Biomedical subjects

J B Little

Publications and source records attributed to J B Little.

At least 127 records · Page 7Linked to original sources

PubMed1990

Exogenous lactate modifies the repair of potentially lethal damage in three human tumor cell lines irradiated in vitro.

Lactate is one of several pathophysiological factors accumulating in the micromilieu of tumors under both hypoxic and well-oxygenized conditions, and thus may affect the recovery of irradiated tumor cells in vivo. In the present study, we investigated the effects of postirradiation incubation with exogenous lactate during confluent holding recovery on the repair of potentially lethal damage in three human tumor cell lines. Recovery was either unaffected or enhanced by low concentrations of exogenous lactate (2-5 mM), whereas it was suppressed by higher concentrations (10-50 mM). With high concentrations, survival in all three cell lines was lower at the end of the confluent holding period than at the beginning, yielding recovery ratios of less than 1.0. The effects differed quantitatively among the three tumor cell lines, and between the tumor cells and the normal diploid fibroblasts (AG 1522) studied previously.

Cell Line↗

PubMed1990

In vitro radiosensitivity of human diploid fibroblasts derived from women with unusually sensitive clinical responses to definitive radiation therapy for breast cancer.

Between January 1985 and December 1986, 811 patients were treated for carcinoma of the breast at the Joint Center for Radiation Therapy by an identical protocol. Of these 811 patients, five patients (0.6%) were identified as having an unusually sensitive clinical response to routine external beam irradiation. This unusual clinical response was characterized by severe skin erythema and edema during the first few weeks of treatment, requiring treatment breaks. Skin fibroblast cell strains were established from these five women as well as from six women with a normal clinical response to breast irradiation (chosen at random from the population of 811 patients). Radiation survival parameters were determined by a colony formation assay from complete survival curves in coded and blinded samples. Cells from the sensitive patients were significantly more sensitive to the cytotoxic effects of radiation in vitro as determined by the parameters D0, D, D10, and n, than were the strains derived from patients with a normal response. We conclude that an unusually severe response to standard fractionated radiotherapy may be associated with greater intrinsic radiation sensitivity of the individual's somatic cells.

Adult↗

PubMed1990

Efficient mutation induction by 125I and 131I decays in DNA of human cells.

To examine the role of radiation energy deposition in DNA on cellular effects, we investigated the ability of 125IdUrd and 131IdUrd to kill cells and induce mutations at the hprt locus. We employed human lymphoblastoid cells proficient (TK6) or deficient (SE30) in the ability to incorporate a thymidine analog into DNA by way of the thymidine kinase (TK) scavenger pathway. Iodine-125 releases a shower of low-energy Auger electrons upon decay which deposit most of their energy within 20 nm of the decay site, whereas 131I is a high-energy beta/gamma emitter that is generally considered to emit sparsely ionizing radiation. Although 125IdUrd incorporated into cellular DNA was very effective at producing toxic and mutagenic effects in TK6 cells, virtually no effect was seen in TK-deficient cells incubated with similar levels of 125IdUrd in the extracellular medium. In response to 131IdUrd treatment, 0.45 X 10(-6) mutants were induced per centigray dose deposited within the nucleus in TK-proficient cells, whereas few mutations were induced in TK-deficient cells at doses up to 38 cGy from 131I decays occurring in the medium. The differences in biological response between TK6 and SE30 cells cannot be explained by differential radiosensitivity or IdUrd sensitization of the cell lines involved. We conclude that both 125I and 131I decays occurring while incorporated into DNA are more effective at inducing cell killing and mutations in human cells than either nonincorporated decays or low-LET radiations. These results suggest that localized energy deposition is an important factor in producing biologically important damage by both of these isotopes, and that residual lesions following the decay of DNA-incorporated radioisotopes may contribute to the toxic and mutagenic effects observed in TK-proficient cells. Furthermore, they emphasize that certain beta/gamma-emitting isotopes such as 131I may be particularly hazardous when incorporated into DNA.

Cell Line↗

PubMed1990

Sensitivity of human diploid fibroblast cell strains from various genetic disorders to acute and protracted radiation exposure.

The cytotoxic effect of acute X irradiation was studied by a colony formation assay in 114 human skin fibroblast cell strains from 31 apparently normal individuals and 83 patients with a variety of genetic disorders possibly associated with in vitro hypersensitivity to ionizing radiation. The effect of protracted exposure to beta radiation from tritiated water (HTO) was examined in parallel experiments in 65 of these strains. The disorders included neurological diseases and syndromes characterized by an increased susceptibility to spontaneous and radiation-induced cancer. Homozygous ataxia telangiectasia and Nijmegen break syndrome cells were highly sensitive to both types of radiation. However, the response of cells from the other genetic disorders fell within the broad range characteristic of normal cell strains. While HTO may be useful as a quantitative method for determining the cytotoxic response of human diploid cells to ionizing radiation, the present results indicate that it does not offer a more sensitive assay than acute X irradiation for detecting minor degrees of hypersensitivity.

Cell Line↗

PubMed1989

Oncogenic point mutations in the human retinoblastoma gene: their application to genetic counseling.

Mutations of the retinoblastoma gene, most of which cannot be detected by conventional Southern blotting, are known to cause both the nonhereditary and hereditary forms of retinoblastoma and have been implicated in the development of other cancers. Nonhereditary retinoblastoma is caused by a somatic mutation. Hereditary retinoblastoma is caused by a germ-cell mutation, most often a new one, and thus there is usually no family history of the disease. Unlike patients with the nonhereditary disease, those with the hereditary form are at risk for additional retinoblastomas, and their progeny are at risk for the tumors. We used a sensitive technique of primer-directed enzymatic amplification, followed by DNA sequence analysis, to identify mutations as small as a single nucleotide change in tumors from seven patients with simplex retinoblastoma (with no family history of the disease). In four patients the mutation involved only the tumor cells, and in three it involved normal somatic cells as well as tumor cells but was not found in either parent; thus, these mutations appeared to be new, germ-cell mutations. In addition, we found point mutations in cells from a bladder carcinoma, a small-cell carcinoma of the lung, and another retinoblastoma. We conclude that the technique that we have described can distinguish hereditary from nonhereditary retinoblastoma and that it is useful in risk estimation and genetic counseling.

Base Sequence↗

PubMed1989

Radiation sensitivity of cell strains from families with genetic disorders predisposing to radiation-induced cancer.

This investigation was designed to test the hypothesis that skin fibroblasts from patients with genetic disorders characterized by hypersusceptibility to X-ray-induced cancer are sensitive to the cytotoxic or clastogenic effects of X-irradiation in vitro. Cell strains were established from 28 specifically ascertained patients from families with nevoid basal cell carcinoma syndrome, retinoblastoma, or other disorders apparently predisposing to radiation-induced cancer. These included 10 patients with a clear personal or family history of radiation-induced tumors. These cell strains were examined for the cytotoxic effects of X-irradiation in 3 distinct series of separate, blinded experiments, along with a group of 9 similarly coded cell bank controls. Cells from 11 of these patients and 6 controls were studied for sensitivity to X-ray-induced chromosomal aberrations. Seven of the 37 cell strains were moderately hypersensitive to radiation-induced cell killing; 2 of these were from patients with radiation-induced tumors and 1 was a cell bank control. These results suggest that such isolated cases of hypersensitivity probably do not relate to the underlying genetic disorder. Overall, the X-ray response of cells from affected individuals in this study showed no systematic difference from that of cells from nonaffected relatives or cell bank controls for either cytotoxicity or clastogenicity.

Adolescent↗

PubMed1989

Sister chromatid exchange in chronic ethylene oxide-exposed primates: unexpected effects of in vitro culture duration, incubation temperature, and serum supplementation.

Ethylene oxide (EtO), a potent monofunctional DNA alkylating agent, has been shown to induce sister chromatid exchanges (SCE) in the peripheral blood lymphocytes of animals and workers exposed to it in vivo. We have previously reported that elevations of SCE persist for 6 years after cessation of EtO exposure in cynomolgus monkeys chronically exposed to EtO; the elevation in mean SCE was entirely attributable to a subpopulation of high SCE frequency cells (HFCs). We now report that the detection of persistent HFCs is dependent on the conditions of cell growth, and that EtO exposure increases the replication indices of lymphocytes from the exposed animals when these cells are examined at early cytogenetic harvest times. Culture of lymphocytes in differing serum supplements, changes in cytogenetic harvest times, and alterations in in vitro incubation temperature all markedly affected mean SCE frequency by influencing the detection of HFCs. The frequency of EtO-induced HFCs was independent of 5-bromodeoxyuridine concentration, used for differential staining of sister chromatids. These observations indicate that the detection of persistent alkylation-induced chromosomal changes, observed long after cessation of in vivo chronic exposure of these animals, is highly dependent upon factors affecting cell growth.

Animals↗

PubMed1989

Sensitization of noncycling human diploid cells to X-irradiation by pretreatment with 9-beta-D-arabinofuranosyladenine.

The interactions between X-irradiation and 9-beta-D-arabinofuranosyladenine (ara-A) were studied in confluent, density-inhibited cultures of normal human diploid fibroblast cell strain AG1522. ara-A (0.1 to 3.0 x 10(-3) M) was added to the cultures either 2 or 24 h prior to irradiation. The cells were subcultured at low density to measure the surviving fraction either immediately after irradiation, or they were returned to the incubator in the continued presence of ara-A for an additional 4 or 24 h. In cells subcultured immediately after irradiation, pretreatment with ara-A greatly enhanced cell killing by X-rays in a concentration-dependent fashion. The D0 of the survival curve decreased from 143 cGy to 74 or 49 cGy following pretreatment with 1.0 or 3.0 x 10(-3) M ara-A, respectively. ara-A by itself had little effect on the cloning efficiency of nonirradiated cells at concentrations up to 10(-3) M, but higher levels were cytotoxic. When subculture was delayed for 4 or 24 h, the enhancement in survival reflecting potentially lethal damage recovery was not reduced by continuing incubation with ara-A at concentrations up to 10(-3) M. Higher levels significantly inhibited potentially lethal damage repair, as well as producing marked sensitization. With 24-h delayed subcultivation, the D0 of the survival curve decreased from 324 cGy to 63 cGy following incubation with 3.0 x 10(-3) M ara-A. These results indicate that pretreatment with nontoxic concentrations of ara-A can markedly sensitize human cells to the lethal effects of X-rays. This effect appears to be independent of its ability to inhibit postirradiation cellular recovery processes.

Cell Line↗

PubMed1989

Molecular analysis of DNA isolated from the different stages of x-ray-induced transformation in vitro.

A major challenge in radiation carcinogenesis is to identify the cellular gene or genes involved in initiating the process. We examined the transforming activities of DNAs obtained from C3H10T1/2 cells during x-ray-induced morphological transformation. DNAs extracted from mass cultures of 10T1/2 cells at different times after irradiation with 600 rad and from type III-transformed foci were transfected into NIH 3T3 cells. The results indicate that certain oncogenes are activated beginning 3 wk after irradiation, well before the appearance of macroscopically visible transformed foci. For DNA isolated from x-ray-transformed 10T1/2 cells (type III foci), the frequencies of transfection were 0.003-0.11 foci/microgram of genomic DNA with NIH 3T3 cells and 0.004-0.04 foci/microgram genomic DNA using 10T1/2 cells as recipients. Southern blot analysis of DNAs obtained from 23 primary transfectants and from 23 x-ray-transformed cell lines indicated no gross rearrangements or amplification of any of the 14 oncogenes screened (v-Ha-ras, v-Ki-ras, N-ras, v-myc, v-raf, v-src, v-fes, v-abl, v-mos, v-erbA, v-erbB, v-myb, v-fos, v-sis). This suggests that x-irradiation may activate as yet unidentified oncogenes. The occurrence of positive transfection 3 wk after irradiation is discussed in terms of the hypothesis that transformation may not occur as a direct consequence of the exposure to x-rays but develops as a rare event in the progeny of the irradiated cells at some later time, as a consequence of the delayed activation of certain genes.

Animals↗

PubMed1989

Efficient immortalization by SV40 T DNA of skin fibroblasts from patients with Wilms' tumor associated with chromosome 11p deletion.

We report that transfection with a plasmid containing the SV40 early region (T) leads to a very high frequency of immortalization in two different strains of human diploid fibroblasts, each with a partial deletion of the short arm of chromosome 11. Immortalization often occurred without a recognizable "crisis" phase. Preimmortal cells showed a high frequency of spontaneous mutations and chromosomal aberrations. These results suggest that a gene involved in the control of senescence of human cells in vitro may be associated with this chromosome.

Antigens, Polyomavirus Transforming↗

PubMed1989

Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage.

We have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9-beta-D-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.

Adenosine Diphosphate Ribose↗

PubMed1989

Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation.

In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, we have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells irradiated with either fast neutrons or accelerated argon ions. Individual mutant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loss and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbA1 locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes.

Blotting, Southern↗

PubMed1989

Identification of ataxia telangiectasia heterozygotes by flow cytometric analysis of X-ray damage.

Flow cytometry was used to identify heterozygotes for the autosomal recessive DNA-repair deficiency disease ataxia telangiectasia (AT). Confluent G0/G1 fibroblasts from 4 homozygotes (at/at), 5 obligate heterozygotes (at/+) and 7 presumed normal controls (+/+) were X-irradiated with 200 Rad and subcultured immediately in medium containing 5-bromodeoxyuridine (BrdU). Cells were harvested 72 h later and stained with fluoresceinated anti-BrdU antibody to identify cells that had entered S phase. They were counterstained with propidium iodide to measure total DNA content. On the basis of relative release from G0/G1, the at/+ strains as a group (33 +/- 3% release) were distinguished from both the presumed +/+ strains (60 +/- 3%) and at/at strains (85 +/- 3%), although the individual values for some strains did show overlap between genotypes. When 10 cell strains were coded and analyzed in 'blind' experiments, all 4 heterozygotes were correctly assigned, although one poorly growing presumed normal line was incorrectly assigned as a heterozygote. By a similar assay in which exponentially growing cultures were pulsed briefly with BrdU 8 h after irradiation with 400 Rad and then harvested immediately, presumed +/+ cells as a group could be distinguished from at/at cells but not from at/- cells. This combination of assays assists in the identification of all 3 AT genotypes. This should be of both basic and diagnostic use, particularly in families known to segregate AT.

Adolescent↗

PubMed1989

A comparison of mutation induction at the tk and hprt loci in human lymphoblastoid cells; quantitative differences are due to an additional class of mutations at the autosomal tk locus.

X-Rays, ethyl methanesulfonate and ICR-191 induced 2 classes of trifluorothymidine-resistant mutants at the autosomal tk locus in human lymphoblastoid cells. These classes were differentiated by their growth rates; some mutants grew with a normal doubling time of 14-18 h (tk-NG), while others grew much more slowly, with doubling times of 21-44 h (tk-SG). Only mutants with normal growth rates were observed at the X-linked hprt locus; the frequencies of mutations induced at hprt were equal to those induced for tk-NG mutants. Thus, more mutations overall (by up to a factor of 6) were induced at tk than at hprt. These results are discussed in relation to recent studies in rodent cells, in which much greater mutation frequencies were observed at autosomal loci.

Aminacrine↗

PubMed1989

Modification of radiosensitivity and recovery from X ray damage in vitro by retinoic acid.

We studied the effects of all-trans retinoic acid (RA) combined with X-irradiation on confluent cultures of human breast cancer (MCF-7) and melanoma (C-143) cell lines, as well as in a normal human diploid fibroblast strain (AG 1522). RA in non-cytotoxic concentrations was a potent inhibitor of confluent holding recovery of potentially lethal damage (PLD repair) for all three cell types. A complete inhibition of recovery was observed at lower RA concentrations in the tumor lines than in the fibroblast strain. Exposure to RA prior to and during irradiation resulted in a radiosensitizing effect that was similar in MCF-7 and AG 1522 cells. The implications of these findings are discussed in terms of the potential value of retinoids as biological response modifiers for the clinical radiotherapy of cancer.

Cell Line↗

PubMed1989

Sister chromatid exchange in painters recently exposed to solvents.

Epidemiologic studies have suggested that exposure to solvent-based materials in the painting trades is associated with an increased cancer risk. In order to determine if the solvent exposures of current union members of the International Brotherhood of Painters and Allied Tradesman (IBPAT) are associated with a genotoxic risk, we have measured the sister chromatid exchange (SCE) frequency in their peripheral blood lymphocytes. Previous reports have shown that chronic solvent exposure of these workers does not elevate SCE frequencies. We now report that acute exposure, estimated by the number of days worked over the month prior to venipuncture, is associated with elevated SCE levels in currently smoking workers. No elevation in SCE was associated with similar recent exposure in nonsmoking, solvent-exposed painters.

Adult↗

PubMed1989

Expression of lethal mutations in progeny of irradiated mammalian cells.

The cloning efficiency of the progeny of CHO and BALB/3T3 cells surviving acute exposures of 100-1000 cGy of X-rays was examined by three different experimental protocols. A dose-dependent decrease in cloning efficiency was observed with both cell types up to 13-15 population doublings after exposure. This decrease persisted for longer times after high radiation doses; for example, the cloning efficiency of the progeny of 3T3 cells 28 population doublings after irradiation with 1000 cGy was 44 per cent of that in parallel non-irradiated controls. Confluent holding under conditions which allowed the repair of potentially lethal damage had no effect on this phenomenon. These results are consistent with the hypothesis that large numbers of lethal mutations may be expressed among the progeny of surviving cells for many generations after irradiation.

Animals↗

PubMed1989

Locus specificity for mutation induction in human cells exposed to accelerated heavy ions.

The relative efficiencies of two types of densely ionizing particles were compared for the induction of mutations at two distinct genetic loci in human cells. Mutations to 6-thioguanine resistance (hgprt locus) or to trifluorothymidine resistance (tk) locus were scored in TK6 human lymphoblastoid cells exposed to graded doses of 40Ar ions (470 MeV/amu, LET = 95-97 keV/microns) or 28Si ions (456 MeV/amu, 61 keV/microns). The autosomal tk locus was more efficiently mutated than the X-linked hgprt locus following heavy particle irradiations. This was predominantly due to the contribution of a class of slowly growing mutants scored at the tk locus. Silicon ions were more efficient per unit dose than argon ions for the induction of mutants at either locus. When the mutant yield for a particular ion was compared with particle fluence, similar numbers of hgprt mutants are induced by equal numbers of 40Ar or 28Si ions. Comparison of the number of tk mutants with particle fluence demonstrates an increased efficiency for 28Si ions over 40Ar. These data suggest that the LET-RBE relationship may be different for individual genetic loci in human cells.

Argon↗