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Biomedical subjects

J B Little

Publications and source records attributed to J B Little.

At least 109 records · Page 6Linked to original sources
PubMed1992

Effect of dose rate on the survival of irradiated human skin fibroblasts.

The survival of cells in density-inhibited, confluent cultures maintained at 37 degrees C was examined following exposure to 137Cs gamma rays at low dose rates (0.023 or 0.153 Gy/h) or to 60Co gamma rays at a single high dose rate (0.70-0.75 Gy/min). Cells from an ataxia telangiectasia (AT) homozygote showed no dose-rate effect, whereas a three- to fivefold increase in D0 was observed for all other cell strains exposed at low dose rates. The magnitude of the dose-rate effect did not differ significantly among cells from persons with hereditary retinoblastoma, basal cell nevus syndrome, or AT-heterozygote compared with normal cell strains, and was not related to the size of the shoulder (extrapolation number) of the survival curve. Furthermore, no differences in the capacity for the repair of potentially lethal damage during confluent holding were observed among these latter cell strains.

Ataxia Telangiectasia↗
PubMed1992

Differential sensitization of human tumor cells by ara-A to X irradiation and its relationship to inherent radioresponse.

We have previously shown that pretreatment of plateau-phase cultures of human tumor cells with ara-A can markedly sensitize them to the cytotoxic effects of X irradiation; the degree of sensitization varied in two different cell lines. The present study was undertaken to determine whether variability in radiosensitization by ara-A occurs at random in human tumor cell lines or if it is related to their intrinsic radiosensitivity (human tumor radioresponse). The interaction between ara-A and X irradiation was examined in plateau-phase cultures of early-passage tumor cell lines of varying radioresponse (D0 range 0.85-3.15 Gy) subcultured immediately after irradiation to measure survival. In six of the eight cell lines studied, pretreatment with ara-A greatly enhanced the lethal effects of X irradiation in a concentration-dependent fashion. Little or no effect was observed in the two radiosensitive cell lines. When ara-A sensitization was plotted as a function of D10 or D, a linear relationship was observed. These data suggest that pretreatment with ara-A is effective in sensitizing radiation-resistant human tumor cells to the lethal effects of X rays, and that this phenomenon may be dependent upon inherent tumor cell radiosensitivity.

Cell Line↗
PubMed1992

Evidence that DNA double-strand breaks initiate the phenotype of delayed reproductive death in Chinese hamster ovary cells.

A persistently reduced cloning efficiency occurs in many of the cloned progeny of Chinese hamster ovary (CHO) cells surviving X irradiation, a stable phenotype we have previously termed delayed reproductive death (Int. J. Radiat. Biol. 60, 483-496, 1991). We now report that this phenotype is also induced by the alkylating agent ethyl methanesulfonate (EMS), but not by irradiation with ultraviolet light. The restriction endonuclease HinfI, which binds at G [symbol: see text] ANTC DNA sequences and generates cohesive-end double-strand breaks, was also efficient in inducing delayed reproductive death. On the other hand, an X-ray-sensitive CHO mutant, xrs-5, which is defective in the repair of DNA double-strand breaks, did not show this phenotype following X irradiation. These results suggest that DNA double-strand breaks, as well as the endogenous repair processes for these breaks, are associated with the induction of the delayed reproductive death phenotype in CHO cells. The possible mechanism for the induction of delayed reproductive death by EMS is discussed.

Animals↗
PubMed1991

A system for assaying homologous recombination at the endogenous human thymidine kinase gene.

A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK+/+ parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or -1 frameshifts. Resulting TK-/- mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. Mutations were characterized by exon-specific polymerase chain reaction amplification and direct sequencing. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by approximately 8 kilobases. These lines undergo spontaneous reversion to TK+ at a frequency of less than 10(-7), and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK+. The mode of reversion to TK+ was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. Our data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

Amino Acid Sequence↗
PubMed1991

Genotoxic and mutagenic effects of the diagnostic use of thallium-201 in nuclear medicine.

In order to investigate possible mutagenic effects of in vivo exposure to low levels of ionizing radiation used in nuclear medicine, we have examined the hypoxanthine guanine phosphoribosyl transferase (hprt) mutant fraction (MF) and chromosome aberration (CA) frequency in 24 nuclear medicine patients before and after injection of thallium-201. The mean MF of the thallium-201-exposed cohort was 5.2 +/- 4.4 x 10(-6) before injection exposure. No significant difference in MF was observed 24 h later. In 11 patients who were studied on a third occasion, 30 days after thallium-201 exposure, there was again no significant difference in post-exposure as compared with the pre-exposure MF. The frequency of CA in peripheral blood lymphocytes was not significantly different, comparing pre- and 24 h to 1 month post-radionuclide exposure. Thus, thallium-201 exposure was not associated with significant elevations in MF or CA frequency in lymphocytes of exposed individuals.

Chromosome Aberrations↗
PubMed1991

Delayed reproductive death in X-irradiated Chinese hamster ovary cells.

The cytotoxic effects of X-irradiation may be delayed for many generations of replication in mammalian cells. In addition to a reduced cloning efficiency, progeny of surviving Chinese hamster ovary (CHO) cells isolated after 12-34 population doublings postirradiation showed a variety of abnormalities including lower attachment ability to culture dishes and slower cell cycle progression. When these progeny were seeded as single cells 12-23 generations after irradiation, they formed a higher fraction of abortive colonies and of non-homogeneous colonies containing giant cells. All of these characteristics were evident whether progeny cells were subcultured by trypsinization or by mitotic selection. These results suggest that residual damage may be carried by surviving progeny of irradiated cells over many mitotic cycles; this damage is eventually expressed in terms of pleomorphic changes leading to delayed reproductive failure.

Animals↗
PubMed1991

Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay.

Epidemiologic application of the human in vivo hypoxanthine-guanine phosphoribosyltransferase (hprt) mutation assay requires screening of mutant colonies to differentiate independent from clonal origin. Previously, sibship was defined by Southern blot analysis of T cell receptor gene rearrangements. We report here a more expedient method to determine these rearrangements utilizing the polymerase chain reaction (PCR) and a DNA single-strand conformation polymorphism technique. The results are consistent with those obtained by Southern blotting in that sibship can be defined easily. A major advantage is that cells may be taken directly from the microtiter plate, eliminating the necessity to expand the clones and isolate genomic DNA. Cell lines which have not undergone receptor gene rearrangements cannot serve as PCR templates and do not interfere with this analysis. Furthermore, background from the large number of nonmutant lymphocytes present in the well does not hinder the analysis of the T cell receptor pattern of a mutant. This technique facilitates rapid screening of a large number of clones in a shorter time than Southern blotting, and is useful for the study of in vivo mutation and the clonal expansion of mutants in populations of T cells.

Base Sequence↗
PubMed1991

Molecular structure of mutations at an autosomal locus in human cells: evidence for interallelic homologous recombination.

A high proportion of spontaneous mutations at the heterozygous thymidine kinase (TK) locus in a human B-lymphoblast cell line involved loss of the entire active allele. Loss of heterozygosity often extended to other loci on chromosome 17q. The authors have developed a system for analysing the role of homologous recombination and gene conversion in such events. A heteroallelic (TK-/-) cell line containing single + 1 frameshifts in exons 4 and 7 was generated by repeated exposures to ICR-191. Revertant mutations to TK+/- were selected and analysed for the presence or absence or each frameshift as well as changes in linked polymorphic markers on 17q. The molecular changes associated with reversion to TK+ can thus be analysed. Preliminary results indicate that homologous recombination can be detected with this system, though it occurs at low frequency (less than 10(-7]. The authors believe this represents the first quantitative assay for measuring recombination between alleles of a specific intact gene in human cells. It should prove useful in evaluating the potency of various classes of mutagens in inducing recombinational and gene conversion events.

Alleles↗
PubMed1991

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles.

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.

Alpha Particles↗
PubMed1990

Molecular genetic analysis of recessive mutations at a heterozygous autosomal locus in human cells.

We have investigated the genotypic changes that lead to expression of a recessive allele at a heterozygous autosomal locus in a human cell line. Mutant clones lacking thymidine kinase activity were derived from a B-cell lymphoblastoid line initially heterozygous at the tk locus, and restriction mapping was performed to detect intragenic structural alterations in the tk gene. In addition, informative molecular markers located elsewhere on chromosome 17 were analysed in order to detect large-scale (multilocus) events. We report that among 325 spontaneous and induced mutants, allele loss was more common than intragenic rearrangements or point mutations; in many cases, loss of heterozygosity appears to have extended well beyond the locus under selection. Cytogenetic analysis of a subset of these mutants showed that expression of the recessive TK-deficient phenotype and the associated loss of heterozygosity for chromosome 17 markers was not typically associated with detectable chromosomal changes.

Blotting, Southern↗
PubMed1990

Sister-chromatid exchanges in lymphocytes from styrene-exposed boat builders.

Sister-chromatid exchanges (SCE) were measured in peripheral blood lymphocytes from 40 workers in the boat-building trade. Twenty of these workers were exposed to significant amounts of styrene. The mean air concentration of styrene in the breathing zone of the boat builders was 209 mg/m3 in the 7 exposed current smokers and 230 mg/m3 in the 13 exposed non-smokers. Urinary styrene metabolites were also measured and the mean mandelic acid/creatinine ratios in the exposed, smokers was 275 mg/g, and in the exposed, non-smokers 323 mg/g. The SCE frequency in lymphocytes from the styrene-exposed group did not differ from that in the controls, although smoking significantly induced SCE in these workers.

Adult↗
PubMed1990

Molecular characterization of hprt mutants induced by low- and high-LET radiations in human cells.

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequence probes for regions of the X chromosome which are linked to hprt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30-48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14-54% of the observed changes. In the case of mutants induced by [125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rare events.

Animals↗
PubMed1990

Cytogenetic effects of extremely low doses of plutonium-238 alpha-particle irradiation in CHO K-1 cells.

CHO K-1 cells were irradiated during the G1 phase with 0.5-6 rad of alpha particles. There was no appreciable cell killing in this low dose range. Significantly increased frequencies of sister-chromatid exchanges were induced by doses as low as 0.5 rad of alpha-particle irradiation, whereas increased numbers of chromosomal aberrations were observed following exposure to 2 rad. These results suggest that very low doses of alpha radiation may lead to radiation-induced genetic alterations.

Alpha Particles↗
PubMed1990

Delayed appearance of lethal and specific gene mutations in irradiated mammalian cells.

We have examined the occurrence of lethal and 6-thioguanine resistant (hprt locus) mutants among the progeny of irradiated CHO and BALB/3T3 cells. The expression of lethal mutations, as measured by a reduced cloning efficiency in the progeny cell population, was detected up to 30 mean population doublings after x-radiation. Preliminary evidence indicates that the expression of mutations at the hprt locus may also be delayed for at least 6-7 population doublings. These results suggest that radiation can induce genetic instability in cells, resulting in an increased rate of spontaneous mutations which persists for many generations of cell division. These findings are discussed in terms of their possible influence on the response of irradiated tumor cell populations in vivo.

Animals↗
PubMed1990

Induction of chromosomal aberrations and sister chromatid exchanges by alpha particles in density-inhibited cultures of mouse 10T1/2 and 3T3 cells.

We examined the dose-response relationships for the induction of cell killing, chromosomal aberrations and sister chromatid exchanges (SCE) by 220 kV X-rays and 5.3 MeV alpha particles from a 238Pu source. The cells were irradiated in density-inhibited, confluent cultures. The D0 values for the X-ray and alpha particle survival curves were 1.7 Gy and 0.7 Gy, and the extrapolation numbers 2.5 and 1.0, respectively, for mouse 3T3 cells. Chromosomal aberrations increased linearly with dose for alpha-radiation and roughly with the square of dose for X-rays in 3T3 cells. At 37 per cent survival, 1.0 chromosomal aberration per cell was induced by X-rays and 1.7 per cell by alpha-radiation, but the fraction of cells without aberrations was similar. In confluent holding recovery experiments there was a 50 per cent reduction in X-ray-induced aberrations during the first 4 h of confluent holding. No decline in alpha-induced aberrations was observed with holding times up to 24 h. The dose-response relationship for the induction of SCE by X-rays increased linearly with doses up to 100 cGy in both 3T3 and 10T1/2 cells, then declined, reaching nearly background levels after 400 cGy. The induction of SCE increased rapidly in these cell lines with doses of 2.5-5.0 cGy of alpha-radiation, then declined. The relative biological effectiveness (RBE) was 15-25 for the induction of SCE by low doses (2.5-5.0 cGy) of alpha particles.

Alpha Particles↗
PubMed1990

Mutation induction by 125iodoacetylproflavine, a DNA-intercalating agent, in human cells.

Survival and the induction of mutations at the hprt and tk loci were measured in TK6 human lymphoblastoid cells following treatment with the DNA-intercalating agent 125iodoacetylproflavine (125IAP). 125IAP was readily taken up into the cells, was localized to the nucleus, and was released rapidly following resuspension of the cells in fresh medium. Treatment with 125IAP for 24 h yielded a D0 of 110 decays/cell and an induced mutant fraction of 0.13 x 10(-6) per decay at the hprt locus and 0.4 x 10(-6) per decay at the tk locus. Molecular analyses of 125IAP-induced hprt mutants by Southern blot revealed a high proportion of large-scale changes at this locus. When these results are compared with those observed with 125IdUrd, 125IAP shows a reduced effectiveness per decay, related perhaps to the non-covalent nature of intercalator binding, resulting in reduced energy deposition in the DNA.

Acridines↗
PubMed1990

Low-dose radiation effects: interactions and synergism.

Experimental data from the author's laboratory are discussed, which demonstrate mutagenic and carcinogenic effects of low dose and low dose-rate exposure to ionizing radiation, and the modification of these effects by various chemical agents. These data are derived from studies of mutagenesis and neoplastic transformation in vitro, as well as on the induction of lung cancer in hamsters. On the basis of these results, several avenues of future research are suggested. These include: 1) the development and application of new human cell systems for studying radiation mutagenesis and transformation; 2) the study of the effects of protraction of exposure to radiation in such systems (emerging evidence suggests the dose-rate effect for mutagenesis may be minimal or absent in human cells); and 3) further examination of the role of secondary factors in radiation carcinogenesis. It appears unlikely that important information in these areas will arise from human epidemiologic studies. Knowledge derived from experimental systems should, however, enhance our understanding of the risks of radiation exposure and possible strategies to reduce them.

Animals↗