Mild hyperthermia-induced apoptosis is dependent on p53 in human lymphoid cells.

Mild hyperthermia is known to enhance apoptosis. The p53 tumor-suppressor gene product has been shown to function in apoptosis in response to genotoxic stress. However, there is little information regarding the mechanism of p53-dependent apoptosis induced by heat stress. In present study, a p53 contribution in mild hyperthermia-induced apoptosis was investigated in human lymphoid system. After 30-minute exposure at 44 degrees C, the accumulation of p53 protein was clearly observed in TK6 and ML-1 cells. Using comet assay, the more significant sensitivity to hyperthermic apoptosis was found in TK6 (wild-type p53) than in WI-L2-NS (mutated in p53). Furthermore, the significantly rapid shifting from early apoptotic phase to late apoptotic was observed in heat-induced p53 TK6 cells. These findings suggest that p53-dependent apoptosis is efficaciously induced by mild hyperthermia as non-genotoxic stress in human lymphoid system.

Critical parameters influencing hyperthermia-induced apoptosis in human lymphoid cell lines.

Brief mild hyperthermia is sufficient to induce apoptosis (programmed cell death) in many cell lines. Here we describe the effects of a number of factors modulating heat shock induced apoptosis outcomes. We report the effects of cell type, heat load, recovery times, cellular growth phase, and protein synthesis on the levels of apoptoses seen in heat stressed cell populations. We observe that a number of cell lines are competent to undergo heat stress induced apoptosis using both the comet assay and cellular and nuclear morphologies. Of the cell lines tested we saw a wide spectrum of sensitivities, ranging from resistant (less than 1% apoptotic after 12 h) to exquisitely sensitive (>95%). By incrementally increasing the heat load from 37-49 degrees C, we observed a gradual increase in apoptosis with a significant change from apoptotic to necrotic death at temperatures beyond 45 degrees C. The kinetics of the apoptotic response to heat shock were also examined. A time dependent increase in apoptotic cell death was seen after initial hyperthermic treatment with most cell types reaching a 'plateau' at 18 h. In addition to these parameters we report that growth phase has a strong influence on the number of apoptoses induced as a result of heat stress. Cultured cells, grown to a plateau, undergo apoptosis at a much higher level than similarly treated cells taken during an exponential phase of growth. Finally, we determined the necessity of protein synthesis for apoptotic competency.

NSAID-induced apoptosis in Rous sarcoma virus-transformed chicken embryo fibroblasts is dependent on v-src and c-myc and is inhibited by bcl-2.

Mounting epidemiological and experimental evidence implicates non-steroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis--a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11-23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, overexpression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.

Key morphologic changes and DNA strand breaks in human lymphoid cells: discriminating apoptosis from necrosis.

Apoptosis is an important form of physiologic cell death displayed by an enormous variety of tissues under divergent conditions. The recent attention toward apoptosis in virtually all aspects of modern biology indicates that rapid and accurate differentiation between apoptosis and necrotic death is of considerable interest. Apoptosis is distinguishable from necrosis on the basis of several criteria. In this study, we undertook to examine the effects of mild hyperthermia (43 degrees C leading to apoptotic death) and severe hyperthermia (50 degrees C leading to necrotic killing) on associated DNA fragmentation. Using laser scanning and fluorescent microscopic evaluation of DNA "comets" in the single cell gel assay, we compared necrotic and apoptotic DNA damage in a variety of human leukemia and lymphoma cell lines at the level of the individual cell. We show that necrotic cells do display detectable DNA damage. We confirm our preliminary report that comet "tail moment" is sufficient to distinguish between necrotic and apoptotic DNA damage, while comet tail length may confuse the two forms. We report that a recovery period is necessary for expression of increasing apoptotic but not necrotic DNA damage. We show that apoptosis increases with prolonged hyperthermia and confirm that the mode of death changes from apoptosis to necrosis with higher heat loads, producing a greater fraction of cells showing damage. In addition, we show that for necrotic cells, DNA tail moment reflects sensitivity to prolonged exposure without a concomitant change in tail length.

Anti-human cytomegalovirus activity and toxicity of sulfonated anthraquinones and anthraquinone derivatives.

Sulfonated anthraquinones and other anthraquinone derivatives were evaluated for anti-human cytomegalovirus (HCMV) activity, cytotoxicity and genotoxicity. Acid blues 40 and 129, acid black 48, alizarin violet R and reactive blue 2 were the most active compounds having selective indices of greater than 30 and EC50 values of 4-30 microM. When tested against a clinical isolate, the 4 compounds were 2- to 5-fold less active. The antiviral activity was distinctly separate from the virucidal activity (> 1000 microM). The compounds were weakly toxic to either log phase or stationary cells in most of the following cytotoxicity assays: neutral red uptake assay, lactic acid dehydrogenase assay, trypan blue exclusion assay and radiolabeled macromolecular precursor uptake assays. Using a genotoxicity assay, the comet assay, only reactive blue 2 and acid black 48 were found to cause DNA strand breakage. This occurred at concentrations of 30 and 170 microM, respectively. These results suggest that these compounds could be a prototype for synthesizing even more effective HCMV-inhibitory anthraquinone derivatives.

Induction of apoptotic cell DNA fragmentation in human cells after treatment with hyperthermia.

The biological significance of apoptosis is becoming increasingly clear. Its relevance in tumor response to treatment as well as recent evidence for its important function as a regulating mechanism in tumorigenesis has also been demonstrated. One of the most prominent biological features of apoptosis is nucleosomal DNA fragmentation. In this communication, we present a study of DNA fragmentation in Raji cells which have been subjected to hyperthermia treatment to induce apoptosis. We found that the induction and onset of fragmentation is swift, and consistent with previous reports that fragmentation must be a rapid event.

The comet assay: a comprehensive review.

The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. Its use has increased significantly in the past few years. This paper is a review of the studies published to date that have made use of the comet assay. The principles of strand break detection using both the alkaline and neutral versions of the technique are discussed, and a basic methodology with currently used variations is presented. Applications in different fields are reviewed and possible future directions of the technique are briefly explored.

Spongiform encephalopathies and prions: an overview of pathology and disease mechanisms.

The etiology of spongiform encephalopathies has been sharply contested for decades. At the heart of the issue is the question of disease origin: Are prion disease representative of primary neurodegenerative genetic disorders, or are they bona fide infectious diseases? This article provides a brief outline of the progress made in the elucidation of prion disease mechanisms in the context of pathological support of the 'protein only' hypothesis. The answer to the above question appears to be that spongiform encephalopathies are uniquely both infectious and genetic neurodegenerative diseases.

The effect of electromagnetic field exposure on the formation of DNA single strand breaks in human cells.

Electromagnetic fields (EMF) have been reported to be associated with human cancers in a number of epidemiological studies. Agents that are associated with cancer affect DNA in an adverse manner. This is a report of a DNA damage study in human cells exposed to EMFs. Single strand breaks in DNA are proposed to be necessary events in both mutagenesis and carcinogenesis. The single cell gel assay is a sensitive and accurate technique that was used in this study for single strand break detection. The EMF exposure system used here appeared to have no direct effect on DNA damage induction in a series of experiments. Moreover, EMF did not have a significant effect in potentiating DNA damage in cells treated with oxidative stresses.

Alkali-labile sites are prevalent in kidney tissue DNA.

Inherent DNA damage in the form of single strand breaks and alkali labile sites is generated by essential intracellular processes, such as DNA replication and repair. Utilizing the in situ DNA unwinding assay and the comet assay (single cell gel), we have observed high levels of alkali induced DNA strand breaks in cells isolated from mouse kidney tissue homogenate. Kidney cell DNA demonstrated a 7.4 x increase in nucleoid expansion and a 7 x increase in comet length compared to negative control cells (thymocytes and splenocytes) in the two assay systems, comparable to epididymal sperm cells which have previously been demonstrated to contain abundant alkali-labile sites. These results argue for the existence of prevalent alkali-labile sites in kidney cell DNA.

Video enhanced laser scanning microscopic analysis of DNA comet formation.

Laser Scanning Microscopy is a sensitive tool that provides a unique method of analyzing biological systems. Coupled with the Single Cell Gel assay, it allows for accurate and reproducible detection of DNA strand breaks. An understanding of the theory of DNA comet formation is lacking. Using dexamethasone induced apoptosis in murine thymocytes as a model for double strand breaks, we used video enhanced laser scanning microscopy to evaluate the leading edge of DNA migration in the single cell gel assay. In this system, comet length increases significantly within the first thirty seconds of electrophoresis, the greatest increase in length is completed within the first minute, and the first two minutes are important in significant increases in DNA migration during DNA comet formation.

Detection of apoptosis induced DNA cleavage in scrapie-infected sheep brain.

The pathogenesis and molecular basis of nerve cell death which accompanies scrapie infections in sheep are not well understood. Degeneration of neurons in culture caused by prion protein fragments has recently been reported to be consistent with mechanisms of cell death by apoptosis or programmed cell death. Apoptosis activation during prion-related encephalopathies has not yet been established in vivo. We report here the detection of DNA damage consistent with apoptosis in the brain cells of sheep infected with scrapie using laser scanning microscopic analysis of the single cell gel assay. We suggest that this DNA fragmentation is the result of the activation of the mechanisms characteristic of apoptotic cell death.

Laser scanning microscopic analysis of DNA damage in frozen tissues.

DNA damage is central to research in many fields, especially cancer research and toxicology. The possible loss of DNA structural integrity during freezing or sustained maintenance at low temperatures may present difficulties in the interpretation of data accumulated in studies of tissues collected over a period of time and subsequently evaluated. Using laser scanning microscopic analysis of the recently developed single-cell gel (SCG) assay to measure DNA strand breaks in individual cells, we found that the basal levels of DNA damage in frozen tissue was higher than fresh tissue, but tissues frozen for greater lengths of time do not appear to contain significantly more DNA damage than those frozen for a short period. Evaluation of DNA damage in tumors stored by or collected using cryopreservation may produce artificially exaggerated levels of damage, which could limit analytical interpretations.

Analysis of single-cell gel electrophoresis using laser-scanning microscopy.

The single-cell gel (SCG) assay has been shown to be a valuable technique to measure DNA-strand breaks in individual cells. Imaging of the assay is enhanced by laser-scanning microscopy (LSM) technology. Cells embedded in low melt point agarose were treated with 12.5 mM H2O2 to induce DNA-strand breaks. Following cell lysis and electrophoresis under alkaline conditions to allow single-stranded break detection, analysis of the resulting comets can provide an accurate method of comparing DNA-damage levels. The migration patterns of stained DNA were quantitated by LSM analysis. A statistically significant time-dependent dose-response relationship was clearly observed. LSM analysis of the SCG technique allows rapid, sensitive, and reproducible quantitation of single-stranded breaks and alkali-labile sites in the DNA of single cells.

Measuring the repair of H2O2-induced DNA single strand breaks using the single cell gel assay.

The single cell gel (SCG) assay is for the detection of DNA single strand breaks and alkali labile sites in single cells. This investigation showed how the technique had an effective application in examining DNA single strand break repair. DNA from cultured human cells was damaged by treating the cells with H2O2, and samples were allowed to repair for various time intervals. The cells were then electrophoresed in a gel on a microscope slide, and the sizes of the individual DNA 'comets' were measured using laser scanning microscopy. Analysis of the comets indicated that actual DNA repair could be monitored over time. The SCG assay will help the study of DNA damage and repair, which will aid our understanding of the complex processes of mutagenesis and carcinogenesis.