The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells

Summary A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of ¹⁴C hypoxanthine and ¹⁴C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10 000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines.Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells.

A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of 14C hypoxanthine and 14C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines. Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.     

The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells.

Exposure of Chinese hamster ovary (CHO) cells clone K1BH4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86-648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency ro 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5-648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0-260 ergs/mm2 is adequately described by f(X)=10(-6) (13.6 + 2.04 X). That the UV-induced mutations to 6-thioguanine resistance affects the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus is supported by the observation that all randomly isolated drug-resistant colonies contained highly reduced or undetectable HGPRT activity.     

Establishment of a dose-response relationship for reverse mutation at the HPRT (hypoxanthine guanine phosphoribosyl transferase) locus in L5178Y mouse lymphoma cells

L5178 mouse lymphoma cells were treated with the mismatching agent 6-hydroxy-aminopurine (HAP), a base analogue known to produce forward and reverse mutations in bacteria. Mutants with the phenotype deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT) were selected in 6-thioguanine (TG)-containing medium and isolated. Reverse mutations to Hhe HPRT-proficient phenotype oc occuredd both spontaneously and after treatment with ethyl nitrosourea (ENU), which suggested that the initial HAP treatment had induced point mutations at the HPRT locus.

Reconstruction experiments, in which a small number of wild-type cells together with different numbers of mutant cells were seeded in HAT medium, indicated that densities up to 10⁶ cells per ml can be used for the selection of revertants. Optimal expression of induced revertants was obtained two days after treatment.

The dose-response relationship for induction of reverse mutations by ENU appears not to deviate from linearity. The highest revertant frequency observed was 3.3 × 10⁻⁵ at an ENU concentration of 1 mM. The spontaneous reversion frequency per generation — based on 3 spontaneous revertants — was estimated to be 1.3 × 10⁻⁹. All revertants were indistinguishable from the parental wild-type line with respect to the activity as well as the electrophoretic mobility of HPRT.     

Hydrogen peroxide induced mutations at the HPRT locus in primary human T-lymphocytes

Reactive oxygen species (ROS) produced by intracellular metabolism are believed to contribute to spontaneous mutagenesis in somatic cells. Hydrogen peroxide (H(2)O(2)) has been shown to induce a variety of genetic alterations, probably by the generation of hydroxyl radicals via the Fenton reaction. The kinds of DNA sequence alterations caused by H(2)O(2) in prokaryotic cells have been studied extensively, whereas relatively little is known about the mutational spectrum induced by H(2)O(2) in mammalian genes. We have used the T-cell cloning assay to study the ability of H(2)O(2) to induce mutations at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in primary human lymphocytes. Treatment of cells for 1 h with 0.34-1.35 mM of H(2)O(2) caused a dose dependent decrease of cell survival and increase of the HPRT mutant frequency (MF). After 8 days of expression time, the highest dose of H(2)O(2) caused a 5-fold increase of MF compared to the untreated control cells. Mutant clones were collected and the genomic rearrangements at the T-cell receptor (TCR) gamma-locus were studied to identify independent mutations. RT-PCR and DNA sequencing was used to identify mutations in the HPRT coding region. Due to a relatively high frequency of sibling clones, only six independent mutations were obtained among the controls, and 20 among the H(2)O(2) treated cells. In both sets, single base pair substitutions were the most common type of mutation (5/6 and 13/20, respectively), with a predominance of transitions at GC base pairs, which is also the most common type of HPRT mutation in T-cells in vivo. Among the single base pair substitutions, five were new mutations not previously reported in the human HPRT mutation database. Overall, the kinds of mutation occurring in T-cells in vivo and H(2)O(2) treated cells were similar, albeit the number of mutants was too small to allow a meaningful statistical comparison. These results demonstrate that H(2)O(2) is mutagenic to primary human T-lymphocytes in vitro and induces mutations of the same kind that is observed in the background spectrum of HPRT mutation in T-cells in vivo.     

Hypoxanthine transport in normal and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient diploid human lymphoblasts

We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of K_m. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process.     

The time dependence of bystander responses induced by iron-ion radiation in normal human skin fibroblasts

Although bystander effects have been shown for some high-LET radiations, few studies have been done on bystander effects induced by heavy-ion radiation. In this study, using a Transwell insert co-culture system, we have demonstrated that irradiation with 1 GeV/nucleon iron ions can induce medium-mediated bystander effects in normal AG01522 human fibroblasts. When irradiated and unirradiated bystander cells were combined in shared medium immediately after irradiation, a two- to threefold increase in the percentage of bystander cells with gamma-H2AX foci occurred as early as 1 h after irradiation and lasted at least 24 h. There was a twofold increase in the formation of micronuclei in bystander cells when they were co-cultured with irradiated cells immediately or 1 or 3 h after irradiation, but there was no bystander effect when the cells were co-cultured 6 h or later after irradiation. In addition, bystander micronucleus formation was observed even when the bystander cells were co-cultured with irradiated cells for only 1 h. This indicates that the crucial signaling to bystander cells from irradiated cells occurs shortly after irradiation. Moreover, both gamma-H2AX focus formation and micronucleus formation in bystander cells were inhibited by the ROS scavengers SOD or catalase or the NO scavenger PTIO. This suggests that ROS and NO play important roles in the initiation of bystander effects. The results with iron ions were similar to those with X rays, suggesting that the bystander responses in this system are independent of LET.     

Cytogenetic changes induced in human diploid fibroblasts by tsA58 SV40 at permissive and restrictive temperatures

Human diploid fibroblasts have been transformed by ts A58 SV40. At the permissive temperature, apparent chromosome and chromatic rearrangements were observed in a high percentage of cells and the frequency of SCE increased. If the transformed phenotype returned to normal at the restrictive temperature these alterations also returned to normal levels. Chromosome banding demonstrated many apparent chromosomal rearrangements in which diffuse staining material was joining intact chromosomes end-to-end and forming pseudostructural abnormalities. Homogeneously staining regions associated with gene amplification or virus-induced alterations in the coiling and stickiness of telomeric regions are possible mechanisms.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Novel mutational spectrum induced by N-methyl-N'-nitro-N-nitrosoguanidine in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts

The kinds and locations of mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 75 independent mutants, derived from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated normal human fibroblasts, were characterized by direct sequencing of mRNA-polymerase chain reaction (mRNA-PCR)-amplified cDNA. Treatment of human cells with low (6 or 8 microM) or high (10 or 12 microM) doses of MNNG resulted in 35-fold or 150-fold average increases in mutation frequency, respectively. A high frequency of mutants lacking a complete exon was observed in both groups. Further characterization of half of these mutants by DNA-PCR amplification of intron-exon boundaries showed that they contained base substitutions. The kinds of base substitutions differed distinctly between these two groups. In the low dose group, a broad mutational spectrum was observed: ten out of the 31 base substitutions were A.T to G.C transitions, six contained G.C to A.T transitions, and the other 15 exhibited transversions. In contrast, the majority (84%) of base substitutions among the high dose group were G.C to A.T transitions; the others (16%) were transversions. All of the 32 G.C to A.T transitions were located on the non-transcribed strand, assuming that the causative premutational lesion was O6-methylguanine. These results indicate preferential repair of lesions located on the transcribed strand. In addition, G.C to A.T and A.T to G.C transitions preferentially occurred at positions with guanine and thymine at the adjacent 5' position, respectively.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.     

Simulation of exon deletion mutations induced by low-LET radiation at the HPRT locus

Friedland, W., Li, W. B., Jacob, P. and Paretzke, H. G. Simulation of Exon Deletion Mutations Induced by Low-LET Radiation at the HPRT Locus. Radiat. Res. 155, 703-715 (2001). The induction of HPRT mutants with exon deletions after irradiation with photons was simulated using the biophysical radiation track structure model PARTRAC. The exon-intron structure of the human HPRT gene was incorporated into the chromatin fiber model in PARTRAC. After gamma and X irradiation, simulated double-stranded DNA fragments that overlapped with exons were assumed to result in exon deletion mutations with a probability that depended on the genomic or the geometric distance between the breakpoints. The consequences of different assumptions about this probability of deletion formation were evaluated on the basis of the resulting fractions of total, terminal and intragenic deletions. Agreement with corresponding measurements was obtained assuming a constant probability of deletion formation for fragments smaller than about 0.1 Mbp, and a probability of deletion formation decreasing with increasing geometric or genomic distance between the end points for larger fragments. For these two assumptions, yields of mutants with exon deletions, size distributions of deletions, patterns of deleted exons, and patterns of deleted STS marker sites surrounding the gene were calculated and compared with experimental data. The yields, size distributions and exon deletion patterns were grossly consistent, whereas larger deviations were found for the STS marker deletion patterns in this comparison.     

12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced gene sequences in human primary diploid fibroblasts and their expression in SV40-transformed fibroblasts

We have isolated cDNA sequences from TPA-treated primary human fibroblasts, which indicate RNA species that are coordinately regulated after treatment of these cells with either ultraviolet light, mitomycin C, the UV-induced factor EPIF, or TPA. The levels of RNA are elevated in Bloom syndrome (cells of two out of three patients). After transformation with SV40 one of the sequences is overexpressed while another one is reduced. Both genes maintain their inducibility by the agents mentioned.