Adenocarcinoma/diagnóstico por imagen , Animales , Anticuerpos Monoclonales , Autorradiografía/métodos , Neoplasias del Colon/diagnóstico por imagen , Humanos , Radioisótopos de Yodo/farmacocinética , Melanoma/diagnóstico por imagen , Melanoma/radioterapia , Ratones , Ratones Desnudos , Radioinmunodetección , Distribución Tisular , Trasplante Heterólogo , Células Tumorales Cultivadas , Radioisótopos de Itrio/farmacocinéticaRESUMEN
Inhomogeneities in activity distributions over distances from 10 to 10(4) microns are observed in many tumors treated with radiolabeled antibodies. Resulting nonuniformities in absorbed dose may have consequences for the efficacy of radioimmunotherapy. Activity variations may be directly studied with quantitative autoradiography (ARG). Converting these data to absorbed dose distributions requires additional information about pharmacokinetics, the use of a point source function and consideration of the complete three-dimensional activity distribution, as obtained from sequential autoradiographic slices. Thermoluminescent dosimetry with specially prepared CaSO4:Dy dosimeters implanted into tissue can directly measure absorbed dose in selected regions. The conditions under which thermoluminescent dosimeters (TLD) are used differ markedly from "normal" use conditions in external beam radiotherapy. Therefore special calibration and quality assurance precautions are needed to assure the precision of this technique. Procedures and pitfalls in the use of both techniques in radioimmunotherapy are described.
Asunto(s)
Neoplasias/radioterapia , Radioinmunoterapia/métodos , Radiometría/métodos , Autorradiografía/métodos , Partículas beta , Humanos , Dosificación Radioterapéutica , Dosimetría Termoluminiscente/métodosRESUMEN
Three-dimensional dose distributions have been calculated for LS174T human colon cancer xenografts in athymic nude mice treated with 131I-labeled 17-1A monoclonal antibody. Autoradiographs were made for fifteen to twenty 32-micron-thick representative serial sections of tumors removed 1 and 4 days postinjection. Film density readings were converted to activity density and entered into a radiotherapy treatment planning system. Three-dimensional dose distributions were obtained by summing the dose contributions due to each voxel of uniform activity. Isodoserate distributions and dose-rate-volume histograms for representative tumors at 1 and 4 days following 131I-labeled 17-1A injection showed a progressive change from a predominantly surface deposition (day 1) to a more volumetric deposition (day 4). Average tumor doses calculated using the assumptions of uniform source distribution and local dose deposition resulted in a poor estimation of the cumulative dose because of the significant time-dependent dose-rate nonuniformities.
Asunto(s)
Neoplasias del Colon/radioterapia , Radioinmunoterapia , Animales , Autorradiografía , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Dosificación Radioterapéutica , Trasplante HeterólogoRESUMEN
Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.
Asunto(s)
Fosforilasa Quinasa/química , Animales , Activación Enzimática , Modelos Moleculares , Neutrones , Fosforilasa Quinasa/efectos de la radiación , Conejos , Dispersión de Radiación , Soluciones , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
The sensitivity and precision of teflon-imbedded CaSO4:Dy microthermoluminescent dosimeters (micro-TLDs) were determined. The micro-TLDs were sectioned from miniature TLDs (200 microns x 400 microns x 5 mm) that were fabricated using standard techniques. In order to measure absorbed dose, the miniature TLDs can be implanted directly into tissues (e.g., tumor xenografts) that have received injections of radiolabeled monoclonal antibodies. Micro-TLDs recovered from tissue sections cut with a microtome can be read out to determine local absorbed dose. The precision of dose estimation was quantified for uniformly irradiated 32-, 96-, and 192-microns TLD chips; coefficients of variation ranged from 22% to 41%, depending on chip size. The coefficients of variation were reduced to less than 12% using individual relative sensitivity factors for each micro-TLD. The spatial resolution of the micro-TLDs was studied by placing miniature TLDs across the sharp penumbral region of a linear accelerator x-ray field. TLDs were sectioned into 32-microns chips which were read out to determine the relative absorbed dose. The sharpness of the penumbra was readily quantified by the micro-TLDs.
Asunto(s)
Radioinmunoterapia/instrumentación , Dosimetría Termoluminiscente/instrumentación , Animales , Humanos , Neoplasias Experimentales/radioterapia , Sensibilidad y Especificidad , Trasplante HeterólogoRESUMEN
Inhibition of growth of LS174T human colon cancer xenografts in athymic nude mice due to 131I-labeled MoAb 17-1A treatment was compared to inhibition due to different single doses of 60Co external radiation. From those data, conditions which produced equivalent radiobiological end points could be identified and compared to dose estimates calculated using a technique analogous to the Medical Internal Radiation Dose (MIRD) Committee formalism. The tumor growth rate in mice injected with a single intraperitoneal administration of 300 microCi of 131I-labeled MoAb was reduced relative to tumor growth in untreated control animals and in mice administered unlabeled MoAb and was found to be similar to the growth rate of tumors given a single 6 Gy dose of 60Co radiation. Furthermore, the growth rate of tumors in mice that received three injections of 300 microCi of 131I-labeled MoAb on days 9, 16 and 28 after tumor cell injection was similar to the growth rate of tumors given a single 60Co dose of 8 or 10 Gy. The biodistribution data for 125I-labeled 17-1A MoAb were used to calculate total doses for the tumor and various normal tissues in animals given a single administration of 131I-labeled 17-1A MoAb. The absorbed radiation dose in tumor was approximately five times higher than in normal tissues. The results of the present study indicate that the tumor growth inhibition produced by the administration of radiolabeled antibody can equal that produced by up to 10 Gy of external beam radiation. In addition, the MIRD calculations allow comparison of this form of low dose radiation to external photon irradiation.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias del Colon/radioterapia , Radioisótopos de Yodo/uso terapéutico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Línea Celular , Radioisótopos de Cobalto/uso terapéutico , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Femenino , Humanos , Radioisótopos de Yodo/administración & dosificación , Radioisótopos de Yodo/farmacocinética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Distribución Tisular , Trasplante HeterólogoRESUMEN
Model peptides with predetermined secondary, tertiary, and quaternary conformation have been successfully designed, synthesized, and characterized in an attempt to mimic the three-dimensional structure of an antigenic determinant. This work is a continuing effort to map the antigenic structure of the protein antigen lactate dehydrogenase C4 (LDH-C4) to develop a contraceptive vaccine. A putative topographic determinant with alpha alpha topology which associates into four-helix bundles was designed on the basis of the framework model of protein folding. An idealized amphiphilic 18-residue sequence (alpha 1) and a 40-residue alpha alpha fold (alpha 3) have been shown to form stable 4-helix structures in solution with a free energy of association on the order of -20.8 kcal/mol (tetramerization of alpha 1) and -7.8 kcal/mol (dimerization of alpha 3). Both alpha 1 and alpha 3 form stable monolayers at the air-water interface. The CD spectra of Langmuir-Blodgett monolayers are characteristically alpha-helical. Both CD and FTIR spectroscopic studies reval a high degree of secondary structure. The SAXS data strongly suggest that the helices are arranged in a four-helix bundle since the radius of gyration of 17.2 A and the vector distribution function are indicative of a prolate ellipsoid of axial dimensions and molecular weight appropriate for the four-helix bundle. The major contribution to the formation and stabilization of alpha 1 and alpha 3 is believed to be hydrophobic interaction between the amphiphilic alpha-helices. The displayed heptad repeat, helix dipole, ion pairs, and the loop sequence may have also contributed to the overall stability and antiparallel packing of the helices. A detailed structural analysis of a relevant topographic immunogenic determinant will elucidate the nature of antigen-antibody interactions as well as provide insight into protein folding intermediates.
Asunto(s)
Epítopos/síntesis química , L-Lactato Deshidrogenasa/inmunología , Secuencia de Aminoácidos , Dicroismo Circular , Análisis de Fourier , Isoenzimas , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/aislamiento & purificación , Conformación Proteica , Soluciones , Espectrofotometría Infrarroja/métodosRESUMEN
Small-angle X-ray and neutron scattering data were used to study the solution structure of calmodulin complexed with a synthetic peptide corresponding to residues 577-603 of rabbit skeletal muscle myosin light chain kinase. The X-ray data indicate that, in the presence of Ca2+, the calmodulin-peptide complex has a structure that is considerably more compact than uncomplexed calmodulin. The radius of gyration, Rg, for the complex is approximately 20% smaller than that of uncomplexed Ca2+.calmodulin (16 vs 21 A), and the maximum dimension, dmax, for the complex is also about 20% smaller (49 vs 67 A). The peptide-induced conformational rearrangement of calmodulin is [Ca2+] dependent. The length distribution function for the complex is more symmetric than that for uncomplexed Ca2+.calmodulin, indicating that more of the mass is distributed toward the center of mass for the complex, compared with the dumbell-shaped Ca2+.calmodulin. The solvent contrast dependence of Rg for neutron scattering indicates that the peptide is located more toward the center of the complex, while the calmodulin is located more peripherally, and that the centers of mass of the calmodulin and the peptide are not coincident. The scattering data support the hypothesis that the interconnecting helix region observed in the crystal structure for calmodulin is quite flexible in solution, allowing the two lobes of calmodulin to form close contacts on binding the peptide. This flexibility of the central helix may play a critical role in activating target enzymes such as myosin light chain kinase.
Asunto(s)
Calmodulina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Animales , Sitios de Unión , Bovinos , Técnicas In Vitro , Estructura Molecular , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Conejos , Dispersión de RadiaciónRESUMEN
In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calmodulina/metabolismo , Glicosilación , Adenilil Ciclasas/metabolismo , Animales , Química Encefálica , Calcio/metabolismo , Bovinos , Dicroismo Circular , Bromuro de Cianógeno , Activación Enzimática , Técnicas In Vitro , Lisina/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Quinasas/metabolismo , Dispersión de Radiación , Rayos XRESUMEN
Fourier transform infrared (FTIR) spectroscopy has been used to examine the conformationally sensitive amide I' bands of calmodulin and troponin C. These are observed to undergo a sequence of spectroscopic changes which reflect conformational rearrangements that take place when Ca2+ is bound. Calmodulin and troponin C show similar though not identical changes on Ca2+ binding, and the effect of Mg2+ on troponin C is quite different from that of Ca2+. Both proteins show absorption maxima in the amide I' region at 1644 cm-1 which is significantly lower in frequency than has been generally observed for proteins that contain a high percentage of alpha-helix. It is proposed that an unusually high proportion of the helices in the structures of these proteins are distorted from the normal alpha-helical configuration such that the carbonyl stretching frequencies are lowered. It is further proposed that the shift to lower frequency is due to backbone carbonyl groups in the distorted helices that form strong hydrogen bonds with solvent molecules. A decrease in intensity at 1654 cm-1, the normal frequency assignment for alpha-helical structure, is observed as Ca2+ binds to calmodulin and troponin C. This suggests that Ca2+ binding results in a net decrease in "normal" alpha-helix conformation. There is a corresponding increase in intensity of the band at 1644 cm-1, possibly due to an increase in distorted helix content, allowing for a net increase in helix consistent with circular dichroism estimates of the Ca2+-dependent changes in helix content in calmodulin.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Magnesio/metabolismo , Troponina/metabolismo , Animales , Encéfalo/metabolismo , Bovinos , Análisis de Fourier , Cinética , Conformación Proteica , Espectrofotometría Infrarroja , Troponina C , PavosRESUMEN
X-ray solution scattering data from skeletal muscle troponin C and from calmodulin have been measured. Modeling studies based on the crystal structure coordinates for these proteins show discrepancies between the solution data and the crystal structure that indicate that if the size and shape of the globular domains are the same in solution as in the crystal, the distances between them must be smaller by several angstroms. Bringing the globular domains closer together requires structural changes in the interconnecting helix that joins them.
Asunto(s)
Calmodulina , Troponina , Animales , Encéfalo/metabolismo , Bovinos , Modelos Moleculares , Músculos/metabolismo , Conformación Proteica , Conejos , Soluciones , Relación Estructura-Actividad , Troponina C , Difracción de Rayos XRESUMEN
While X-ray crystallographic data on cytochrome c show the reduced and oxidized forms to have very similar structures, there is a considerable body of data, mostly from solution studies, that indicates the reduced form is more stable and that the interior of the protein is less accessible to solvent in this state. These observations have led to the hypothesis that while the time-averaged structure is preserved between the two forms, the dynamics of the two forms are different. The oxidized form has been proposed to undergo more large-amplitude, low-frequency motions than the reduced form. The crystal structure data were derived from crystals grown in high salt concentrations, but the solution studies were done at relatively low ionic strength. Small-angle X-ray scattering has been used to examine the effects of the ionic strength and oxidation state on the solution structure of cytochrome c. We find that the radius of gyration and the maximum linear dimension of oxidized cytochrome c are significantly larger than those for reduced cytochrome c, in 5 mM phosphate buffer at pH 7.3, and further that this difference is suppressed by addition of 200 mM sodium chloride. We conclude that there is a real structural difference between the two forms at low ionic strength in solution and that this difference is likely to contribute to the observed differences in accessibility and compressibility.