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J L Dempsey

Publications and source records attributed to J L Dempsey.

14 recordsLinked to original sources
PubMed1993

In vivo mutations at the H-2 locus in mouse lymphocytes.

An experimental system has been developed to study in vivo autosomal mutations in murine splenic lymphocytes. Mutant lymphocytes were isolated by immunocytotoxicity using monoclonal antibodies directed against the k and d alleles of the K and D H-2 histocompatibility loci and were enumerated using limiting-dilution cloning. Genomic allele loss in mutant clones was detected using allele-specific primers in a polymerase chain reaction. Mutant clones were classified on the basis of phenotypic and genotypic criteria into "no change", deletion or recombination mutants. The geometric mean mutation frequency in 102 mice was 2.42 x 10(-4). Detailed phenotypic and genotypic study of 87 mutant clones from 4 mice revealed "no change" mutants in 83%, mutants due to deletion in 7% and mutants due to recombination in 7%. Anomalous results were obtained in 3% of mutant clones. The development of an animal model for study of in vivo mutations at an autosomal locus will further advance study of mutations, particularly those involving chromosomal changes such as mitotic recombination.

Animals↗
PubMed1993

Effect of dietary restriction on in vivo somatic mutation in mice.

Dietary restriction is the one experimental variable which has been shown reproducibly to retard ageing. The effect of dietary restriction on in vivo mutation was studied in mice by feeding experimental mice 60% of the amount eaten by control mice and measuring mutations in lymphocytes at the hypoxanthine phosphoribosyl transferase locus at 4 weeks, 6 months and 12 months of age. Dietary restriction markedly decreased the age-associated accumulation of mutations observed in control mice, which suggests that somatic mutation is involved in the ageing process and that the majority of in vivo mutations result from dietary factors.

Aging↗
PubMed1987

Effect of age on lymphocyte proliferation.

The relationship of lymphocyte proliferative capacity to age was studied using lymphocytes from neonates, from young adults aged 20-30 and from healthy individuals aged 70-90. Mass cultures expanded exponentially and eventually died after a final expansion of 10(19)-10(52). They therefore showed a Hayflick effect, but in contrast to reported findings for other cell types there was no relationship between age and the magnitude of the final expansion. Cytogenetic and molecular studies showed that monoclonality developed in all mass cultures. Study of individual clones also showed exponential growth followed by cessation. The magnitude of the expansion, 10(4)-10(35), was substantially less than that observed for mass cultures, but was related to age. We conclude that lymphocytes have a heterogeneous proliferative potential, that the overall proliferative potential declines with age but that rare cells having extended proliferative potential continue to be present into old age. The development of monoclonality during the course of mass cultures has implications for the interpretation of findings from such cultures since observations drawn from the later stages of culture reflect the properties of rare cells having high proliferative potential and do not necessarily reflect the properties of the overall population.

Adult↗
PubMed1986

Measurement of in vivo mutant frequency in lymphocytes in the mouse.

A limiting-dilution cloning technique for quantifying in vivo mutations at the hypoxanthine phosphoribosyl transferase locus in mouse splenocytes was developed. Mouse splenocytes were cultured in round-bottom microwells with irradiated feeder cells, concanavalin A, and a source of interleukin 2 at five cells/well in the absence of thioguanine, and at 5 X 10(4) cells/well in the presence of 2.5 micrograms/ml thioguanine; mutant frequency was calculated as the ratio of the cloning efficiencies with or without thioguanine. The geometric mean (95% range) for the mutant frequency in 20 mice was 1.54 X 10(-6) (4.7 X 10(-7) -2.6 X 10(6)) and whole-body X-irradiation resulted in a dose-related increase in mutant frequency of up to approximately 20 times the baseline level. The in vivo murine mutation assay should be a useful system for genotoxicity testing and may be of particular value in establishing risk estimates for human populations exposed to genotoxins.

Animals↗
PubMed1985

Methods for study of mutations and mutagenesis in human lymphocytes.

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.

Clone Cells↗
PubMed1985

Increased mutation frequency following treatment with cancer chemotherapy.

The relationship between somatic mutation and cancer was studied by measuring in vivo mutation frequency and in vitro mutability using lymphocytes from 28 untreated adult patients with solid tumors, 14 untreated patients with lymphoma, and 27 patients with solid tumors or lymphoma who had been treated with chemotherapy and/or radiotherapy. In vivo mutation frequency in untreated patients did not differ from that of controls, except perhaps in patients with lymphoma, who showed a slight increase. Lymphocytes from untreated patients with solid tumors or lymphoma did not show a greater increase in mutations induced after X-radiation or UV radiation than did lymphocytes from controls. For all the untreated patients, the geometric mean mutation frequency was 6.72 X 10(-6), and it was significantly increased to 19.57 X 10(-6) following chemotherapy and 34.40 X 10(-6) following chemotherapy and radiotherapy. The results suggest that excessive systemic exposure to mutagens or inherent susceptibility to mutagenesis are not important etiological factors in at least the majority of patients with cancer. The mutations produced by treatment may be related to the late side effects of therapy such as second neoplasms.

Adult↗
PubMed1984

Mutation frequency in human lymphocytes increases with age.

Several theories of ageing predict that somatic mutations should increase with age. This prediction was tested for human lymphocytes using a recently developed clonal technique for enumeration of mutations, and an increase of 1.6% per year in mutations with age was detected.

Adolescent↗
PubMed1984

Mutations in human lymphocytes: effect of X- and UV-irradiation.

The mutagenic effects of X- and UV-irradiation on human lymphocytes were studied using a highly efficient cloning technique. The hypoxanthine-guanine phosphoribosyl-transferase enzyme locus was used to study mutation induction, with mutant cells being selected by their ability to form a clone in the presence of the purine analogue 6-thioguanine. Mutation dose-response curves for X- and UV-irradiation were established by studying lymphocytes from 11 individuals on day 10 after irradiation. The mean mutation frequency of unirradiated lymphocytes was 2.9 X 10(-6) and there were dose-dependent increase to 9.5 X 10(-5) after 400 rad of X-irradiation, and to 5.6 X 10(-5) after 125 erg/mm2 of the UV. The expression time of X-ray-induced mutations was 3-7 days. Dose-responses were obtained for mutation frequency and survival following X-irradiation of proliferating and non-proliferating lymphocytes from 8 individuals. Compared with non-proliferating lymphocytes, the proliferating lymphocytes developed fewer mutations but had a greater mortality after irradiation

Dose-Response Relationship, Radiation↗
PubMed1983

Detection of the carrier state for an X-linked disorder, the Lesch-Nyhan syndrome, by the use of lymphocyte cloning.

Using a limiting dilution technique, we found that the frequency of thioguanine resistant (TGR) lymphocyte clones was less than 5.0 X 10(-5) in 14 normal individuals, between 9.0 X 10(-3) and 8.9 X 10(-2) in seven heterozygotes for Lesch-Nyhan syndrome, and 0.88 and 0.87 in two hemizygotes. TGR clones from heterozygotes were expanded and had the hemizygote phenotype as evidenced by low hypoxanthine incorporation and severely deficient hypoxanthine-guanine-phosphoribosyl-transferase activity. Enumeration of TGR lymphocyte clones provides a simple technique for detection of heterozygosity for Lesch-Nyhan syndrome. A similar approach using lymphocyte cloning may be suitable for detection of the carrier state for other X-linked disorders.

Clone Cells↗
PubMed1983

Evidence that thioguanine-resistant lymphocytes detected by autoradiography are mutant cells.

The autoradiographic detection of thioguanine-resistant (TGR) cells has been proposed as a method for measurement of in vivo mutation frequency. This proposition was tested by combining the autoradiographic technique with the technique of long-term lymphocyte culture. Irradiation of lymphocytes on day 0 following by culture showed a dose-related increase the frequency of TGR cells with an expression time of approx. 7 days. There was a suggestion that mis-sense mutations as well as non-sense mutations and deletions were increased. Incubation with aminopterin for 10 days prevented the increase in TGR cells after irradiation and probably decreased the frequency of TGR cells prior to irradiation. The results suggest that many or most of the TGR cells measured autoradiographically are mutant cells. However, the variability observed with the method imposes limitations on its use and suggests that it will be of more value in studying populations rather than individuals.

Aminopterin↗
PubMed1982

Enumeration of thioguanine-resistant lymphocytes using autoradiography.

Strauss and Albertini (1979) proposed a method whereby the frequency of thioguanine-resistant lymphocytes could be measured by an autoradiographic technique and suggested that it would provide a measure of in vivo mutation frequency. A number of variables influencing the method were studied and concentration of thioguanine and duration of assay found to be particularly important. The modified method finally arrived at involved culture of PHA-stimulated lymphocytes for 64 h in the presence of 40 microgram/ml of thioguanine, lysis of cells with Nonidet and collection of nuclei on Nucleopore membranes. The frequency of TG-resistant cells was 1.5 x 10(-6) - 1.7 x 10(-5) in young adults and it showed an increase with age. The technique was also adapted for use with continuously cultured lymphocytes, by using a concentration of 60 microgram/ml of thioguanine and an incubation time of 72 h. The frequency of TG-resistant continuously cultured cells was less than 10(-6) - 2 x 10(-5). Consideration of the effect of different variables on the assay suggested that the above estimates for TG-resistant lymphocytes were more valid than the previously published substantially higher estimates.

Autoradiography↗
PubMedDate not supplied

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique.

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.

Adult↗