Fas Pathway Research Articles

Pigment epithelium-derived factor promotes Fas-induced cardiomyocyte apoptosis via its receptor phospholipase A2

AimsCardiovascular diseases cause significant morbidity and mortality worldwide. Recently, our research team demonstrated that a multifunctional cytokine, pigment epithelium-derived factor (PEDF), plays a critical role in regulating myocardial infarction. However, few researchers have studied the molecular mechanisms by which PEDF and its receptors influence the pathophysiology of cardiovascular disease. We tested the hypothesis that PEDF affects cardiomyocyte apoptosis under hypoxic conditions and determined the role that its receptors phospholipase A2 (PLA2) and laminin receptor play in this process. Main methodsCardiomyocytes were isolated from neonatal mice and treated with PEDF under normoxic and hypoxic conditions; then, apoptosis was assessed using Annexin V/PI staining and flow cytometry. Western blotting and immunofluorescence staining were used to detect PEDF receptor expression, and siRNA knockdown of PEDF receptors was performed to determine which receptor was involved in mediating cardiomyocyte apoptosis. Key findingsOur results demonstrated that PEDF increased cardiomyocyte apoptosis during hypoxia via Fas and that PEDF receptors were expressed on cardiomyocyte cell membranes. Furthermore, siRNA experiments indicated that the PEDF receptor PLA2 was responsible for inducing cardiomyocyte apoptosis via the Fas pathway. SignificancePEDF promoted Fas-induced cardiomyocyte apoptosis via its receptor PLA2.

ATR-Dependent Phosphorylation of FANCM at Serine 1045 Is Essential for FANCM Functions

Fanconi anemia (FA) is a genome instability syndrome that has been associated with both cancer predisposition and bone marrow failure. FA proteins are involved in cellular response to replication stress in which they coordinate DNA repair with DNA replication and cell-cycle progression. One regulator of the replication stress response is the ATP-dependent DNA translocase FANCM, which we have shown to be hyperphosphorylated in response to various genotoxic agents. However, the significance of this phosphorylation remained unclear. Here, we show that genotoxic stress-induced FANCM phosphorylation is ATR-dependent and that this modification is highly significant for the cellular response to replication stress. We identified serine (S1045) residue of FANCM that is phosphorylated in response to genotoxic stress and this effect is ATR-dependent. We show that S1045 is required for FANCM functions including its role in FA pathway integrity, recruiting FANCM to the site of interstrand cross links, preventing the cells from entering mitosis prematurely, and efficient activation of the CHK1 and G2-M checkpoints. Overall, our data suggest that an ATR-FANCM feedback loop is present in the FA and replication stress response pathways and that it is required for both efficient ATR/CHK1 checkpoint activation and FANCM function.

Neuroprotection of S-nitrosoglutathione against ischemic injury by down-regulating Fas S-nitrosylation and downstream signaling

S-nitrosoglutathione (GSNO) has been reported to protect against ischemic brain injury, however, the underlying mechanisms remain to be elucidated. In the present study, we aimed to investigate the effects of GSNO pre-treatment on the S-nitrosylation of Fas and subsequent events in the Fas pathway, and reveal the correlation between Fas S-nitrosylation and nNOS activation in the rat hippocampal CA1 region after global cerebral ischemia. The results showed that GSNO pre-treatment not only facilitated the survival of hippocampal CA1 pyramidal neurons, but also abolished the activation of pro-apoptotic Caspase-8, Bid, Caspase-9 and Caspase-3. The S-nitrosylation of Fas increased sustainedly after global ischemia, and GSNO blocked such an increase. Global cerebral ischemia/reperfusion promoted the binding between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein 95 that has been reported to activate nNOS, and GSNO inhibited the post-ischemic nNOS activation and NO release. A selective nNOS inhibitor 7-nitroindazole diminished the ischemia/reperfusion-induced Fas S-nitrosylation, suggesting a critical role of endogenous NO from nNOS activation in Fas S-nitrosylation. In addition, pre-administration of GSNO decreased the translocation of Fas to membrane, the formation of CD95hi on the membrane, the internalization of Fas aggregates to plasma, as well as the assembly of DISC/hiDISC. These results indicate that GSNO-induced nNOS inactivation associates with the down-regulation of Fas S-nitrosylation and consequent Fas signal cascade, which is responsible for the GSNO-mediated neuronal survival after brain ischemia. The understanding of GSNO neuroprotection provides a novel strategy to find potential therapeutic targets for ischemic stroke.

Crosstalk between Fas and JNK Determines Lymphocyte Apoptosis after Ionizing Radiation

Radiation simultaneously activate Fas and JNK pathway in lymphocytes but their precise interaction is not clearly understood. Activation of Fas pathway is required for radiation induced apoptosis, however induction of JNK pathway may or may not contribute in apoptosis. Here we report that Fas, Fas associated death domain and total JNK are activated in a dose- and time-dependent radiation exposure. A biphasic pattern of phospho-JNK was found at lower doses (1 and 2 Gy), however at higher doses of radiation phospho-JNK was continuously activated. Interestingly, Fas ligand expression remained biphasic at all the doses of radiation. Our results suggest that the Fas pathway is the major player in radiation-induced apoptosis, with JNK playing a contributory role. We also observed that Fas ligand expression by radiation is dependent on JNK activation. We also propose that radiation activates JNK pathway, but sustained activation is required for maximal induction of apoptosis at later times. Our findings define a mechanism for crosstalk between JNK and Fas pathway in radiation-induced apoptosis, which may lead to the development of new therapeutic strategies.

Activated apoptotic and anti‐survival effects on rat hearts with fructose induced metabolic syndrome

Consumption of fructose has been linked to the development of metabolic syndrome, whereas the cardiomyopathic changes and cardiac apoptosis of dietary high-fructose intake have not yet been clarified. The purpose of this study was to evaluate the effects of high-fructose on cardiac apoptotic and survival pathways. Thirty-two Wistar rats were randomly divided into a control group (CON), which received a standard chow diet, and a fructose-induced metabolic syndrome group (FIMS), which received a 50% fructose-content diet for 13 weeks. Histopathological analysis, TUNEL assays and Western blotting were performed on the excised hearts from both groups. The blood pressure, glucose, insulin, triglyceride and cholesterol levels were significantly increased in the FIMS group, compared with the CON group. The abnormal myocardial architecture, enlarged interstitial space and increased cardiac TUNEL-positive apoptotic cells were observed in the FIMS group. The TNF-α, TNF receptor 1, Fas ligand, Fas receptor, FADD, and activated caspase-3 and 8 protein levels (Fas pathway) and the Bax, Bak, Bax/Bcl-2, Bak/Bcl-xL, cytosolic cytochrome c, and activated caspase-3 and nine protein levels (mitochondria pathway) were increased in the FIMS group compared with those in the CON group. The IGFI, IGFI-R, p-PI3K, p-Akt, Bcl-2 and Bcl-xL protein levels (survival pathway) were all significantly decreased in the FIMS group compared with those in the CON group. High-fructose intake elevated blood pressure and glucose levels; moreover, high-fructose diet activated cardiac Fas-dependent and mitochondria-dependent apoptotic pathways and suppressed the survival pathway, which might provide one possible mechanism for developing heart failure in patients with metabolic syndrome.

Brief Report: Human Pluripotent Stem Cell Models of Fanconi Anemia Deficiency Reveal an Important Role for Fanconi Anemia Proteins in Cellular Reprogramming and Survival of Hematopoietic Progenitors

Fanconi anemia (FA) is a genomic instability disorder caused by mutations in genes involved in replication-dependant-repair and removal of DNA cross-links. Mouse models with targeted deletions of FA genes have been developed; however, none of these exhibit the human bone marrow aplasia. Human embryonic stem cell (hESC) differentiation recapitulates many steps of embryonic hematopoietic development and is a useful model system to investigate the early events of hematopoietic progenitor specification. It is now possible to derive patient-specific human-induced pluripotent stem cells (hiPSC); however, this approach has been rather difficult to achieve in FA cells due to a requirement for activation of FA pathway during reprogramming process which can be bypassed either by genetic complementation or reprogramming under hypoxic conditions. In this study, we report that FA-C patient-specific hiPSC lines can be derived under normoxic conditions, albeit at much reduced efficiency. These disease-specific hiPSC lines and hESC with stable knockdown of FANCC display all the in vitro hallmarks of pluripotency. Nevertheless, the disease-specific hiPSCs show a much higher frequency of chromosomal abnormalities compared to parent fibroblasts and are unable to generate teratoma composed of all three germ layers in vivo, likely due to increased genomic instability. Both FANCC-deficient hESC and hiPSC lines are capable of undergoing hematopoietic differentiation, but the hematopoietic progenitors display an increased apoptosis in culture and reduced clonogenic potential. Together these data highlight the critical requirement for FA proteins in survival of hematopoietic progenitors, cellular reprogramming, and maintenance of genomic stability.

Role of TNF-associated cytokines in renal tubular cell apoptosis induced by hyperoxaluria

Crystal-cell interaction has been reported as one of the most crucial steps in urinary stone formation. Hyperoxaluria-induced apoptotic changes in renal tubular epithelial cells is the end-stage of this interaction. We aimed to evaluate the possible pathways responsible in the induction of apoptosis within the involved cells by assessing the receptoral expression of three different pathways. 16 male Spraque-Dowley rats were divided into two groups: Group 1 (n:8) received only distilled water; Group 2 (n:8) received 0.75% ethylene glycol (EG) in their daily water to induce hyperoxaluria for 2weeks. After 24h urine collection, all animals were euthenized and right kidneys were removed and fixed for immunohistochemical evaluation. Oxalate and creatinine levels (in 24h-urine) and FAS, tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor-2 expressions (in tissue) have been assessed. In addition to TNF (p=0.0007) expression; both FAS (p=0.0129 ) and FASL (p=0.032) expressions significantly increased in animals treated with EG. The expressions of TRAIL (p=0.49) and TRAIL-R2 (p=0.34) receptors did not change statistically after hyperoxaluria induction. Although a positive correlation with cytokine expression density and 24h-urinary oxalate expression (mgoxalate/mgcreatinine) has been assessed with TNF (p=0.04, r=0.82), FAS (p=0.05, r=0.80), FAS-L (p=0.04, r=0.82); no correlation could be demonstrated between TRAIL and TRAIL R2 expressions. Our results indicate that apoptosis induced by oxalate is possibly mediated via TNF and FAS pathways. However, TRAIL and TRAIL-R2 seemed to have no function in the cascade. Correlation with urinary oxalate levels did further strengthen the findings.

Abstract 4221: Mutations in Rad51C in colon tumors.

Abstract Fanconi anemia (FA) is a rare inherited chromosomal instability genetic disorder characterized by congenital musculoskeletal abnormalities, bone marrow failure and cancer susceptibility. At the genetic level, monoubiquitination of FANCD2 and I by an FA core complex (FANCA, B, C, E, F, G M and L) is impaired. Monoubiquitinated FANCD2 and I co-localize with DNA damage response proteins such as Rad51C (FANCO) to mediate homologous recombination (HR) repair. Defects in Rad51C have been documented as the cause of FA complementation group O (FANCO) disorder.In cells the effects include chromosomal instability, hypersensitivity to DNA damaging agents and defective DNA damage repair. Germline mutations for Rad51C gene in patients have been reported in breast and ovarian cancers. Although germline mutations for Rad51C gene are known to cause an FA like phenotype, somatic mutations for Rad51C in colon or other tumors have not been previously described. We evaluated 40 formalin fixed paraffin embedded (FFPE) colorectal adenocarcinomas by an immunofluorescence based triple staining method (FATSI) that we have developed to assess for potential somatic functional deficiency of the FA pathway. Thirteen of the 40 (33%) FFPE samples had been noted to lack FANCD2 foci formation. Among the 40 tumors, we analyzed 31 available frozen tumors that included all 13 foci negative tumors and matching non-tumor tissues for mutations in Rad51C coding region. The tumor and non-tumor tissue samples were procured following an IRB protocol of the Ohio State University. RNA and DNA were isolated from the fresh frozen tissue samples with TRIzol reagent. Direct sequencing of PCR amplicons was performed. Two of the 31 tumor DNA samples contained point mutation at the codon 319 (from CGA to TGA, a stop codon) which technically results in a truncated FANCO protein. The Rad51C protein domain shows amino acid 125 to132 (exon 2 to 5) is important for exerting single stranded DNA dependent ATPase activity, a functional nuclear localization signal located from aa 366 to 370 (exon-9). The mutation substitutes arginine at codon 319 to a premature stop codon terminating the protein abruptly, without producing a full length Rad51C protein. Thus the altered protein is unable to localize in the nucleus. Of interest, both tumors with RAD51C mutations lacked formation of FANCD2 foci by FATSI staining. In both cases, the mutation was absent from the matched non-tumor tissues, suggesting a somatic mutation. The underlining molecular alterations that cause deficiency in FANCD2 foci formation in other 11 foci negative tumors are still under investigation currently. Given that the FA pathway plays essential role in response to DNA interstrand cross-link damage agent, and that cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, our findings have the potential significance of identifying a subpopulation among colorectal cancer patients specially susceptible to these type of treatments. Citation Format: Arjun Kalvala, Li Gao, Brittany Barnwel, Greg Otterson, Miguel A Villalona Calero, Wenrui Duan. Mutations in Rad51C in colon tumors. [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 4221. doi:10.1158/1538-7445.AM2013-4221

Fanconi anemia proteins interact with CtBP1 and modulate the expression of the Wnt antagonist Dickkopf-1

AbstractFanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure, and increased susceptibility to cancer. Of the fifteen FA proteins, Fanconi anemia group C (FANCC) is one of eight FA core complex components of the FA pathway. Unlike other FA core complex proteins, FANCC is mainly localized in the cytoplasm, where it is thought to function in apoptosis, redox regulation, cytokine signaling, and other processes. Previously, we showed that regulation of FANCC involved proteolytic processing during apoptosis. To elucidate the biological significance of this proteolytic modification, we searched for molecular interacting partners of proteolytic FANCC fragments. Among the candidates obtained, the transcriptional corepressor protein C-terminal binding protein-1 (CtBP1) interacted directly with FANCC and other FA core complex proteins. Although not required for stability of the FA core complex or ubiquitin ligase activity, CtBP1 is essential for proliferation, cell survival, and maintenance of chromosomal integrity. Expression profiling of CtBP1-depleted and FA-depleted cells revealed that several genes were commonly up- and down-regulated, including the Wnt antagonist Dickkopf-1 (DKK1). These findings suggest that FA and Wnt signaling via CtBP1 could share common effectors.

FcRγ promotes T cell apoptosis in Fas-deficient mice

Deficiency of Fas or its ligand leads to lymphocyte accumulation, lymphadenopathy, splenomegaly, and autoimmunity in mice and humans. Although the Fas pathway is important for limiting the number of peripheral T cells, inactivation of other pro-apoptotic molecules can also perturb T cell homeostasis independently of and/or in concert with Fas deficiency. Here, we show that combined deficiency of Fas and the Fc receptor common γ signaling chain (FcRγ) results in worsened T cell accumulation in comparison with mice lacking Fas alone, with a particularly marked increase in the number of TCRαβ+CD4−CD8− double negative (DN) T cells. LPR FcRγ−/− mice exhibited reduced survival due to progressive lymphadenopathy. We further investigated the mechanisms whereby FcRγ deficiency promotes lymphoproliferative disease in Fas-mutant mice. Interestingly, there were no significant differences in T cell proliferation between LPR FcRγ+/+ and LPR FcRγ−/− mice in vivo and in vitro. However, FcRγ deletion resulted in significantly decreased peripheral T cell apoptosis. Importantly, the observed increase in apoptosis was restricted to a subset of FcRγ+ T cells. These T cells, but not those lacking FcRγ expression, exhibited increased activation of caspases 3 and 9, indicating a role for FcRγ in driving their apoptosis. FcRγ+ DN T cells also showed enhanced sensitivity to TCR restimulation-induced cell death (RICD) despite lacking Fas, suggesting that TCR stimulation of autoreactive T cells in vivo may serve to eliminate FcRγ+ T cells and thus exert partial control over lymphoproliferative disease. Hence, our data reveal a novel role for FcRγ in promoting peripheral T cell apoptosis in Fas-deficient mice, which may be of significant value in understanding autoimmune lymphoproliferative syndromes.

Cutaneous T-Cell Lymphoma and Defective Apoptosis: Fas Pathway

Apoptosis maintains cellular homeostasis and eliminates unneeded T cells. Fas, a transmembrane death receptor, induces apoptosis by activation of

Upregulation of myelin and lymphocyte protein (MAL) after traumatic spinal cord injury in rats

Myelin and lymphocyte protein (MAL) was identified as a tetraspan proteolipid that is highly expressed by oligodendrocytes and Schwann cells as a component of compact myelin. It has also been reported to be a tumour suppressor and induces apoptosis through the Fas pathway. However, its expression and function in spinal cord injury are still unclear, especially in gray matter. In this study, we performed a spinal cord contusion injury (SCI) model in adult rats and detected the dynamic changes of MAL expression in spinal cord. Western blot and immunohistochemistry analysis revealed that MAL was present in gray and white matter of normal spinal cord. It gradually increased, got a peak at 1 day, and then declined to basal levels after spinal cord injury. Double immunofluorescence staining showed that MAL immunoreactivity was found in neurons and oligodendrocytes. Interestingly, MAL expression was increased predominantly in neurons rather than oligodendrocytes. We also examined the expression profiles of active caspase-3, whose changes were correlated with the expression profiles of MAL. Moreover, co-localization of MAL with active caspase-3 was detected. In conclusion, this is the first description of MAL expression changes in gray matter after spinal cord injury. Our results prompted that MAL might participate in CNS pathophysiology after SCI.

Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity

Abstract 648 IntroductionFanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination.Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes.It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussionIn FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study.To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway.To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues.Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. Disclosures:No relevant conflicts of interest to declare.

Regulation of PCNA Mono-Ubiqutination by FANCD2 and Rad51 in Response to Hydroxyurea.

AbstractAbstract 2377FANCD2 is a key player in FA pathway. It has been shown that FANCD2 can interact with PCNA and with Rad18, the ubiquitin ligase responsible for PCNA mono-ubiquitination. The mono-ubiquitination of PCNA is very important for its function in translesion synthesis. We found that in response to DNA damage agent hyxdroxyurea (HU) the interaction of FANCD2 with Rad18 or Rad51, and the interaction of Rad51 with Rad18 or PCNA was enhanced. FANCD2 is required for increased interaction between Rad51 and Rad18 indicating that FANCD2, Rad51 and Rad18 form a complex in response to HU. Rad18 was required for PCNA mono-ubiquitination in response to HU. FANCD2 deficient cells failed to enhance the interaction between Rad18 and Rad51. Furthermore, PCNA mono-ubiquitination was impaired in FANCD2 deficient cells in response to HU. FANCD2 mono-ubiquitination deficient mutant partially rescued PCNA mono-ubiquitination. The partial mono-ubiquitination of PCNA in response to HU in FANCA deficient mutant confirmed the role of non-ubiquitinated FANCD2 in PCNA mono-ubiquitination. The normal mono-ubiquitination of PCNA in FANCJ deficient mutant confirmed that the effect of FANCD2 in PCNA mon-ubiquitination is not due to FA pathway deficiency. Rad51 was also involved in regulating PCNA mono-ubiquitination in response to HU. Rad51 siRNA knock down cells showed decreased PCNA mono-ubiquitination in response to HU. The role of Rad51 in regulating PCNA mono-ubiquitination did not require BRCA1, indicating that this function is independent of HR. More importantly FANCD2 deficient cells were hypersensitive to HU, whereas FANCD2 mono-ubiquitination deficient mutant cells, FANCD2 corrected cells, FANCA deficient cells and FANCJ deficient cells were not hypersensitive to HU. Our data indicate that FANCD2 plays an important role in PCNA mono-ubiquitination and translesion synthesis partially in a mono-ubiquitination independent manner. Rad51 also plays an important role in PCNA mono-ubiquitination and translesion synthesis in a HR independent fashion. Disclosures:No relevant conflicts of interest to declare.

Diminished Oxidative Stress Responses in Bone Marrow Stromal Cell Lines Derived From Fanconi Anemia (Fanc-D2−/−) Mice

Abstract 4398Fanconi Anemia (FA) is an inherited cancer susceptibility syndrome associated with a defect in one or more of the 15 FA pathway gene products leading to bone marrow failure, increased cellular sensitivity to DNA cross-linking agents and increased risk of myeloid leukemia. Underlying DNA repair vulnerabilities render FancD2−/−, a homozygous deletion recombinant negative mice, sensitive to whole body irradiation. We quantitated hematopoiesis in long term bone marrow cultures, and the radiosensitivity and oxidative responses of derived marrow stromal and IL-3 dependent hematopoietic progenitor cell lines derived from FancD2−/− mice on a C57BL/6 background (generously provided by Drs. A. D’Andrea and K. Parmar, Dana-Farber cancer Inst., Boston, MA). Long-term bone marrow culture stromal cell lines derived from FancD2−/− were compared to FancD2+/− or FancD2+/+. FancD2−/− long term marrow cultures showed decreased cobblestone islands, representative of hematopoietic stem cell generating foci, and production of non-adherent cells compared to wild type FancD2+/+ or heterozygous FancD2 +/− cultures (p = 0.0148) and produced fewer colony forming cells on day 7 and 14 compared to FancD2 +/− or FancD2 +/+ cell lines (p < 0.0001). FancD2−/− stromal cell lines showed decreased DNA repair by comet assay after irradiation to 5Gy (P<0.0001). Following irradiation to 5 or 10 Gy, FancD2−/− cells had an increased cell doubling time (44.1 + 0.7 and 54.2 + 7.8) compared to FancD2 +/+ cells (35.5 + 1.5hrs and 33.6 + 1.3,hrs p = 0.006 and 0.051, respectively). FancD2−/− cells had decreased baseline antioxidant levels compared to the FancD2 +/+ cells (0.55 + 0.11 and 0.91 + 0.17 trolox units, respectively). FancD2−/− cells at 24 hr following 10 Gy irradiation had 0.39 + 0.03 trolox units compared to 0.76 + 0.07 trolox units in FancD2 +/+ cells (p = 0.006). In clonogenic irradiation survival curve assays, FancD2−/− marrow stromal cells were radiosensitive (Do = 1.43 + 0.06 Gy, n = 4.98 + 0.65) compared to FancD2 +/+ (Do 1.70 + 0.09 Gy and n = 8.33 + 0.72, p = 0.0395 and 0.0040, respectively) or FancD2 +/− cells (Do = 1.67 + 0.08 and n = 3.63 + 0.44, p = 0.0348 and 0.1365, respectively). Furthermore, FancD2 +/− cells were more radiosensitive compared to FancD2 +/+ cells ((n = 3.63 + 0.44 or 8.33 + 0.72), p = 0.0003). Thus, the FancD2 gene controls several parameters of sensitivity to oxidative stress and ionizing radiation as demonstrated in mouse long-term marrow cultures and derived cell lines. Disclosures:No relevant conflicts of interest to declare.

TOSO promotes β-cell proliferation and protects from apoptosis

Decreased β-cell mass reflects a shift from quiescence/proliferation into apoptosis, it plays a crucial role in the pathophysiology of diabetes. A major attempt to restore β-cell mass and normoglycemia is to improve β-cell survival. Here we show that switching off the Fas pathway using Fas apoptotic inhibitory protein (Faim/TOSO), which regulates apoptosis upstream of caspase 8, blocked β-cell apoptosis and increased proliferation in human islets. TOSO was clearly expressed in pancreatic β-cells and down-regulated in T2DM. TOSO expression correlated with β-cell turnover; at conditions of improved survival, TOSO was induced. In contrast, TOSO downregulation induced β-cell apoptosis. Although TOSO overexpression resulted in a 3-fold induction of proliferation, proliferating β-cells showed a very limited capacity to undergo multiple rounds of replication. Our data suggest that TOSO is an important regulator of β-cell turnover and switches β-cell apoptosis into proliferation.

Induction of Apoptosis in Glioma Cells and Upregulation of Fas Expression Using the Human Interferon-β Gene

We investigated whether IFN-β inhibits the growth of human malignant glioma and induces glioma cell apoptosis using the human IFN-β gene transfected into glioma cells. A eukaryonic expression vector (pSV2IFNβ) for IFN-β was transfected into the glioma cell line SHG44 using liposome transfection. Stable transfection and IFN-β expression were confirmed using an enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was also assessed by Hoechst staining and electron microscopy. In vivo experiments were used to establish a SHG44 glioma model in nude mice. Liposomes containing the human IFN-β gene were injected into the SHG44 glioma of nude mice to observe glioma growth and calculate tumor size. Fas expression was evaluated using immunohistochemistry. The IFN-β gene was successfully transfected and expressed in the SHG44 glioma cells in vitro. A significant difference in the number of apoptotic cells was observed between transfected and non- transfected cells. Glioma growth in nude mice was inhibited in vivo, with significant induction of apoptosis. Fas expression was also elevated. The IFN-β gene induces apoptosis in glioma cells, possibly through upregulation of Fas. The IFN-β gene modulation in the Fas pathway and apoptosis in glioma cells may be important for the treatment of gliomas.

Overcoming reprogramming resistance of Fanconi anemia cells

AbstractFanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs.

DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of cognate CD8+ T cells independently of the PD-1 pathway (123.19)

Abstract Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of the pancreatic islet beta cells. Cytotoxic CD8+ T cells specific for beta cell antigens are a main contributor to T1D development. CD8+ T cells specific for insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes have been implicated in T1D development in both patients and NOD mice. Targeted delivery to steady-state dendritic cells (DCs) of a beta cell peptide mimotope linked to anti-DEC-205 leads to deletional tolerance of transferred cognate CD8+ T cells in NOD mice. Treatment with anti-DEC-205 linked to NRP-V7, a superagonist mimotope of IGRP206-214, selectively deleted endogenous IGRP206-214-specific T cells, a highly prevalent specificity in NOD mice, and showed promise in T1D prevention. Simultaneous treatments with anti-DEC-205/InsI9, a mimotope peptide of the CD8+ T cell insulin epitope InsB15-23, and anti DEC-205/NRP-V7 are being held to study the effect of targeting multiple CD8+ T cell specificities on T1D prevention. Investigation of the operative tolerance mechanisms revealed that antibody blockade of the PD-1 pathway, often implicated in DC- mediated induction of peripheral tolerance, could not inhibit the deletion of transferred T cells by DEC-205-mediated targeting of their antigen to steady- state DC. Involvement of the CTLA-4 or Fas pathways will be probed to further study the molecular mechanism of tolerance induction. .

Abstract SY11-01: Targeting the Fanconi anemia/BRCA pathway in cancer therapy

Abstract SY11-01: Targeting the Fanconi anemia/BRCA pathway in cancer therapy