FA Protein Research Articles

APOE Genotype Disclosure and Lifestyle Advice in a Randomized Intervention Study with Finnish Participants

BackgroundThe APOE ε4 allele is associated with higher risks of cardiovascular diseases and Alzheimer disease than ε3 and ε2. ObjectivesWe studied the effectiveness of dietary and lifestyle guidance and personal genetic risk information [ε4 carrier (ε4+); ε4 noncarrier (ε4−)] as motivators for a healthier lifestyle. MethodsA total of 188 healthy Finnish volunteers (82.4% women; mean ± SD age: 51.0 ± 5.6 y; BMI: 26.0 ± 3.6 kg/m2; total cholesterol: 5.2 ± 0.9 mmol/L) participated in our randomized intervention study. The participants were genotyped for APOE and divided into intervention (INT; INTε4+, n = 33; INTε4−, n = 57) and control groups (CTRL; CTRLε4+, n = 36; CTRLε4−, n = 62). Blood samples, measured observations, and questionnaire data were obtained at baseline and at 1 and 1.5 y. INT participants received their ε4 carrier status at baseline. Monthly Internet-based guidance based on the Finnish Dietary guidelines was provided for all. ResultsThe proportion of SFAs in plasma over time fluctuated less in INTε4+ than in the other groups (P-interaction < 0.05; primary outcome). The lifestyle guidance increased vegetable consumption from 3.5 to 3.6 portions/d, improved the dietary fat quality score by 5.3%, increased the plasma n–3 (ω-3) FA proportion by 7.3%, and decreased the consumption of high-fat/high-sugar foods from 7.3 to 6.5 portions/wk and total- and LDL-cholesterol concentrations by 4.3% and 6.1%, respectively, in the entire participant population (P < 0.05; secondary outcome). Compared with the ε4− participants, ε4+ participants had 2.4% higher plasma n–6 (ω-6) FA, lower C-peptide (3.9 compared with 4.2 nmol/L × h) and sensitive C-reactive protein values, and decreased plasma malondialdehyde concentrations over time (P < 0.05; secondary outcome). ConclusionsLifestyle guidance given to healthy Finnish participants yielded small but beneficial changes. The INTε4+ group did not seem markedly more responsive to the guidance than the other groups.This trial was registered at http://clinicaltrials.gov as NCT03794141.

Autophagy Protects Integrity of Tumor Suppressors From Replication Stress

Several studies support the role of autophagy in maintenance of genome integrity and cancer suppression. However, it remains unclear how autophagy deficiency contributes to mutagenic or epigenetic alterations during the early stages of carcinogenesis. In this study, we show that compromised autophagy led to deregulated activity of Fanconi anemia tumor suppressor pathway, which plays a major role in the suppression of genomic alterations caused by replication stress. We found that expression of FA protein FANCF is significantly reduced in autophagy-deficient cells in a manner that epigenetic alteration drives the defect. Comprehensive genome-wide analysis revealed that autophagy deficiency causes copy number variations at common fragile sites and multiple other loci encoding tumor suppressors, leading to their deregulation. Our results suggest that autophagy deficiency drives genetic and epigenetic catastrophe escalated by the defect in FA-pathway, disrupts proper control of other tumor suppressors and promotes tumorigenesis at the early stage.

Characterization and effects of DDG on the intake and digestibility of finishing bulls in feedlots

The aim of this study was to characterize four corn and sorghum co-products (DDG) in terms of their protein and carbohydrate fractions; we also evaluated the effects of substituting the protein source of the conventional supplement by DDG on consumption and nutrient digestibility in confined finishing cattle. Thirty-six male Nellore cattle with a mean age of 24 months were used. The treatments were: FA: concentrate with corn as an energy source and cottonseed meal as a protein source; DDG50: concentrate with a 50% substitution of the FA protein source by DDG; DDG100: concentrate with 100% substitution of the FA protein source by DDG. The experimental design was completely randomized with three treatments and three replicates (pens) containing four animals per pen. We found that the use of DDG in the finishing phase did not interfere with the animals’ food intake or the digestibility of the nutrients (p &gt; 0.05). Nutrients were used by the animals; therefore, DDGs may be viable substitutes of cottonseed meal. We conclude that the bromatological composition of this co-product is influenced a lot during processing; therefore, the nutritional values of this co-product present in the composition tables may not be true.

A Study of Dietary and BMI Changes Over Time in Mothers of Children with Food Allergies.

Few follow-up surveys have been conducted with regard to the changes in diet of mothers of children with food allergy. We examined changes in food and BMI over time in the mothers of children with food allergies. A total of 146 mothers completed a diet survey twice, with the first conducted in 2013-2016 and the second in 2018, and the dietary changes were examined. Furthermore, among the 120 mothers who eliminated eggs from their diet in the first survey, 98 continued to eliminate eggs and 22 reintroduced eggs during the second survey, and the change over time was examined. Additionally, factors related to BMI were analyzed. We observed a change in the amount of egg intake over time within each group. As the number of children who consumed eggs as the causative food declined, the amount of eggs consumed by the concerned mothers significantly increased (median: 7.8 g/1,000 kcal→12.7 g/1,000 kcal) (p<0.01), even in children who continued to not consume eggs. We found a negative correlation between BMI in mothers of children with FA and vegetable protein. The mothers indicated that their awareness on food allergy improved, which we believe led to increased consumption of foods that had been restricted thus far. BMI was believed to be related to synchronization with the elimination-substitution diet.

Source of supplemental dietary fat interacts with relative proportion of forage source in Holstein dairy cows: Production responses, milk fat composition, and rumen fermentation

The effects of alfalfa hay to corn silage (AH:CS) ratio in high-concentrate diets containing 2 sources of fat supplements on production responses and milk fat secretion of dairy cows were evaluated using 8 lactating multiparous Holstein cows in a duplicated Latin square with a 2 × 2 factorial arrangement of diets with 21-d experimental periods. Diets comprised AH:CS ratios of 70:30 or 30:70 supplemented with either calcium salts of palm fatty acids (FA; CaS-FA, 3.5% of dietary dry matter (DM)) or C16:0 enriched palm fat (Palm-FA; 3.0 % of dietary DM). Dry matter intake (DMI) was greater (1.74 kg/d) in cows fed high compared with low AH:CS ratio. Similarly, the high AH:CS ratio had greater production of milk yield, 3.5% fat-corrected milk, and energy-corrected milk compared with low AH:CS ratio whereas fat sources did not differ in any of these parameters with the exception of greater milk yield by CaS-FA compared with Palm-FA. Milk protein yield was the greatest in the high AH:CS ratio + CaS-FA diet. Milk protein content was greater for diets containing the high AH:CS ratio and also for Palm-FA vs. CaS-FA diets. Milk fat yield was not affected by the treatments, but milk fat content was greater for Palm-FA diets. Feed efficiency (milk yield/DMI) was the lowest for the high AH:CS ratio + Palm-FA diet. The molar proportion of propionate was greater for diets containing high AH: CS ratio, but otherwise volatile FA were not affected by treatments. The high AH:CS ratio had greater de novo synthesized FA (< C16:0) in milk fat without affecting mixed-origin FA (C16:0 plus cis-9 C16:1) or preformed FA (> C16) compared with Palm-FA. CaS-FA had greater proportions and yields of de novo synthesized and mixed-origin FA, and lower preformed FA including many C18 biohydrogenation intermediates such as trans-10 C18:1 and trans-10, cis-12 C18:2 which are due to greater intake of n-6 C18:2 indicating altered ruminal biohydrogenation. CaS-FA also had greater unsaturated FA in milk fat compared with Palm-FA. Overall, the source of fat supplement interacted with AH:CS ratio affecting milk FA profile and protein yield but not milk production of dairy cows.

Fanconi Anemia complementation group C protein in metabolic disorders.

Given importance of 22-Fanconi Anemia (FA) proteins together to act in a signaling pathway in preventing deleterious clinical symptoms, e.g. severe bone marrow failure, congenital defects, an early onset of aging and cancer, studies on each FA protein become increasingly attractive. However, an unbiased and systematic investigation of cellular effects resulting from each FA protein is missing. Here, we report roles of FA complementation C group protein (FANCC) in the protection from metabolic disorders. This study was prompted by the diabetes-prone feature displayed in FANCC knockout mice, which is not typically shown in patients with FA. We found that in cells expressing FANCC at different levels, there are representative alterations in metabolites associated with aging (glycine, citrulline, ornithine, L-asparagine, L-tyrosine, L-arginine, L-glutamine, L-leucine, L-isoleucine, L-valine, L-proline and L-alanine), Diabetes Mellitus (DM) (carbon monoxide, collagens, fatty acids, D-glucose, fumaric acid, 2-oxoglutaric acid, C3), inflammation (inosine, L-arginine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, hypoxanthine, L-methionine), and cancer ( L-methionine, sphingomyelin, acetyl-L-carnitine, L-aspartic acid, L-glutamic acid, niacinamide, phospho-rylethanolamine). We also found that FANCC can act in an FA-pathway-independent manner in tumor suppression. Collectively, featured-metabolic alterations are readouts of functional mechanisms underlying reduced tumorigenicity driven by FANCC, demonstrating close links among cancer, aging, inflammation and DM.

Hematopoietic PBX-interacting protein (HPIP) is over expressed in breast infiltrative ductal carcinoma and regulates cell adhesion and migration through modulation of focal adhesion dynamics.

The scaffolding protein, hematopoietic PBX-interacting protein (HPIP/PBXIP1), regulates cell migration necessary for cancer cell dissemination. However, the mechanism that governs this process remains unknown. We show here that HPIP expression is associated with stages of breast cancer where cell dissemination results in poor patient outcome. Our investigation finds a novel association of HPIP with focal adhesion kinase (FAK) regulating FA dynamics. Interestingly, this interaction that led to activation of FAK protein was mediated by the C-terminal domain of HPIP and not the typical integrin-binding motif. Further, short hairpin RNA-mediated knockdown of FAK expression significantly reduced HPIP-induced cell migration indicating participation of FAK pathway. Live-cell time-lapse imaging and biochemical analysis further established the role of HPIP in microtubule-induced FA disassembly. We also found that HPIP-mediated MAPK activation led to phosphorylation and subsequent activation of calpain2, and the activated calpain2 in turn proteolyses FA protein, talin. Interestingly, HPIP is also proteolysed by calpain2 in breast cancer cells. The proteolysis of HPIP and talin by calpain2, and the activation of calapin2 by HPIP-mediated MAPK phosphorylation, is a novel regulatory axis to modulate the cell migration signal. Together, we have determined HPIP as a novel activator of FAK and a new substrate of calpain2. These molecular interactions between HPIP and FAK, and HPIP and calpain2 regulate cell adhesion and migration through modulation of FA dynamics.

Abstract 2380: Separate but important roles of αSpII and FANCD2 in the FA pathway after DNA interstrand crosslink damage

Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, an increased incidence of cancer, genomic instability, congenital abnormalities and a defect in ability to repair DNA interstrand crosslinks (ICLs). We have previously shown that FA cells have a deficiency in the structural protein, nonerythroid alpha spectrin (SpII), which is critical for repair of DNA ICLs and binds to crosslinked DNA. The FA protein, FANCD2, after monoubiquitination (FANCD2-Ub), has also been shown to be critical for ICL repair. However, the relationship between SpII and FANCD2 and whether they are involved in the same steps or events in this process is not known. The present study was undertaken to address these questions and to examine the role of SpII in the function of non-ubiquitinated FANCD2 (non-Ub FANCD2) and FANCD2-Ub in the FA pathway after ICL damage. Immunofluorescence microscopy was used to determine whether these proteins co-localize in nuclear foci after cells are damaged with an ICL agent, 8-methylpsoralen plus UVA light or mitomycin C. Time course measurements showed that formation of FANCD2 foci in normal human cells is different from those of SpII, FANCA and the ICL repair protein, XPF. FANCD2 foci were visible 2 hours after damage, plateaued at 16 hours and were still present at 72 hours. In contrast, SpII, FANCA and XPF foci were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. SpII foci, over this time course, did not co-localize with FANCD2 foci after ICL damage and this is corroborated by co-immunoprecipitation studies which show that non-Ub FANCD2 and FANCD2-Ub dissociate from SpII after ICL damage. Studies knocking down expression of SpII by siRNA show that SpII is not needed for monoubiquitination of FANCD2 or its localization to chromatin and nuclear foci after ICL damage. This indicates that SpII and FANCD2-Ub are involved in different steps in the repair process, functioning sequentially, independently and/or in parallel to each other within the FA network. However, in FA-A cells, where FANCD2 is present but is not monoubiquitinated and does not form nuclear foci after ICL damage, restoration of SpII levels to normal by knocking down µ-calpain, a protease which cleaves SpII, leads to formation of FANCD2 foci. However, FANCD2 is not monoubiquitinated. This indicates that SpII is needed for formation of non-Ub FANCD2 nuclear foci after ICL damage and suggests that this may be an important process in the ICL repair response, particularly since we have previously shown that restoration of SpII levels in FA-A cells to normal leads to reversal of a number of the phenotypic deficiencies in these cells. Thus both FANCD2-Ub and non-Ub FANCD2 may be involved in distinct but important steps in the repair process; SpII appears to be of critical importance in the role of non-Ub FANCD2 in the FA pathway and in maintenance of genomic stability after ICL damage. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. Separate but important roles of αSpII and FANCD2 in the FA pathway after DNA interstrand crosslink damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2014-2380

The Fanconi anemia/BRCA pathway is involved in DNA interstrand cross-link repair of adriamycin-resistant leukemia cells

The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.

Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats

BackgroundPerinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult.MethodsIn rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines.ResultsPrenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls.ConclusionsFA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy.

Cytokinesis Failure In Fanconi Anemia Pathway Deficient Hematopoietic Cells

AbstractAbstract 878Fanconi Anemia (FA) is a human genomic instability disorder characterized by progressive bone marrow failure, congenital abnormalities and high predisposition to cancer. Bone marrow failure in FA children is attributed partly to the excessive apoptosis and subsequent failure of the hematopoietic stem cell compartment. Understanding the mechanisms of bone marrow failure may allow better diagnosis and treatment for FA and other aplastic anemia patients. There are fourteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O). The FA pathway, regulated by these FA gene products, mediates DNA repair and promotes normal cellular resistance to DNA crosslinking agents. Recent studies suggest that besides maintaining genomic stability, the FA pathway may also play a role in mitosis since FANCD2 and FANCI, the two key FA proteins, are localized to the extremities of ultra-fine DNA bridges (UFBs) linking sister chromatids during cell division (Chan et al, Nat Cell Biol, 11:753-760, 2009; Naim and Rosselli, Nat Cell Biol, 11:761-768, 2009). Whether FA proteins play a direct role in cell division is still unclear. To dissect the mechanisms of bone marrow failure in FA, we have investigated the requirement of FA pathway during mitosis. Initially, we investigated the number of DNA bridges occurring during mitosis in FA-deficient and proficient cells by immunofluorescence and Hoechst staining. FA-deficient patient cell lines (FANCG-deficient and FANCD1/BRCA2-deficient cells) as well as Hela cells with shRNA-mediated knockdown of the FA pathway, displayed an increase in UFBs compared to the FA proficient cells during mitosis. The UFBs were coated by BLM (the RecQ helicase mutated in Bloom syndrome) in early mitosis. In contrast, the FA protein, FANCM, was recruited to the bridges at a later stage. Since the DNA bridges occluding the cleavage furrow potentially induce cytokinesis failure, we assessed FA-deficient cells for multinucleation. The increased number of DNA bridges correlated with a higher rate of binucleated cells in FA deficient Hela cell lines and FA patient-derived fibroblast cells. Moreover, an increase in binucleated cells was also detectable in FA-deficient primary murine bone marrow hematopoietic stem cells (Fancd2-/- cells and Fancg-/- cells) compared to the wild-type cells undergoing proliferation and in FA patient-derived bone marrow stroma cells compared to the stroma cells from normal human bone marrow. Interestingly, the increase in binucleated cells in FA-deficient murine hematopoietic stem cells correlated with the increase in apoptotic cells. Binuclearity, scored by immunostaining for microtubules and Hoechst staining for DNA, was the result of cytokinesis failure as observed by live cell imaging. Therefore, we investigated whether the FA-deficient cells are sensitive to the cytokinesis inhibitors. FA-deficient murine bone marrow lineage negative cells (Fancd2-/- cells) or FA human fibroblast cells were exposed to VX-680 (an inhibitor of Aurora kinases regulating cytokinesis) in culture for 72 hrs and cell survival was assessed. VX-680 caused increased toxicity (reduced cell viability and increased apoptosis) on FA-deficient cells in comparison to the wild-type cells. Enhanced inhibition of clonogenic growth of murine FA-deficient bone marrow cells (Fancd2-/- cells) compared to the wild-type cells was also observed by exposure to VX-680. These data indicated that FA pathway-deficient hematopoietic cells are hypersensitive to cytokinesis inhibitors. Collectively, our results underscore the importance of the FA pathway in mitosis and suggest that the cytokinesis failure observed in FA deficient hematopoietic cells could contribute to bone marrow failure in Fanconi anemia patients. Disclosures:No relevant conflicts of interest to declare.

Screening for large genomic rearrangements of the BRIP1 and CHK1 genes in Finnish breast cancer families

In search for susceptibility genes that could explain an additional portion of familial breast cancer clustering in Finland, we set out to evaluate the presence of large genomic rearrangements in two candidate genes, BRIP1 and CHK1. BRIP1 is a BRCA1 associated protein that is mutated in a fraction of familial breast cancer and Fanconi anemia cases. To date, the role of large BRIP1 deletions in breast cancer susceptibility is not well-characterized. CHK1 is a critical maintainer of cell cycle checkpoints and genomic stability, and is also involved in the BRCA1 and FA protein signalling pathways. Although CHK1 is a very important protein for cell cycle and DNA integrity maintenance control, no mutations in this gene has yet been associated with predisposition to cancer. For the present study, blood DNA from affected index persons of 111 Northern Finnish breast cancer families was assessed for possible constitutional exonic deletions or amplifications in the BRIP1 and CHK1 genes by using the multiplex ligation-dependent probe amplification method. Our results showed that exonic deletions or amplifications affecting the BRIP1 and CHK1 genes seem not to contribute to hereditary breast cancer susceptibility in the Finnish population. To our knowledge, this is the first attempt to determine the existence of large CHK1 deletions in familial breast cancer or in any disease with a hereditary background.

Oxidative-Stress Specific Interaction Between FANCD2 and FOXO3a.

Abstract 1089Poster Board I-111Fanconi anemia (FA) is a human genomic instability syndrome that is uniquely sensitive to oxidative stress. Members of the FA protein family are involved in repair of genetic damage caused by DNA cross-linkers. The molecular pathway in which the FA proteins function in oxidative stress response has not been defined. Here we report functional interaction between the FA protein FANCD2 and the forkhead transcription factor FOXO3a in response to oxidative stress. FOXO3a was colocalized to FANCD2 foci in cells subjected to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA, but could be restored after complementation with a functional FANCA. Consistent with this, a non-monoubiquitinated FANCD2 mutant failed to bind FOXO3a. While both DNA cross-linker mitomycin C and ionizing radiation induced monoubiquitination of FANCD2, neither was able to induce the association of FANCD2 and FOXO3a. This indicates that the FOXO3a-FANCD2 interaction is oxidative stress specific. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway converging FANCD2 and FOXO3a identified in this study is likely to contribute to cellular anti-oxidant defense. DisclosuresNo relevant conflicts of interest to declare.

Fanconi Anemia and the Response of Human Hematopoietic Cells to DNA Damage

Given their critical role in the generation and renewal of the hematopoietic system, we hypothesize that hematopoietic stem and primitive cells employ unusually stringent DNA damage responses to maintain genome stability. We also propose that Fanconi anemia proteins prevent bone marrow failure through their participation in these DNA repair and damage response pathways. We are testing both hypotheses by characterizing cellular responses to DNA double-strand breaks (DSBs) in 3 lineage-negative populations of hematopoietic cells isolated from human umbilical cord blood: hematopoietic stem cell-containing primitive CD34+ CD38− CD45RA−, early progenitor CD34+ CD38+ and late progenitor CD34− CD38+.Two key events in the initial cellular response to DSBs are phosphorylation of H2AX histones in chromatin at or near DSBs, and association of 53BP1 with gamma-H2AX at DSB sites. Once DSBs are detected, they then are repaired by two major pathways: error-prone non-homologous end-joining (NHEJ), utilized throughout the cell cycle, and error-free homologous recombination (HR), used when sister chromatids are present during S/G2 phases of the cell cycle.To study the response to and repair of DSBs in our sorted hematopoietic populations, we exposed quiescent and cytokine-stimulated haematopoietic cells and human fibroblasts to ionizing radiation (IR). Irradiation with 3 Gy rapidly induced formation of 53BP1 foci in all cell types, both cycling and non-cycling. In contrast, X-irradiation only induced foci of the Fanconi anemia protein FANCD2 in cycling cells. The majority of the induced FANCD2 foci colocalized with foci of gamma-H2AX.The subsequent persistence of 53BP1 foci at X-ray induced damage sites differed significantly between the lin− hematopoietic cells and the fibroblasts. During the first 6 hours following IR, cycling primitive and progenitor hematopoietic cells resolved 53BP1 foci more slowly than cycling human primary fibroblasts. In addition, X-ray induced 53BP1 foci persisted even longer in quiescent primitive CD34+ CD38− and early progenitor CD34+ CD38+ cells than in their cycling counterparts, while foci resolution kinetics were similar in quiescent and cycling late progenitor CD34− CD38+ cells and in fibroblasts. We also used a neutral comet assay to determine the kinetics of DSB rejoining following exposure to 15 Gy. We found that resolution of 53BP1 foci is not directly linked to rejoining of DSBs in lin− hematopoietic cells, as they rejoin DSBs more rapidly than primary fibroblasts, yet resolve 53BP1 foci more slowly. The effect is most prominent in primitive CD34− CD38− cells. Both cycling and non-cycling fibroblasts displayed similar kinetics of DSB rejoining. However, quiescent lin− hematopoietic cells displayed persistent breaks compared to cycling counterparts or to fibroblasts. Of particular note, quiescent lin− hematopoietic cells still had not rejoined a significant fraction of induced DSBs 24 hours post irradiation.Our results indicate that human primitive and progenitor hematopoetic cells differ from fibroblasts in their DNA damage responses and DSB repair kinetics. Our findings also suggest that, unlike fibroblasts, progression through the cell cycle plays a key role in the rapid repair of DSBs in hematopoietic cells. Because Lin− hematopoietic cells are more efficient at rejoining DSBs when cycling, we suggest that error-free HR repair plays an important role in DSB repair in these cells. Our detection of DNA damage-induced FANCD2 foci only in cycling lin− hematopoietic cells provides indirect support for this conclusion, as FA proteins are thought to be involved in HR repair. We are currently testing this hypothesis by determining the effect of knocking down FA protein expression on DSB repair and DNA damage responses in our three hematopoietic populations.

A Cell-Free Assay To Screen for Compounds That Modulate the Fanconi/BRCA Pathway.

Fanconi anemia is a multi-gene cancer susceptibility and bone marrow failure syndrome. In the current model, at least eight proteins (FANCA, -B-C,-E, -F, -G, -L, -M) are part of a nuclear complex that is required for the S phase and DNA-damage dependent monoubiquitination of FANCD2. This event is thought to functionally link the FA complex proteins to major breast cancer susceptibility proteins BRCA1, BRCA2 (FANCD1), and the BRCA1-associated helicase Brip1(FANCJ). An understanding of the function of the FA protein network is incomplete not only because some FA proteins are still unidentified, but also because the functions of individual proteins may be interdependent and are difficult to assess out of context with the entire FA network. We recently developed a cell-free system to evaluate the function of the Fanconi/BRCA pathway proteins in an S phase context in Xenopus egg extracts (Sobeck, et al. 2006). Egg extracts are naturally cell-cycle synchronized and mimic the complex interplay of proteins that support cellular DNA replication and regulated DNA damage checkpoint activation. Intricate protein interactions can be assayed in egg extracts, even without knowing each of the components if there is a measurable endpoint. We tested the hypothesis that the mobility shift of FANCD2 could be used as an endpoint in cell-free assays to determine FA pathway function. We found that an antibody specific for the Xenopus FANCD2 protein detected a single band of the expected size in western blots of proteins separated by SDS-PAGE from unstimulated egg extracts. Addition of DNA substrates to extracts resulted in the appearance of a slower mobility form of FANCD2, consistent with the monoubiquitinated FANCD2-L isoform observed in human cells following DNA damage. We measured inhibition or stimulation of xFANCD2-L in the presence of a series of candidate compounds.We found compounds that inhibit FANCD2-L, including curcumin, which was also identified in a cell-based assay as an inhibitor of FANCD2-L (Chirnomas, et al., 2006). Thus, this cell-free assay successfully mirrors the outcome obtained with a small molecule inhibitor of the FA/BRCA pathway in cell-based assays. This new approach is an improvement relative to cell-based screens because the extracts are fully synchronized, which maximizes the sensitivity of detection of S-phase events. Moreover, cell-free screens are rapid, inexpensive and well suited for semi- or high-throughput methods to identify small molecules that modulate the FA/BRCA DNA-damage response pathway.

Analyzing seed weight, fatty acid composition, oil, and protein contents in Vernonia galamensis germplasm by near‐infrared reflectance spectroscopy

AbstractVernonia galamensis is a potential new industrial oilseed crop from the Asteraceae family. The interest in this species is due to the presence of a high vernolic acid content of its seed oil, which is useful in the oleochemical industry for paints and coatings. The development of a rapid, precise, robust, nondestructive, and economical method to evaluate quality components is of major interest to growers, processors, and breeders. NIR reflectance spectroscopy (NIRS) is routinely used for the prediction of quality traits in many crops. This study was conducted to establish a rapid analytical method for determining the quality of intact seeds of V. galamensis. A total of 114 Vernonia accessions were scanned to determine seed weight, FA composition, oil, and protein contents using NIRS. Conventional chemical analysis for FA composition, total oil, and protein contents were performed by GC, Soxhlet extraction, and the Dumas combustion method, respectively. Calibration equations were developed and tested through cross‐validation. The coefficient of determination in cross‐validation for FA ranged from 0.47 (linoleic acid) to 0.55 (vernolic acid), and for oil, protein, and seed weight from 0.71 (oil) to 0.86 (seed protein). It was concluded that NIRS calibration equations developed for seed weight and seed quality traits can be satisfactorily used as early screening methods in V. galamensis breeding programs.

Fanconi anemia protein complex: mapping protein interactions in the yeast 2- and 3-hybrid systems

AbstractFanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive bone marrow failure and cancer predisposition. Eight FA complementation groups have been identified. The FANCA, FANCC, FANCE, FANCF, and FANCG proteins form a nuclear complex required for the monoubiquination of the FANCD2 protein. To investigate the architecture of the FA protein complex, the yeast 2-hybrid system was used to map contact points of the FANCA/FANCG, FANCC/FANCE, and FANCF/FANCG interactions. FANCG was shown to interact with both the amino-terminus of FANCA and the carboxyl-terminal region of FANCF. A FANCG mutant truncated at the carboxyl-terminus retained the ability to interact with FANCA. The interaction between FANCG and FANCF was ablated by a Leu71Pro mutant of FANCG. A central region of FANCE was sufficient for FANCC binding. A Leu554Pro mutant of FANCC failed to interact with FANCE. To further examine complex assembly, the yeast 3-hybrid system was used to investigate the ability of FANCG to act as a molecular bridge in mediating interaction between other FA proteins. FANCG was able to mediate interaction between FANCA and FANCF, as well as between monomers of FANCA. Direct interaction between FANCE and FANCD2 was also demonstrated in the yeast 2-hybrid system. This interaction involving an amino-terminal region of FANCD2 may provide a link between the FA protein complex and its downstream targets. (Blood. 2003;102:136-141)

Reply to “Involvement of oxidative stress in Fanconi's anaemia: from phenotype to FA protein functions”

Reply to “Involvement of oxidative stress in Fanconi's anaemia: from phenotype to FA protein functions”

Involvement of oxidative stress in Fanconi's anaemia: from phenotype to FA protein functions

Involvement of oxidative stress in Fanconi's anaemia: from phenotype to FA protein functions

A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA

AbstractFanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FAgenes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA− cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.