The lines irs1, irs2 and irs3, derived from V79-4 hamster cells, are sensitive to DNA-damaging agents including ionizing radiations. However, unlike some other radiosensitive lines, the irs lines show no apparent defect in the repair of DNA strand breaks. We have now assessed the mis-repair of DNA damage in the irs lines by measuring spontaneous and X-ray induced frequencies of mutation in the HPRT gene. irs1 was found to be hypermutable, showing instability in spontaneous mutant frequency and an elevation of the radiation-induced frequency relative to the parental line. In contrast, irs2 and irs3 showed similar mutational responses to the parental line. The results support other lines of evidence suggesting that irs1 has a mis-repair phenotype. The irs2 line has previously been shown to have a phenotype similar to cells from the human disorder ataxia-telangiectasia and this similarity is maintained in their mutational response to X-rays. The irs lines were also tested for ability to undergo V(D)J recombination, since this process has recently been found to be defective in some radiosensitive lines with impaired double-strand break repair. Using an extrachromosomal vector containing a V(D)J rearrangement cassette, correct recombination was shown to occur at similar frequencies to parental V79-4 cells in each of the three irs lines. Thus the irs lines indicate that processes other than DNA double-strand break repair also control radiosensitivity, in particular those processes which may affect the regulation of DNA repair.

A direct comparison was carried out of the biological effectiveness of protons and alpha-particles of the same linear energy transfer (LET) under identical conditions with a variety of in vitro biological systems. Monolayers of mammalian cells were irradiated with accelerated beams of protons (1.2 and 1.4 MeV) and alpha-particles (30 and 35 MeV) corresponding to LETs of 23 and 20 keV microns-1 for each particle type. For V79-4 cells it was observed that the linear term of the dose-response for cell inactivation by protons was significantly greater than that for alpha-particles of the same LET. For HeLa and HeLa S3 cells, also, the linear term appeared to be greater for protons, but this was not observed with more limited data for C3H 10T1/2 cells. The result for V79 cells is in agreement with the report of Belli et al. (1989) who observed that the biological effectiveness of protons rose sharply between 17 and 30 keV microns-1 in strong contrast to alpha-particles which reached a peak effectiveness at greater than 100 keV microns-1. These results place new constraints on the biologically relevant features of the microscopic structure of radiation tracks, and have implications for the mechanistic and practical comparison between radiations.

A versatile irradiator has been constructed for in vitro irradiation of mammalian cells with alpha-particles of well-defined energy, LET, direction, dose and dose rate. It is based on approximately 1.2 x 10(9) Bq of 238Pu (on a platinum disc) contained in a He-filled chamber. In a standard configuration, monolayers of cells grown in 10 Hostaphan-based dishes are irradiated with 3.26 +/- 0.22 MeV alpha-particles (LET 121 keV microns-1) at selectable dose rates from approximately 2 Gy min-1 down to less than 10(-4) Gy min-1 (i.e. fluence rates of 1 x 10(7) cm-2 min-1 to 3 x 10(2) cm-2 min-1). Single dishes can be irradiated at dose rates up to 24 Gy min-1 (fluence rate 1 x 10(8) cm-2 min-1). Incident energy and LET can be varied from 0.8 to 4.2 MeV and 266 to 102 keV microns-1, respectively. The irradiator has full incubation and gassing facilities for protracted irradiations. The irradiator is particularly suitable for in vitro analytical studies of the biological effects of alpha-particles of energies and LETs similar to those which cells may receive in vivo from radionuclides such as radon and the actinides. It has been used successfully for investigations of a variety of alpha-particle-induced effects in different cell types irradiated either as attached monolayers or as very thin suspensions.

A quantitative comparison has been carried out between laser scanning confocal microscopy on living cells and standard electron microscope methods on fixed samples. It was estimated from these measurements that there was about 10-20% reduction in thickness in fixed samples of monolayer V79-4 hamster cells. Precise information on the true thickness of living cells, as irradiated, is required for full interpretation of radiobiological data with poorly penetrating radiations, including ultrasoft X-rays. The confocal microscope allows rapid measurements on unperturbed living samples.

A KrF laser-based repetitively pulsed (5 Hz) plasma X-ray source producing photons at hυ≈0.85 keV (iron, L-shell) from a precision steel rotating target has been developed. This source has been used for exposures of several minutes duration to study soft X-ray induced cell killing and DNA damage effects in cultured Chinese hamster cells. To minimise the debris from the laser plasma source the target chamber contained helium at 300 torr with a slow flow. The biological material was exposed outside the target chamber by X-rays emerging through a beryllium vacuum window into a helium-filled beam-line. The results showed that the source was very effective at killing V79 cells and at inducing DNA damage in V79 cells but not in AA8 cells.

A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV-gpt and HSVtk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSVtk-neo gene.

A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells (Brown and Thacker, 1984), were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from γ-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT (cross-reacting material, CRM), using an antibody raised against partially purified V79-4 HPRT enzyme. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the γ-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency, either spontaneously or after treatment with the powerful mutagen ethyl nitrosourea (ENU). All except 2 of the EMS-induced mutants reverted with ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of γ-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. The EMS-induced mutations are likely to be mostly point mutations, with at least 40% of the missense type, while γ-ray-induced mutations may arise mostly through larger genetic changes.

Four X-ray-sensitive mutants of CHO cells, described previously by Jeggo and Kemp (1983), showed enhanced sensitivity to both 60Co γ-rays and 238Pu α-particles relative to the responses of the parent line. The enhanced response to a densely ionising radiation (α-particles) was less than that to X- or γ-rays, suggesting that these mutants are deficient mainly in the repair of damage from relatively sparsely ionising radiation tracks. Plateau-phase cultures of the parental CHO cells showed considerable recovery upon irradiation with low-dose-rate γ-rays, compared to irradiation at ‘high’ dose rates, but little or no recovery was seen for the mutants. Similarly, preliminary data on recovery during post-irradiation holding of plateau-phase cultures show that this process is also absent in the mutants. These responses have several similarities to those of cells from patients with the radiosensitive disorder ataxia telangiectasia (AT), and are discussed with reference to AT cells and other radiosensitive mutants.

The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. However, still higher frequencies of mutation were found for the hamster HGPRT gene when it had been stably transferred into the same hamster cell line. It is suggested, therefore, that transferred DNA may integrate in sequences which are more 'reactive' than most of the genome. Cell-free extracts of 10 TG-resistant mutants of XGPRT-proficient sublines showed no measurable XGPRT activity. High molecular weight DNA from XGPRT-proficient sublines used in the mutation studies hybridized with nick-translated pSV2-gpt DNA, showing two distinct bands when cut with the restriction enzyme Eco R1. This suggests that a single copy of pSV2-gpt DNA was integrated in these sublines. DNA from most spontaneous and mutagen-induced TG-resistant mutants had lost these two hybridizing bands, but one spontaneous mutant was found with rearranged pSV2-gpt sequence.

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.

HGPRT enzyme activity in mutant colonies selected in 6-thioguanine can be assessed directly in petri dishes using an autoradiographic method. The application of this method to large-scale quantitative mutation experiments verifies that: (a) the maximum induced frequency of mutation of HGPRT-deficiency can be measured at short expression times, such as 3 days after treatment, when cells are respread into relatively low thioguanine concentrations, and (b) ionizing radiation induces predominantly mutants with zero HGPRT activity. Other potential applications of this method are discussed.

Cell killing and the frequency of mutation to thioguanine resistance (HGPRT enzyme deficiency) were measured after irradiation of cultured hamster cells with 250-kV X rays and with /sup 238/Pu ..cap alpha.. particles. The frequency of mutants induced by these ..cap alpha.. particles, for a given level of cell killing, was approximately twice that induced by X rays. This agrees with expectations from our previously published data on mutation induction by radiations of high linear energy transfer, but disagrees with another recently published report of the relative mutagenicity of /sup 238/Pu ..cap alpha.. particles. Possible reasons for this discrepancy are discussed.

Inactivation and mutation to thioguanine-resistance of V79 hamster cells were studied after irradiation with accelerated helium, boron or nitrogen ions covering a range of linear energy transfer from 28 to 470 keV micrometers-1. For all radiation qualities a dose-dependent increase in mutant frequency was found for doses giving surviving fractions greater than about 0.20. The effectiveness per unit dose for both inactivation and mutation induction increased with the linear energy transfer of the radiation to a maximum in the range 90-200 keV micrometer-1. However, the maximum mutagenic effectiveness relative to gamma-rays was about two or more times that for inactivation. It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.

The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5·10−5 per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1·10−6 in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with γ-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than γ-rays for the induction of thioguanine-resistant mutants in these cells.