Peter E. Bryant

Light-induced cell separation in a tailored optical landscape

We demonstrate passive optical sorting of cell populations in the absence of any externally driven fluid flow. Specifically, we report the movement of erythrocytes and lymphocytes in an optical landscape, consisting of a circularly symmetric light pattern created by a Bessel light beam. These distinct cell populations move, spontaneously and differentially, across the underlying periodic optical landscape. Thus, we were able to separate lymphocytes from a mixed population of cells containing erythrocytes and then collect the lymphocytes in a microcapillary reservoir. We also demonstrate an enhanced form of this separation that exploits the polarizability of silica microspheres by attaching spheres coated with antibodies to cell surface markers to a subpopulation of lymphocytes. These techniques may be applied using standard laboratory apparatus.

Telomere length, chromatin structure and chromosome fusigenic potential.

Abstract. Telomeres are specialized structures at chromosome ends that are thought to function as buffers against chromosome fusion. Several studies suggest that telomere shortening may render chromosomes fusigenic. We used a novel quantitative fluorescence in situ hybridization procedure to estimate telomere length in individual mammalian chromosomes, and G-banding and chromosome painting techniques to determine chromosome fusigenic potential. All analysed Chinese hamster and mouse cell lines exhibited shorter telomeres at short chromosome arms than at long chromosome arms. However, no clear link between short telomeres and chromosome fusigenic potential was observed, i.e. frequencies of telomeric associations were higher in cell lines exhibiting longer telomeres. We speculate that chromosome fusigenic potential in mammalian cell lines may be determined not only by telomere length but also by the status of telomere chromatin structure. This is supported by the observed presence of chromatin filaments linking telomeres in Chinese hamster chromosomes and of multibranched chromosomes oriented end-to-end in the murine severe combined immunodeficient (SCID) cell line. Multibranched chromosomes are the hallmark of the human ICF (Immune deficiency, Centromeric instability, Facial abnormalities) syndrome, characterized by alterations in heterochromatin structure.

Trapping and manipulation of low-index particles in a two-dimensional interferometric optical trap

We demonstrate optical trapping and manipulation of low-index spheres in two dimensions, using the pattern produced by two interfering plane waves. This technique shows, for what is believed to be the first time, alignment of an array of hollow spheres and simultaneous manipulation of high- and low-index particles in the horizontal plane. Furthermore, rodlike particles (up to 30microm in length) are manipulated simultaneously with the low-index particles. This technique offers a practical method for manipulating bubbles, low-index droplets, or rodlike biological samples.

Photoporation and cell transfection using a violet diode laser

The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

Adaptive response: some underlying mechanisms and open questions

Organisms are affected by different DNA damaging agents naturally present in the environment or released as a result of human activity. Many defense mechanisms have evolved in organisms to minimize genotoxic damage. One of them is induced radioresistance or adaptive response. The adaptive response could be considered as a nonspecific phenomenon in which exposure to minimal stress could result in increased resistance to higher levels of the same or to other types of stress some hours later. A better understanding of the molecular mechanism underlying the adaptive response may lead to an improvement of cancer treatment, risk assessment and risk management strategies, radiation protection, e.g. of astronauts during long-term space flights. In this mini-review we discuss some open questions and the probable underlying mechanisms involved in adaptive response: the transcription of many genes and the activation of numerous signaling pathways that trigger cell defenses - DNA repair systems, induction of proteins synthesis, enhanced detoxification of free radicals and antioxidant production.

High Chromosomal Sensitivity of Chinese Hamster Xrs 5 Cells to Restriction Endonuclease Induced DNA Double-strand Breaks

The cytogenetic effects of restriction endonucleases (RE) and X-rays were examined in the radiosensitive mutant Chinese hamster cell line xrs 5 and its normal parental line CHO K1. Cells were permeabilized with Sendai virus and exposed to Pvu II and Eco RV which induce blunt-ended double-strand breaks (dsb) in the DNA of cells, or Bam H1 and Eco R1 which induce cohesive-ended dsb with a four-base overlap. Treated cells were then assayed for the presence of metaphase chromosomal aberrations by sampling at multiple fixation times and in experiments where cells were exposed to graded series of RE concentrations. Exposure to X-rays or RE causing blunt-ended dsb was found to be between two and three times more effective in xrs 5 than in CHO K1 cells. We interpret this higher chromosomal sensitivity of xrs 5 cells as reflecting the reported defect in dsb repair in xrs 5. Both xrs 5 and CHO K1 cells yielded less aberrations after exposure to Bam H1 or Eco R1 than after exposure to Pvu II or Eco RV, confirming our previous results and demonstrating that cohesive-ended dsb are less damaging than blunt-ended dsb. Multiple fixation time experiments showed that the higher sensitivity of xrs 5 was evident at several different sampling times after treatment. Similarly the low yield of aberrations after exposure of cells to Bam H1 was evident at all sampling times. Overdispersion of chromosomal aberrations was observed in samples exposed to RE. This is thought to be due to a non-uniform permeabilization of the cell population to RE. Our results indicate that RE-induced dsb are handled by cells in a similar way to those arising during X-ray exposure.

Kinetics of Chromatid Aberrations in G2 Ataxia-telangiectasia Cells Exposed to X-rays and Ara A

The cytogenetic effects of X-rays alone or in combination with 9-beta-D-arabinofuranosyladenine (ara A) were studied in an immortalized fibroblastic line of ataxia-telangiectasia (A-T) cells. The average length of G2 in this line was determined by autoradiographic labelling (labelled mitoses) to be approximately 5 h. Samples of A-T cells treated with or without ara A, 4 h prior to fixation were irradiated at 1/2-hourly intervals, from 1.5 h to 3.5 h before fixation and then examined for the presence of metaphase chromatid aberrations. It is postulated that the kinetics of disappearance (rejoining) of chromatid deletions with postirradiation incubation time reflects the underlying repair of dsb. This rejoining was found to be inhibited by ara A. Thus the frequency of deletions in the presence of ara A should represent the frequency of deletions in the absence of dsb repair. The rejoining kinetics for deletions in A-T was similar to that found in a previous study of normal human fibroblasts (Mozdarani and Bryant 1987). The number of deletions in X-irradiated A-T cells at 1.5 h before fixation was found to be higher by a factor of approximately 2 than that found previously in normals, indicating that in A-T a higher rate of conversion of dsb into chromatid deletions occurs. The frequency of exchanges induced in G2 A-T cells was similarly enhanced but, unlike the situation in normal cells, ara A was found to cause only a slight increase in this frequency.

A component of DNA double-strand break repair is dependent on the spatial orientation of the lesions within the higher-order structures of chromatin

By the use of a modified neutral filter elution procedure variations in the repair of DNA dsb have been observed between the ionizing radiation sensitive mutant xrs-5 and the parent cell line CHO-K1. Conventional neutral filter elution requires harsh lysis conditions to remove higher-order chromatin structures which interfere with elution of DNA containing dsb. By lysing cells with non-ionic detergent in the presence of 2 mol dm-3 salt, histone-depleted structures that retain the higher-order nuclear matrix organization, including chromatin loops, can be produced. Elution from these structures will only occur if two or more dsb lie within a single-looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of dsb in loops containing multiple dsb are repaired with slow kinetics whilst dsb occurring in loops containing single dsb are repaired with fast kinetics. Xrs-5 cells are defective in the repair of multiply damaged loops. This work indicates that the spatial orientation of dsb in the higher-order structures of chromatin are a possible factor in the repair of these lesions.

Induction of chromosomal damage by restriction endonuclease in CHO cells porated with streptolysin O

A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.

The Induction of DNA Double-strand Breaks in CHO Cells by Pvu II: Kinetics Using Neutral Filter Elution (pH 9·6)

Chinese hamster CHO K1 cells were treated with the restriction endonuclease Pvu II during electroporation and assayed for DNA double-strand breaks (dsb). Dsb were measured by the non-denaturing filter elution technique (pH 9.6) at various times up to 24 h after restriction endonuclease (RE) treatment. The frequency of dsb following electroporation in the presence of 200 units/ml Pvu II increased over the post-treatment incubation period. This was found not to be due to cell or DNA degradation, indicating that Pvu II remains active for at least 24 h inside the cell. We suggest that these kinetics of dsb result from a competition between incision (by Pvu II) and dsb repair.