Michael J. Borrelli

Time-temperature analysis of cell killing of BHK cells heated at temperatures in the range of 43.5°C to 57.0°C☆

Abstract Baby hamster kidney (BHK) cells were heated at temperatures in the range of 43.5°C to 57.0°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 D 0 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°C to 57.0°C; namely, a 1.8-fold increase in exposure time was required for a 1°C decrease in temperature in order to obtain isosurviival. 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.

Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.

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.

On the path to the heat shock response: destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification.

This review discusses the initial events that occur during oxidative stress that induce the synthesis of heat shock proteins. The focus is on non-native oxidation or modification of protein thiols and the destablization that can result. Proteins that contain non-native modified thiols can become destablized such that they unfold into molten globule-like intermediates at or below 37 degrees C, relieving Hsf-1 negative regulation, and inducing Hsp transcription.

Successful Microbubble Sonothrombolysis Without Tissue-Type Plasminogen Activator in a Rabbit Model of Acute Ischemic Stroke

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 &mgr;m MB+US (n=8); and (6) tagged albumin 3 &mgr;m MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm2) 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 &mgr;m MB was 5×109 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 &mgr;m MB+US (0.7%±0.9%; P=0.018), and tagged 3 &mgr;m 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.

Microbubbles Improve Sonothrombolysis In Vitro and Decrease Hemorrhage In Vivo in a Rabbit Stroke Model

Introduction: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. Materials and Methods: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. Results:In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P < 0.0001). 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 P < 0.0183 and P = 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P < 0.0001), 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. Conclusion:Combining tPAand 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.

A method for freezing synchronous mitotic and G1 cells

A modification of the protocol developed by Kawamoto, J C & Barrett, J N, Brain res (1986), in press for freezing primary neuron cultures in a solution containing low sodium and high lactate and potassium concentrations was used to freeze synchronous mitotic and G1 CHO cells. After thawing, the cells behaved as if they had never been frozen with respect to cell growth, cell division, plating efficiency, and hyperthermic sensitivity.

A simple method for making ion-selective microelectrodes suitable for intracellular recording in vertebrate cells

A simple procedure for manufacturing Cl-, K+, and pH liquid membrane ion-sensitive microelectrodes is presented in detail. Electrodes suitable for recording from the specimen of interest are back-filled with a small amount of silane solution and heated for 5 min on a hot plate at a temperature between 400 and 500 degrees C, after which they are injected with the ion-sensitive resin. The procedure is adaptable to many different glass stocks, e.g., single-barreled, double-barreled, or theta glass, and can be used to produce electrodes having a wide range of tip sizes for recording either extracellular or intracellular ion activities. Another advantage of the method is speed; up to 10 electrodes can be prepared simultaneously, permitting over 40 functional electrodes to be made per hour.

Evidence that the feeder effect in mammalian cells is mediated by a diffusible substance

Chinese hamster ovary (CHO), baby hamster kidney (BHK), and NG108-15 neuroblastoma cells were heated and then plated for the colony-formation assay either with or without feeder cells present. Besides these cell lines, four other cell types were used as feeders. All cell lines functioned equally well as feeders for each of the heated cell lines. In some experiments the heated and feeder cells were separated by semipermeable membranes. This separation had no effect on the feeder effect, indicating that cell-to-cell contact was not requisite. The feeder effect appears to be mediated by a low molecular weight, diffusible substance produced by the feeder cells.

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.

Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation

Abstract A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the proteins distribution within living cells.