Boar spermatozoa successfully predict mitochondrial modes of toxicity: implications for drug toxicity testing and the 3R principles.

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker & JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P<0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r=0.559) and proportions of motile (r=0.55) or progressively motile (r=0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

Study of molecular events in cells by fluorescence correlation spectroscopy.

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

High light field confinement for fluorescent correlation spectroscopy using a solid immersion lens.

In this paper we present recent single molecule detection experiment using a solid immersion lens (SIL) for fluorescent correlation spectroscopy measurements. We compared the performance of the SIL in combination with an air objective (40x, numerical aperture (NA)=1.15) with a water immersion objective (40x, NA=0.6) in a confocal microscope system (ConfoCorr 1). Important parameters for single molecule experiments such as collection efficiency and excitation field confinement were investigated. Although the two set-ups have similar numerical aperture the measurements demonstrated higher field confinement and better collection efficiency for the SIL system in comparison to the conventional confocal set-up. Adding spherical aberrations shifts the sample volume up to 4 microm away from the plane surface of the SIL and conserves a diffraction limited focal volume. In this case the FCS autocorrelation demonstrates a free 3D diffusion of dye molecules in a highly confined light field.

Suppression of exonucleolytic degradation of double-stranded DNA and inhibition of Exonuclease III by PNA.

Degradation of double-stranded DNA by Exonuclease III in the presence of complementary anti-parallel PNA was studied. It was found for the first time that the PNA suppresses the degradation of dsDNA in a sequence-specific manner as well as inhibits the activity of Exonuclease III in a non-specific way.

Insulin binding monitored by fluorescence correlation spectroscopy.

AIM/HYPOTHESIS: The characteristics of insulin binding to its receptors have been extensively studied by the radioligand binding assay. We used fluorescence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its receptor. METHODS: Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intensity fluctuations and autocorrelation functions for Rh-Ins, free in the incubation medium and bound to the cell membrane, were studied at single-molecule detection sensitivity in a 0.2 fL confocal volume. RESULTS: Measurements at the cell membrane revealed Rh-Ins binding with at least two diffusion components (diffusion times tauD1 = 0.8 ms, tauD2 = 20 ms) and corresponding weight fractions of y1 = 0.43 and y2 = 0.42. Specificity of the binding was shown by the dislocation of bound Rh-Ins when excess unlabelled insulin was added. Scatchard analysis showed a nonlinear plot, revealing two binding processes with different affinities (Kass approximately 2 x 10(10) M(-1) and approximately 1 x 10(9) M(-1), respectively). CONCLUSION/INTERPRETATION: The fluorescence correlation spectroscopy results show two classes of binding sites with different affinities for insulin, or interactions between receptor sites consistent with negative cooperativity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensitivity (single molecule detection), FCS, provides additional data allowing a more precise evaluation of the kinetics of ligand-receptor interactions at low expression levels in living cells.

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects.

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.

Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells.

Fluorescence correlation spectroscopy (FCS) allows the study of interactions of fluorescently labeled ligand with receptors in living cells at single-molecule detection sensitivity. From the autocorrelation functions of fluorescence intensity fluctuations, the diffusion time of molecules through the confocal volume is analyzed, and from that, the molecular weights of free and bound molecules can be calculated. We have applied FCS to study the receptor diversity for the neuropeptide galanin (GAL) in cultured cells. FCS measurement of the fluorophore rhodamine-labeled GAL (Rh-GAL) has been performed in 0.2-fL confocal volume elements of the laser beam. The analysis of autocorrelation functions of Rh-GAL in solution above cells and at cell membranes demonstrates that the diffusion time of unbound Rh-GAL is 0.16 ms, whereas diffusion times of membrane-bound Rh-GAL are 22 and 700 ms. Because both of the diffusion times (22 and 700 ms) are much longer as compared to that of unbound Rh-GAL, they correspond to slow-diffusing complexes when Rh-GAL is bound to the cell membranes. Addition of excess nonlabeled GAL is accompanied by competitive displacement. Full saturation of the GAL binding is obtained at nanomolar concentrations. Scatchard analysis of binding data reveal one binding process, assuming one binding site per Rh-GAL (n = 1). On the other hand, the appearance of two diffusion times, 22 and 700 ms, suggests the existence of two subpopulations of GAL receptor complexes or two subtypes of GAL receptor not detected before. This makes an important point that FCS permits the identification of receptors, which were not possible to detect before by conventional binding techniques. The inhibitory effect of pertussis toxin on the GAL binding considers a G-protein-involved allosteric system, important for the clarification of essential steps in the G-protein-related signal transduction. This study is of pharmaceutical significance, since it will provide insights into how FCS can be used as a rapid technique for studying ligand-receptor interactions in living cells, which is one step forward for large-scale drug screening in cell cultures.

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen.

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.

Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy.

We investigated the specific binding of epidermal growth factor (EGF) to its membrane-bound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodamine-labeled EGF (Rh-EGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace Rh-EGF binding, demonstrating no cross-reaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K(ass) of 1.5 x 10(9) M(-1). The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k(diss) = 2.9 x 10(-4) s(-1). The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligand-receptor interactions in cell cultures. This is a step forward toward large-scale drug screening in cell cultures.

UV-Fluorescence correlation spectroscopy of 2-aminopurine.

We have built a fluorescence correlation spectroscopy (FCS) microscope for ultraviolet excitation (280-300 nm) and emission. With UV excitation the fluorescence of 'natural fluorophores' such as the modified nucleotide 2-aminopurine can be analyzed. The sensitivity of a natural fluorophore toward conformational changes can reveal dynamics in biomolecules. UV-FCS is well suited for detection of intensity fluctuations related to such conformational dynamics. Here we show UV-FCS measured on p-Quarterphenyl and on 2-aminopurine (2-AP). The triplet state rate constants and the excitation cross section for 2-AP were estimated to k23 = 1 x 10(6) s(-1), k31 = 3 x 10(5) s(-1), and sigma(exc) = 2 x 10(-17) cm2.

Quantitative two-color fluorescence cross-correlation spectroscopy in the analysis of polymerase chain reaction.

We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target.

The standard deviation in fluorescence correlation spectroscopy.

The standard deviation (SD) in fluorescence correlation spectroscopy (FCS) has been mostly neglected in applications. However, the knowledge of the correct SD is necessary for an accurate data evaluation, especially when fitting theoretical models to experimental data. In this work, an algorithm is presented that considers the essential features of FCS. It allows prediction of the performance of FCS measurements in various cases, which is important for finding optimal experimental conditions. The program calculates the SD of the experimental autocorrelation function online. This procedure leads to improved parameter estimation, compared to currently used theoretical approximations for the SD. Three methods for the calculation of the SD are presented and compared to earlier analytical solutions (D. E. Koppel. 1974. Phys. Rev. A. 10:1938-1945.), calculation directly from fluorescence intensity values, by averaging several FCS measurements, or by dividing one measurement into a set of shorter data packages. Although the averaging over several measurements yields accurate estimates for the SD, the other two methods are considerably less time consuming, can be run online, and yield comparable results.

C-peptide binding to human cell membranes: importance of Glu27.

In addition to its established role in proinsulin folding, C-peptide has a function in regulation of cellular activity. The 31-residue peptide influences renal, vascular, and metabolic functions in patients with insulin-dependent diabetes mellitus. Binding to cells has been demonstrated for C-peptide, which can be displaced by its C-terminal pentapeptide. We have now used fluorescence correlation spectroscopy to investigate structural requirements on the pentapeptide part for C-peptide binding. All pentapeptide residues, E(27)GSLQ(31), were individually replaced with Ala and the capacity of the resulting peptides to displace rhodamine-labelled full-length human C-peptide from human renal tubular cell membranes was determined. This showed that Glu27 is essential for displacement, while replacement of Gly28 with Ala has little effect, and replacement of any of the three most C-terminal residues had intermediate effects. Morevover, free Glu displaces full-length C-peptide to about 50%, while free Ala, C-peptide(1-26), and the truncated pentapeptide, corresponding to the tetrapeptide G(28)SLG(31), have no displacing capacity. The peptides EVARQ (corresponding to the rat C-terminal pentapeptide) and ELGGGPGAG (corresponding to positions 11-19 of human C-peptide) do not displace human C-peptide. These results indicate that Glu27 of C-peptide is critically involved in binding to cellular targets.

Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects.

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.

Fluorescently labeled model DNA sequences for exonucleolytic sequencing.

We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper Földes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project.

Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide.

The enzymatic incorporation of deoxyribonucleoside triphosphates by a thermostable, 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase was studied for PCR-based high-density labeling of 217-bp "natural" DNA in which fluorescent-dUTP was substituted completely for the normal dTTP. The amplified DNA carried two different sorts of tethered dye molecules. The rhodamine-green was used for internal tagging of the DNA. Since high-density incorporation of rhodamine-green-X-dUTP led to a substantial reduction (quenching) of the rhodamine-green fluorescence, a second "high" quantum yield label, Cy5, was inserted via a 5'-tagged primer in order to identify the two-color product. A theoretical concept of fluorescence auto- and cross-correlation spectroscopy developed here was applied to quantify the DNA sequence formed in terms of both the number of two-color fluorescent molecules and the number of covalently incorporated rhodamine-green-X-dUMP residues. The novel approach allowed to separate optically the specific DNA product. After complete, exonucleolytic degradation of the two-color DNA we determined 82-88 fluorescent U* labels incorporated covalently out of 92 maximum possible U* incorporations. The heavily green-labeled DNA was then isolated by preparative mobility-shift electrophoresis, re-amplified in a subsequent PCR with normal deoxyribonucleoside triphosphates, and re-sequenced. By means of this novel methodology for analyzing base-specific incorporations that was first developed here, we found that all fluorescent nucleotides and the normal nucleotides were incorporated at the correct positions. The determined labeling efficiency of 0.89-0.96 indicated that a fraction of the substrate analog was not bearing the fluorophore. The results were used to guide developments in single-molecule DNA sequencing. The labeling strategy (principal approach) for PCR-based high-density tagging of DNA, which included an appropriate thermostable DNA polymerase and a suitable fluorescent dye-dNTP, was developed here.

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

p53 Latency. C-terminal domain prevents binding of p53 core to target but not to nonspecific DNA sequences.

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

Specific binding of proinsulin C-peptide to intact and to detergent-solubilized human skin fibroblasts.

Proinsulin C-peptide exerts physiological effects on kidney and nerve function, but the mechanisms involved remain incompletely understood. Using fluorescence correlation spectroscopy, we have studied binding of rhodamine-labelled human C-peptide to intact human skin fibroblasts and to detergent-solubilised extracts of fibroblasts, K-562, and IEC-6 cells. Specificity was shown by displacement of rhodamine-labelled human C-peptide with unlabelled human C-peptide. C-peptide was found to bind to the cell membranes of intact fibroblasts with an association constant of 3 x 10(9) M(-1), giving full saturation at about 0.9 nM, close to the physiological C-peptide plasma concentration. Treatment of all investigated cells with the zwitter-ionic detergent Chaps was found to release macromolecules that bind specifically to C-peptide. The binding in Chaps extracts of fibroblasts was sensitive to time but remained reproducible for up to 2 h at room temperature. Lysophosphatidylcholine, Triton X-100, beta-octylglucopyranoside, SDS, or cholate gave extracts with only low or nonspecific binding. It is concluded that C-peptide binding components can be solubilised from cells, and that Chaps appears to be a suitable detergent.

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.