Electroporation-induced damage in mammalian cell DNA.

Electroporation induced damage in the DNA of HL60 cells has been investigated by alkaline elution techniques. DNA damage is minimised by reducing the total charge applied (i.e., voltage x capacitance). Reduction of either of these electrical parameters, however, compromises the induced permeability of the cells to small molecules. The data presented concerning the effects of voltage and capacitance on DNA damage and the permeability of cells can be used to specify optimum conditions for electroporation in which DNA damage is minimised. The duration for which the current is applied can be seen to have a significant effect on the level of DNA damage. A modest temperature rise may occur when an electric charge is passed through electroporation buffer, but this event alone does not induce DNA damage in cells. The effect of voltage upon the permeability of HL60 cells to fluorescent-labelled molecules of varying molecular weight is reported.

Radiation-induced myeloid leukaemia in CBA/H mice: a non-immunogenic malignant disease in syngeneic mice.

In vivo growth characteristics of myeloid leukaemia induced by whole-body irradiation of CBA/H male mice were examined in the strain of origin by procedures expected to enhance or depress immunological responses. Syngeneic growth in vivo (survival time and frequency of takes) was not modified by attempted active immunization with radiation-inactivated cells or by sublethal whole-body irradiation of recipients before inoculation of small numbers of clonogenic cells. Since the growth stimuli involved in in vivo repair of severely damaged normal haemopoietic tissue also did not modify the growth of the radiation-induced leukaemia cells in syngeneic passage, their growth in vivo in the irradiated primary hosts can be regarded as autonomous by the stage at which leukaemia was diagnosed. Challenge inocula in the "immunization" experiments were 1-9 clonogenic cells from 4 different passaged lines and in the whole-body radiation experiments, 1-10(3) clonogenic cells derived from 11 different primary hosts and 4 different passaged lines.

Kinetics and mechanism of DNA repair. Evaluation of caged compounds for use in studies of u.v.-induced DNA repair.

Experiments are described in which the feasibility of using caged dideoxy and other nucleoside triphosphate analogues for trapping breaks induced by u.v. radiation damage to mammalian cell DNA is evaluated. These nucleotide analogues that have a photolabile 1-(2-nitrophenyl)ethyl-protecting group attached to the gamma-phosphate are placed in situ by permeabilizing cells by exposure to hypo-osmotic medium. The nucleoside triphosphate is released from the cage by a 351 nm u.v. laser pulse whence it may incorporate in the growing chain of DNA induced by the excision-repair process and terminate chain elongation. If the photoreleased dideoxynucleoside triphosphate is isotopically labelled in the alpha-phosphate position the break is trapped and labelled. Incorporation of radioactivity into trichloroacetic acid insoluble material in these experiments confirms their potential for use in studies of the kinetics of mammalian cell DNA repair.

The kinetics and mechanism of repair of UV induced DNA damage in mammalian cells. The use of 'caged' nucleotides and electroporation to study short time course events in DNA repair.

Using 'caged' DNA break trapping agents as well as the equivalent uncaged reagents and an automated apparatus, we have measured time courses of incorporation of radiolabelled nucleotides into HL60 cellular DNA in the early stages after 248 UV laser damage. These time courses show two distinctive phases, one between 0 and 120 seconds and another after 120 secs following damage. The first phase consists of a transient which shows a rapid initial incorporation of radiolabel followed by a sharp fall in incorporated label. This occurs with TTP as well as ddATP, which suggests that an excision activity which results in removal of recently incorporated bases is not solely provoked by the incorporation of an unnatural base, but also by the incorporation of an incorrectly paired base in a phase of what may be low fidelity repair. The second phase consists of a more steady state of incorporation. Both phases are dose dependent and show higher incorporation at higher doses. The transient is most apparent at does which cause some lethality. It may represent a form of emergency or 'panic' repair where it seems that there may be an immediate effort to maintain strand continuity in the damaged DNA. Results of experiments with polymerase inhibitors suggest that a polymerase which is sensitive to aphidicholin and which shows some sensitivity to dideoxythymidine is active during the transient phase of repair. Since excision of newly incorporated radiolabel takes place very rapidly during the first phase this would imply that a polymerase with an associated proof-reading nuclease is active at this stage. Polymerases alpha, delta, and epsilon all have this property but delta and epsilon have a higher sensitivity to dideoxythymidine than does alpha. Since the transient burst phase shows significant inhibition by dideoxythymidine, it is more likely that delta or epsilon are active at this stage. The putative panic response discussed in relation to proof reading mechanisms in aminoacyl-tRNA and DNA synthesis.

Kinetics and mechanism of DNA repair. Preparation, purification and some properties of caged dideoxynucleoside triphosphates.

Caged dideoxyribosylthymine triphosphate, dideoxyadenosine triphosphate and arabinosylcytosine triphosphate were prepared in high yield by reaction with 1-(2-nitrophenyl)diazoethane at pH 4 and room temperature for 24 h. Synthesis of caged alpha-32P-labelled dideoxyadenosine triphosphate (approx. 5000 Ci/mmol) in 85% yield was achieved by a modification of the method used for the synthesis of the unlabelled compounds. ATP was shown to be an excellent buffer in the synthesis of alpha-32P-labelled material, and in caged form to be an effective carrier in h.p.l.c. purification. Preparative h.p.l.c. was used to achieve purification of unlabelled caged compounds to greater than 98% purity and 32P-labelled material to 97% purity. Photolysis of unlabelled and 32P-labelled caged compounds by using XeF-excimer laser irradiation at 351 nm was characterized by using difference spectrophotometry and h.p.l.c. analysis. The stability of caged dideoxyadenosine [a-32P]triphosphate in the presence of cultured mammalian cells was evaluated; the adenosine derivative is essentially stable for 1 h.

Kinetics and mechanism of DNA repair: an automated programmable apparatus for fast time-resolved studies of the repair of mammalian DNA after u.v. irradiation.

An automated apparatus, designed and constructed for use in fast time-resolved studies of mammalian DNA repair after u.v. irradiation, is described. The methodology makes use of caged DNA-break-trapping reagents, e.g. [alpha-32P]dideoxyadenosine triphosphate [see Meldrum, Shall, Trentham and Wharton (1990) Biochem J. 266, 885-890] and the apparatus incorporates excimer lasers both for the delivery of u.v. damage and for the photoactivation of the caged reagents. The design is based on a sample-changing turntable which permits the sequential irradiation and quenching of samples. A maximum of eight samples can be processed in a single experiment, the sequence of events being programmed on a microcomputer, which permits a very generalized experimental sequence. Pipettes for addition of cells, nucleotides and quenching agent are driven pneumatically. A pair of pneumatically operated platinum electrodes allows electroporation of cells for loading of negatively charged reagents prior to irradiation. The time resolution of the apparatus is dependent upon the experimental scheme used and can be very short (e.g. 1 ms) when caged reagents are used; a more usual period is 1 s for the shortest incubation.

Optimisation of electroporation for biochemical experiments in live cells.

To introduce into cells small molecules, which do not permeate the cell membrane naturally, electroporation is the fastest and most efficient technique. Although it is not completely benign, the speed at which a full population of cells can be permeated gives it a strong advantage over all other cell permeation techniques. Here we describe the potential damaging effects of electroporation and how to derive conditions which avoid these and assure its use for biochemical experiments in live cells.

Gap junction communication dynamics and bystander effects from ultrasoft X-rays.

Gap junctions provide a route for small molecules to pass directly between cells. Toxic species may spread through junctions into 'bystander' cells, which may be exploited in chemotherapy and radiotherapy. However, this may be prevented by junction closure, and therefore an understanding of the dose-dependency of inhibition of communication and bystander effects is important. Low-energy ionising radiation (ultrasoft X-rays) provides a tool for the study of bystander effects because the area of exposure may be carefully controlled, and thus target cells may be clearly defined. Loss of gap junction-mediated intercellular communication between irradiated cells was dose-dependent, indicating that closure of junctions is proportional to dose. Closure was associated with hyperphosphorylation of connexin43. Inhibition of communication occurred in bystander cells but was not proportional to dose. Inhibition of communication at higher radiation doses may restrict the spread of inhibitory factors, thus protecting bystander cells. The reduction in communication that takes place in bystander cells was dependent on cells being in physical contact, and not on the release of signalling factors into the medium.