Edwin H. Goodwin

Alpha-particle-induced sister chromatid exchange in normal human lung fibroblasts: evidence for an extranuclear target.

We investigated the relationship between nuclear hits by alpha particles and the subsequent occurrence of sister chromatid exchanges (SCEs) in normal human diploid lung fibroblasts (HFL1). Cells were exposed to 238Pu alpha particles at doses ranging from 0.4-12.9 cGy and subsequently analyzed for SCEs. A significant increase in SCE frequency was observed even at the lowest dose examined. The extent of induction of SCEs in the HFL1 cells showed dose dependency in the very low dose range, i.e. 0.4-2.0 cGy. Thereafter, induction of SCEs was independent of dose. Based on measurements of the nuclear areas of the HFL1 cells in conjunction with target theory calculations, the lowest dose resulted in an approximately 8.6-fold increase in the percentage of cells showing excessive SCEs over the theoretically expected percentage of cells whose nuclei were calculated to be traversed by one or more alpha particles. The extent of the discrepancies between theoretically expected and experimentally observed frequencies of SCEs became progressively reduced with increasing radiation dose. We additionally determined that SCEs induced by the alpha particles have no significant dependency on the time of cell collection after exposure to a selected dose of alpha particles, thereby confirming that the differences between the theoretically predicted and observed SCE frequencies were not due to an artifact of the time of cell sampling for the SCE measurements. These results obtained with normal human cells are similar to those of other investigators who observed excessive SCEs in immortalized rodent cells beyond that which could be attributed exclusively to nuclear traversals by alpha particles. Such consistent findings point to the existence of an alternative, extranuclear target through which alpha particles cause DNA damage, as detected by SCE analysis. The existence of an extranuclear compartment as a target for alpha particles may have important implications for the susceptibility of lung cells to the DNA-damaging effects of alpha-particle exposure due to the inhalation of radon progeny.

Alpha Particles Induce the Production of Interleukin-8 by Human Cells

The pulmonary microenvironment is a primary target for alpha particles like those emitted by inhaled radon and its progeny. While exposure to alpha particles has recently been associated with the generation of extracellular and intracellular reactive oxygen species (ROS; Cancer Res. 57, 3963-3971, 1997), little is known about how exposure to alpha particles may affect the generation of oxidative stress-related mediators in the respiratory tract. Interleukin-8 (IL8) is a cytokine recognized for its potent role as a chemoattractant and activator of polymorphonuclear leukocytes. Oxidative stress can up-regulate expression of the gene that encodes IL8 (IL8) in a variety of cell types. In this study, we set out to investigate a potential linkage between the generation of ROS and production of IL8 in alpha-particle-irradiated normal human lung fibroblasts. ELISA revealed that exposure of the fibroblasts to low doses of alpha particles (3.6-19 cGy) caused significant increases in generation of the IL8 protein as early as 30 min after irradiation. Northern blot analyses revealed that such increases were associated with increased IL8 mRNA levels. Cells exposed to alpha particles in the presence of antioxidants, i.e. superoxide dismutase and dimethyl sulfoxide, resulted in significant decreases in extracellular IL8 protein levels. Similar results were obtained with cells treated with dexamethasone, an inhibitor of transcription. Our results indicate that alpha-particle-induced increases in production of IL8 occur temporally in parallel with elevated production of ROS. Conceivably, such production of IL8 induced by alpha particles may contribute to an inflammatory response in the lower respiratory tract. Additionally, the promitogenic effects of IL8 may be a factor in hyperplastic responses in the airway epithelial cells to inhaled radon and radon progeny and perhaps other stresses associated with ROS.

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.

Strand-specific fluorescence in situ hybridization: the CO-FISH family

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5′→3′ towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5′→3′ orientation of DNA sequences with respect to the pter→qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.

Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice

The Brca2 tumor‐suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51‐interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51‐interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2lex1) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2lex1 mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2lex1 mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy‐terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51.

Transmission of Radiation-Induced Acentric Chromosomal Fragments to Micronuclei in Normal Human Fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.

Hyper telomere recombination accelerates replicative senescence and may promote premature aging

Werner syndrome and Bloom syndrome result from defects in the RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and display premature aging phenotypes. Similarly, XFE progeroid syndrome results from defects in the ERCC1-XPF DNA repair endonuclease. To gain insight into the origin of cellular senescence and human aging, we analyzed the dependence of sister chromatid exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telomeric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, BLM, or ERCC1-XPF. Consistent with our other studies, we found evidence of elevated T-SCE in telomerase-negative but not telomerase-positive backgrounds. In telomerase-negative WRN-deficient cells, T-SCE—but not G-SCE—frequencies were significantly increased compared with controls. In contrast, SCE frequencies were significantly elevated in BLM-deficient cells irrespective of genome location. In ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed from controls. A theoretical model was developed that allowed an in silico investigation into the cellular consequences of increased T-SCE frequency. The model predicts that in cells with increased T-SCE, the onset of replicative senescence is dramatically accelerated even though the average rate of telomere loss has not changed. Premature cellular senescence may act as a powerful tumor-suppressor mechanism in telomerase-deficient cells with mutations that cause T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular senescence may be a factor contributing to accelerated aging in Werner and Bloom syndromes, but not XFE progeroid syndrome.

The Dose-Dependent Fragmentation of Chromatin in Human Fibroblasts by 3.5-MeV α Particles from 238Pu: Experimental and Theoretical Considerations Pertaining to Single-Track Effects

The technique of premature chromosome condensation (PCC) was used to examine the dose-response relationship for the production of interphase (G0) chromosome fragments in noncycling normal human fibroblasts following exposure to 238Pu alpha particles, with special emphasis on the low-dose region. The dose response was convincingly linear from 0.2 to 3.0 Gy. Analysis of further data collected over a dose range of 1.1 to 22.4 cGy provided no evidence of deviation from linearity in this low-dose region. The fact that this lower dose range extends into the region where single-particle effects are dominant suggests that a linear extrapolation of this response from higher to lower doses is valid. Ratios of coefficients for the induction of fragments produced by 238Pu alpha particles versus 60Co gamma rays gave an RBE of 2.34 +/- 0.09. Distributions of fragments among 60Co gamma-irradiated cells were consistent with a Poisson expectation of random damage. In contrast, overdispersion appeared to be a general feature of 238Pu alpha-particle-induced fragmentation, a phenomenon explainable under the assumption that single-particle traversals are capable of producing multiple PCC fragments. Data obtained were used to estimate practical and theoretical lower-dose limits of detection of initial chromatin breaks provided by current PCC methodology.

Dose Responses for Chromosome Aberrations Produced in Noncycling Primary Human Fibroblasts by Alpha Particles, and by Gamma Rays Delivered at Sublimiting Low Dose Rates

Abstract Cornforth, M. N., Bailey, S. M. and Goodwin, E. H. Dose Responses for Chromosome Aberrations Produced in Noncycling Primary Human Fibroblasts by Alpha Particles, and by Gamma Rays Delivered at Sublimiting Low Dose Rates. Radiat. Res. 158, 43–53 (2002). As the total dose of X or γ rays is delivered at lower and lower rates, the yield of chromosome aberrations progressively diminishes. Simultaneously, the shape of the dose response changes from one exhibiting pronounced upward curvature at high dose rates to one approaching linearity at low dose rates. Although the maximum sparing effect caused by lowering the dose rate can be predicted from classical cytogenetic theory, it has yet to be verified experimentally. Here, noncycling normal human fibroblasts were exposed to graded doses of 137Cs γ rays at chronic dose rates of 6.3 and 2.8 cGy h−1, dose rates that we reasoned should be lower than those required to achieve maximal sparing. This was indeed shown to be the case, after it was determined that the two chronic dose rates produced identical linear dose responses of 0.05 total aberrations per cell Gy−1. Consistent with cytogenetic theory, this value was statistically indistinguishable from the linear coefficient derived from a fit to aberration frequencies produced by high-dose-rate exposure. Exposure to 238Pu α particles also produced a linear dose response for total aberrations, whose slope—with respect to 137Cs γ rays as a reference radiation—implied a maximum RBE of 35 ± 2.

The non-targeted effects of radiation are perpetuated by exosomes.

Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein-RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA-protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE, spread these effects to naïve cells, and perpetuate GI in the affected cells.