Biomedical subjects

R Hilf

Publications and source records attributed to R Hilf.

At least 55 records · Page 3Linked to original sources

PubMed1992

Characterization of insulin-like growth factor receptors in the insulin-responsive R3230AC mammary adenocarcinoma.

To ascertain whether insulin-like growth factors (IGF-1 and IGF-2) affect the estrogen- and insulin-responsive R3230AC mammary carcinoma, studies of IGF-1 and IGF-2 receptors were conducted on primary-culture tumor cells and on membranes purified from whole tumors. By saturation binding analysis, cells in culture displayed one class of IGF-1 binding sites with an affinity constant, Kd, of 5.5 +/- 0.7 x 10(-9) M, whereas in membrane preparations, high and low affinity IGF-1 binding sites, with Kd's of 5 +/- 1.7 x 10(-9) M and 1 +/- 0.4 x 10(-7) M, respectively, were detected. Specificity of binding was demonstrated by 85% displacement of 125I-IGF-1 with 1,000-fold excess unlabeled IGF-1 and 50% displacement with 6.5 x 10(-9) M IGF-1 or 3 microM insulin. Binding sites for IGF-2 were also demonstrable in cultured cells, having a Kd of 7 +/- 0.8 x 10(-10) M, and 50% displacement was obtained with 1.5 x 10(-9) M IGF-2 or 1.5 x 10(-8) M IGF-1. Cross-linking experiments on cultured cells confirmed the presence of IGF-1 and IGF-2 receptors. In purified tumor membranes, IGF binding proteins (M(r) 28,000-32,000) were also detected; their labeling was not displaced by 10(-5) M insulin. In vitro, the tumor cells secrete one or more IGF binding proteins into the medium. Despite the fact that these cells expressed specific IGF receptors, their growth was apparently independent of these growth factors, since neither IGF-1 nor IGF-2 was mitogenic for R3230AC cells in vitro. Nevertheless, IGF-1 caused concentration-related significant increases in the plating efficiency of these cells. Further studies are necessary to determine the functional role of these growth factors, their receptors, and their binding proteins in the biology of this rodent mammary tumor.

Adenocarcinoma↗

PubMed1992

What differentiates antiestrogen-liganded vs estradiol-liganded estrogen receptor action?

This paper provides an annotated summary of the current literature detailing what is known about the interaction of antiestrogen-liganded estrogen receptor (ER) vs estradiol-liganded ER with DNA in vitro and in vivo. It describes the current models of the mechanism by which antiestrogen-liganded ER blocks transcription of certain estrogen-regulated genes.

Breast Neoplasms↗

PubMed1992

EGF receptors in R3230AC rat mammary carcinomas: characteristics and regulation in vitro and in vivo.

Epidermal growth factor (EGF), at 10(-11) M and 10(-10) M, stimulated [methyl-3H]thymidine incorporation into DNA and cell growth of R3230AC mammary adenocarcinomas in primary cultures, whereas at higher concentrations (10(-9) M and 10(-8) M) EGF inhibited DNA synthesis and cell growth in vitro. To determine whether these responses were receptor-mediated, 125I-EGF binding to freshly dissociated cells and primary cultures of R3230AC cells was measured and found to be time- and temperature-dependent. Specificity of EGF binding was demonstrated by 50% displacement occurring at an EGF concentration of 0.46 nM. Using 125I-EGF concentrations from 0.05 nM to 10 nM, saturable binding sites were documented; Scatchard analysis of these data produced curvilinear plots, suggesting the presence of high affinity (0.44-0.93 nM) and low affinity (1.5-4.8 nM) sites. 125I-EGF was rapidly internalized by cultured R3230AC tumor cells. By 30 min, 73% (25 degrees C) and 77% (37 degrees C) of total 125I-EGF was internalized (resistance to acid/salt extraction), whereas at 4 degrees C, cells internalized only 20% of EGF over a 3 hr incubation period. Following incubation with 125I-EGF for 2 hr at 4 degrees C, 25 degrees C, or 37 degrees C, the majority of cell-associated radioactivity eluted with intact 125I-EGF. However, when the material that dissociated from R3230AC cells after the 2 hr incubation was analyzed, 38% (25 degrees C) and 46% (37 degrees C) of the radioactivity migrated as lower molecular weight products, indicating that 125I-EGF was partially degraded intracellularly by R3230AC cells in primary culture. Pre-incubation of cells in primary culture with EGF (1-100 nM) for 30 min at 37 degrees C, led to "down-regulation" of EGF receptors; 1 nM EGF reduced the specific binding of 125I-EGF by 54% with higher concentrations (10 nM and 100 nM) reducing it further. Scatchard analysis of down-regulated cells showed a reduced number of high affinity binding sites with no change in the Kd of binding. Sialoadenectomy of rats had no effect on R3230AC tumor growth or EGF receptor levels in the tumor, liver, or uterus. Experiments to determine whether perturbations of the insulin milieu or ovariectomy would alter EGF receptors were performed. 125I-EGF binding was significantly elevated in tumors from diabetic rats (152% increase vs controls) and binding was returned to control (77% of intact rats) levels after administration of insulin to diabetic rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗

PubMed1991

Photosensitized release of von Willebrand factor from cultured human endothelial cells.

Cultured endothelial cells from the human umbilical vein were incubated with low concentrations (1 microgram/ml) of the photosensitizer Photofrin II. Following a sublethal light exposure, a light dose-dependent release of von Willebrand factor (vWf) into the culture medium was observed. Analysis of the multimeric composition of the released protein indicated that it originated from the intracellular pool of large vWf multimers stored in the Weibel-Palade bodies. This release was detected as early as 1 h postirradiation. Release was inhibited at low temperature and was dependent upon the presence of extracellular calcium. Photosensitization resulted in an influx of calcium whose time course paralleled vWf release from the cells. Since vWf mediates platelet adhesion to the vascular subendothelium, it is possible that its photochemically stimulated release in vivo could contribute to platelet thrombus formation observed in tissue following photodynamic therapy.

Cysteine↗

PubMed1991

Estrogen receptor alters the topology of plasmid DNA containing estrogen responsive elements.

We have recently used DNA containing estrogen responsive element (ERE) sequences for affinity purification to prepare calf uterine estrogen receptor (ER) at near homogeneity. The capacity of this purified ER to alter DNA topology upon binding was examined. Although the ER is not a topoisomerase, the presence of ER changes the distribution of topoisomers generated by incubation of plasmid DNA with excess wheat germ topoisomerase I. This effect is larger in plasmids containing a consensus ERE sequence. Two dimensional gel electrophoretic analysis suggested that interaction of ER and ERE causes negative supercoiling in regions of the plasmid accessible to topoisomerase I, resulting from overwinding of DNA contacting the ER. The extent of topological alteration was dependent on ER concentration. We suggest that the observed conformational changes in the DNA could have a role in regulation of transcription.

Animals↗

PubMed1991

Photosensitizing activities of picket fence porphyrins in vitro and in vivo.

A set of structurally distinct o-substituted tetraphenylporphyrins, the picket fence porphyrins, were evaluated for their ability to photosensitize tumor mitochondria in vitro, in vivo-in vitro, and tumor implants in situ. Differential photosensitized inactivation efficiencies toward mitochondrial enzymes in vitro are reported for the 12 compounds studied as a function of side chain length and isomer structure. Fluorescence studies in aqueous solution coupled with mitochondrial uptake studies indicate that the observed range of inactivation efficiencies are due to different inherent solubilization properties for the picket fence porphyrins. Studies with the most soluble compound, 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin, using an in vivo-in vitro protocol indicate that a more effective photosensitization can be obtained by using an interval of 4 h between photosensitizer administration and irradiation as compared to 24 h for Photofrin II. Irradiation of tumors in vivo 4 h following administration of 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin, resulted in a mean tumor doubling time more than eight times longer than that observed for untreated tumors. 31P NMR spectroscopy in situ indicated that photodynamic therapy using 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin induced a rapid and significant reduction in high energy phosphate metabolites.

Animals↗

PubMed1991

Gross and microscopic changes in the viscera induced by photodynamic therapy applied to the lower abdomen of intact rats.

Photodynamic therapy (PDT) is a promising approach to the treatment of cancer. Preferential retention of the photosensitizer by malignant tissue has been considered a hallmark of this treatment modality. However, photosensitivity can be observed in normal, non-neoplastic tissues, and the present study investigated the effects of PDT treatment on the abdomen of intact rats. A circular region (1 cm diameter) on the shaved abdomen of Fischer rats, pretreated 24 h prior with Photofrin II, was irradiated for 30 min at 632 nm. Control animals received either photoradiation or Photofrin II administration. Subsequent lesions were observed in the irradiated skin, its associated abdominal wall, and the underlying gut in rats receiving Photofrin II and laser irradiation. All tissues were not equally sensitive to PDT treatment. Gut lesions were consistently more severe than were skin and abdominal wall injuries. By 24 hr after treatment, the gut manifested a transmural hemorrhagic necrosis, while the irradiated skin and abdominal wall were edematous, with an inflammatory infiltrate in the dermis and around occasional swollen myocytes. These results indicate that superficial lesions induced by PDT may not be reliable indicators of the extent of deeper PDT tissue damage. Further, it may be possible to take advantage of this discrepancy in tissue sensitivity and treat deep tissues through less sensitive superficial tissues.

Abdominal Muscles↗

PubMed1991

Oxygen consumption and diffusion effects in photodynamic therapy.

Effects of oxygen consumption in photodynamic therapy (PDT) are considered theoretically and experimentally. A mathematical model of the Type II mechanism of photooxidation is used to compute estimates of the rate of therapy-dependent in vivo oxygen depletion resulting from reactions of singlet oxygen (1O2) with intracellular substrate. Calculations indicate that PDT carried out at incident light intensities of 50 mW/cm2 may consume 3O2 at rates as high as 6-9 microM s-1. An approximate model of oxygen diffusion shows that these consumption rates are large enough to decrease the radius of oxygenated cells around an isolated capillary. Thus, during photoirradiation, cells sufficiently remote from the capillary wall may reside at oxygen tensions that are low enough to preclude or minimize 1O2-mediated damage. This effect is more pronounced at higher power densities and accounts for an enhanced therapeutic response in tumors treated with 360 J/cm2 delivered at 50 mW/cm2 compared to the same light dose delivered at 200 mW/cm2. The analysis further suggests that the oxygen depletion could be partially overcome by fractionating the light delivery. In a transplanted mammary tumor model, a regimen of 30-s exposures followed by 30-s dark periods produced significantly longer delays in tumor growth when compared to the continuous delivery of the same total fluence.

Animals↗

PubMed1990

Effects of various photoradiation regimens on the antitumor efficacy of photodynamic therapy for R3230AC mammary carcinomas.

Clinical photodynamic therapy consists of the systemic administration of a derivative of hematoporphyrin (Photofrin II) followed by exposure of malignant lesions to continuous visible laser irradiation. We investigated the effects of various modifications of laser light delivery on the efficacy of photodynamic therapy in controlling R3230AC mammary tumor growth. We observed a significant delay in growth (from initial to 2 times initial volume) of tumors exposed to periodic irradiation (100 mW/cm2/0.25 h, 1-h dark interval, 100 mW/cm2/0.25 h; total fluence, 180 J/cm2), compared to untreated controls or to tumors receiving continuous irradiation at the same total fluence. Other periodic light treatment regimens, consisting of 3-, 6-, or 24-hr dark intervals, delayed tumor growth but not significantly more than continuous irradiation at the same fluence. A biochemical basis was sought by comparing continuous versus periodic irradiation for effects on mitochondrial or cytosolic enzymes in vivo. Although both cytochrome c oxidase and pyruvate kinase activities were reduced dramatically during the first 24 h by continuous or periodic irradiation schemes, recovery of enzyme activity to initial levels took longer after the periodic irradiation protocol (168 h), compared to the continuous irradiation regimen (72 h). We observed a significantly greater delay in the growth of tumors exposed to 50 mW/cm2/2 h continuously, compared to controls or to tumors exposed to the same total fluence but with light delivered at 100 or 200 mW/cm2. The data presented here indicate that the efficacy of photodynamic therapy could be significantly increased by modifications in the delivery of photoradiation.

Animals↗

PubMed1990

In situ assessment of tumor vascularity using fluorine NMR imaging.

In situ fluorine NMR imaging has been used to measure vascularity in subcutaneously implanted mammary tumors. Oxyferol, a perfluorinated blood substitute comprised of an emulsion of 25% w/v perfluorotributylamine, was used as a tracer. Following iv administration, this perfluorocarbon emulsion remains primarily in the vasculature during the image acquisition period. The distribution of the PFTA in the 19F NMR image gives a map of tissue regions with intact vascularity. This technique has been used to demonstrate decreased blood flow in necrotic regions of R3230AC mammary tumors in which vasculature had been damaged either as a result of spontaneous necrosis or by photodynamic therapy (PDT). Damage to tumor vascularity following PDT was observed prior to the development of necrosis.

Animals↗

PubMed1990

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues.

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.

Animals↗

PubMed1990

Photodynamic therapy for human malignant mesothelioma in the nude mouse.

Photodynamic therapy (PDT) utilizes a photoactivatable preparation, Photofrin II, which selectively localizes in cancerous tissue and produces substances toxic to that tissue when activated by light. Whether PDT would be able to selectively destroy human malignant mesothelioma was investigated by using a human-derived malignant mesothelioma tumor subcutaneously implanted in nude mice. Human malignant mesothelioma was grown subcutaneously to a size of 0.2-0.4 cm3. Selective retention of Photofrin II was studied by measuring light-induced inhibition of cytochrome c oxidase activity in tumor, heart, and lung. Photofrin II was retained in greater quantities in tumor than in heart or lung at 24 hr after injection. Using laser light at 630 nm under varying conditions, tumor growth was measured every 2 days following PDT for 18 days. All PDT regimens were successful in destroying malignant mesothelioma. Photofrin II at 5 mg/kg was superior to 2 mg/kg (P less than 0.005), light delivered at 50 mW/cm2 x 2 hr was superior to that delivered at 200 mW/cm2 x 30 min (P less than 0.05), and a total fluence of 180 J/cm2 was equivalent to 360 J/cm2 in affecting tumor growth. Ten of 12 mice treated at 50 mW/cm2 became tumor-free and remained so for 30 days following treatment. We concluded that PDT was effective against human malignant mesothelioma in a nude mouse model without adversely affecting the animal. A role for PDT in treating patients with malignant mesothelioma may exist.

Animals↗

PubMed1990

Chalcogenapyrylium dyes as photochemotherapeutic agents. 2. Tumor uptake, mitochondrial targeting, and singlet-oxygen-induced inhibition of cytochrome c oxidase.

Cationic selena- and tellurapyrylium dyes 1d-g and 1i were found to inhibit cytochrome c oxidase upon irradiation of isolated mitochondrial suspensions treated with 10 microM solutions of dye. The amount of inhibition by these dyes was found to be related to oxygen concentration and inversely related to the concentration of added imidazole, a singlet-oxygen trap, suggesting that singlet oxygen is responsible, at least in part, for the inhibition of the enzyme. Dyes 1d-g and 1i, containing either selenium or tellurium, produce singlet oxygen with a quantum efficiency, phi (1O2), between 0.005 and 0.09 in methanol. Dyes 1a-c, containing the lighter chalcogens oxygen and sulfur, have values of phi (1O2) that are less than 0.0008 in methanol and do not inhibit cytochrome c oxidase in irradiated mitochondrial suspensions. Dyes 1c and 1d have nearly identical spectral and redox properties. Only the selenapyrylium dye 1d inhibits the enzyme, suggesting that neither ground-state nor excited-state electron transfer is important in inhibition of the enzyme. Electron micrographs of human U251 glioma cells, treated in vitro with 1i and light, showed pronounced morphology changes in the mitochondrial membranes relative to electron micrographs of untreated cells. Epifluorescence microscopy of the treated cells showed granular yellow-green fluorescence presumably from photooxidized dye in the mitochondria.

Adenocarcinoma↗

PubMed1990

Increased efficacy of photodynamic therapy of R3230AC mammary adenocarcinoma by intratumoral injection of Photofrin II.

Photodynamic therapy consists of the systemic administration of a derivative of haematoporphyrin (Photofrin II) followed 24-72 h later by exposure of malignant lesions to photoradiation. We investigated the efficacy of this treatment after direct intratumoral injection of Photofrin II. This direct treatment regimen resulted in higher rates of inhibition of mitochondrial cytochrome c oxidase (5.13% J-1 cm-2 x 10(-1) and succinate dehydrogenase (3.14% J-1 cm-2 x 10(-1] in vitro at 2 h after intratumoral injection compared to rates of inhibition obtained after intraperitoneal drug administration: 0.51 and 0.42% J-1 cm-2 x 10(-1), respectively. A significant delay in tumour growth in vivo was observed in animals that received intratumoral injections 2 h before photoradiation compared to animals injected intraperitoneally at either 2 or 24 h before photoradiation. The treatment protocols were compared with control groups, consisting of Photofrin II administration intratumorally or intraperitoneally without photoradiation, or photoradiation in the absence of Photofrin II. These data indicate that the intratumoral injection regimen with Photofrin II enhanced the efficacy of photodynamic therapy. The greater delay in tumour growth observed after intratumoral administration of Photofrin II suggests a mechanism favouring direct cell damage.

Adenocarcinoma↗

PubMed1990

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements.

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.

Animals↗

PubMed1989

Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography.

Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.

Animals↗

PubMed1989

Effects of host hormonal status on binding of activated estrogen receptor to nuclei from R3230AC and 7,12-dimethylbenz[a]anthracene-induced mammary tumors.

The effects of various hormonal perturbations that alter growth of two different rat mammary tumors in vivo were investigated by study of the interactions of [3H]estradiol-charged estrogen receptors ([3H]ER) with tumor nuclei in vitro. Nuclei from the transplantable R3230AC adenocarcinoma were isolated after ovariectomy, estrogen treatment, or progesterone treatment. Saturable specific binding of [3H]ER to nuclei was assayed in this in vivo-like system. Scatchard analysis of [3H]ER-nuclear binding data indicated that these perturbations did not affect affinity, which ranged from Kd 1.0 to 2.4 nM. However, the number of [3H]ER-binding sites/nucleus was altered according to the treatment: intact rats, 94,500 +/- 4,200; ovariectomy, 70,400 +/- 3,200; ovariectomy plus estradiol, 82,100 +/- 5,800; and ovariectomy plus progesterone, 73,900 +/- 2,500. Nuclei from primary tumors induced by 7,12-dimethylbenz(a)anthracene displayed similar affinities for [3H]ER, although these tumors had fewer binding sites per nucleus. Animals bearing 7,12-dimethylbenz(a)-anthracene-induced tumors were either ovariectomized or made diabetic by administration of streptozotocin, perturbations that cause regression of the majority of tumors. The number of [3H]ER binding sites per nucleus, in tumors classified according to growth characteristics in host animals subsequent to hormonal perturbation, was: intact growing 36,300 +/- 3,400; ovex regressing, 15,400 +/- 3,400; ovex, estrogen-treated growing, 28,100 +/- 2,700; diabetic regressing, 19,500 +/- 2,400; diabetic static, 32,100 and diabetic growing, 42,000 +/- 7,100. These results indicate that (a) the number of nuclear ER-binding sites can be reduced by hormonal interventions that cause tumor regression and (b) endogenous ovarian hormones may play a role in regulating nuclear ER binding.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1989

Antiestrogen(4-hydroxytamoxifen)-charged estrogen receptor binding to nuclei from normal and neoplastic rat mammary tissues is not affected by host hormonal status.

Interaction of [3H]4-hydroxytamoxifen-charged estrogen receptor [( 3H]AER) with nuclei was compared to that of [3H]17 beta-estradiol-charged estrogen receptor [( 3H]ER) in vitro. Specificity of [3H]AER binding was demonstrated since more than 90% of [3H]AER binding was displaced by ten-fold excess estradiol-charged ER. For R3230AC tumors, the number of [3H]AER binding sites was approximately 40% lower than the number of [3H]ER binding sites. There were no differences in affinity of binding of these receptors complexes (Kd range 0.7-1.6 nM). In contrast 0.7-1.6 nM). In contrast to a reduction of [3H]ER binding after ovariectomy, no difference in the number of [3H]AER binding sites was seen among tumors from intact, ovex, or estrogen-treated ovex rats. These results suggest that [3H]AER bind to 60% of the sites that bind [3H]ER, and that neither tissue type nor host ovarian status affects the number of nuclear [3H]AER binding sites.

Animals↗