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Supplementary Methods from Novel Chemical Enhancers of Heat Shock Increase Thermal Radiosensitization through a Mitotic Catastrophe Pathway

Supplementary Figures 1-9 from Novel Chemical Enhancers of Heat Shock Increase Thermal Radiosensitization through a Mitotic Catastrophe Pathway

Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(±)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation. [Cancer Res 2007;67(2):695–701]

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Background: The combination of biological variation and nanomaterial heterogeneity makes elucidating the mechanisms of interactions between cells and nanoparticles extremely complicated. Accurate nanoparticle quantification can be extremely challenging, and cellular response can change based on the location of the nanoparticle and the cell type under investigation. These complications are only augmented by the additional of external stimuli. These limitations have yielded a wide range of studies that show effects, but often provide little mechanistic insight. Results: Gold nanomaterials were stably immobilized onto glass coverslips treated with mercaptosilane to control both the average number of nanoparticles that interact with cells and their spatial orientation relative to the cell membrane. Surfaces were characterized optically and by electron microscopy to confirm their surface density and uniformity. The thermal response of Au nanocage-coated surfaces to near infrared laser irradiation was measured in cell culture medium and modelled computationally. The modelling showed a vastly higher thermal dose than would be predicted by bulk temperature measurements. Adherent or non-adherent cell lines were cultured directly on the nanocage-coated surface or in the medium, respectively, in culture wells and laser irradiation was applied. Survival of cells growing in suspension correlated with the bulk temperature increase in the culture medium, as measured by viability assay. Conversely, adherent cells exhibited a much greater susceptibility than expected from the bulk temperature measurement, which is ostensibly related to the close interaction with the nanoparticles on their growth substrate and induction of substantially greater thermal dose upon laser exposure. Conclusions: This platform is designed to be a new tool to determine how many particles need to be in contact with a cell to induce desired physical or biological effects. Here we demonstrate the delivery of precise thermal doses following laser irradiation. The anticipated biological effects based on bulk measurements vastly underestimated the effects that were observed, which is ascribed to the proximity of the nanoparticle to the cell and the extraordinary high surface temperature of the particle. This platform could be expanded to a variety of nanoparticles, external stimuli, and cell types to enable more deliberate and optimized application of nanomedicine.

A simple and reproducible procedure was developed to measure the volume of liquid microinjected into cells. A calibration curve of droplet fluorescence intensity versus volume was constructed by injecting a fluorescent dextran solution through a 125–150 µm diameter micropipette into an oil-filled culture dish to create a spray of varied-sized droplets. The droplets retained a spherical shape because they were in an oil medium and they settled onto a glass surface coated with a superhydrophobic surface. Fluorescent micrographs of the droplets were obtained and analyzed with Image-J software to quantify the fluorescence intensity and radius of each spherical droplet to produce the calibration curve. Subsequently, Dut-145 human prostate carcinoma cells were microinjected with the same fluorescent dextran solution and fluorescent micrographs of the cells were obtained using the identical exposure conditions used to photograph the droplets. The measured fluorescence intensity of the microinjected cells was entered into the formula for the regression line that was fit to the calibration curve allowing determination of the volume of solution injected into each cell. Thus, a mixture consisting of known concentrations of a test material of test material (macromolecules, drugs, etc.) and a fluorescent dextran, volumetric, tracer can be used to quantify the relationship between the amount of a microinjected material and subsequent effects on cells.

In search of the sequence of pathogenic events leading to glucocorticoid-induced osteonecrosis, we determined the molecular, biomechanical, cellular, and vascular changes in the femur of C57BL/6 mice receiving prednisolone for 14, 28, or 42 days. The femoral head, but not the distal femur, of mice treated for 14 days showed a decrease in the expression of the hypoxia-inducible factor (Hif)-1α and vascular endothelial growth factor (VEGF), the number of osteoblasts, and bone formation rate and strength and showed an increase in osteoclasts. These changes were accompanied by conversion of the normal dendritic vasculature to pools of edema as detected by magnetic resonance imaging, providing robust diagnostic evidence of early osteonecrosis. At that time point, there were no detectable changes in bone density, cortical or cancellous bone architecture, midshaft or distal cancellous bone, or osteocyte apoptosis. In mice treated for 28 days, femoral head cancellous density, cortical width, and trabecular thickness decreased, and by 42 days the femoral heads had full-depth cortical penetrations and cancellous tissue osteonecrosis. These results indicate that the femoral head is a particularly sensitive anatomical site to the adverse effects of glucocorticoid excess on bone and that decreases of Hif-1α and VEGF expression, bone vascularity, and strength precede the loss of bone mass and microarchitectural deterioration, thus rendering the femoral head vulnerable to collapse., Glucocorticoid-induced osteonecrosis of the murine femoral head is due to site-specific decreases in Hif-1α and VEGF expression, vascularity, and strength before loss of bone mass.

Strategies to increase the proportion of neural stem cells that differentiate into neurons are vital for therapy of neurodegenerative disorders. In vitro, the extracellular matrix composition and topography have been found to be important factors in stem cell differentiation. We have developed a novel artificial extracellular matrix (aECM) formed by attaching gold nanocages (AuNCs) to glass coverslips. After culturing rat neural stem cells (rNSCs) on these gold nanocage-coated surfaces (AuNC-aECMs), we observed that 44.6% of rNSCs differentiated into neurons compared to only 27.9% for cells grown on laminin-coated glass coverslips. We applied laser irradiation to the AuNC-aECMs to introduce precise amounts of photothermally induced heat shock in cells. Our results showed that laser-induced thermal stimulation of AuNC-aECMs further enhanced neuronal differentiation (56%) depending on the laser intensity used. Response to these photothermal effects increased the expression of heat shock protein 27, 70, and 90α in rNSCs. Analysis of dendritic complexity showed that this thermal stimulation promoted neuronal maturation by increasing dendrite length as thermal dose was increased. In addition, we found that cells growing on AuNC-aECMs post laser irradiation exhibited action potentials and increased the expression of voltage-gated Na+ channels compared to laminin-coated glass coverslips. These results indicate that the photothermal response induced in cells growing on AuNC-aECMs can be used to produce large quantities of functional neurons, with improved electrochemical properties, that can potentially be transplanted into a damaged central nervous system to provide replacement neurons and restore lost function.

IV injection of dodecafluoropentane emulsion (DDFPe) increases oxygen transportation and reduces brain infarct volume in a rabbit stroke model. Tissue distribution of the parent perfluorocarbon dodecafluoropentane (DDFP) is unknown but is critical to understanding the mechanism by which DDFPe is effective in treating ischemia and for determining safe dosing. Previous studies showed a DDFP blood half-life of2 min yet therapeutic effects lasted90 min after injection. We describe DDFP distribution in brain, kidney, liver, spleen, and lung following nine dosing regimens in New Zealand White (NZW) rabbits. Single and multi-dose schedules were administered to NZW rabbits (n = 27). A single DDFPe dose (0.6 ml/kg) group was sacrificed 2 min after dosing and eight multi-dose groups (4 doses of 0.3 or 0.6 ml/kg and 15 doses of 0.1, 0.3, or 0.6) were sacrificed 90 min after final injections. Tissues were flash frozen and analyzed with headspace sampling/GC-MS. DDFP brain concentration increased with increasing dose in the 15 dose groups (4.70, 8.34, and 14.3 μg/g) and indicative of linear pharmacokinetics within this dose range. The DDFP lung concentration was not reflective of increasing dose or dose frequency. The total clearance of DDFP was consistent with previous reports showing 98% of DDFP is cleared within 2 h of administration.

The aim of this study was to determine the uptake of intravenously administered N-[11CH3]-dimethylaminoparthenolide (DMAPT) into orthotopic 9LSF glioblastoma brain tumors in Fisher 344 rats from positron emission tomography (PET) imaging studies. [11C]methyl iodide (11CH3I) was utilized as a [11C]-labeling reagent to label the precursor methylaminoparthenolide (MAPT) intermediate. From PET imaging studies it was found that brain uptake of N-[11CH3]DMAPT into brain tumor tissue was rapid (30min), and considerably higher than that in the normal brain tissue.

Background: Dodecafluoropentane emulsion (DDFPe), given IV one hour after stroke, has been shown to greatly reduce the percent stroke volume (%SV) in rabbits. With repeated doses its effect continued for 24 hours. Purpose: Test DDFPe as neuroprotective agent in permanent occlusion rat stroke models in Sprague Dawley (SD) and Spontaneously Hypertensive Rats (SHR) measuring both %SV and neurological assessment scores (NAS). Methods: The male rats received either saline (control), or one or four doses (1x or 4x) of DDFPe (0.6ml/kg IV) one hour post stroke. Treatment groups were SD (n=26) (control, 1x and 4x; n=12, 7 and 7) and SHR (n=14) (control, 1x and 4x; n=7, 3 and 4). The 4x doses were given at 1.5 hour intervals. At six hours post stroke, the rats received a NAS using standard tests for balance, reflexes, and motor performance. Then rats were euthanized and brains removed for TTC evaluation of %SV. Results: For %SV analysis strain differences were not significant therefore strains were combined. DDFPe significantly decreased %SV in 1x and 4xDDFPe groups compared to control groups (2.59±1.81 and 0.98±0.88 vs. 9.24±6.06, p≤0.001 each; p≤0.0001 for the overall test for treatment effect). The 1x versus 4xDDFPe groups were not significantly different (p=0.40). In NAS analysis both strains showed significant improvement with 4xDDFPe therapy vs. controls, (SD: 5.00+2.45 vs. 9.36+3.56, p=0.01; SHR: 7.75+4.43 vs. 12.14+3.08, p=0.05). Differences between the 1x DDFPe group and controls were not significant (SD: 8.43+3.69; SHR: 9. 33+3.51). Conclusion: DDFPe treatment provides significant neuroprotection when assessed six hours post stroke.

Thrombi, e.g. blood clots, in circulatory system pose acute health risk, globally. This research investigated roles of biodegradable starch slurry in advancing biomass machining efficiency. Hard clots (fibrin-rich) prepared from rabbit blood were exposed in vitro concomitantly to ultrasound (1 MHz) and starch slurry. Starch slurry particles (diameter ∼250 nm) yielded a 200% increase in material removal (sonothrombolysis) efficiency. Mechanistic participation of starch, a non-Newtonian material, at the interface of biomass-ultrasonic radiation is discussed. Overall in subtractive biomanufacturing, the role of biodegradable slurry is critical for enhancing material removal efficiency.

Dodecafluoropentane emulsion (DDFPe) in 250 nm nanodroplets seems to swell modestly to accept and carry large amounts of oxygen in the body at29 °C. Small particle size allows oxygen delivery even into hypoxic tissue unreachable by erythrocytes. Using permanent cerebral embolic occlusion in rabbits, we assessed DDFPe dose response as a neuroprotectant at 7 and 24 h post-embolization without lysis of arterial obstructions and investigated blood pharmacokinetics. New Zealand White rabbits (N = 56) received cerebral angiography and embolic spheres (diameter = 700-900 μm) occluded middle and/or anterior cerebral arteries. Intravenous DDFPe dosing (2 % w/v emulsion) began at 60 min and repeated every 90 min until sacrifice at 7 or 24 h post-embolization. Seven-hour groups: (1) control (embolized without treatment, N = 6), and DDFPe treatment: (2) 0.1 ml/kg (N = 7), (3) 0.3 ml/kg (N = 9), (4) 0.6 ml/kg (N = 8). Twenty-four-hour groups: (5) control (N = 16), and DDFPe treatment: (6) 0.1 ml/kg (N = 10). Infarcts as percent of total brain volume were determined using vital stains on brain sections. Other alert normal rabbits (N = 8) received IV doses followed by rapid arterial blood sampling and GC-MS analysis. Percent infarct volume means significantly decreased for all DDFPe-treated groups compared with controls, p = 0.004 to0.03. Blood DDFP (gas) half-life was 1.45 ± 0.17 min with R = 0.958. Mean blood clearance was 78.5 ± 24.9 ml/min/kg (mean ± SE). Intravenous DDFPe decreases ischemic stroke infarct volumes. Blood half-life values are very short. The much longer therapeutic effect,90 min, suggests multiple compartments. Lowest effective dose and maximum effective therapy duration are not yet defined. Rapid development is warranted.

The aim of this study was to determine the uptake of intravenously administered N-[11CH3]dimethylaminoparthenolide (DMAPT) into orthotopic 9LSF glioblastoma brain tumors in Fisher 344 rats from positron emission tomography (PET) imaging studies. [11C]Methyl iodide (11CH3I) was utilized as a [11C]-labeling reagent to label the precursor methylaminoparthenolide (MAPT) intermediate. From PET imaging studies it was found that brain uptake of N-[11CH3]DMAPT into brain tumor tissue was rapid (30 minutes), and considerably higher than that in the normal brain tissue.

Background and Purpose— Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue-type plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with 3 types of MB using a rabbit embolic stroke model. Methods— New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3-day-old cylindrical clots (0.6×4.0 mm). Groups included: (1) control (n=11) embolized without treatment; (2) tPA (n=20); (3) tPA+US (n=10); (4) perflutren lipid MB+US (n=16); (5) albumin 3 μm MB+US (n=8); and (6) tagged albumin 3 μm MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm 2 ) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3 μm MB was 5×10 9 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on hematoxylin and eosin sections. Results— Infarct volume percent was lower for rabbits treated with lipid MB+US (1.0%±0.6%; P =0.013), 3 μm MB+US (0.7%±0.9%; P =0.018), and tagged 3 μm MB+US (0.8%±0.8%; P =0.019) compared with controls (3.5%±0.8%). The 3 MB types collectively had lower infarct volumes ( P =0.0043) than controls. Infarct volume averaged 2.2%±0.6% and 1.7%±0.8% for rabbits treated with tPA alone and tPA+US, respectively ( P =nonsignificant). Conclusions— Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer’s disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes—regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.

Tissue plasminogen activator (tPA) is the thrombolytic standard of care for acute ischemic stroke, but intracerebral hemorrhage (ICH) remains a common and devastating complication. We investigated using ultrasound (US) and microbubble (MB) techniques to reduce required tPA doses and to decrease ICH.Fresh blood clots (3-5 hours) were exposed in vitro to tPA (0.02 or 0.1 mg/mL) plus pulsed 1 MHz US (0.1 W/cm²), with or without 1.12 × 10⁸/mL MBs (Definity or albumin/dextrose MBs [adMB]). Clot mass loss was measured to quantify thrombolysis. New Zealand white rabbits (n = 120) received one 3- to 5-hour clot angiographically delivered into the internal carotid artery. All had transcutaneous pulsed 1 MHz US (0.8 W/cm²) for 60 minutes and intravenous tPA (0.1-0.9 mg/kg) with or without Definity MBs (0.16 mL/mg/kg). After killing the animals, the brains were removed for histology 24 hours later.In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P0.0001). At 0 and 0.02 mg/mL, tPA clot loss was greater with adMBs compared with Definity (P ≤ 0.05). With MB, the tPA dose was reduced 5-fold with good efficacy. In vivo, both Definity MB and tPA groups had less infarct volume compared with controls at P0.0183 and P = 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P0.0001), and ICH incidence outside of strokes was significantly lower (P = 0.005) compared with no MB. However, infarct volume in Definity MB versus tPA was not different at P = 0.19.Combining tPA and MB yielded effective loss of clot with very low dose or even no dose tPA, and infarct volumes and ICH were reduced in acute strokes in rabbits. The ability of MBs to reduce tPA requirements may lead to lower rates of hemorrhage in human stroke treatment.

Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(±)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation. [Cancer Res 2007;67(2):695–701]

A series of novel carbamate and carbonate dimers of melampomagnolide B (MMB) have been synthesized by reaction of the MMB-triazole carbamate synthon 6 with various terminal diamino and dihydroxy alkanes. The resulting dimeric products 7b, 7c and 7f were selected and evaluated for anticancer activity against a panel of 60 human hematological and solid tumor cell lines. The most active compounds, 7b, 7c and 7f, exhibited GI50 values in the range 250-780 nM against the majority of leukemia cell lines in the tumor cell panel. Specifically, compounds 7b and 7f exhibited potent growth inhibition against non-small cell lung cancer cell lines NCI-H522 (GI50 = 160 nM) and HOP-92 (GI50 = 170 nM), respectively. Also, compound 7f also potently inhibited the growth of melanoma cell lines LOX IMVI, MALME-3M, and UACC-62 (GI50 values = 170, 190 and 190 nM, respectively); breast cancer cell line MDA-MB-468 (GI50 = 190 nM); colon cancer cell line HCT-116 (GI50 = 190 nM); and renal cancer cell line RXF 393 (GI50 = 160 nM). Compound 7f and the simple dicarbonate dimer of MMB (8) showed anticancer activity 300-fold and 1 × 106-fold, respectively, more cytotoxic than 7f and DMAPT at a concentration of 10 μM against rat 9L-SF gliosarcoma cells. The dimeric compounds 7a-7j & 8 were also screened for antileukemic activity against M9-ENL1 acute myelogenous leukemia (AML) cells and primary AML cell specimens. These compounds exhibited two to twelve-fold more potent antileukemic activity (EC50 = 0.5-2.9 μM) against the M9-ENL1 cell line when compared to parthenolide (EC50 = 6.0 μM). The dimeric analogues were also active against the primary AML cell specimens in the nanomolar to lower micromolar range and exhibited two to ten-fold more potent antileukemic activity (EC50 = 0.86-4.2 μM) when compared to parthenolide (EC50 = 2.5-16 μM). Thus, dimer 7f exhibited promising anticancer activity against a variety of both hematological and solid human tumor cell lines, while dimer 8 was superior to 7f against 9L-SF gliosarcoma, M9-EML1 and AML cells. These two novel dimeric analogs of MMB warrant further investigation with regard to their mechanism of action, especially as it relates to the activity of dimeric forms of active monomeric molecules and the implications this may have on structure-activity relationships and drug design.

This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41 degrees C for either 30, 60, or 120 min. Maximal SERCA1a activity (micromol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41 degrees C compared with 37 degrees C. HSP70 protected against thermal inactivation of SERCA1a activity at 37 degrees C but not at 41 degrees C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys515 in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were co-transfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40 degrees C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions both in vitro and in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain.

Dodecafluoropentane emulsion (DDFPe), an oxygen transport agent, has been shown to reduce infarct volume in animal models of acute ischemic stroke (AIS). Our study assesses the effect of DDFPe on MRI markers of infarct evolution in the early hours after vascular occlusion in a rat AIS model. We hypothesized that DDFPe will delay the development of MRI markers of AIS and/or reduce the extent of infarction.Permanent, unilateral surgical occlusion of the middle cerebral and common carotid arteries was performed in control (n=4) and treatment (n = 10) rats. The treatment group received 1 IV dose of 2% w/v DDFPe at 0.6 mL/kg at 1 hour post-occlusion versus none. Diffusion-weighted (DWI) and inversion recovery (IR) MRI sequences were obtained over the 4 hours following occlusion. Infarct extent was quantified by number of abnormal MRI slices per sequence for each group and time point. Student's T-test was applied.DDFPe-treated rats demonstrated reduced infarct extent versus controls over combined time points on IR at 5.43 ± 0.40 (mean ± standard error) abnormal slices vs. 7.38 ± 0.58 (P = 0.01) and on DWI at 5.21 ± 0.54 vs. 9.00 ± 0.95 (P0.01). Development of abnormal MRI signal was delayed in the treatment group.DDFPe delays and reduces MRI markers of AIS in the early hours following vascular occlusion in a rat stroke model. Further investigation of DDFPe as a neuroprotectant is warranted.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

A number of protein reactive compounds, including the thiol reagents diamide and arsenite, are known inducers of heat shock protein (HSP) synthesis and thermotolerance. These compounds are thought to damage cellular protein, which has been proposed to serve as the signal for induction. The specific mechanism of protein damage and its relation to thermal denaturation are unknown. The Ca2+-ATPase of sarcoplasmic reticulum, a membrane protein that contains 24 cys residues, was used to determine the effect of diamide, arsenite, N-ethylmaleimide (NEM), and the cys-specific probes Br-DMC and IAEDANS, which label one or two specific cys residues, respectively, on protein conformation and stability. The Ca2+-ATPase was chosen because diamide has been shown to affect the thermal properties of a class of membrane proteins of CHO cells (Freeman et al., 1995). The labeling of one or two thiols has no effect on activity or conformation, while more extensive reaction (but with less than approximately five to eight groups titrated) results in destabilization of the Ca2+-ATPase such that it denatures thermally at 37 degrees C. Higher levels of titration result in greater destabilization such that the protein is no longer stable at room temperature, with the production of a state similar to the thermally denatured state as assayed by activity, differential scanning calorimetry, ANS binding, and light scattering. The fractional denaturation induced by these thiol reagents, determined by the decrease in the heat absorbed during thermal denaturation, is directly proportional to inactivation of ATPase activity. Thus, inactivation of the Ca2+-ATPase by thiol reagents occurs because of denaturation not through oxidation of essential thiols. These results indicate that these thiol-specific heat shock inducers function by two mechanisms: (1) destabilization of proteins such that they thermally denature at 37 degrees C and (2) direct denaturation, apparently driven by thermal processes at room temperature, following more extensive reaction which results in extreme destabilization. We suggest that these are general mechanisms by which heat shock inducers damage proteins.

While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca2+-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response. J. Cell. Physiol. 171:143–151, 1997. © 1997 Wiley-Liss, Inc.

The objective of this study was to unequivocally demonstrate thermotolerance expression in mammalian cells in the absence of stress-induced synthesis of heat shock proteins (HSPs). Mitotic cells were selected as an experimental system since their genome was in the form of condensed chromosomes and ostensibly incapable of being transcribed; thus, obviating stress-induced HSP gene expression. Asynchronous Chinese hamster ovary (CHO) cells were treated with 0.2 microgram/ml nocodazole to accumulate cells in mitosis for harvest by mitotic shakeoff. Cells were maintained in mitosis with nocodazole during thermotolerance induction, thermotolerance development, and all challenge hyperthermia exposures. Although the heat shock transcription factor was activated by the thermotolerance inducing heat shock, as indicated by gel mobility shift assay, no increase in steady-state HSP mRNA levels was detected, as expected. Preferential synthesis of HSPs from extant mRNA was not detected during thermotolerance development and cellular levels of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins remained constant, as determined by Western Blot analyses. The magnitude and induction threshold of expressed thermotolerance was not diminished when cells were incubated with 10.0 micrograms/ml cycloheximide during thermotolerance development confirming that new protein synthesis was not requisite. Parallel experiments were performed using nonmitotic cells in which protein synthesis was inhibited during thermotolerance development with 10.0 micrograms/ml cycloheximide. As with mitotic cells, high levels of thermotolerance were attained without detectable increases in the cellular content of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins. The results of this study demonstrated that high levels of thermotolerance could be expressed in mitotic cells without stress-induced, preferential synthesis of HSPs, and support the contention that a substantial fraction of thermotolerance expressed in nonmitotic cells also occurs independently of induced HSP synthesis.

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0 degrees C, are present in both the nucleus and cytoplasm.

Introduction: Neuroprotective strategies in ischemic stroke include oxygen delivery to sustain penumbra and prevent hypoxic cell death. Hyperbaric oxygen, blood substitutes, and liquid fluorocarbon-based oxygen carriers have often failed in treatment of stroke and other ischemic disorders. Dodecafluoropentane emulsion (DDFPe, boiling point 29°C) shifts to quasi-gas phase at body temperature, which allows absorption and transportation of very high levels of oxygen. Exceptionally small particle size, 250-300 nm, may allow oxygen delivery even through occluded vessels, by diffusion into hypoxic tissue unreachable by whole blood. In a preliminary stroke study in rabbits, DDFPe reduced infarct volumes in all experimental groups by 80% or more. Hypothesis: Repeated doses of DDFPe can reduce infarct volume for up to 24 hours after permanent cerebral artery occlusion in rabbits. Methods: New Zealand White rabbits (N=55) received cerebral angiography from a femoral artery approach. Embolic microspheres (diameter=700-900 μm) were injected into the internal carotid artery, permanently occluding the middle cerebral and/or anterior cerebral arteries. Rabbits were randomly assigned to treatment groups and sacrifice times as in Table 1. In all treated groups, intravenous DDFPe dosing with a 2% w/v emulsion began at 1 hour post-embolization and was repeated every 90 minutes until sacrifice at either 7 or 24 hours post-embolization. Following sacrifice, infarcts were measured as a percent of brain volume using vital stains on brain sections. Results: Percent infarct volume means significantly decreased for all DDFPe treated groups compared with controls (Table 1). Conclusion: Intravenous DDFPe begun 1 hour after stroke onset protects the brain from ischemic injury in the rabbit model of permanent embolic stroke. Decreased infarct volumes represent salvaged brain tissue. This effect can be observed for 24 hours with repeated doses.

Introduction: Intravenous dodecafluoropentane emulsion (DDFPe) has proven ability to transport oxygen in ischemic and anemic animal models. Therapeutic effects in rabbits and pigs last at least 90 minutes, although in human studies dodecafluoropentane (DDFP) is eliminated rapidly from the blood through respiration. DDFPe neuroprotection reduces infarct volumes >80% in our rabbit embolic stroke model. Since little is known about DDFPe activity in rabbits, blood pharmacokinetics were investigated in healthy rabbits to determine dosing regimens. Hypothesis: We hypothesize that DDFP pharmacokinetics in the rabbit are similar to those in humans, with blood half-life values near 2.2±1.2 min, and clearance values near 49.6±10.8 mL/min/kg. Methods: Bilateral ear catheters, one intravenous and one intraarterial, were introduced into New Zealand White rabbits (N=8, 4.2±0.9 kg) for DDFPe administration and blood sampling, respectively. Rabbits were divided into 3 DDFPe dosage groups: 1) single bolus (0.6 mL/kg) with blood sampling for 4 hrs (N=4), 2) 15 doses (0.1 mL/kg) with sampling for 24 hrs (N=2), 3) 5 doses (0.6 mL/kg) with sampling for 7 hrs (N=2). Repeated doses of 2% w/v DDFPe were administered every 90 min. All samples were kept frozen until thawed before analysis by headspace gas chromatography-mass spectrometry. Results: An exponential regression closely approximated the blood concentration vs. time plot of single bolus sampling with an average R 2 of 0.96±0.02, thus compartmental modeling was used for pharmacokinetic analysis. The blood half-life was 1.9±0.6 min, with a clearance of 90.1±45.3 mL/min/kg. In all multi-dose rabbits, blood concentration of DDFP did not reach a steady state. The concentration vs. time data exhibited a repeated sequence of individual bolus data, with DDFP reaching undetectable levels 10 min after each dose. Conclusion: Blood elimination half-life and clearance rates of DDFPe in rabbits are comparable to humans, further indicating that the rabbit stroke model is appropriate for investigation of the neuroprotective potential of DDFPe. The time disparity between blood levels and therapeutic oxygen transport ability of DDFPe suggests multiple compartments and merits further investigation.

The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation-induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted in immediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60 degrees C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of < or = 48 degrees C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediate and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide-treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein.

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.

Sonothrombolysis (STBL) uses ultrasound to lyse thrombi that cause stroke, myocardial infarct and other vascular occlusion diseases; and efficacy is improved markedly by combining tPA and/or microbubbles (MBs) with ultrasound. The efficacy of in vitro STBL varies considerably depending upon how blood clots are formed. However, the relationship between the physical and chemical nature of blood clots and STBL efficacy has not been quantified. This study measured the effects that clot age, density and thrombin content had on STBL efficacy and the ability of tPA and MBs to complement STBL. Blood clots with different characteristics were produced by mixing pooled rabbit plasma with rabbit blood cells, adding varied amounts of thrombin and calcium, followed by incubation at 37 o C in glass tubes for 3 h to 72 h. Blood clots were then cured at 5 o C for 24 h to 72 h. The volume and mass density of the clots were measured, 9-11 mg pieces were cut from the main clot, weighed and then insonated with 1 MHz, pulsed (20% duty factor) ultrasound in a Mylar chamber through which fresh rabbit serum containing tPA and or MBs was flowed continuously past the clot. STBL efficacy was measured as % of clot mass lysed in 15 min. For clots incubated 3 h to 24 h at 37 o C with 5 mM Ca + 2 and 5 U of thrombin, maximal STBL with ultrasound plus tPA was observed with 0.1 mg/ml tPA for all ultrasonic intensities tested. Using MBs (1.0-2.5 x10 8 /ml) produced a comparable level of STBL. More effective STBL was produced by combining tPA with MBs, even with reduced tPA concentrations (0.006-0.01 mg/ml). However, increasing clot thrombin, up to 60 U, proportionally reduced STBL efficacy with tPA or MBs, and combining tPA with MBs no longer produced an additive STBL effect. This was also true for clots incubated with 25 mM Ca + 2 that did not contract and thus had a fourfold lower mass density. Increasing thrombin levels produced clots whose STBL efficacy was markedly lower with either tPA or MBs, and for which combining tPA with MBs produced no additive STBL effect. Hence, the level of thrombin used during clot formation (and putatively the degree of fibrin networks) was the major factor in determined STBL efficacy and whether or not tPA combined with MBs were additive in potentiating STBL. Surprisingly, the mass density of the clot alone had no effect on STBL efficacy.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C (PKC) inhibitor, on the development of thermotolerance and expression of heat shock genes (HSP70 and HSP28) was investigated in human colon carcinoma HT-29 cells. After acute heating at 45 degrees for 15 min, cells became resistant to a challenge heat shock. The development of thermotolerance was suppressed by adding H-7 after heat shock. Northern blots show that the levels of HSP70 and HSP28 mRNA increased rapidly and reached maximal values within 6 hr. H-7 suppressed the accumulation of HSP70 and HSP28 mRNA as well as their protein synthesis, and the level of suppression was concentration dependent. However, little effect was observed if the drug was added 1 hr before and during heat shock. These results suggest that PKC is involved in the regulation of heat shock gene expression after acute heat shock.

Acetaminophen (APAP) toxicity is responsible for approximately half of all cases of acute liver failure in the United States. The mouse model of APAP toxicity is widely used to examine mechanisms of APAP toxicity. Non-invasive approaches would allow for serial measurements in a single animal to study the effects of experimental interventions on the development and resolution of hepatocellular necrosis. The following study examined the time course of hepatic necrosis using small animal magnetic reasonance imaging (MRI) following the administration of 200 mg/kg ip APAP given to B6C3F1 male mice. Mice treated with saline served as controls (CON). Other mice received treatment with the clinical antidote N-acetylcysteine (APAP+NAC). Mouse liver pathology was characterized using T1 and T2 weighted sequences at 2, 4, 8 and 24 h following APAP administration. Standard assays for APAP toxicity (serum alanine aminotransaminase (ALT) levels and hemotoxylin and eosin (H&E) staining of liver sections) were examined relative to MRI findings. Overall, T2 sequences had a greater sensitivity for necrosis and hemorrhage than T1 (FLASH) images. Liver injury severity scoring of MRI images demonstrated increased scores in the APAP mice at 4, 8 and 24 h compared to the CON mice. APAP+NAC mice had MRI scores similar to the CON mice. Semi-quantitative analysis of hepatic hemorrhage strongly correlated with serum ALT. Small animal MRI can be used to monitor the evolution of APAP toxicity over time and to evaluate the response to therapy.

Purpose To assess the efficacy of dodecafluoropentane emulsion (DDFPe), a nanodroplet emulsion with significant oxygen transport potential, in decreasing infarct volume in an insoluble-emboli rabbit stroke model. Materials And Methods New Zealand White rabbits (N = 64; weight, 5.1 ± 0.50 kg) underwent angiography and received embolic spheres in occluded internal carotid artery branches. Rabbits were randomly assigned to groups in 4-hour and 7-hour studies. Four-hour groups included control (n = 7, embolized without treatment) and DDFPe treatment 30 minutes before stroke (n = 7), at stroke onset (n = 8), and 30 minutes (n = 5), 1 hour (n = 7), 2 hours (n = 5), or 3 hours after stroke (n = 6). Seven-hour groups included control (n = 6) and DDFPe at 1 hour (n = 8) and 6 hours after stroke (n = 5). DDFPe dose was a 2% weight/volume intravenous injection (0.6 mL/kg) repeated every 90 minutes as time allowed. After euthanasia, infarct volume was determined by vital stains on brain sections. Results At 4 hours, median infarct volume decreased for all DDFPe treatment times (pretreatment, 0.30% [ P = .004]; onset, 0.20% [ P = .004]; 30 min, 0.35% [ P = .009]; 1 h, 0.30% [ P = .01]; 2 h, 0.40% [ P = .009]; and 3 h, 0.25% [ P = .003]) compared with controls (3.20%). At 7 hours, median infarct volume decreased with treatment at 1 hour (0.25%; P = .007) but not at 6 hours (1.4%; P = .49) compared with controls (2.2%). Conclusions Intravenous DDFPe in an animal model decreases infarct volumes and protects brain tissue from ischemia, justifying further investigation.

1. 1.An electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce HSP70 or anti-HSP70 antibody into Chinese hamster ovary cells. 2. 2.There was a 1.5- or 1.9-fold increase in the amount of intracellular HSP70 by electroporation in the presence of 0.75 or 1.5 mg/ml, respectively. 3. 3.Cells electroporated with HSP70 became resistant to hyperthermic killing; i.e. the survival increased 6–7-fold from 1.2 × 10 −2 to 7–8 × 10 −2 heating at 45.5°C for 20 min. 4. 4.In contrast, introduction of 1 mg/ml anti-HSP70 antibody sensitized cells to hyperthermic killing; i.e. the reciprocal of the survival slope was decreased from 1.68 to 1.25 min. 5. 5.Thus, our results support the hypothesis that HSP70 plays an important role in heat resistance.

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.

Pretreatment plus concomitant treatment with 10 micrograms/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c-myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c-myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c-myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide-induced reduction of actinomycin D bound to the acid precipitable fraction of the cells.

We investigated the correlation between the development of acute thermotolerance and the phosphorylation, synthesis, and expression of the HSP28 family in murine L929 cells. Following heating at 43 degrees C for 30 min, thermotolerance developed rapidly in exponential-phase cells and reached its maximum 4-9 h after heat shock. Maximal thermal resistance was maintained for 24 h and then gradually decayed. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, HSP28 synthesis during incubation at 37 degrees C for 12 h following heat shock was not detected by [3H]-leucine labeling followed by two-dimensional polyacrylamide gel electrophoresis. In addition, Northern blots failed to demonstrate expression of the HSP28 gene. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was observed during incubation at 37 degrees C after heat shock. These results demonstrate that HSP28 synthesis and its phosphorylation are not required to develop acute thermotolerance in L929 cells.

Current rabbit stroke models often depend on symptoms as endpoints for embolization and produce wide variation in location, size, and severity of strokes. In a further refinement of an angiographic embolic stroke model, localized infarctions were correlated to neurologic deficits with the goal to create a rabbit model for long-term studies of therapies after stroke.New Zealand White rabbits (4-5 kg; N = 71) had selective internal carotid artery (ICA) angiography and a single clot was injected. At 24 hours, neurologic assessment score (NAS) was measured on an 11-point scale (0, normal; 10, dead). Brains were removed and stained to identify stroke areas. All animals with single strokes (n = 31) were analyzed by specific brain structure involvement, and NAS values were correlated.Stroke incidence differed by location, with cortex, subcortical, and basal ganglia regions highest. The middle cerebral artery (MCA), at 52%, and anterior cerebral artery (ACA), at 29%, were most commonly involved, with the largest stroke volumes in the ACA distribution. Brainstem and cerebellum strokes had disproportionately severe neurologic deficits, scoring 2.25 +/- 1.0 on the NAS, which represented a significant (P.02) difference versus cortex (0.5 +/- 0.2), subcortical (1.3 +/- 0.4), and basal ganglia (0.5 +/- 0.3), all in the frontal or parietal regions.MCA and ACA distributions included 81% of strokes. These sites were relatively silent (potentially allowing longer-term survival studies) whereas others in the posterior circulation produced disproportionately severe symptoms. Symptoms were not reliable indicators of stroke occurrence, and other endpoints such as imaging may be required. These are important steps toward refinement of the rabbit stroke model.

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.

Analysis of gene expression following stimulation of growth-arrested cells has been the main approach for identification of growth-associated genes. Since the activation of these gene sequences is dependent on both the stimulatory agent and the state of quiescence of the cell, the activation and role of the same genes may be entirely different in non-growth arrested, actively proliferating cells. We have addressed the question of growth-associated gene expression during active growth by analyzing gene expression during G-1 of cells which have just exited mitosis without first leaving the cell cycle. We were able to isolate, by a non-inductive, drug free system, a population of highly synchronized Swiss 3T3 cells within mitosis (greater than 90%) in numbers sufficient to determine the pattern of expression of a large number of representative growth-associated genes. Our results show that after replating the mitotic cells into conditioned medium: (1) growth-associated gene expression is not constant during G-1 of actively proliferating cells, and (2) while a number of genes (e.g., JE, c-myc, ODC, p53, and histone) exhibited patterns of expression similar to that reported in the quiescent systems, others (e.g., nur-77, vimentin, calcyclin) exhibited patterns which were completely different. From these results, we can begin to construct a temporal map of G-1 progression during active growth.

Protein denaturation resulting from temperatures between 42.0 degrees C and 50 degrees C has been observed and implicated as the lethal lesion for hyperthermic cell killing. A logical corollary is that protection against hyperthermic killing requires stabilization of cellular proteins against thermal denaturation. To test this, Chinese hamster ovary cells were treated with the heat protector cycloheximide and then subjected to differential scanning calorimetry to measure protein denaturation. Cycloheximide stabilized proteins that denatured between 42 degrees C and 52 degrees C in control cells by increasing their transition (denaturation) temperature by an average of 1.3 degrees C. In addition, cycloheximide reduced the cytotoxicity of actinomycin D and adriamycin, suggesting that protein stabilization protects cells against stresses other than hyperthermia.

Background: Vascular endothelial growth factor (VEGF)-165 promotes cardiomyogenesis in chronic myocardial ischemia and nonreperfused myocardial infarction (MI). It is unknown whether this effect is present in reperfused MI. We sought to investigate the effect of VEGF-165 gene therapy on cardiomyogenesis after reperfused MI. Methods and Results: Twenty-four Yucatan minipigs underwent thoracotomy and a vascular clamp was placed in the left circumflex artery. Reperfusion was reestablished after 90 minutes, and VEGF-165 gene therapy or placebo was administered. A replication-deficient recombinant human adenovirus serotype 5 was used for gene transfer (Ad5-VEGF165). The same viral vector devoid of VEGF gene (Ad5-β-Galactosidase) was used as placebo. Two administration routes were tested, intramyocardial (IM) injection and circumflex intracoronary (IC) infusion. The pigs were assigned to one of the following groups: IM Ad5-VEGF165 (n = 6), IM Ad5-βGal (n = 6), IC Ad5-VEGF165 (n = 6), and IC Ad5-βGal (n = 6). All pigs received 5-bromo-2′-deoxyuridine (BrdU) 250 mg IV twice a week to label cells undergoing DNA replication. The hearts were explanted at 4 weeks. BrdU-labeled cardiomyocytes in the peri-infarct area were counted by a pathologist blinded to group assignment. The number of BrdU-labeled cardiomyocytes per million cells was 4-fold higher in the group receiving IM VEGF-165 (64 ± 11.4) vs. IM placebo (16 ± 10.6), P = 0.034. No difference in infarct size or ventricular function was observed between the groups. Conclusions: IM VEGF-165 gene therapy promotes cardiomyogenesis in reperfused MI. However, no benefit in infarct size or cardiac function was observed at 4 weeks. The origin of these cells remains unknown and needs to be determined.

Liposomes are unilamellar spherical membranes made of lipids. Their ability to evade the immune system and to transport water soluble substances encased in their inner cavity make them excellent carriers for drug delivery, especially for cancer treatment. To increase their efficacy, liposomes must be endowed with mechanisms for fast and controlled release of incorporated material after reaching their target. Here we show that controlled drug release from liposomes may be achieved through the use of X-ray as a triggering stimulus.

Baby hamster kidney (BHK) cells were heated at temperatures in the range of 43.5 degrees C to 57.0 degrees C to determine the time-temperature relationship of cell killing. The cells were grown on 0.025 mm thick pieces of mylar to minimize warm-up times. After heating, the cells were plated for the colony formation assay. The endpoints of 1%, 10%, or 90% isosurvival, or the D0 values of the survival curves were used to construct plots of the logarithm of the reciprocol of the exposure time versus the reciprocol of the absolute temperature. The data for each endpoint resulted in a straight line plot, indicating that the time-temperature relationship for cell killing remained constant from 43.5 degrees C to 57.0 degrees C; namely, a 1.8-fold increase in exposure time was required for a 1 degree C decrease in temperature in order to obtain isosurvival. Heated BHK cells were also examined using electron microscopy. The threshold level of altered morphology was the dissociation of polyribosomal structure and the formation of electron-dense granules within the mitochondria. The time-temperature relationship for the induction of this altered morphology was identical to that for the 90% isosurvival endpoint. Hence, the appearance of altered morphology appears to be related to cell killing.