Feng Wu

Arsenite-Induced Alterations of DNA Photodamage Repair and Apoptosis After Solar-Simulation UVR in Mouse Keratinocytes in Vitro

Our laboratory has shown that arsenite markedly increased the cancer rate caused by solar-simulation ultraviolet radiation (UVR) in the hairless mouse skin model. In the present study, we investigated how arsenite affected DNA photodamage repair and apoptosis after solar-simulation UVR in the mouse keratinocyte cell line 291.03C. The keratinocytes were treated with different concentrations of sodium arsenite (0.0, 2.5, 5.0 μM) for 24 hr and then were immediately irradiated with a single dose of 0.30 kJ/m2 UVR. At 24 hr after UVR, DNA photoproducts [cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6-4PPs)] and apoptosis were measured using the enzyme-linked immunosorbent assay and the two-color TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay, respectively. The results showed that arsenite reduced the repair rate of 6-4PPs by about a factor of 2 at 5.0 μM and had no effect at 2.5 μM. UVR-induced apoptosis at 24 hr was decreased by 22.64% at 2.5 μM arsenite and by 61.90% at 5.0 μM arsenite. Arsenite decreased the UVR-induced caspase-3/7 activity in parallel with the inhibition of apoptosis. Colony survival assays of the 291.03C cells demonstrate a median lethal concentration (LC50) of arsenite of 0.9 μM and a median lethal dose (LD50) of UVR of 0.05 kJ/m2. If the present results are applicable in vivo, inhibition of UVR-induced apoptosis may contribute to arsenite’s enhancement of UVR-induced skin carcinogenesis.

Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

AIMnTo evaluate the ability of anti-ricin A-chain antibodies, delivered intracellularly, to protect against ricin-induced cytotoxicity in RAW264.7 cells.nnnMETHODSnAnti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide. RAW264.7 cells were incubated with these antibodies either before or after ricin exposure. The changes in cytotoxicity were estimated by MTT assay. Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy.nnnRESULTSnInternalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies. Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells.nnnCONCLUSIONnIntracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for post-exposure treatment of ricin intoxication.

Alterations in Gene Expression in Rat Skin Exposed to 56Fe Ions and Dietary Vitamin A Acetate

Abstract Zhang, R., Burns, F. J., Chen, H., Chen, S. and Wu, F. Alterations in Gene Expression in Rat Skin Exposed to 56Fe Ions and Dietary Vitamin A Acetate. Radiat. Res. 165, 570– 581 (2006). The purpose of the present work was to examine gene expression patterns in rat skin exposed to a beam of 56Fe ions, a surrogate for the high-energy, heavy-ion galactic radiation background, as a basis for obtaining a better understanding of the possible mechanism(s) behind the radioprotective activity of vitamin A. A 2 × 4-cm rectangle of dorsal rat skin was exposed to 1.01 GeV/nucleon 56Fe ions generated by the Alternating Gradient Synchrotron at Brookhaven National Laboratory. Gene expression patterns were monitored in either the presence or absence of a 250-ppm dietary supplement of vitamin A acetate in powdered lab chow. Although vitamin A and other retinoids show anti-carcinogenic activity in several animal models, the underlying changes in gene expression have not been examined extensively. At either 1 or 7 day after irradiation, a 1-cm square of irradiated and control rat skin was excised and analyzed using the Affymetrix rat microarray (RG_U34A) system. Microarray responses were displayed and processed by GeneSpring 7.0 and GOTree software. At 1 day after 3 Gy of 56Fe-ion irradiation, the expression of 110 genes was significantly up-regulated (P < = 0.05) in comparison to levels in control rat skin, while no genes were altered by the vitamin A acetate supplement alone. Combined with 56Fe-ion radiation, the vitamin A acetate supplement blocked the expression of 88 (80%) of the 110 genes and eliminated 16 of 18 gene categories that were significantly altered (all increased) by the 56Fe-ion radiation. Categories with large numbers of genes eliminated by the retinoid included response to stress, 33 genes; response to biotic stimulus, 38 genes; signal transduction, 35 genes; and regulation of cellular/physiological process, 40 genes. Even for immune response and response to biotic stimulus, the only two categories that remained significantly altered in the presence of the vitamin, the combined number of altered genes was reduced from 74 to 13. No significant alterations in gene expression were found at 7 days relative to the numbers in controls. The results indicate that at 1 day dietary vitamin A acetate strongly interfered with 56Fe-ion-induced gene expression within the broad categories of stimulus- and stress-related genes, implying that the latter gene categories likely play a role in the radioprotective action of the vitamin.

Molecular Characterization of the Peripheral Airway Field of Cancerization in Lung Adenocarcinoma

Field of cancerization in the airway epithelium has been increasingly examined to understand early pathogenesis of non-small cell lung cancer. However, the extent of field of cancerization throughout the lung airways is unclear. Here we sought to determine the differential gene and microRNA expressions associated with field of cancerization in the peripheral airway epithelial cells of patients with lung adenocarcinoma. We obtained peripheral airway brushings from smoker controls (n=13) and from the lung contralateral to the tumor in cancer patients (n=17). We performed gene and microRNA expression profiling on these peripheral airway epithelial cells using Affymetrix GeneChip and TaqMan Array. Integrated gene and microRNA analysis was performed to identify significant molecular pathways. We identified 26 mRNAs and 5 miRNAs that were significantly (FDR <0.1) up-regulated and 38 mRNAs and 12 miRNAs that were significantly down-regulated in the cancer patients when compared to smoker controls. Functional analysis identified differential transcriptomic expressions related to tumorigenesis. Integration of miRNA-mRNA data into interaction network analysis showed modulation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the contralateral lung field of cancerization. In conclusion, patients with lung adenocarcinoma have tumor related molecules and pathways in histologically normal appearing peripheral airway epithelial cells, a substantial distance from the tumor itself. This finding can potentially provide new biomarkers for early detection of lung cancer and novel therapeutic targets.

Linking Gamma-H2AX Foci and Cancer in Rat Skin Exposed to Heavy Ions and Electron Radiation.

AbstractThis study uses acute doses of three test radiations, [40Ar ions (L = 125 keV&mgr;−1), 20Ne ions (L = 25 keV&mgr;−1) and electron radiation] to examine a potential quantitative link between rat skin cancer induction and gamma-H2AX foci in rat keratinocytes exposed in vitro to radiations with comparable L values. Theory provided a testable link between cancer yield and gamma-H2AX foci yields: YCa(D,L)rat−1 = (NF)2−1YAX(D,L)keratinocyte−1 (eqn 1), where YCa(D,L) is cancers(rat) −1 at 1.0 y, YAX(D,L) is in vitro gamma-H2AX foci(keratinocyte) −1, D is radiation dose, L is linear energy transfer, N is irradiated keratinocytes in vivo, and F is the error rate of end joining. An explicit expression for cancer yield was derived based on cancers arising in the ion track region in proportion to D and L (first term) and independently in proportion to D2 in the delta ray region in between the ion tracks (second term): YCa(D,L) = CCaLD + BCaD2 (eqn 1a). Parameters quantified include: CCa = 0.000589 ± 0.000150 cancers-micron[rat(kev)Gy]−1; BCa = 0.0088 ± 0.0035 cancers(ratGy2)−1, F = (8.18 ± 0.91) × 10−10; N = (8.8 ± 1.2) × 107 and (NF)2−1 = 0.036 ± 0.006 cancer keratinocyte(rat H2AX foci)−1. Verification of eqns (1) and (1a) and the constancy of F support the hypothesis that end-rejoining errors play a major role in radiation carcinogenesis in rat skin. Cancer yields per rat were consistently predictable based on gamma-H2AX foci yields in keratinocytes in vitro such that 27.8 H2AXfoci(keratinocyte)−1 predicted 1.0 cancer(rat)−1 at 1 y.

d-limonene prevents ultraviolet irradiation: Induced cyclobutane pyrimidine dimers in Skh1 mouse skin

d-limonene prevents ultraviolet irradiation: Induced cyclobutane pyrimidine dimers in Skh1 mouse skin

Abstract 4864: Aroma terpenes prevent UVB-induced sunburn in Skh1 mouse skin.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnUnderstanding how aroma terpenes prevent sunburn and/or skin cancer in mice could lead to more effective and safer ways of blocking sun damage to human skin. In our previous study, β-damascenone, an aroma terpene, protected against sunburn by activating both keratinocyte and sebaceous gland pathways that fortified and thickened the cornified envelope and sebum layers of mouse epidermis. In the current study, the sunburn preventive activity of d-limonene was examined to confirm that these related compounds work similarly. In addition to sunburn, immunohistochemical analyses of proliferation (PCNA) and a damage-response gene N-Myc Down Regulated Gene 1 (NDRG1) were investigated in control versus d-limonene-treated Skh-1 mice. The mice were treated either topically on the dorsum or orally with different concentrations of d-limonene in liponate (100%, 10%, 1% and 0%) followed by exposure of the dorsum to solar simulation ultraviolet radiation (UVR) of 1.5 kJ/m2 from fluorescent ‘sunlamps’. Either 4 topical 5 μL doses or a single dose of 20 μL (0.95 μg/g body weight) of 100% d-limonene provided complete protection from UVR-induced sunburn. The protection was less but still significant at 10% d-limonene but was absent at 1% d-limonene in comparison to UVR alone. PCNA-labeling showed a marked proliferation increase of epidermal basal cells, outer root sheath cells of hair follicles and sebaceous gland cells by separate exposures to d-limonene and UVR relative to controls. The combined treatment of d-limonene followed by UVR produced fewer PCNA-positive cells in epithelial tissue relative to UVR-only mice. The NDRG1 protein (an indirect measure of DNA damage) was overwhelmingly (∼85% of keratinocytes) induced by UVR indicating heavy DNA damage. The UVR-induced NDRG1 index remained at ∼85% at 1% d -limonene, but was significantly reduced at 10% d-limonene to ∼24% and to less than 1% at 100% d -limonene indicating nearly complete elimination of cellular damage as a pure compound; a finding that is consistent with complete sunburn prevention. The thickness of the cornified envelope plus sebum layers nearly doubled within 5 days after administration of the d-limonene corresponding to the time of UVR application. Overall the results indicate that d-limonene protected against sunburn by activating keratinocyte/sebaceous gland-based pathways that fortified and thickened the cornified envelope plus sebum layers on the skin surface by releasing elevated levels of UVR-absorbing proteins that decreased UVR dose to underlying cutaneous tissues.nnCitation Format: Ahmed N. Uddin, Ivica Labuda, Feng Wu, KamMeng Tchou-Wong, Fredric J. Burns. Aroma terpenes prevent UVB-induced sunburn in Skh1 mouse skin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2013-4864