Daryl W. Fairbairn

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.

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.

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°C, we observed a gradual increase in apoptosis with a significant change from apoptotic to necrotic death at temperatures beyond 45°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.

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.

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.

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.

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.

Thymidine kinase: the future in breast cancer prognosis

Abstract The ability to precisely describe tumour aggressiveness in human breast cancer is inadequate. Current prognostic indicators, although useful, cannot accurately predict tumour behavior. As a result, multivariate analyses are required to obtain any significant value from a wide rangeastic indicators. Prognostic indications are generally determined from the primary tumour, but real prognostic value is dependent upon the activity of potentially metastatic lesions. We present in this paper evidence for the use of thymidine kinase, an enzyme indicative of cellular proliferation, as a tumour marker with individual prognostic value. It has been previously established that thymidine kinase levels in both tumours and sera of patients with breast cancer have been shown in clinical studies to be positively correlated with tumour stage. The monitoring of serial serum samples has also proven to be of prognostic value in predicting response to therapy. Measurements of thymidine kinase levels in primary tumours have demonstrated utility as a predictor of tumour recurrence. We propose that the development of a more accurate method of measuring thymidine kinase activity may aid significantly in the management of breast cancer, while eliminating the current detection problems of radio-labeling assay systems. This can be done by the inclusion of an immunologically based detection system.

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.

A method for rescue of hybridomas from freezer failure

Dear Editor: The cryopreservation of eukaryotic cell lines is a routine laboratory technique. Freezing agents such as glycerol or dimethyl sulfoxide (DMSO) are commonly used to preserve cells through the freezing process and alleviate damage such as mechanical injury by ice crystal formation, concentration of electrolytes, pH changes, dehydration and denaturation of proteins (2). These lethal effects are minimized by addition of cryopreservation agents such as glycerol or DMSO, which lowers the freezing point and allows for a slower cooling rate. Cell freezing is usually a slow process which requires several hours of temperature reduction before ceils are ready to be submerged in liquid nitrogen. The thawing process is usually performed very quickly at 37 ° C and under constant agitation in order to remove the cryopreservative since it is toxic to ceils (3). Quick removal of the cryopreservative is important to insure viability of freshly thawed cells. Due to a freezer failure in our laboratory, several valuable hybridoma cells were in danger of being permanently lost. The 7 0 ° C freezer had been down for at least 48 hours before the power failure was noticed. All the cryovials containing hybrid cell lines were warm to the touch before rescue attempts were begun. The hybrids were frozen for no longer than two months at a density of 1 × 107 cells per vial in 1 ml of 90% fetal calf serum (FCS) and 10% glycerol. There were 240 vials of hybridoma clones which were contained in the freezer which we attempted to rescue. In order to facilitate our rescue attempts, we devised a supplemented media to increase our cell recovery (Media B). Various other groups have altered RPMI 1640 in order to best suit the needs of the particular cell lines which they are culturing (4,5,7). All ingredients unless otherwise noted were obtained from Sigma