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Biomedical subjects

John B Little

Publications and source records attributed to John B Little.

At least 19 recordsLinked to original sources
PubMed2006

Abnormal gene expression profiles in unaffected parents of patients with hereditary-type retinoblastoma.

The hereditary form of retinoblastoma (Rb) is associated with a germ line mutation in one RB allele and is characterized by the occurrence of multiple, bilateral Rb tumors and a predisposition to the development of second cancers. In an earlier study, we observed an unexpected hypersensitivity to ionizing radiation in skin fibroblasts derived from unaffected parents of children with hereditary Rb. In at least four of these five families, there was no family history of Rb, indicating a new germ line mutation. We hypothesize that the increased parental cell sensitivity to radiation may reflect the presence of an as yet unrecognized genetic abnormality occurring in one or both parents of children with Rb. In the present study, we use DNA microarray technology to determine whether differences in gene expression profiles occurred in the unaffected parents of patients with hereditary Rb relative to normal individuals. Microarray analyses were validated by quantitative reverse transcription-PCR measurements. A distinct difference was observed in the patterns of gene expression between unaffected Rb parents and normal controls. By use of the prediction analysis for microarrays and principal component analysis methodologies, significant differences between the two groups were identified when as few as nine genes were analyzed. Further study of this phenomenon may offer a new insight into the genetic mechanisms of Rb and perhaps more broadly in cancer biology.

Adult↗
PubMed2006

Cellular radiation effects and the bystander response.

This report reviews briefly some of the findings reported over the past 2 years that enhance our understanding of the radiation-induced bystander effect. These developments include: technicological advances; the role of oxidative stress; the effect of cytoplasmic irradiation; cell-to-cell communication; and evidence that Connexin 43 mediated intercellular communication is induced by radiation exposure. A few overriding unanswered questions are discussed. These include: what is the signal(s) transmitted from irradiated to bystander cells; what is the relationship between the bystander response and other non-targeted effects of radiation; are there beneficial effects associated with the bystander response; and what is the significance of the bystander effect for radiation protection?

Animals↗
PubMed2006

Cancer survivorship--genetic susceptibility and second primary cancers: research strategies and recommendations.

Cancer survivors constitute 3.5% of the United States population, but second primary malignancies among this high-risk group now account for 16% of all cancer incidence. Although few data currently exist regarding the molecular mechanisms for second primary cancers and other late outcomes after cancer treatment, the careful measurement and documentation of potentially carcinogenic treatments (chemotherapy and radiotherapy) provide a unique platform for in vivo research on gene-environment interactions in human carcinogenesis. We review research priorities identified during a National Cancer Institute (NCI)-sponsored workshop entitled "Cancer Survivorship--Genetic Susceptibility and Second Primary Cancers." These priorities include 1) development of a national research infrastructure for studies of cancer survivorship; 2) creation of a coordinated system for biospecimen collection; 3) development of new technology, bioinformatics, and biomarkers; 4) design of new epidemiologic methods; and 5) development of evidence-based clinical practice guidelines. Many of the infrastructure resources and design strategies that would facilitate research in this area also provide a foundation for the study of other important nonneoplastic late effects of treatment and psychosocial concerns among cancer survivors. These research areas warrant high priority to promote NCI's goal of eliminating pain and suffering related to cancer.

Antineoplastic Agents↗
PubMed2006

Induction of apoptosis in human tumor cells after exposure to Auger electrons: comparison with gamma-ray exposure.

To clarify the contribution of apoptosis to cell death in four human solid tumor cell lines, clonogenic cell survival (indicator of radiosensitivity) and induction of caspase-3 (CASP-3)/caspase-3-like proteases (CASP-3LP) and the production of DNA fragmentation (markers for apoptosis) were studied in RKO, LS174T, MCF7 and TE671 cells exposed to DNA-incorporated Auger-electron-emitting (125)I (5-[(125)I]iodo-2'-deoxyuridine) or gamma-radiation. Clonogenic survival was assessed by colony-forming assay, CASP-3/CASP-3LP induction with a fluorogenic substrate and DNA fragmentation by ligation-mediated polymerase chain reaction. For (125)I, log dose-survival curves had no shoulder [high-linear-energy-transfer (LET)-like] and decreased exponentially at different rates in various cell lines. Induction of CASP-3/CASP-3LP in radiosensitive RKO and LS174T cells was threefold greater than that in radioresistant TE671 and MCF7 cells. Nucleosomal laddering in (125)I-radiosensitive cell lines was dose-dependent, and no laddering was detected in radioresistant lines. For gamma-radiation, the survival curve for LS174T cells was monoexponential and that for the other lines exhibited a distinct shoulder (low-LET-like). The most radiosensitive cell line, LS174T, showed the highest induction of CASP-3/CASP-3LP, and the most radioresistant line, TE671, showed the lowest induction. Although DNA laddering was not detectable in TE671 cells, it was observed in other lines, being most prominent in LS174T cells. We conclude that apoptosis initiated by DNA-incorporated (125)I is dose-dependent, correlates with cell radiosensitivity and takes place through a CASP-3-mediated pathway, whereas that after gamma-irradiation probably occurs via a CASP-3-independent pathway and/or a CASP-3-mediated pathway and does not correlate with cell radiosensitivity.

Apoptosis↗
PubMed2006

Lauriston S. Taylor lecture: nontargeted effects of radiation: implications for low-dose exposures.

Traditional thinking has been that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. This implies that (1) biological effects occur only in irradiated cells, (2) radiation traversal through the nucleus of the cell is a prerequisite to produce a biological response, and (3) DNA is the target molecule in the cell. Evidence has been emerging, however, for non-DNA targeted effects of radiation; that is, effects including mutations, chromosomal aberrations, and changes in gene expression that occur in cells that in themselves receive no radiation exposure. Two of these phenomena will be described in this paper. The first is radiation-induced genomic instability whereby biological effects, including elevated frequencies of mutations and chromosomal aberrations, arise in the distant descendants of irradiated cells. The second phenomenon has been termed the "bystander effect," whereby in a mixed population of irradiated and nonirradiated cells, biological effects arise in those cells that receive no radiation exposure. The damage signals are transmitted from cell to cell through gap junction channels, and the genetic effects observed in bystander cells appear to result from an upregulation of oxidative stress. The possible influence of these nontargeted effects of radiation on the response to low-dose exposures is discussed.

Bystander Effect↗
PubMed2005

Cellular mechanisms for low-dose ionizing radiation-induced perturbation of the breast tissue microenvironment.

Radiation exposure is an important form of environmental carcinogen and has been associated with increased risk of breast cancer. Epigenetic events, especially those involving alterations in the breast stromal microenvironment, may play an important role in radiation-induced carcinogenesis but remain not well understood. We here show that human mammary stromal fibroblasts respond to protracted low-dose ionizing radiation exposures by displaying a senescence-like phenotype. Using a three-dimensional coculture system to model the interactions of different mammary cell types with their neighbors and with their environment, we provide a direct experimental proof that ionizing radiation-induced senescence-like fibroblasts significantly perturb the mammary stromal microenvironment, which is highlighted by impaired formation of pseudopodia networks due to marked cytoskeletal alterations in senescence-like fibroblasts and increased extracellular matrix degradation because of the up-regulation of multiple secreted matrix metalloproteinases. Within such a perturbed environment, mammary ductal morphogenesis is completely disrupted and epithelial cells instead grow into enlarged cystic structures, which further develop and become disorganized cell masses on inactivation of cellular death pathways. Breast carcinoma cells growing in such an environment are enabled to fully express their malignant potential as evidenced by the alpha6beta4 integrin/phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway-dependent invasive growth. Our results suggest that ionizing radiation, in addition to causing gene mutations in epithelial cells, can contribute to breast carcinogenesis by perturbing the tissue microenvironment that leads to dysregulated cell-cell and cell-matrix interactions.

Animals↗
PubMed2004

Low dose exposure to sodium arsenite synergistically interacts with UV radiation to induce mutations and alter DNA repair in human cells.

Inorganic arsenic is a known human carcinogen, yet its mechanism of action remains poorly understood. Epidemiological data suggest that arsenic exposure interacts with UV radiation exposure to increase the risk of skin cancer. Studies have suggested that arsenic is able to impair DNA repair enzymes and alter the repair of UV-induced DNA damage. Here we have tested the hypothesis that arsenite [As(III)] and UV interact synergistically to enhance mutagenesis. TK6 human lymphoblastoid cells that are functionally heterozygous at the thymidine kinase (TK) locus were pre-exposed to As(III) alone and in combination with UV. Our data suggest that As(III) is mutagenic only at high doses at the TK locus. As(III) enhanced UV mutagenesis in a more than additive fashion. To investigate the mechanism underlying this synergy we assessed the removal of UV-induced dimers in TK6 cells using the T4 endonuclease-incorporated Comet assay. Pre-treatment with As(III) specifically inhibited the repair of UV-induced pyrimidine dimer-related DNA damage. Taken together, these data suggest that pre-treatment of human cells with arsenic impairs the nucleotide excision repair pathway and leads to enhanced UV mutagenesis.

Arsenites↗
PubMed2004

The radiation-induced bystander effect: evidence and significance.

A multitude of biological effects observed over the past two decades in various in vivo and in vitro cell culture experiments have indicated that low dose/low fluence ionizing radiation has significantly different biological responses than high dose radiation. Exposure of cell populations to very low fluences of alpha-particles or incorporated radionuclides results in significant biological effects occurring in both the irradiated and nonirradiated cells in the population. Cells recipient of growth medium from irradiated cultures can also respond to the radiation exposure. This phenomenon, termed the 'bystander response', has been postulated to impact both the estimation of risks of exposure to ionizing radiation and radiotherapy. Amplification of radiation-induced cytotoxic and genotoxic effects by the bystander effect is in contrast to the observations of adaptive responses, which are generally induced following exposure to low dose, low linear energy transfer radiation and which tend to attenuate radiation-induced damage. In this article, the evidence for existence of radiation-induced bystander effects and our current knowledge of the biochemical and molecular events involved in mediating these effects are described. Potential similarities between factors that mediate the radiation-induced bystander and adaptive responses are highlighted.

Alpha Particles↗
PubMed2003

Cancer risks attributable to low doses of ionizing radiation: assessing what we really know.

High doses of ionizing radiation clearly produce deleterious consequences in humans, including, but not exclusively, cancer induction. At very low radiation doses the situation is much less clear, but the risks of low-dose radiation are of societal importance in relation to issues as varied as screening tests for cancer, the future of nuclear power, occupational radiation exposure, frequent-flyer risks, manned space exploration, and radiological terrorism. We review the difficulties involved in quantifying the risks of low-dose radiation and address two specific questions. First, what is the lowest dose of x- or gamma-radiation for which good evidence exists of increased cancer risks in humans? The epidemiological data suggest that it is approximately 10-50 mSv for an acute exposure and approximately 50-100 mSv for a protracted exposure. Second, what is the most appropriate way to extrapolate such cancer risk estimates to still lower doses? Given that it is supported by experimentally grounded, quantifiable, biophysical arguments, a linear extrapolation of cancer risks from intermediate to very low doses currently appears to be the most appropriate methodology. This linearity assumption is not necessarily the most conservative approach, and it is likely that it will result in an underestimate of some radiation-induced cancer risks and an overestimate of others.

Biophysical Phenomena↗
PubMed2003

Expression of CONNEXIN43 is highly sensitive to ionizing radiation and other environmental stresses.

To gain a greater understanding of the mechanisms underlying the cellular responses to low-dose ionizing radiation, gene expression profiles were examined by microarray analysis of cDNA from confluent human diploid fibroblast cultures exposed to very low fluences of alpha-particles. The data, supported by Northern and Western analyses, indicate that radiation induces a significant up-regulation of CONNEXIN43 expression. This phenomenon was observed in a variety of irradiated cell types. These findings are consistent with our previous observations that connexin43 (cx43)-mediated gap-junction intercellular communication is involved in the bystander response observed in cell cultures exposed to fluences of alpha-particles by which only a very small fraction of the cell nuclei is traversed by a particle track (E. I. Azzam et al., Proc. Natl. Acad. Sci. USA, 98: 473-478, 2001). Increased mRNA levels in cells from irradiated cultures correlated with increased cx43 protein levels by approximately 4 h after irradiation. The induction of cx43 was observed by mean alpha-particle doses as low as 0.16 cGy, and also in cells exposed to gamma-rays, t-butyl hydroperoxide, and hyperthermia. Exposure to these stresses also resulted in post-translational modification of cx43; increased phosphorylation and hyperphosphorylation of the protein was observed. Up-regulation of cx43 expression in ionizing radiation exposed cells correlated with functional communication through gap junctions, as evidenced by dye transfer from irradiated to nonirradiated cells. In contrast, the response after UV radiation varied and was cell type-dependent. Overall, these data suggest a critical role for genes involved in intercellular communication in mediating the cellular responses to a variety of stresses.

Alpha Particles↗
PubMed2003

Oxidative metabolism, gap junctions and the ionizing radiation-induced bystander effect.

Evidence accumulated over the past two decades has indicated that exposure of cell populations to ionizing radiation results in significant biological effects occurring in both the irradiated and nonirradiated cells in the population. This phenomenon, termed the 'bystander response', has been shown to occur both in vitro and in vivo and has been postulated to impact both the estimation of risks of exposure to low doses/low fluences of ionizing radiation and radiotherapy. Several mechanisms involving secreted soluble factors, oxidative metabolism and gap-junction intercellular communication have been proposed to regulate the radiation-induced bystander effect. Our current knowledge of the biochemical and molecular events involved in the latter two processes is reviewed in this article.

Animals↗
PubMed2003

Genomic instability and bystander effects: a historical perspective.

Data have been emerging over the past two decades concerning two phenomena in which important biological effects of ionizing radiation arise in cells that in themselves receive no radiation exposure. In the first, radiation-induced genomic instability, biological effects occur in the progeny of the irradiated cell after many generations of cell division. In the second, radiation-induced bystander effects, they arise in cells that receive no radiation exposure as a consequence of damage signals transmitted from neighboring irradiated cells; transmission may be mediated either by direct intercellular communication through gap junctions, or by factors released into the surrounding medium. In both phenomena, the biological effects appear to be associated with an upregulation of oxidative metabolism. The present paper is designed to review the historical background leading to our current knowledge of these two phenomena, and to indicate some future directions for research that will allow us to assess better their importance in the health effects of exposure to ionizing radiation.

Animals↗
PubMed2003

Genomic instability and radiation.

Genomic instability is a hallmark of cancer cells, and is thought to be involved in the process of carcinogenesis. Indeed, a number of rare genetic disorders associated with a predisposition to cancer are characterised by genomic instability occurring in somatic cells. Of particular interest is the observation that transmissible instability can be induced in somatic cells from normal individuals by exposure to ionising radiation, leading to a persistent enhancement in the rate at which mutations and chromosomal aberrations arise in the progeny of the irradiated cells after many generations of replication. If such induced instability is involved in radiation carcinogenesis, it would imply that the initial carcinogenic event may not be a rare mutation occurring in a specific gene or set of genes. Rather, radiation may induce a process of instability in many cells in a population, enhancing the rate at which the multiple gene mutations necessary for the development of cancer may arise in a given cell lineage. Furthermore, radiation could act at any stage in the development of cancer by facilitating the accumulation of the remaining genetic events required to produce a fully malignant tumour. The experimental evidence for such induced instability is reviewed.

Genes, p53↗
PubMed2003

Involvement of the nonhomologous end joining DNA repair pathway in the bystander effect for chromosomal aberrations.

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of alpha particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4-9% of wild-type cells and 36-55% of Xrcc5-/- cells, whereas only 2-3% of the nuclei were traversed by an alpha particle and thus received any radiation exposure. G22p1-/- cells responded similarly to Xrcc5-/- cells, whereas Prkdc-/- and Adprt-/- cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5-/- and G22p1-/- cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc-/- cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

Alpha Particles↗
PubMed2002

Bystander effect for chromosomal aberrations induced in wild-type and repair deficient CHO cells by low fluences of alpha particles.

We have previously shown that when confluent cultures of mammalian cells are exposed to very low fluences of alpha particles, fluences whereby as few as 1% of the cell nuclei are traversed by a single particle, genetic effects including specific gene mutations and sister chromatid exchanges (SCE) are induced in neighboring, non-irradiated "bystander" cells. The present investigation was designed to examine the induction of chromosomal aberrations in wild-type CHO cells and its DNA double strand break repair deficient mutant xrs-5 by a broad range of alpha particle fluences yielding mean doses of 0.17-200cGy. The dose-response curve for the induction of aberrations was curvilinear for both cell lines, with a greater effect occurring at very low fluences owing to aberrations arising in bystander cells. These aberrations were predominantly of the chromatid-type. With such fluences, the number of cells with induced aberrations per nucleus irradiated increased up to 4-fold in CHO cells and 15-fold in xrs-5 cells over that expected if aberrations occurred only in irradiated cells. These results are discussed in terms of the hypothesis that the primary DNA damage in bystander CHO cells is oxidative base damage leading to a relatively small bystander effect for gross chromosomal aberrations as compared with mutations or SCE; the larger bystander effect in xrs-5 cells is the result of oxidative damage and non-repaired DNA strand breaks which may result from opposed oxidative lesions.

Alpha Particles↗
PubMed2002

Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures.

The role of oxidative metabolism in the up-regulation/activation of stress-induciblesignaling pathways as well as induction of micronucleus formation in bystander cells was investigated. By immunoblotting and in situ immunofluorescence, active Cu-Zn superoxide dismutase (SOD) enzyme and active catalase enzyme were shown to inhibit the up-regulation of p21(Waf1) as well as the induction of micronucleus formation in bystander cells from confluent cultures of normal human diploid fibroblasts irradiated with 0.3-3 cGy of alpha-particles. Enzyme activity assays indicated that exogenous SOD became significantly associated with the cells. Reactive oxygen species apparently derived from a flavin-containing oxidase enzyme [presumably an NAD(P)H-oxidase] appeared to be major contributors to the bystander-induced up-regulation of p53 and p21(Waf1) as well as micronucleus formation, as evidenced by the inhibition of these effects with diphenyliodonium. Rapid activation of nuclear factor kappaB, Raf-1, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase and their downstream effectors activator protein 1, ELK-1, p90RSK, and activating transcription factor 2 was also observed in cultures exposed to very low fluences of alpha-particles. Significant attenuation in the activation of these kinases and transcription factors occurred in irradiated cultures treated with either SOD or catalase. Overall, these results support the hypothesis that superoxide and hydrogen peroxide produced by flavin-containing oxidase enzymes mediate the activation of several stress-inducible signaling pathways as well as micronucleus formation in bystander cells from cultures of human cells exposed to low fluences of alpha-particles.

Alpha Particles↗
PubMed2002

Unexpected sensitivity to radiation of fibroblasts from unaffected parents of children with hereditary retinoblastoma.

The response to ionizing radiation was examined in diploid skin fibroblasts derived from 5 patients with hereditary type retinoblastoma as well as their parents. Unexpected sensitivity to cell killing, as measured by clonogenic survival, as well as enhanced radiation-induced G(1) arrest were observed in at least 1 parental fibroblast strain in all 5 families. In all cases, parental strains were equally or more radiosensitive than the probands. The mutation of the retinoblastoma gene (RB) determined in 4 of 5 probands was either absent from the parental cells, as expected from the negative family histories, or identical, in 1 father who was a known carrier. In the fifth family, the family history was negative for retinoblastoma. We hypothesize that the increased parental cell sensitivity to radiation suggests the presence of an as yet unrecognized genetic event occurring in 1 or both parents of children with retinoblastoma. Whether it increases mutability of the RB locus or other loci or interacts with RB is conjectural.

Cell Survival↗