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1.
Biomolecules ; 14(5)2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38785926

ABSTRACT

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis.


Subject(s)
Epithelial Cells , Esophagus , Organoids , Animals , Organoids/radiation effects , Organoids/pathology , Mice , Esophagus/radiation effects , Esophagus/pathology , Epithelial Cells/radiation effects , Epithelial Cells/pathology , Epithelial Cells/metabolism , DNA Damage , Esophageal Squamous Cell Carcinoma/pathology , Linear Energy Transfer , Esophageal Neoplasms/pathology , Esophageal Neoplasms/metabolism , Cell Differentiation/radiation effects , Tumor Suppressor p53-Binding Protein 1/metabolism , MAP Kinase Signaling System/radiation effects , Radiation Tolerance
2.
Curr Protoc ; 3(12): e949, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38050649

ABSTRACT

Siglecs (sialic acid-binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor-type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi-quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec-Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec-Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec-Fc fusion protein Support Protocol 1: Sialidase treatment of mammalian cells Support Protocol 2: Preparation of recombinant Siglec-Fc fusion protein via transient transfection of mammalian cells.


Subject(s)
N-Acetylneuraminic Acid , Sialic Acid Binding Immunoglobulin-like Lectins , Animals , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , N-Acetylneuraminic Acid/metabolism , Ligands , Flow Cytometry , Neuraminidase/metabolism , Antigens, CD/genetics , Antigens, CD/metabolism , Recombinant Fusion Proteins , Recombinant Proteins , Polysaccharides , Mammals/metabolism
3.
JACS Au ; 3(7): 1864-1875, 2023 Jul 24.
Article in English | MEDLINE | ID: mdl-37502146

ABSTRACT

The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation. An immature smaller form of full-length PTPRA was found to be deficient in O-glycosylation, most likely due to failure to transit the Golgi. N-glycosylation, on the other hand, is dispensable for cell surface expression and contributes less than the extensive O-glycosylation to the overall solution structure of the ectodomain. The glycosylation information is combined with the overall structural features of the ectodomain derived from small-angle X-ray scattering and high-speed atomic force microscopy monitoring to establish a dynamic structural model of the densely glycosylated PTPRA ectodomain. The observed high structural flexibility, as manifested by continuous transitioning from fully to partially extended and fold-back conformations, suggests that the receptor-type phosphatase is anchored to the membrane and kept mostly at a monomeric state through an ectodomain shaped and fully shielded by glycosylation.

4.
Eur J Med Chem ; 232: 114146, 2022 Mar 15.
Article in English | MEDLINE | ID: mdl-35149460

ABSTRACT

Enzymatic synthesis that is commenced by the sugar nucleotide regeneration system (SNRS) protocol can minimize 1) the consumption of exorbitant sugar nucleotides, 2) the amount of transferases required, and 3) byproduct feedback inhibition. In this study, LacNAc extensions/modifications of the N-linked mannose core were carried out efficiently with SNRS with high yields and purities on all branches in a uniform manner. In addition, we demonstrate that with SNRS, bacterial glycosyltransferases exhibit a wide acceptor tolerance for bi- and triantennary mannose core structures as substrates for target oligosaccharides. The synthesized small library of mannose core-based glycans and linear O-glycans were screened for their binding affinity against h-Siglecs 2, 4, 7, 9, 14, 15, and m-Siglec-15 to explore their structure-based binding preferences. Microarray data revealed that each Siglec showed few distinct yet overlapping specificities. An increase in branching from mono to di or tri antennary did not necessarily lead to increasing affinity. Glycans with the disialoside sequence α(2,3)α(2,8)/α(2,6)α(2,8) showed high specificity and affinity for Siglec-7, and sLex α(2,3) exhibited a strong affinity for Siglec-9. Explicit recognition of α(2,6)α(2,3)- linear and α(2,3)α(2,6)-branched glycans by Siglecs-2, 4, and 15, respectively, suggests that these structures can act as potential candidates for the further development of high-affinity ligands.


Subject(s)
Polysaccharides , Sialic Acid Binding Immunoglobulin-like Lectins , Nucleotides , Polysaccharides/chemistry , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sugars
5.
Int J Radiat Oncol Biol Phys ; 109(5): 1521-1532, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33232771

ABSTRACT

PURPOSE: Computed tomographic (CT) scans in adolescents have increased dramatically in recent years. However, the effects of cumulative low-dose exposures on the development of radiation sensitive organs, such as the mammary gland, is unknown. The purpose of this work was to define the effects of dose rate on mammary organ formation during puberty, an especially sensitive window in mammary development. We used a fractionated low-dose x-ray exposure to mimic multiple higher dose CT scans, and we hypothesized that fractionated exposure would have less of an effect on the number of mammary gland defects compared with an acute exposure. METHODS AND MATERIALS: Female mice were subjected to fractionated low-dose x-ray exposure (10 cGy/d for 5 days), acute x-ray exposure (1 × 50 cGy), or sham exposure. As the wide genetic diversity in humans can play a role in a person's response to irradiation, 2 genetically diverse mouse strains differing in radiation sensitivity (BALB/c-sensitive; C57BL/6-resistant) were used to investigate the role of genetic background on the magnitude of the effect. RESULTS: Unexpectedly, our data reveal that multiple low-dose exposures produce greater immune and mammary defects for weeks after exposure compared with controls. The most pronounced defects being increased ductal branching in both strains and a greater percentage of terminal end buds in the BALB/c strain of mice exposed to fractionated radiation compared with sham. Radiation-induced defects near the terminal end bud were also increased in both strains. CONCLUSIONS: The findings suggest that fractionated low-dose exposures are potentially more damaging to organ development compared with an equivalent, single acute exposure and that genetic background is an important parameter modifying the severity of these effects.


Subject(s)
Dose Fractionation, Radiation , Mammary Glands, Animal/radiation effects , Sexual Maturation , Abnormalities, Radiation-Induced/etiology , Age Factors , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/radiation effects , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/radiation effects , Female , Immunity, Cellular/radiation effects , Mammary Glands, Animal/growth & development , Mice , Mice, Inbred BALB C/genetics , Mice, Inbred C57BL/genetics , Morphogenesis/radiation effects , Radiation Exposure/adverse effects , Radiation Injuries, Experimental/etiology , Radiation Tolerance/genetics , Tomography, X-Ray Computed/adverse effects
6.
Life Sci Space Res (Amst) ; 25: 28-41, 2020 May.
Article in English | MEDLINE | ID: mdl-32414491

ABSTRACT

During space travel astronauts will be exposed to a very low, mixed field of radiation containing different high LET particles of varying energies, over an extended period. Thus, defining how human cells respond to these complex low dose exposures is important in ascertaining risk. In the current study, we have chosen to investigate how low doses of three different ion's at various energies uniquely change the kinetics of three different phospho-proteins. A normal hTERT immortalized fibroblast cell line, 82-6, was exposed to a range of lower doses (0.05-0.5 Gy) of radiation of different qualities and energies (Si 1000 MeV/u, Si 300 MeV/u, Si 173 MeV/u, Si 93 MeV/u, Fe 1000 MeV/u, Fe 600 MeV/u, Fe 300 MeV/u, Ti 300 MeV/u, Ti 326 MeV/u, Ti 386 MeV/u), covering a wide span of LET's. Exposed samples were analyzed for the average intensity of signal as a fold over the geometric mean level of the sham controls. Three phospho-proteins known to localize to DNA DSBs following radiation (γH2AX, pATF2, pSMC1) were studied. The kinetics of their response was quantified by flow cytometery at 2 and 24 h post exposure. These studies reveal unique kinetic patterns based on the ion, energy, fluence and time following exposure. In addition, γH2AX phosphorylation patterns are uniquely different from phospho-proteins known to be primarily phosphorylated by ATM. This latter finding suggests that the activating kinase(s), or the phosphatases deactivating these proteins, exhibit differences in their response to various radiation qualities and/ or doses of exposure. Further studies will be needed to better define what the differing kinetics for the kinases activated by the unique radiation qualities plays in the biological effectiveness of the particle.


Subject(s)
Heavy Ions , Linear Energy Transfer , Phosphoproteins/radiation effects , Signal Transduction/radiation effects , Cell Line , DNA Damage/radiation effects , Dose-Response Relationship, Radiation , Humans , Phosphorylation/radiation effects
7.
Methods Mol Biol ; 2132: 85-98, 2020.
Article in English | MEDLINE | ID: mdl-32306317

ABSTRACT

Siglecs are transmembrane receptor-like vertebrate lectins that recognize glycans containing sialic acid. Most Siglecs also interact with intracellular signal transduction molecules, and modulate immune responses. Recombinant soluble Siglecs fused with the fragment crystallizable (Fc) region of immunoglobulin G (Siglec-Fc) are a versatile tool for the investigation of Siglec functions. We describe protocols for the production of recombinant Siglec-Fc, the analysis of expression of Siglec ligands by flow cytometry, and the identification of the Siglec ligand candidates based on proximity labeling.


Subject(s)
Sialic Acid Binding Immunoglobulin-like Lectins/genetics , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sialic Acids/metabolism , Biotin/chemistry , Flow Cytometry , Humans , Ligands , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/chemistry , Signal Transduction , Staining and Labeling
8.
Life Sci Space Res (Amst) ; 20: 101-112, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30797427

ABSTRACT

There exists a wide degree of genetic variation within the normal human population which includes disease free individuals with heterozygote defects in major DNA repair genes. A lack of understanding of how this genetic variation impacts cellular phenotypes that inform cancer risk post heavy ion exposure poses a major limitation in developing personalized cancer risk assessment astronauts. We initiated a pilot study with Human Mammary Epithelial Cell strains (HMEC) derived from wild type, a p16 silenced derivative of wild type, and various genetic variants that were heterozygote for DNA repair genes; BRCA1, BRCA2 and ATM. Cells strains were exposed to different high and low LET radiation qualities to generate both simple and complex lesions and centrosome aberrations were examined as a surrogate marker of genomic instability and cancer susceptibility post different exposures. Our results indicate that centrosome aberration frequency is higher in the genetic variants under study. The aberration frequency increases with dose, complexity of the lesion generated by different radiation qualities and age of the individual. This increase in genomic instability correlates with elevated check-point activation post radiation exposure. These studies suggest that the influence of individual genetics on cell cycle regulation could modify the degree of early genomic instability in response to complex lesions and potentially define cancer predisposition in response to HZE exposure. These results will have significant implications in estimating cancer susceptibility in genetically variant individuals exposed to HZE particles.


Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/pathology , Breast/pathology , Chromosome Aberrations , Cosmic Radiation , Genetic Variation , Breast/metabolism , Breast/radiation effects , Breast Neoplasms/genetics , Breast Neoplasms/radiotherapy , Cells, Cultured , DNA Damage , Female , Humans , Phenotype , Phosphoproteins , Pilot Projects
9.
Aging (Albany NY) ; 9(3): 665-686, 2017 02 28.
Article in English | MEDLINE | ID: mdl-28245431

ABSTRACT

Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by one's age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations and changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.


Subject(s)
Aging/pathology , Breast Neoplasms/pathology , Centrosome/radiation effects , Epithelial Cells/radiation effects , Stem Cells/radiation effects , Adult , Aged , Cells, Cultured , DNA Damage/radiation effects , Disease Susceptibility , Epithelial Cells/pathology , Female , Genomic Instability , Humans , Middle Aged , Stem Cells/physiology
10.
Life Sci Space Res (Amst) ; 9: 19-47, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27345199

ABSTRACT

Robust predictive models are essential to manage the risk of radiation-induced carcinogenesis. Chronic exposure to cosmic rays in the context of the complex deep space environment may place astronauts at high cancer risk. To estimate this risk, it is critical to understand how radiation-induced cellular stress impacts cell fate decisions and how this in turn alters the risk of carcinogenesis. Exposure to the heavy ion component of cosmic rays triggers a multitude of cellular changes, depending on the rate of exposure, the type of damage incurred and individual susceptibility. Heterogeneity in dose, dose rate, radiation quality, energy and particle flux contribute to the complexity of risk assessment. To unravel the impact of each of these factors, it is critical to identify sensitive biomarkers that can serve as inputs for robust modeling of individual risk of cancer or other long-term health consequences of exposure. Limitations in sensitivity of biomarkers to dose and dose rate, and the complexity of longitudinal monitoring, are some of the factors that increase uncertainties in the output from risk prediction models. Here, we critically evaluate candidate early and late biomarkers of radiation exposure and discuss their usefulness in predicting cell fate decisions. Some of the biomarkers we have reviewed include complex clustered DNA damage, persistent DNA repair foci, reactive oxygen species, chromosome aberrations and inflammation. Other biomarkers discussed, often assayed for at longer points post exposure, include mutations, chromosome aberrations, reactive oxygen species and telomere length changes. We discuss the relationship of biomarkers to different potential cell fates, including proliferation, apoptosis, senescence, and loss of stemness, which can propagate genomic instability and alter tissue composition and the underlying mRNA signatures that contribute to cell fate decisions. Our goal is to highlight factors that are important in choosing biomarkers and to evaluate the potential for biomarkers to inform models of post exposure cancer risk. Because cellular stress response pathways to space radiation and environmental carcinogens share common nodes, biomarker-driven risk models may be broadly applicable for estimating risks for other carcinogens.


Subject(s)
Biomarkers/metabolism , Cosmic Radiation/adverse effects , Neoplasms, Radiation-Induced/diagnosis , Dose-Response Relationship, Radiation , Evaluation Studies as Topic , Humans , Neoplasms, Radiation-Induced/etiology , Neoplasms, Radiation-Induced/metabolism , Risk Assessment
11.
J Cell Biochem ; 117(3): 671-83, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26297932

ABSTRACT

Nonerythroid α spectrin (αIISp) and the Fanconi anemia (FA) protein, FANCD2, play critical roles in DNA interstrand cross-link (ICL) repair during S phase. Both are needed for recruitment of repair proteins, such as XPF, to sites of damage and repair of ICLs. However, the relationship between them in ICL repair and whether αIISp is involved in FANCD2's function in repair is unclear. The present studies show that, after ICL formation, FANCD2 disassociates from αIISp and localizes, before αIISp, at sites of damage in nuclear foci. αIISp and FANCD2 foci do not co-localize, in contrast to our previous finding that αIISp and the ICL repair protein, XPF, co-localize and follow a similar time course for formation. Knock-down of αIISp has no effect on monoubiquitination of FANCD2 (FANCD2-Ub) or its localization to chromatin or foci, though it leads to decreased ICL repair. Studies using cells from FA patients, defective in ICL repair and αIISp, have elucidated an important role for αIISp in the function of non-Ub FANCD2. In FA complementation group A (FA-A) cells, in which FANCD2 is not monoubiquitinated and does not form damage-induced foci, we demonstrate that restoration of αIISp levels to normal, by knocking down the protease µ-calpain, leads to formation of non-Ub FANCD2 foci after ICL damage. Since restoration of αIISp levels in FA-A cells restores DNA repair and cell survival, we propose that αIISp is critical for recruitment of non-Ub FANCD2 to sites of damage, which has an important role in the repair response and ICL repair.


Subject(s)
Cell Nucleus/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Spectrin/metabolism , Ubiquitinated Proteins/metabolism , Cell Line , Chromatin/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Humans , Protein Transport , Ubiquitination
12.
Radiat Res ; 184(1): 105-19, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26114329

ABSTRACT

During space travel, astronauts are exposed to a wide array of high-linear energy transfer (LET) particles, with differing energies and resulting biological effects. Risk assessment of these exposures carries a large uncertainty predominantly due to the unique track structure of the particle's energy deposition. The complex damage elicited by high charge and energy (HZE) particles results from both lesions along the track core and from energetic electrons, δ rays, generated as a consequence of particle traversal. To better define how cells respond to this complex radiation exposure, a normal hTERT immortalized skin fibroblast cell line was exposed to a defined panel of particles carefully chosen to tease out track structure effects. Phosphorylation kinetics for several key double-strand break (DSB) response proteins (γ-H2AX, pATF2 and pSMC1) were defined after exposure to ten different high-LET radiation qualities and one low-LET radiation (X ray), at two doses (0.5-2 Gy) and time points (2 and 24 h). The results reveal that the lower energy particles (Fe 300, Si 93 and Ti 300 MeV/u), with a narrower track width and higher number and intensity of δ rays, cause the highest degree of persistent damage response. The persistent γ-H2AX signal at lower energies suggests that damage from these exposures are more difficult to resolve, likely due to the greater complexity of the associated DNA lesions. However, different kinetics were observed for the solely ATM-mediated phosphorylations (pATF2 and pSMC1), revealing a shallow induction at early times and a higher level of residual phosphorylation compared to γ-H2AX. The differing phospho-protein profiles exhibited, compared to γ-H2AX, suggests additional functions for these proteins within the cell. The strong correspondence between the predicted curves for energy deposition per nucleosome for each ion/energy combination and the persistent levels of γ-H2AX indicates that the nature of energy distribution defines residual levels of γ-H2AX, an indicator of unrepaired DSBs. Our results suggest that decreasing the energy of a particle results in more complex damage that may increase genomic instability and increase the risk of carcinogenesis.


Subject(s)
Cosmic Radiation , Activating Transcription Factor 2/analysis , Ataxia Telangiectasia Mutated Proteins/physiology , Cell Cycle/radiation effects , Cells, Cultured , DNA Damage , Fibroblasts/radiation effects , Histones/analysis , Humans , Linear Energy Transfer , Nucleosomes/radiation effects
13.
PLoS One ; 7(7): e41249, 2012.
Article in English | MEDLINE | ID: mdl-22844446

ABSTRACT

Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFß1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFß1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFß1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFß Receptor 1 (TGFßR1) kinase inhibitor, can efficiently block TGFß1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFß1, but also in the absence of TGFß1.


Subject(s)
Epithelial Cells/cytology , Epithelial Cells/radiation effects , Epithelial-Mesenchymal Transition/radiation effects , Proton Therapy , Cell Line , Dose-Response Relationship, Radiation , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/drug effects , Esophagus/cytology , Humans , Phenotype , Phosphorylation/drug effects , Phosphorylation/radiation effects , Signal Transduction/drug effects , Signal Transduction/radiation effects , Smad2 Protein/metabolism , Smad7 Protein/metabolism , Transforming Growth Factor beta1/pharmacology , Up-Regulation/drug effects , Up-Regulation/radiation effects
14.
Radiat Oncol ; 7: 96, 2012 Jun 19.
Article in English | MEDLINE | ID: mdl-22713703

ABSTRACT

BACKGROUND: Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. RESULTS: Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. CONCLUSIONS: These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.


Subject(s)
Cell Cycle/radiation effects , Cell Survival/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA Repair , Linear Energy Transfer , Nuclear Proteins/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Proliferation , DNA-Binding Proteins/metabolism , Endonucleases , Flow Cytometry , HEK293 Cells , Humans , Kinetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Time Factors , Tumor Suppressor Proteins/metabolism
15.
Biochemistry ; 49(26): 5570-81, 2010 Jul 06.
Article in English | MEDLINE | ID: mdl-20518497

ABSTRACT

We have previously shown that there is a deficiency in the structural protein, nonerythroid alpha spectrin (alphaIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of alphaIISp and correlates with a decreased level of repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of alphaIISp is its susceptibility to cleavage by the protease, mu-calpain. We hypothesized that an increased level of mu-calpain cleavage of alphaIISp in FA cells leads to an increased level of breakdown of alphaIISp and that knocking down expression of mu-calpain in FA cells should restore levels of alphaIISp and correct a number of the phenotypic defects observed. The results showed that there is increased mu-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in alphaIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to mu-calpain. We hypothesize that this binding may lead to inhibition of mu-calpain activity in normal cells. Knocking down mu-calpain by siRNA in FA-A cells restored levels of alphaIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicate that FA proteins may play an important role in maintaining the stability of alphaIISp in the cell by regulating its cleavage by mu-calpain. Thus, by reducing the level of breakdown of alphaIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.


Subject(s)
Calpain/metabolism , Chromosomal Instability/drug effects , DNA Repair/drug effects , RNA, Small Interfering/pharmacology , Spectrin/metabolism , Calpain/antagonists & inhibitors , Calpain/genetics , Cell Line , Fanconi Anemia/pathology , Humans , Protein Binding
16.
Biochem Biophys Res Commun ; 381(2): 288-93, 2009 Apr 03.
Article in English | MEDLINE | ID: mdl-19217883

ABSTRACT

Nonerythroid alpha-spectrin (alphaIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that alphaIISp plays in normal human cells and in the repair defect in FA, alphaIISp was knocked down in normal cells using siRNA. Depletion of alphaIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced alphaIISp and XPF nuclear foci. Thus depletion of alphaIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.


Subject(s)
Carrier Proteins/physiology , Chromosomal Instability/genetics , DNA Repair/genetics , Fanconi Anemia/genetics , Microfilament Proteins/physiology , Carrier Proteins/genetics , Cell Line, Tumor , Cross-Linking Reagents/pharmacology , DNA/drug effects , DNA/genetics , Gene Knockdown Techniques , Humans , Microfilament Proteins/genetics , RNA, Small Interfering/genetics , Transfection
17.
Biochemistry ; 48(2): 254-63, 2009 Jan 20.
Article in English | MEDLINE | ID: mdl-19102630

ABSTRACT

The structural protein nonerythroid alpha spectrin (alphaIISp) plays a role in the repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), in which there is a defect in ability to repair such cross-links. We have proposed a model in which alphaIISp, whose stability is dependent on FA proteins, acts as a scaffold to aid in recruitment of repair proteins to sites of damage. In order to get a clearer understanding of the proposed role of FA proteins in maintaining stability of alphaIISp, yeast two-hybrid analysis was carried out to determine whether FA proteins directly interact with alphaIISp and, if so, to map the sites of interaction. Four overlapping regions of alphaIISp were constructed. FANCG interacted with one of these regions and specifically with the SH3 domain in this region of alphaIISp. The site of interaction in FANCG was mapped to a motif that binds to SH3 domains and contains a consensus sequence with preference for the SH3 domain of alphaIISp. This site of interaction was confirmed using site-directed mutagenesis. Two FA proteins that did not contain motifs that bind to SH3 domains, FANCC and FANCF, did not interact with the SH3 domain of alphaIISp. These results demonstrate that one of the FA proteins, FANCG, contains a motif that interacts directly with the SH3 domain of alphaIISp. We propose that this binding of FANCG to alphaIISp may be important for the stability of alphaIISp in cells and the role alphaIISp plays in the DNA repair process.


Subject(s)
Fanconi Anemia Complementation Group G Protein/metabolism , Spectrin/chemistry , Spectrin/metabolism , src Homology Domains/genetics , Amino Acid Motifs , Amino Acid Sequence , Consensus Sequence , Escherichia coli/genetics , Fanconi Anemia Complementation Group G Protein/genetics , Genes, Reporter , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Spectrin/genetics , Two-Hybrid System Techniques , beta-Galactosidase/metabolism
18.
Biochemistry ; 46(50): 14359-68, 2007 Dec 18.
Article in English | MEDLINE | ID: mdl-18020456

ABSTRACT

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.


Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , DNA/metabolism , Apoptosis , Base Sequence , Blotting, Western , Cell Line , DNA/chemistry , DNA/genetics , DNA Damage , Electrophoresis, Polyacrylamide Gel , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Humans , Molecular Sequence Data
19.
Cell Biol Int ; 30(11): 866-78, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16889989

ABSTRACT

Nonerythroid alpha-spectrin (alphaSpIISigma( *)) is a structural protein that has been identified in the nucleus of mammalian cells and shown to be involved in DNA repair. It is also deficient in cells from the clinically diverse genetic disorder Fanconi anemia (FA). In order to get a clearer understanding of the role of alphaSpIISigma( *) in DNA repair, and whether it may have other important functions in the nucleus, studies were undertaken to identify specific alphaSpIISigma( *) protein binding partners in the nucleus. The results demonstrate that multiple proteins co-immunoprecipitate with alphaSpIISigma( *) from nuclear extracts from normal human lymphoblastoid and HeLa cells. These can be grouped into five categories: structural proteins, proteins involved in DNA repair, chromatin remodeling proteins, FA proteins, and transcription and RNA processing factors. These studies indicate that alphaSpIISigma( *) may play a role in a number of diverse and important processes in the nucleus and that a deficiency in this protein, as occurs in FA, could affect a number of critical cellular pathways.


Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Spectrin/metabolism , Chromatin Assembly and Disassembly , DNA Repair , Electrophoresis , Fanconi Anemia/metabolism , HeLa Cells , Humans , Immunoprecipitation , Nuclear Proteins/analysis , Nuclear Proteins/isolation & purification , Protein Binding , RNA Processing, Post-Transcriptional , Recombination, Genetic , Transcription, Genetic
20.
J Cell Sci ; 116(Pt 5): 823-35, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12571280

ABSTRACT

The events responsible for repair of DNA interstrand cross-links in mammalian cells, the proteins involved and their interactions with each other are poorly understood. The present study demonstrates that the structural protein nonerythroid alpha spectrin (alphaSpIISigma*), present in normal human cell nuclei, plays an important role in repair of DNA interstrand cross-links. These results show that alphaSpIISigma* relocalizes to nuclear foci after damage of normal human cells with the DNA interstrand cross-linking agent 8-methoxypsoralen plus ultraviolet A (UVA) light and that FANCA and the known DNA repair protein XPF localize to the same nuclear foci. That alphaSpIISigma* is essential for this re-localization is demonstrated by the finding that in cells from patients with Fanconi anemia complementation group A (FA-A), which have decreased ability to repair DNA interstrand cross-links and decreased levels of alphaSpIISigma*, there is a significant reduction in formation of damage-induced XPF as well as alphaSpIISigma* nuclear foci, even though levels of XPF are normal in these cells. In corrected FA-A cells, in which levels of alphaSpIISigma* are restored to normal, numbers of damage-induced nuclear foci are also returned to normal. Co-immunoprecipitation studies show that alphaSpIISigma*, FANCA and XPF co-immunoprecipitate with each other from normal human nuclear proteins. These results demonstrate that alphaSpIISigma*, FANCA and XPF interact with each other in the nucleus and indicate that there is a close functional relationship between these proteins. These studies suggest that an important role for alphaSpIISigma* in the nucleus is to act as a scaffold, aiding in recruitment and alignment of repair proteins at sites of damage.


Subject(s)
Cell Nucleus/metabolism , Cross-Linking Reagents/pharmacology , Proteins/metabolism , Blotting, Western , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/radiation effects , DNA Adducts/metabolism , DNA Damage , DNA Repair , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Radiation , Fanconi Anemia Complementation Group A Protein , Fluorescent Antibody Technique, Indirect , Humans , Methoxsalen/pharmacology , Nuclear Proteins/metabolism , Precipitin Tests/methods , Spectrin/metabolism , Time Factors , Ultraviolet Rays
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