James R. Lepock

Cellular effects of hyperthermia: relevance to the minimum dose for thermal damage

The specific mechanism of cell killing by hyperthermia is unknown, but the high activation energy of cell killing and other responses to hyperthermia suggest that protein denaturation is the rate-limiting step. Protein denaturation can be directly monitored by differential scanning calorimetry and in general there is a good correlation between protein denaturation and cellular response. Approximately 5% denaturation is necessary for detectable killing. Protein denaturation leads to the aggregation of both denatured and native protein with multiple effects on cellular function.

Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro

Mutations in Cu/Zn superoxide dismutase (SOD) are associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). There is considerable evidence that mutant SOD has a gain of toxic function; however, the mechanism of this toxicity is not known. We report here that purified SOD forms aggregates in vitro under destabilizing solution conditions by a process involving a transition from small amorphous species to fibrils. The assembly process and the tinctorial and structural properties of the in vitro aggregates resemble those for aggregates observed in vivo. Furthermore, the familial ALS SOD mutations A4V, G93A, G93R, and E100G decrease protein stability, which correlates with an increase in the propensity of the mutants to form aggregates. These mutations also increase the rate of protein unfolding. Our results suggest three possible mechanisms for the increase in aggregation: (i) an increase in the equilibrium population of unfolded or of partially unfolded states, (ii) an increase in the rate of unfolding, and (iii) a decrease in the rate of folding. Our data support the hypothesis that the gain of toxic function for many different familial ALS-associated mutant SODs is a consequence of protein destabilization, which leads to an increase in the formation of cytotoxic protein aggregates.

Sonication of proteins causes formation of aggregates that resemble amyloid

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimers disease, Huntingtons disease, Parkinsons disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green‐gold birefringence upon binding the dye Congo red, and cause a red‐shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication‐induced aggregates have high β‐content, and proteins with significant native α‐helical structure show increased β‐structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication‐induced aggregates, including fibrils with diameters of 5–20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye‐binding and structural characteristics, as well as the ability of the sonication‐induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.

Involvement of membranes in cellular responses to hyperthermia.

A number of major questions remain unanswered regarding the relationship between cellular membranes and the viability of cells exposed to hyperthermia. Foremost, are membranes involved at all in hyperthermic killing, radiosensitization, or thermotolerance of mammalian cells after heating to hyperthermic temperatures in the range of 41-45?C? If so, which membranes are implicated: plasma, nuclear, mitochondrial, lysosomal, endoplasmic reticular, etc? Damage might be restricted to only one area of a membrane due to membrane heterogeneity (1). In particular, what specific molecular alterations occur in membranes when they are heated to hyperthermic temperatures? In this communication some of the known structural and functional changes in membranes due to heating will be summarized.

Simultaneous Two-photon Excitation of Photofrin in Relation to Photodynamic Therapy

Abstract Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-γ) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-γ) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-γ excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-γ PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-γ killing of vascular endothelial cells in vitro. The 2-γ absorption properties of Photofrin were investigated in the 750–900 nm excitation wavelength range. It was shown that 2-γ excitation dominates over 1-γ excitation above 800 nm. The 2-γ absorption spectrum of Photofrin in the 800–900 nm excitation wavelength range was measured. The 2-γ cross section decreased from about 10 GM (1 GM = 10−50 cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-γ contribution was negligible. Cell death was monitored with the use of 2-γ scanning laser microscopy. The light doses required for killing were high (6300 J cm−2 for ∼50% killing), but 2-γ cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-γ PDT due to its low 2-γ cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-γ cross sections (>100 GM), which will be required for 2-γ PDT of AMD (and other conditions) to be clinically practical.

How do cells respond to their thermal environment

Changes in growth temperature induce both activating and inactivating responses from cells, with the magnitude of the temperature change being among the factors that influence which type of response dominates. Aside from upregulated enzyme activity, induction of thermotolerance is the most widely studied and best understood activating response that cells exhibit following heat shock. Inactivating responses to heat shock that are of biomedical interest include heat radiosensitization and cytotoxicity. Interestingly, the activation energy for inducing thermotolerance, heat cytotoxicity, and radiosensitization all fall within a similar range of 120–146 kcal per mole. The relatively high activation energy for each of these responses suggests that they all involve a heat-induced molecular transition as a trigger, and several lines of research suggest strongly that protein denaturation is the common transition that triggers all three responses. Low levels of protein denaturation are sufficient to attract the 90 kDa heat shock protein (HSP90) such that it frees up heat shock factor 1, which then trimerizes to form an active transcription factor that upregulates expression of heat shock proteins. Upregulation of heat shock proteins and other heat-induced events result in the development of thermotolerance, which protects cells from subsequent exposure to heat shock and other stresses. A more severe heat shock increases protein denaturation proportionately and leads to aggregation of both denatured and native proteins. This results in inactivation of protein synthesis, cell cycle progression, and DNA repair processes such that cells either die or are sensitized to radiation and other cytotoxic events. The ultimate fate of cells following a heat shock depends upon the summation of the activation and inactivation events that are induced, which appears to be governed by the resultant magnitude of protein denaturation and aggregation. Treatments that stabilize cellular proteins against denaturation and aggregation reduce the magnitude of inactivating responses while increasing that of activating responses for a given heat shock (time at temperature), while treatments that sensitize proteins to denaturation and aggreation have the converse effect. These findings support the conclusion that the determinant of the cellular response to heat shock is the amount of heat-induced protein denaturation and aggregation and not the time at temperature.

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.

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of cotyledon senescence on the composition and physical properties of membrane lipid

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.