Auger effects on bromo-deoxyuridine-monophosphate irradiated with monochromatic X-rays around bromine K-absorption edge

Summary In order to study the enhanced effect by Auger cascade, samples of bromo-deoxyuridine-monophosphate (Br-dUMP) in aqueous solutions were irradiated with monochromatic X-rays at 13.49 keV and 13.43 keV, just above and below the K-absorption edge of bromine, using synchrotron radiation as a source. Radiolytic products such as deoxyuridine-monophosphate (dUMP), uracil and bromo-uracil (Br-uracil) were isolated using high performance liquid chromatography. Their amounts were quantitatively analysed as a function of the absorbed dose in the solutions containing Br-dUMP for the energy of the X-rays.G values for these products were calculated on the basis of the absorbed energy. As the results, the ratios of G values of radiolytic products from Br-dUMP between X-rays of 13.49 keV and 13.43 keV were 2.2 for dUMP, 1.02 for Br-uracil and 1.23 for uracil, suggesting clearly the energy dependent enhancement. On the other hand, little significant difference between X-rays of 13.49 keV and 13.43 keV was observed for theG values of uracil released from dUMP irradiated in aqueous solutions. It seemed to confirm that the Auger electrons from K-shell of bromine atoms might play the main role for energy-dependent enhancement at induction of these radiolytic products.     

Increase in efficacy of antibiotic therapy of anthrax under experimental conditions]

The results of experimental therapy of antraxis infected mice with cefazoline (kefzol) and ampicillin incapsulated into liposomes are presented. Protective activity of the same free antibiotics combinated with amixine and leukinferone was evaluated also. Treatment with liposomal cefazoline enhanced mice survival upto 60 per cent, and life period upto 1.3 +/- 0.3 days. After liposomal ampicillin administration for 3 times the same indices were 60 per cent and 6.5 +/- 0.9 days, after 2 times administration--80 per cent and 14 +/- 1.8 days when compared to the groups of the animals treated with free antibiotics. It was shown that administration of ampicillin with amixine or with leukinferone provided enhanced mice survival upto 20 per cent, administration of cefazoline with leukinferone--upto 30 per cent.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Long-term effects of low-level 239Pu contamination on murine bone-marrow stem cells and their progeny

The effects of long-term internal contamination with 13.3 kBq kg-1 239Pu injected intravenously were studied in 10-week-old ICR (SPF) female mice. Radiosensitivity of spleen colony-forming units (CFU-S) and 125IUdR incorporating into proliferating cells of vertebral bone marrow and spleens were determined in plutonium-treated and control animals one year after nuclide injection. The CFU-S in 239Pu-treated mice were more sensitive to X-rays (D0 = 0.52 +/- 0.01 Gy) than in controls (D0 = 0.84 +/- 0.02 Gy). 125IUdR incorporation into bone marrow and spleen cells was reduced after plutonium contamination. At one year following plutonium injection, the occurrence of chromosome aberrations was evaluated in metaphase figures of femoral bone marrow cells. The frequency of aberrations increased early after plutonium treatment, at later intervals it tended to decrease but not below the control level. While the relative numbers of vertebral marrow CFU-S decreased significantly, but only to 86 per cent of normal, cellularity of vertebral bone marrow, peripheral blood counts and survival of 239Pu-treated mice did not differ from the control data.     

Relative biological effectiveness of accumulated 125IdU and 125I-estrogen decays in estrogen receptor-expressing MCF-7 human breast cancer cells

The therapeutic potential for delivering a cytotoxic dose of radiation (using the decay of Auger-electron emitters) to the cell nucleus of cancer cells that express estrogen receptors (ERs) by radiolabeled estrogen was investigated in the ER-expressing human breast cancer cell line, MCF-7. The radiolabeled estrogen/ER complex irradiates the cell nucleus by binding specific DNA sequences called estrogen response elements (EREs). Cell clonogenicity and induction of DNA double-strand breaks (DSBs) by gamma radiation or accumulation of (125)I-iododeoxyuridine ((125)IdU) or E-17alpha[(125)I]iodovinyl-11betamethoxyestradiol ((125)IVME2) decays were determined. MCF-7 cells were efficiently killed by accumulation of (125)IdU (D(0) = 30 decays per cell) and (125)IVME2 decays (D(0) = 28 decays per cell). DNA DSBs were induced by the accumulation of (125)IdU (approximately 3750 decays per cell required to reduce the mean value of the elution profile to 50%) or (125)IVME2 decays (approximately 465 decays per cell required to reduce the mean value to 50%). For survival of MCF-7 cells after gamma irradiation, the D(0) was 1 Gy, and approximately 65 Gy was required to reduce the mean value to 50% for induction of DSBs. The RBE values for cell killing and induction of DSBs by (125)IVME2 relative to gamma radiation were 4.8 and 18.8, respectively. The RBE values for cell killing and induction of DSBs by (125)IdU relative to gamma radiation were 4.5 and 2.3, respectively. Cell killing in a manner similar to that induced by high-LET radiation and the high RBE for induction of DSBs by (125)IVME2 in the ER-expressing MCF-7 cells provide a biological rationale for the use of Auger electron-emitting radionuclides covalently bound to estrogen to deliver a cytotoxic dose of radiation to ER-positive cancers.     

Absence of synergistic enhancement of non-thermal effects of ultrasound on cell killing induced by ionizing radiation

The present study was performed to elucidate the role of non-thermal effects (cavitation and direct effects) of ultrasound, in simultaneous combination with X-irradiation on the cytotoxicity of mouse L cells. Firstly, mouse L cells were exposed to X-rays and ultrasound (1 MHz continuous wave, spatial peak temporal average intensity; 3.7 W/cm2) simultaneously at 37 degrees C under O2 or Ar saturated conditions to examine the cavitational effect of ultrasound. Secondly, cells were exposed to X-rays and ultrasound at 37 degrees C under N2O saturated conditions, which suppresses the cavitation, to examine the direct effects of ultrasound. The cavitational effect under O2 and Ar saturated conditions induced an exponential decrease in cell survival, and resulted in an additive effect on cell killing with the combination of X-rays and ultrasound. The direct effect in the N2O conditions induced no cell killing and did not modify the cell killing induced by X-rays. These results suggested that the non-thermal effects of ultrasound did not interact synergistically with X-rays for cell killing.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine

We have studied two X-ray-sensitive mutants xrs 5 and xrs 6 (derived from the CHO-K1 cell line), known to be defective in repair of double-strand breaks, for cell killing and frequency of the chromosomal aberrations induced by X-irradiation. The survival experiments showed that mutants are very sensitive to X-rays, the D0, for the wild-type CHO-K1 was 6-fold higher than D0 value for the mutants. The modal number of chromosomes (2 n = 23) and the frequency of spontaneously occurring chromosomal aberrations were similar in all 3 cell lines. X-Irradiation of synchronized mutant cells in G1-phase significantly induced both chromosome- and chromatid-type of aberrations. The frequency of aberrations in xrs mutants was 12-fold more than in the wild-type CHO-K1 cells. X-Irradiation of G2-phase cells also yielded higher frequency of aberrations in the mutants, namely 7-8-fold in xrs 5 and about 3.5-fold in xrs 6 compared to the wild-type CHO-K1 cells. There was a good correlation between relative inability to repair of DNA double-strand breaks and induction of aberrations. The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase on the frequency of X-ray-induced chromosomal aberrations in these 3 cell lines was also studied. 3AB potentiated the frequency of aberrations in G1 and G2 in all the cell types. In the mutants, 3AB had a potentiating effect on the frequency of X-ray-induced chromosomal aberrations only at low doses. X-Ray-induced G2 arrest and its release by caffeine was studied by cytofluorometric methods. The relative speed with which irradiated S-G2 cells progressed into mitosis in the presence of caffeine was CHO-K1 greater than xrs 5 greater than xrs 6. Caffeine could counteract G2 delay induced by X-rays in CHO-K1 and xrs 5 but not in xrs 6. Large differences in potentiation by caffeine were observed among these cells subjected to X-rays and caffeine post-treatment for different durations. These responses and possible reasons for the increased radiosensitivity of xrs mutants are discussed and compared to ataxia telangiectasia (A-T) cells and a radiosensitive mutant mouse lymphoma cell line.     

Radiation-induced lethality and mutation in a repair-deficient CHO cell line

A U.V.-sensitive, DNA repair-deficient mutant of Chinese hamster ovary cells was tested for its response to the lethal effects of X-irradiation and simulated solar light, and to the mutagenic actions of X-rays. A slight sensitivity to killing by X-rays and a greater sensitivity to solar light was observed relative to the wild-type CHO cells. More mutations were induced at a given dose of X-rays in the sensitive cell line than in the wild-type. These results are interpreted in terms of overlap in the repair processes which take place after U.V. damage in mammalian cells with those that take place after other types of radiation damage.     

Responses of synchronous L5178Y S/S cells to heavy ions and their significance for radiobiological theory

Synchronous suspensions of the radiosensitive S/S variant of the L5178Y murine leukaemic lymphoblast at different positions in the cell cycle were exposed aerobically to segments of heavy-ion beams (20Ne, 28Si, 40Ar, 56Fe and 93Nb) in the Bragg plateau regions of energy deposition. The incident energies of the ion beams were in the range of 460 +/- 95 MeV u-1, and the calculated values of linear energy transfer (LET infinity) for the primary nuclei in the irradiated samples were 33 +/- 3, 60 +/- 3, 95 +/- 5, 213 +/- 21 and 478 +/- 36 keV microns-1, respectively; 280 kVp X-rays were used as the baseline radiation. Generally, the maxima or inflections in relations between relative biological effectiveness (RBE) and LET infinity were dependent upon the cycle position at which the cells were irradiated. Certain of those relations were influenced by post-irradiation hypothermia. Irradiation in the cell cycle at mid-G1 to mid-G1 + 3 h, henceforth called G1 to G1 + 3 h, resulted in survival curves that were close approximations to simple exponential functions. As the LET infinity was increased, the RBE did not exceed 1.0, and by 478 keV microns-1 it had fallen to 0.39. Although similar behaviour has been reported for inactivation of proteins and certain viruses by ionizing radiations, so far the response of the S/S variant is unique for mammalian cells. The slope of the survival curve for X-photons (D0: 0.27 Gy) is reduced in G1 to G1 + 3 h by post-irradiation incubation at hypothermic temperatures and reaches a minimum (Do: 0.51 Gy) at 25 degrees C. As the LET infinity was increased, however, the extent of hypothermic recovery was reduced progressively and essentially was eliminated at 478 keV microns-1. At the cycle position where the peak of radioresistance to X-photons occurs for S/S cells, G1 + 8 h, increases in LET infinity elicited only small increases in RBE (at 10% survival), until a maximum was reached around 200 keV microns-1. At 478 keV microns-1, what little remained of the variation in response through the cell cycle could be attributed to secondary radiations (delta rays) and smaller nuclei produced by fragmentation of the primary ions.     

Modulation of macrophage activation and resistance by suppressor T lymphocytes in chronic murine Schistosoma mansoni infection

Activated macrophages (M phi) appear responsible for at least part of the concomitant resistance in mice infected with Schistosoma mansoni. We found that as murine S. mansoni progressed from acute (8 to 12 wk of infection) to chronic (16 to 24 wk) stages, acquired resistance decreased (57% resistance to challenge with cercariae at 8 wk vs 28% by 24 wk, p less than 0.05), as did macrophage activation (21% +/- 2 killing of schistosomula by 8 wk M phi vs 8% +/- 2 for 24 wk M phi, p less than 0.01). T cells from the spleens of 8 wk-infected mice were capable of activating M phi from naive animals when stimulated with worm antigens (24% +/- 2 killing vs 8% +/- 2 induced by normal T cells, p less than 0.01); T cells obtained from 24 wk-infected mice did not activate M phi (13% +/- 2 killing). Furthermore, T cells from 24 wk-infected animals suppressed activation of M phi by 8 wk T cells. The addition of 10(5) 24 wk T cells to 3 X 10(5) antigen-stimulated 8 wk T cells reduced subsequent M phi killing from 27% +/- 4 to 13% +/- 2 (p less than 0.05). Week 24 T cells (3 X 10(5] reduced this additionally to 9% +/- 1 (p less than 0.01), a value no greater than that of unstimulated M phi. The subpopulation of T cells responsible for suppression of M phi activation was Lyt-2+1- nonadherent T cells. Cyclophosphamide treatment of chronically infected mice resulted in enhanced resistance (41%), M phi activation (18% +/- 1 killing), and T cell activation of naive M phi (10% +/- 1 killing). Thus, during chronic S. mansoni infection, resistance to reinfection wanes in parallel to and perhaps because of development of suppressor T cells that interfere with T-dependent M phi activation.     

Lowered pH eliminates the enhanced hyperthermic killing of E. coli induced by procaine or exposure to N2 gassing

Survival of E. coli K1060 is enhanced when they are heated at 47 degrees C in pH 6 medium as compared to pH 7.4. At pH 6 nitrogen bubbling and 10 mM procaine did not increase hyperthermic killing. The membrane content of phosphatidylethanolamine is about 80 per cent of the total of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin phospholipids. The polar headgroup of this lipid is highly sensitive to pH changes in this pH range. The changes in electrostatic charge with its secondary effects on membrane-protein relationships may explain resistance to hyperthermic killing. Thus, the difference in response to lowered pH of bacterial, compared to mammalian cells may be revealing membrane-related phenomena critical to hyperthermic killing. Also increased levels of cardiolipid were observed in linolenic acid grown cells which could be an 'attempt' to stabilize their membranes.     

Survival of V79 cells following simultaneous irradiation with X-rays and neutrons in air or hypoxia

V79 Chinese hamster cells have been irradiated with X-rays and neutrons given simultaneously. The oxygen enhancement ratio and r.b.e. were measured as a function of the proportion of the dose due to the neutrons, which varied from 0 to 100 per cent. These were compared with the values calculated assuming the two types of radiation act independently, following an approach suggested by Curtis. The o.e.r. was less than the predicted value when the neutrons contributed less than about 40 per cent of the total dose. The r.b.e. also did not vary as predicted on the basis of independent action. The 'oxygen gain factor' reached half its maximum value when the proportion of the dose due to neutrons was only about 27 per cent. The results imply that there may be interaction between the damage caused by X-rays and neutrons and that beams having only 20 to 30 per cent of their dose due to high l.e.t. radiation, could be of therapeutic benefit.     

DNA damage and biological expression of adenovirus: a comparison of liquid versus frozen conditions of exposure to gamma rays

Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Isolation and characterization of mitomycin-C-sensitive mouse lymphoma cell mutants

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.     

The response of mouse skin to combined hyperthermia and X-rays.

The effects of combined hyperthermia and X-irradiation were studied in the skin of the mouse ear. Ears were heated for 1 hour by immersion in a waterbath at temperatures ranging from 37 degrees C--43 degrees C. These heat treatments had little visible effect alone, but when combined with X-rays, enhanced the radiation response. Enhancement depended on the degree of heating. When heat was given immediately after X-rays, the radiation dose to cause a given skin reaction had to be reduced by about 10 per cent for 37 degrees C and about 40 per cent of 43 degrees C. The timing and sequence of the two treatments were important. Heat after X-rays was less effective than heat before X-rays. When heat followed X-rays, the enhancing effect was lost completely if the interval exceeded 4 hours. When heat preceded X-rays, the effect was lost more slowly, depending on temperature. The implications of this for the treatment of cancer by combined therapy are discussed.     

Lethal and mutagenic effects of 252Cf radiation in cultured human cells

HeLa MR cells were exposed to radiation emitted from a man-made spontaneously fissioning isotope, californium-252. The neutron to gamma-ray ratio in the radiation dose was measured to be 2.0. The extrapolation number of the dose-survival curve was 1.3 and the Do was 200 cGy. A dose-dependent increase in mutation to 6-TGr (6-thioguanine resistant) was observed. The relative biological effectiveness (r.b.e.) for cell killing of the neutrons from 252Cf, calculated relative to high-dose-rate X-rays, was 2.6 at 50 per cent survival. The r.b.e. for mutation induction was 2.7 at a mutation frequency of 5 X 10(-5) per surviving cell.     

Relationships between chromosome damage, cell cycle delay and cell killing induced by bleomycin or X-rays

The extent of mitotic delay and chromosome aberration induction by X-rays and bleomycin has been compared in normal human foetal fibroblasts at doses giving approximately equal levels of cell killing, assayed as colony-forming ability. Bleomycin induced much less G2 delay and chromosome damage than X-rays. We conclude that the major mechanism of cell killing by bleomycin does not involve chromosome damage but the cells pass through a number of division cycles before dying and a common DNA lesion is involved in G2 delay and chromosome damage.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Studies on phenotypic complementation of ataxia-telangiectasia cells by chromosome transfer.

Cells derived from patients with the cancer-prone inherited disorder ataxia-telangiectasia (A-T) show an abnormal response to ionizing radiation-induced DNA damage, such as an increased cell killing and a diminished inhibition of DNA synthesis. The enhanced killing of A-T (group D) cells by X-rays can be corrected by multiple cDNAs, mapping to different chromosomes (6, 11, 17, and 18). In order to examine whether genes located on these chromosomes complement AT-D cells, normal neo-tagged chromosomes 6, 11, 17, and 18 were introduced into AT-D cells by microcell-mediated chromosome transfer. However, correction of the enhanced killing of AT-D cells by X-rays could only be achieved by chromosome 11 and by none of the other chromosomes tested. The enhanced killing of A-T (complementation group C) cells was also corrected by chromosome 11. Usually, but not in all microcell hybrid clones, chromosome 11 also corrected the radioresistant DNA synthesis (RDS) phenotype of AT-D and AT-C cells. These results (i) confirm findings by others suggesting assignment of the ATD and ATC genes to chromosome 11, (ii) demonstrate that several genes can modify the cellular radiation response when they are taken out of their normal genomic context and/or control, and (iii) indicate that the RDS phenotype and the enhanced cell killing in A-T are independent pleiotropic features resulting from the primary mutations in A-T. Also, our findings underscore that, in establishing cDNAs as candidate genes for A-T, microcell-mediated chromosome transfer studies are needed to exclude nonspecific correcting effects of these candidate cDNA genes.