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J W Simons

Publications and source records attributed to J W Simons.

At least 127 records · Page 7Linked to original sources
PubMed1984

A re-appraisal of the BHK 21 clone-13 cell transformation system with respect to the assay conditions and the mutational origin of the transformed phenotype.

Two BHK 21/13 cell lines (BHK-ICI and BHK-A3) were used for studies on cell transformation. The BHK-ICI cells formed micro-colonies in agar while the BHK-A3 cells did not. The size distribution of colonies in agar derived from BHK-ICI cells was homogeneous and did not permit a distinction between untransformed micro-colonies and transformed macro-colonies. Moreover, the transformation frequency in BHK-ICI cells was influenced substantially by cell density, as densities greater than 500 cells/ml reduced the observed transformation frequency, whereas no reduction in transformation frequency was observed for BHK-A3 cells for densities up to 10(4) cells/ml. Therefore, we recommend the use of a BHK cell line that does not form micro-colonies for the transformation assay. In contrast with published data, obtained at high cell density with BHK cells that form micro-colonies, induction of cell transformation in BHK-A3 cells by treatment with 4NQO did not behave as a mutational trait. (i) Continuous treatment did not lead to a steady increase in transformation frequency but to a reduction in transformation frequency after the second day of exposure. (ii) A continuous treatment up to 24 h proved to be about five times more effective in inducing cell transformation than a one hour acute treatment. The results support the suitability of the BHK cells as an assay for cell transformation if the adverse effects of cell density are met and the formation of micro-colonies is prevented. The use of BHK-A3 cells at a density of 10(4)/ml fulfills these requirements.

4-Nitroquinoline-1-oxide↗
PubMed1983

Ascertainment of the effect of differential growth rates of mutants on observed mutant frequencies in X-irradiated mammalian cells. An indication for the occurrence of X-ray-induced untargeted mutagenesis.

As it is not known to what extent differential growth rates of induced mutants lead to over- and under-representation of mutants in treated populations and thereby affect the determination of mutant frequencies, the mutation induction in X-irradiated L5178Y mouse lymphoma cells was determined via two methods. The first method involves the standard protocol which may suffer from the effect of differential growth rates, while the second method is based upon the fluctuation test in which the differential growth rates can be actually measured. It appeared that the standard protocol led to a mutant frequency that was similar to the mutant frequency determined in the fluctuation test. Therefore, the standard protocol appears to lead to only a minor under-estimation if any. Substantial heterogeneity in growth rates of induced mutants was observed, but the mutants with a selective advantage appear largely to compensate for the mutants that are lost because of selective disadvantage. It was calculated that the chance for isolating the same mutant twice from a treated population had been increased 2.2-fold because of the observed differential growth rates. Therefore, our data indicate that the standard protocol does not lead to serious errors in the determination of mutant frequencies and in the sampling of mutants. The fluctuation tests were also used to determine the spontaneous mutation frequency per cell per generation. The mutation rate appeared more than 10-fold enhanced in X-irradiated cells which may be attributed to the induction of a process of untargeted mutagenesis in mammalian cells.

Animals↗
PubMed1982

Effect of temperature on mutation in cultured human skin fibroblasts.

Because clothing enhances the gonadal temperature in the human male and because as a consequence of that the spontaneous mutation rate might be increased, a study was undertaken to determine the effect of temperature on mutation towards HPRT deficiency in human diploid skin fibroblasts. Culturing of the cells in HAT medium containing azaserine, to remove pre-existing mutants, was highly mutagenic. Some results suggested that azaserine acts as an indirect mutagen. The mutational process in the presence of azaserine was influenced by temperature, a rise in temperature from 33 to 37 degrees C leading to a more than 10-fold increase in mutation rate per cell generation. This temperature dependence, taken as a starting point for the estimation of the consequences of the rise in gonadal temperature, could be responsible for an increase of 135% in the incidence of a sex-linked lethal (Lesch-Nyhan) in the human population. Epidemiological data on the frequency of Lesch-Nyhan disease and on the ratio of paternal and maternal mutations leading to Lesch-Nyhan disease do not contradict the findings in the cultured cells.

Cells, Cultured↗
PubMed1982

Phenotypic drug resistance in mammalian cells in vitro.

When mammalian cells are cultured at low concentrations of toxic drugs, they often become phenotypically resistant. We studied whether this phenotypic resistance is due to selection of preexisting variants. The drugs 8-azaguaine (AG) and 6-thioguanine (TG) were used and, as a parameter for resistance, the incorporation of hypoxanthine was determined. Preexisting variation among clones in the uptake of hypoxanthine was found, and this variation has a hereditary component. This transmission of aberrant incorporation of hypoxanthine does not appear a stable trait, and the aberrant cell lines returned gradually to the original steady state. There are indications that within a cell population cells with altered levels of incorporation of hypoxanthine arise continuously and at a high frequency. Treatment with marginally toxic concentrations of AG or TG indicates that, at least for AG, survival is not related to the preexisting variation in hypoxanthine uptake. The observed phenomena could be of importance for the selection of drugs to be used in cancer chemotherapy.

Animals↗
PubMed1982

Study on the variation in X-ray sensitivity of human diploid skin fibroblasts. Normal radiosensitivity of cells derived from patients with Huntington's disease.

9 cell strains derived from patients with Huntington's disease and 9 from age- and sex-matched controls were investigated for X-ray sensitivity. No differences in radiosensitivity were observed for the two groups. The two groups taken together reveal a dependence of radiosensitivity on intrinsic cloning efficiency which in turn correlates with donor age. A difference in radiosensitivity between males and females is also indicated although at the borderline of significance. As a parameter for radiosensitivity the dose needed to obtain 0.1% survival appears superior to the Do.

Adult↗
PubMed1982

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

L5178Y 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-thoiguanine(TG)-containing medium and isolated. Reverse mutations to the HPRT-proficient phenotype occurred 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 10(6) 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 x 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 x 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.

Animals↗
PubMed1982

Attempts to detect ataxia telangiectasia (AT) heterozygotes by cytogenetical techniques.

AT cells are known to be more sensitive than normal human cells to ionizing radiation as well as other agents, as judged by cell survival, induced chromosomal aberrations, and micronuclei. The inhibition of DNA replication following X-irradiation is less in AT cells than in normal cells. Four different cell lines heterozygous for AT were investigated for their response to various treatments in order to see whether they behaved in an intermediate way. In most of the tests, the heterozygous AT cells behaved like normal cells. In the frequencies of induced chromosomal aberrations following X-rays, there was heterogeneity in response, with one cell line showing a definite intermediate response.

Adult↗
PubMed1981

Effects of ethionine on the replicational fidelity in V79 chinese hamster cells.

The possibility that ethionine, the ethyl analog of methionine and potent liver carcinogen, exerts its action by blocking the methylation of DNA and thereby rendering the post-replicative methylation instructed error-avoidance system inoperative was investigated. While the results are not directly supportive for the existence of such a repair system in V79 Chinese hamster cells, effects of ethionine were found. Following the exposure of ethionine-treated cells to EMS an increase in cell killing and a decrease in mutation induction was observed. The base analog, 6-hydroxyaminopurine, was shown to be clearly mutagenic in the mammalian cells and in the presence of ethinine a drastic decrease in mutant frequency was observed. Ethionine itself did not appear mutagenic over the entire dose range tested (1-1000 micrograms/ml).

2-Aminopurine↗
PubMed1980

Determination of the expression time and the dose--response relationship for mutations at the HGPRT (hypoxanthine-guanine-phosphophoribosyl transferase) locus induced by X-irradiation in human diploid skin fibroblasts.

The length of the expression time for mutants resistant to 8-azaguanine or 6-thioguanine induced by X-rays was determined in human diploid skin fibroblasts. The cells were seeded in the selective medium over a period of 14 days after treatment. Direct expression of at least a part of the mutants was observed at day 0, and an increase of the mutant frequency over the entire cultivation period appeared to be due to spontaneous mutation. The dose-response relationship does not appear to deviate from linearity. The mutation rate per R had a mean value of 2.1 x 10(-7) which is about twice the value of the mutation rate found in rodent cells for the same locus.

Azaguanine↗
PubMed1979

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in V-79 Chinese hamster cells. A first approach to a risk estimate in photochemotherapy.

The effect of 8-methoxypsoralen (8-MOP) and long-wave ultraviolet irradiation (UVA) on cell killing and mutation induction was studied in V-79 Chinese hamster cells. No effect was observed after treatment with 8-MOP alone (50 microgram/ml, 4 h), UVA alone (9000 J/m2), or 8-MOP metabolized by rat-liver microsomes. Combined treatment with 8-MOP and UVA induced both cell killing and mutation. This was also observed under conditions approaching patient treatment with PUVA photochemotherapy with respect to the concentration of 8-MOP in the skin and the amount of UVA received by the epidermal cells. A simple relation proved to apply for mutation induction under different treatment conditions: 5.5 X 10(-8) per J/m2 per microgram 8-MOP/ml. On this basis the mutation induction in dividing cells per session of PUVA-photochemotherapy amounts to 12.4 X 10(-5), which is probably an over-estimation.

Animals↗
PubMed1979

Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in diploid human skin fibroblasts: an improved risk estimate in photochemotherapy.

Cell killing and the induction of mutation were studied in dividing and non-dividing human skin fibroblasts as a result of treatment by 8-methoxypsoralen (8-MOP) and long-wave UV irradiation (UVA). The cytotoxic effect was highly dependent upon the duration of the UVA exposure. The frequency of mutations increased linearly with the UVA dose at concentrations of 10 and 0.25 microliter 8-MOP/ml, the latter representing the concentration in the skin during PUVA treatment. The number of mutations induced per unit dose (= per microgram 8-MOP/ml per joule UVA/m2) was calculated: for dividing cells this value was 3.3 X 10(-8) per cell and for non-dividing cells 0.6 X 10.8(-8) per cell. On the basis of these values the expected number of induced mutants in the human skin per session of photochemotherapy is 1.2 X 10(-5), and per 30 years of maintenance therapy 1.3 X 10(-2) per cell. A comparison was made between this frequency and the frequency to be expected from spontaneous mutation. In addition the significance of absence in patients of SCE induction by photochemotherapy is discussed.

Cell Survival↗
PubMed1979

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts.

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.

Cell Line↗
PubMed1978

Mutagenicity and carcinogenicity of 8-MOP/UVA in cell cultures.

HGPRT-deficient mutants of chinese hamster cells and human skin fibroblasts are selected with 6-thioguanine after treatment with the combination 8-MOP and UVA. Calculation based on this study indicate that 1.7 X 10(-5) mutants will be induced in the epidermis per session of PUVA therapy. Induction of cell-transformation was observed in C3H and 3T3 mouse cells.

Animals↗
PubMed1976

Linear dose--response relationships after prolonged expression times in V-79 Chinese hamster cells.

The expression time for induced mutants resistant to 6-thioguanine, in V-79 Chinese hamster cells, was determined by respreading the cells in the selective medium, at various times after treatment. The length of the expression time for mutants induced by X-rays, ethyl methane sulphonate and ultraviolet irradiation was dose dependent. For the highest dose used this was 7 to 8 days, beyond which there was no further changes in mutant frequency. The dose-response relationship of these agents does not appear to deviate from linearity; this permits the calculation of mutation rate per unit dose. For X-rays this value was 1.35 - 10(-7) per rad per locus, for ethyl methane sulphonate, 2.2 - 10(-2) per mole per locus and for ultraviolet irradiation, 6.3 - 10(-6) per erg per mm2 per locus. The effectiveness of the 3 different mutagens for the induction of mutations was compared by calculating the increase in mutant frequency per unit of decrease in survival (Do). These increments in frequency were: 5.6 - 10(-5) for X-rays, 69.5 - 10(-5) for ethyl methane sulphonate and 16.1 - 10(-5) for ultraviolet irradiation.

Cell Line↗
PubMed1975

Studies on the proliferative capacity of mouse spleen cells in serial transplantation.

The ageing of mouse spleen cells in vivo was investigated by determining the size distribution of cell volumes. It was found that ageing is accompained by an increase in cell volume which is in agreement with published data on ageing for a number of cell types both in vivo and in vitro. After the first transplantation into irradiated hosts the size distribution of spleen cells proves to be more heterogeneous which may affect the proliferative capacity. After the fifth transplantation, when the cell population is three years old, the parameters of the volume distributions of spleen cells agree better with the data from middle-aged mice than with the data from old-aged mice. Further it was found that at least six serial transplantations are possible if the interval between passages is long. Therefore our data do not show an intrinsic limited proliferative capacity of these cells.

Age Factors↗
PubMed1975

Ploidy level and mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) deficiency in Chinese hamster cells.

The X-ray induction of 8-azaguanine (AG) resistent mutants in two sets of diploid and tetraploid Chinese hamster cells (DON and V79) was investigated. It was found that (i) the induced mutant frequencies in diploid and tetraploid cells appeared to be of the same order of magnitude and (ii) all mutants showed almost complete loss of hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) activity except that in the tetraploid V79 cells 50--100% of activity was retained. The gene--dosage effect for glucose-6-phosphate dehydrogenase (G6PD) in these cells make it possible to determine the number of chromosomes bearing the HGPRT-gene in mutants by measuring the G6PD activity per cell. The results show that the spontaneous and induced mutants from the diploid V79 and DON as well as the tetraploid DON cells retained the G6PD activity of the parental cells, whereas the induced mutants from the tetraploid V79 cells had about 35% of the parental G6PD activity. With 6-thioguanine (TG) as selective agent, the induced mutant frequencies in diploid and tetraploid DON cells and in diploid V79 cells appeared to be of the same order of magnitude but no mutants could be recovered from tetraploid V79 cells in a single step. TG-resistant tetraploid V79 cells could only be obtained from the AG-resistant mutants after a second selection. The HGPRT activity was lost in these mutants and some of them showed an increase in G6PD activity. The combined data cannot be explained on the basis of a single genetic mechanism.

Alleles↗