Paul F. Wilson

Farnesoid X Receptor Modulates Renal Lipid Metabolism, Fibrosis, and Diabetic Nephropathy

OBJECTIVE—Recent studies indicate an important role for nuclear receptors in regulating lipid and carbohydrate metabolism, fibrosis, and inflammation. Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily. FXR is highly expressed in the liver, intestine, adrenal gland, and kidney. The primary bile acids are the highest affinity endogenous ligands for FXR. The effects of FXR agonists in diabetic kidney disease, the main cause of end-stage renal disease, however, have not been determined. RESEARCH DESIGN AND METHODS—To identify the effect of FXR activation in modulation of diabetic nephropathy, we treated 1) C57BL/6J mice on low-fat diet or high-fat diet with FXR agonists (GW4064 or cholic acid) for 1 week; 2) C57BLKS/J-db/db mice and their lean mates with GW4064 for 1 week; and 3) C57BL/6J-db/db mice and their lean mates with cholic acid for 12 weeks. RESULTS—We found that FXR agonists modulate renal sterol regulatory element–binding protein-1 (SREBP-1) expression and lipid metabolism and renal expression of profibrotic growth factors, proinflammatory cytokines, and oxidative stress enzymes and decrease glomerulosclerosis, tubulointerstitial fibrosis, and proteinuria. In renal mesangial cells, overexpression of FXR or treatment with GW4064 also inhibited SREBP-1c and other lipogenic genes, transforming growth factor-β, and interleukin-6, suggesting a direct role of FXR in modulating renal lipid metabolism and modulation of fibrosis and inflammation. CONCLUSIONS—These results therefore indicate a new and important role for FXR in the kidney and provide new therapeutic avenues for the treatment of diabetic nephropathy.

Repression of mutagenesis by Rad51D-mediated homologous recombination

Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR proficiency. We constructed and characterized a knockout of the paralog Rad51D in widely studied CHO cells. The rad51d mutant (clone 51D1) displays sensitivity to a diverse spectrum of induced DNA damage including γ-rays, ultraviolet (UV)-C radiation, and methyl methanesulfonate (MMS), indicating the broad relevance of HRR to genotoxicity. Spontaneous chromatid breaks/gaps and isochromatid breaks are elevated 3- to 12-fold, but the chromosome number distribution remains unchanged. Most importantly, 51D1 cells exhibit a 12-fold-increased rate of hprt mutation, as well as 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. Xrcc3 irs1SF cells from the same parental CHO line show similarly elevated mutagenesis at these three loci. Collectively, these results confirm the a priori expectation that HRR acts in an error-free manner to repress three classes of genetic alterations (chromosomal aberrations, loss of gene function and increased gene expression), all of which are associated with carcinogenesis.

Role of Homologous Recombination in the Alpha-Particle-Induced Bystander Effect for Sister Chromatid Exchanges and Chromosomal Aberrations

Abstract Nagasawa, H., Peng, Y., Wilson, P. F., Lio, Y-C., Chen, D. J., Bedford, J. S. and Little, J. B. Role of Homologous Recombination in the Alpha-Particle-Induced Bystander Effect for Sister Chromatid Exchanges and Chromosomal Aberrations. Radiat. Res. 164, 141–147 (2005). The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of α particles such that <1% of the nuclei were traversed by an α particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.

Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of (137)Cs gamma-rays. Quiescent G(0)/G(1)-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV approximately 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV approximately 0.6; mean+/-SD of 1.00+/-0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.

VARIATIONS IN RADIOSENSITIVITY AMONG INDIVIDUALS: A POTENTIAL IMPACT ON RISK ASSESSMENT?

To have an impact on risk assessment for purposes of radiation protection recommendations, significantly broad variations in carcinogenic radiosensitivity would have to exist in significant proportions in the human population. Even if we knew all the genes where mutations would have major effects, individual genome sequencing does not seem useful, since we do not know all these genes, nor can we be certain of the phenotypic effect of polymorphisms discovered. Further, sequencing would not reveal epigenetic changes in gene expression. Another approach to develop phenotypic biomarkers for cells or tissues for which variations in radiation response may reflect the variations in carcinogenic sensitivity. To be useful, experimental evidence for such a correlation would be crucial, and it is also evident that correlations may be tissue or tumor specific. Some cellular markers are discussed that have shown promise in this regard. They include chromosome aberration induction and DNA repair assays that are sufficiently sensitive to measure after modest or low doses or dose rates. To this end we summarize here some of these assays and review the results of a number of experiments from our laboratory that show clear differences in DNA repair capacity reflected by gamma-H2AX foci formation in cells from a high proportion (perhaps 1/3) of apparently normal individuals. A low dose-rate assay was used to amplify such differences. Another promising assay combines G(2) chromosomal radiosensitivity with the above gamma-H2AX foci on mitotic chromosomes. There are other potentially useful assays as well.

Cardiovascular Risks Associated with Low Dose Ionizing Particle Radiation

Previous epidemiologic data demonstrate that cardiovascular (CV) morbidity and mortality may occur decades after ionizing radiation exposure. With increased use of proton and carbon ion radiotherapy and concerns about space radiation exposures to astronauts on future long-duration exploration-type missions, the long-term effects and risks of low-dose charged particle irradiation on the CV system must be better appreciated. Here we report on the long-term effects of whole-body proton (1H; 0.5 Gy, 1 GeV) and iron ion (56Fe; 0.15 Gy, 1GeV/nucleon) irradiation with and without an acute myocardial ischemia (AMI) event in mice. We show that cardiac function of proton-irradiated mice initially improves at 1 month but declines by 10 months post-irradiation. In AMI-induced mice, prior proton irradiation improved cardiac function restoration and enhanced cardiac remodeling. This was associated with increased pro-survival gene expression in cardiac tissues. In contrast, cardiac function was significantly declined in 56Fe ion-irradiated mice at 1 and 3 months but recovered at 10 months. In addition, 56Fe ion-irradiation led to poorer cardiac function and more adverse remodeling in AMI-induced mice, and was associated with decreased angiogenesis and pro-survival factors in cardiac tissues at any time point examined up to 10 months. This is the first study reporting CV effects following low dose proton and iron ion irradiation during normal aging and post-AMI. Understanding the biological effects of charged particle radiation qualities on the CV system is necessary both for the mitigation of space exploration CV risks and for understanding of long-term CV effects following charged particle radiotherapy.

Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in γ-irradiated CHO cells

The repair of DNA double-strand breaks (DSBs) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and non-homologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after gamma-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction ( approximately 20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had approximately 2-fold higher chromatid-type CAs and a remarkable approximately 25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al., DNA Repair 4, 782-792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2.

Biological Characterization of Low-Energy Ions with High-Energy Deposition on Human Cells

During space travel, astronauts are exposed to cosmic radiation that is comprised of high-energy nuclear particles. Cancer patients are also exposed to high-energy nuclear particles when treated with proton and carbon beams. Nuclear interactions from high-energy particles traversing shielding materials and tissue produce low-energy (<10 MeV/n) secondary particles of high-LET that contribute significantly to overall radiation exposures. Track structure theories suggest that high charge and energy (HZE) particles and low-energy secondary ions of similar LET will have distinct biological effects for cellular and tissue damage endpoints. We investigated the biological effects of low-energy ions of high LET utilizing the Tandem Van de Graaff accelerator at the Brookhaven National Laboratory (BNL), and compared these to experiments with HZE particles, that mimic the space environment produced at NASA Space Radiation Laboratory (NSRL) at BNL. Immunostaining for DNA damage response proteins was carried out after irradiation with 5.6 MeV/n boron (LET 205 keV/μm), 5.3 MeV/n silicon (LET 1241 keV/μm), 600 MeV/n Fe (LET 180 keV/μm) and 77 MeV/n oxygen (LET 58 keV/μm) particles. Low-energy ions caused more persistent DNA damage response (DDR) protein foci in irradiated human fibroblasts and esophageal epithelial cells compared to HZE particles. More detailed studies comparing boron ions to Fe particles, showed that boron-ion radiation resulted in a stronger G2 delay compared to Fe-particle exposure, and boron ions also showed an early recruitment of Rad51 at double-strand break (DSB) sites, which suggests a preference of homologous recombination for DSB repair in low-energy albeit high-LET particles. Our experiments suggest that the very high-energy radiation deposition by low-energy ions, representative of galactic cosmic radiation and solar particle event secondary radiation, generates massive but localized DNA damage leading to delayed DSB repair, and distinct cellular responses from HZE particles. Thus, low-energy heavy ions provide a valuable probe for studies of homologous recombination repair in radiation responses.

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.

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.