Anti-Tn Monoclonal Antibody Ameliorates Hyperoxia-Induced Kidney Injury by Suppressing Oxidative Stress and Inflammation in Neonatal Mice.

The Tn antigen, an N-acetylgalactosamine structure linked to serine or threonine, has been shown to induce high-specificity, high-affinity anti-Tn antibodies in mice. Maternal immunization with the Tn vaccine increases serum anti-Tn antibody titers and attenuates hyperoxia-induced kidney injury in neonatal rats. However, immunizing mothers to treat neonatal kidney disease is clinically impractical. This study is aimed at determining whether anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury in neonatal mice. Newborn BALB/c mice were exposed to room air (RA) or normobaric hyperoxia (85% O2) for 1 week. On postnatal days 2, 4, and 6, the mice were injected intraperitoneally with PBS alone or with anti-Tn monoclonal antibodies at 25 µg/g body weight in 50 µL phosphate-buffered saline (PBS). The mice were divided into four study groups: RA + PBS, RA + anti-Tn monoclonal antibody, O2 + PBS, and O2 + anti-Tn monoclonal antibody. The kidneys were excised for histology, oxidative stress, cytokine, and Western blot analyses on postnatal day 7. The O2 + PBS mice exhibited significantly higher kidney injury scores, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor-κB (NF-κB) expression, and cytokine levels than did the RA + PBS mice or RA + anti-Tn mice. Anti-Tn monoclonal antibody treatment reduced kidney injury and cytokine levels to normoxic levels. The attenuation of kidney injury was accompanied by a reduction of oxidative stress and NF-κB expression. Therefore, we propose that anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury by suppressing oxidative stress and inflammation in neonatal mice.

Immunization with anti-Tn immunogen in maternal rats protects against hyperoxia-induced kidney injury in newborn offspring.

BACKGROUND: Neonatal hyperoxia increases oxidative stress and adversely disturbs glomerular and tubular maturity. Maternal Tn immunization induces anti-Tn antibody titer and attenuates hyperoxia-induced lung injury in neonatal rats. METHODS: We intraperitoneally immunized female Sprague-Dawley rats (6 weeks old) with Tn immunogen (50 µg/dose) or carrier protein five times at biweekly intervals on 8, 6, 4, 2, and 0 weeks before the delivery day. The pups were reared for 2 weeks in either room air (RA) or in 85% oxygen-enriched atmosphere (O2), thus generating four study groups, namely carrier protein + RA, Tn vaccine + RA, carrier protein + O2, and Tn vaccine + O2. On postnatal day 14, the kidneys were harvested for the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), and collagen expression and histological analyses. RESULTS: Hyperoxia reduced body weight, induced tubular and glomerular injuries, and increased 8-OHdG and NF-κB expression and collagen deposition in the kidneys. By contrast, maternal Tn immunization reduced kidney injury and collagen deposition in neonatal rats. Furthermore, kidney injury attenuation was accompanied by a reduction in 8-OHdG and NF-κB expression. CONCLUSION: Maternal Tn immunization protects against hyperoxia-induced kidney injury in neonatal rats by attenuating oxidative stress and NF-κB activity. IMPACT: Hyperoxia increased nuclear factor-κB (NF-κB) activity and collagen deposition in neonatal rat kidney. Maternal Tn immunization reduced kidney injury as well as collagen deposition in neonatal rats. Maternal Tn immunization reduced kidney injury and was associated with a reduction in 8-hydroxy-2'-deoxyguanosine and NF-κB activity. Tn vaccine can be a promising treatment modality against hyperoxia-induced kidney injury in neonates.

Anti-Tn Monoclonal Antibody Attenuates Hyperoxia-Induced Lung Injury by Inhibiting Oxidative Stress and Inflammation in Neonatal Mice.

Maternal immunization with Tn vaccine increases serum anti-Tn antibody titers and attenuates hyperoxia-induced lung injury in neonatal rats. This study determined whether anti-Tn monoclonal antibody can protect against hyperoxia-induced lung injury in neonatal mice. Newborn BALB/c mice were exposed to room air (RA) or normobaric hyperoxia (85% O2) for 1 week, creating four study groups as follows: RA + phosphate-buffered saline (PBS), RA + anti-Tn monoclonal antibody, O2 + PBS, and O2 + anti-Tn monoclonal antibody. The anti-Tn monoclonal antibody at 25 µg/g body weight in 50 µl PBS was intraperitoneally injected on postnatal days 2, 4, and 6. Hyperoxia reduced body weight and survival rate, increased mean linear intercept (MLI) and lung tumor necrosis factor-α, and decreased vascular endothelial growth factor (VEGF) expression and vascular density on postnatal day 7. Anti-Tn monoclonal antibody increased neonatal serum anti-Tn antibody titers, reduced MLI and cytokine, and increased VEGF expression and vascular density to normoxic levels. The attenuation of lung injury was accompanied by a reduction in lung oxidative stress and nuclear factor-κB activity. Anti-Tn monoclonal antibody improves alveolarization and angiogenesis in hyperoxia-injured newborn mice lungs through the suppression of oxidative stress and inflammation.

Maternal Tn Immunization Attenuates Hyperoxia-Induced Lung Injury in Neonatal Rats Through Suppression of Oxidative Stress and Inflammation.

Hyperoxia therapy is often required to treat newborns with respiratory disorders. Prolonged hyperoxia exposure increases oxidative stress and arrests alveolar development in newborn rats. Tn antigen is N-acetylgalactosamine residue that is one of the most remarkable tumor-associated carbohydrate antigens. Tn immunization increases the serum anti-Tn antibody titers and attenuates hyperoxia-induced lung injury in adult mice. We hypothesized that maternal Tn immunizations would attenuate hyperoxia-induced lung injury through the suppression of oxidative stress in neonatal rats. Female Sprague-Dawley rats (6 weeks old) were intraperitoneally immunized five times with Tn (50 µg/dose) or carrier protein at biweekly intervals on 8, 6, 4, 2, and 0 weeks before the day of delivery. The pups were reared in room air (RA) or 2 weeks of 85% O2, creating the four study groups: carrier protein + RA, Tn vaccine + RA, carrier protein + O2, and Tn vaccine + O2. The lungs were excised for oxidative stress, cytokine, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, and histological analysis on postnatal day 14. Blood was withdrawn from dams and rat pups to check anti-Tn antibody using western blot. We observed that neonatal hyperoxia exposure reduced the body weight, increased 8-hydroxy-2-deoxyguanosine (8-OHdG) expression and lung cytokine (interleukin-4), increased mean linear intercept (MLI) values, and decreased vascular density and VEGF and PDGF-B expressions. By contrast, Tn immunization increased maternal and neonatal serum anti-Tn antibody titers on postnatal day 14, reduced MLI, and increased vascular density and VEGF and PDGF-B expressions to normoxic levels. Furthermore, the alleviation of lung injury was accompanied by a reduction in lung cytokine and 8-OHdG expression. Therefore, we propose that maternal Tn immunization attenuates hyperoxia-induced lung injury in neonatal rats through the suppression of oxidative stress and inflammation.

Sclerostin vaccination mitigates estrogen deficiency induction of bone mass loss and microstructure deterioration.

Sclerostin (SOST) is a Wnt signaling inhibitor detrimental to osteogenic differentiation and bone mineral acquisition. While control of SOST action delays the pathogenesis of skeletal disorders, the effects of SOST vaccination on the estrogen deficiency-induced bone deterioration remain elusive. In this study, we generated a SOST-Fc fusion protein which was composed of a SOST peptide Pro-Asn-Ala-Ile-Gly along with an IgG Fc fragment. SOST-Fc vaccination increased serum anti-SOST antibody levels and reduced serum SOST concentrations in mice. In vitro, anti-SOST serum attenuated the SOST-induced inhibition of osteogenic gene expression in osteoblast cultures. Administration with SOST-Fc increased serum levels of bone formation marker osteocalcin and alleviated the ovariectomy escalation of serum resorption markers CTX-1 and TRAP5b concentrations. It remarkably lessened the estrogen deficiency-mediated deterioration of bone mineral density, morphometric characteristics of trabecular bone, and mechanical strength of femurs and lumbar spines. The SOST-Fc-treated skeletal tissue exhibited moderate responses to the adverse actions of ovariectomy to bone mineral accretion, osteoclast surface, trabecular separation, and fatty marrow histopathology. SOST-Fc treatment increased serum osteoclast-inhibitory factor osteoprotegrin levels in conjunction with strong Wnt3a, ß-catenin, and TCF4 immunostaining in osteoblasts, whereas it weakened the estrogen deficiency enhancement of osteoclast-promoting factor receptor activator of nuclear factor-κB ligand. Taken together, blockade of SOST action by SOST-Fc vaccination sustains Wnt signaling, which harmonizes bone mineral accretion and resorption reactions and thereby ameliorates ovariectomy-induced bone loss. This study highlights SOST-Fc fusion protein as a new molecular therapeutic potential for preventing from osteoporotic disorders.

Tn (N-acetyl-d-galactosamine-O-serine/threonine) immunization protects against hyperoxia-induced lung injury in adult mice through inhibition of the nuclear factor kappa B activity.

Prolonged hyperoxia exposure leads to inflammation and acute lung injury. Since hyperoxia activates nuclear factor kappa B (NF-κB) and proinflammatory mediators in lung fibroblasts and murine lungs, and proinflammatory cytokines upregulate Tn (N-acetyl-d-galactosamine-O-serine/threonine) expression in human gingival fibroblasts. We hypothesized connections exist between Tn expression and inflammation regulation. Thus, we immunized adult mice with Tn antigen to examine whether Tn vaccine can protect against hyperoxia-induced lung injury by inhibiting NF-κB activity and cytokine expression through the action of anti-Tn antibodies. Five-week-old female C57BL/6NCrlBltw mice were subcutaneously immunized with Tn antigen four times at biweekly intervals, and one additional immunization was performed at 1 week after the fourth immunization. Four days after the last immunization, mice were exposed to room air (RA) or hyperoxia (100% O2) for up to 96 h. Four study groups were examined: carrier protein + RA (n = 6), Tn vaccine + RA (n = 6), carrier protein + O2 (n = 6), and Tn vaccine + O2 (n = 5). We observed that hyperoxia exposure reduced body weight, increased alveolar protein and cytokine (interleukin-6 and tumor necrosis factor-α) levels, increased mean linear intercept (MLI) values and lung injury scores, and increased lung NF-κB activity. By contrast, Tn immunization increased serum anti-Tn antibody titers and reduced the cytokine levels, MLI values, and lung injury scores. Furthermore, the alleviation of lung injury was accompanied by a reduction in NF-κB activity. Therefore, we proposed that Tn immunization attenuates hyperoxia-induced lung injury in adult mice by inhibiting the NF-κB activity.

The cytokine-cosmc signaling axis upregulates the tumor-associated carbohydrate antigen Tn.

Tn antigen (GalNAc-α-O-Ser/Thr), a mucin-type O-linked glycan, is a well-established cell surface marker for tumors and its elevated levels have been correlated with cancer progression and prognosis. There are also reports that Tn is elevated in inflammatory tissues. However, the molecular mechanism for its elevated levels in cancer and inflammation is unclear. In the current studies, we have explored the possibility that cytokines may be one of the common regulatory molecules for elevated Tn levels in both cancer and inflammation. We showed that the Tn level is elevated by the conditioned media of HrasG12V-transformed-BEAS-2B cells. Similarly, the conditioned media obtained from LPS-stimulated monocytes also elevated Tn levels in primary human gingival fibroblasts, suggesting the involvement of cytokines and/or other soluble factors. Indeed, purified inflammatory cytokines such as TNF-α and IL-6 up-regulated Tn levels in gingival fibroblasts. Furthermore, TNF-α was shown to down-regulate the COSMC gene as evidenced by reduced levels of the COSMC mRNA and protein, as well as hypermethylation of the CpG islands of the COSMC gene promoter. Since Cosmc, a chaperone for T-synthase, is known to negatively regulate Tn levels, our results suggest elevated Tn levels in cancer and inflammation may be commonly regulated by the cytokine-Cosmc signaling axis.

Targeting of Topoisomerase I for Prognoses and Therapeutics of Camptothecin-Resistant Ovarian Cancer.

DNA topoisomerase I (TOP1) levels of several human neoplasms are higher than those of normal tissues. TOP1 inhibitors are widely used in treating conventional therapy-resistant ovarian cancers. However, patients may develop resistance to TOP1 inhibitors, hampering chemotherapy success. In this study, we examined the mechanisms associated with the development of camptothecin (CPT) resistance in ovarian cancers and identified evodiamine (EVO), a natural product with TOP1 inhibiting activity that overcomes the resistance. The correlations among TOP1 levels, cancer staging, and overall survival (OS) were analyzed. The effect of EVO on CPT-resistant ovarian cancer was evaluated in vitro and in vivo. TOP1 was associated with poor prognosis in ovarian cancers (p = 0.024). EVO induced apoptosis that was detected using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The tumor size decreased significantly in the EVO treatment group compared with the control group (p < 0.01) in a xenograft mouse model. Effects of drugs targeting TOP1 for prognosis and therapy in CPT-resistant ovarian cancer are anticipated. EVO with TOP1 can be developed as an antiproliferative agent for overcoming CPT resistance in ovarian cancers.

Cancer risk in HBV patients with statin and metformin use: a population-based cohort study.

Chronic infection with hepatitis B virus (HBV) often causes chronic inflammation of the liver with an increased incidence of hepatocellular carcinoma (HCC). HBV-infected individuals may also have an increased incidence of nonliver cancers. Taking statin or metformin may decrease inflammation and infiltration, which may, as a result, reduce the risk of liver cancer or other major cancers in patients with HBV infection. The purpose of this study was to evaluate the hypothesis that statin and metformin could reduce the incidence of liver cancer (HCC) or nonliver cancers in patients with HBV.Using the Taiwan Longitudinal Health Insurance Database 2000 to 2008, this cohort study comprised patients with a recorded diagnosis of HBV (N = 71,847) between January 1, 2000 and December 31, 2008. Each patient was followed-up until the end of 2008. The occurrence of HCC or a nonliver cancer was evaluated in patients who either were or were not taking statin or metformin. Cox proportional hazard regressions were used to evaluate the cancer incidence after adjusting for known confounding factors.In total, 71,824 HBV-infected patients comprised the study cohort. Our study showed that either metformin or statin use was associated with a reduction in the incidence of cancer. This was most prominent in patients taking both statin and metformin. The adjusted hazard ratios (HRs) for patients using only statin were 0.52 (95% confidence interval [CI], 0.48-0.57) for all cancers, 0.28 (95% CI, 0.23-0.35) for liver cancer, and 0.63 (95% CI, 0.57-0.70) for nonliver cancers. Patients taking only metformin had risk-adjusted HRs of 0.82 (95% CI, 0.75-0.90) for all cancers, 0.97 (95% CI, 0.84-1.14) for liver cancer, and 0.75 (95% CI, 0.67-0.84) for nonliver cancers. A dose-dependent effect of statin use for chemoprevention was observed for all cancers, including both liver cancer and nonliver cancers. A dose-dependent effect of metformin was also seen in liver cancer and nonliver cancers without stratification into different cumulative daily doses of statin use.This population-based cohort study investigated the protective effect of statin and metformin against cancer events in patients with HBV infection. Our study demonstrated that either statin or metformin served as independent chemopreventive agents with a dose-response effect in reducing the incidence of cancer with a dose-response effect of the agents and an additive or synergistic effect of combining statin and metformin use in reducing the incidence of many cancers.

CFS-1686 causes cell cycle arrest at intra-S phase by interference of interaction of topoisomerase 1 with DNA.

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.

Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique.

Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

Reciprocal relationship of Tn/NF-κB and sTn as an indicator of the prognosis of oral squamous cell carcinoma.

AIMS: In order to determine whether the expression of tumour-associated carbohydrate antigens (Tn/sTn) and a representative inflammation marker, nuclear factor-κB (NF-κB), is associated with the invasiveness of oral squamous cell carcinoma (OSCC), this study has attempted to investigate the correlation of the aforementioned markers with the well-established invasive pattern grading score (IPGS) and clinicopathological parameters. METHODS AND RESULTS: Specimens from 143 OSCC patients with classified clinicopathological parameters and IPGS were stained immunohistochemically using anti-Tn, sTn and NF-κB antibodies. Our results showed that the expression of both Tn and NF-κB was correlated positively with staging (P = 0.036; P = 0.015), recurrence (P < 0.001; P < 0.001) and distant metastasis (P = 0.005; P = 0.009), as well as with IPGS, while the expression of sTn was correlated inversely. In addition, poor survival was associated with overexpression of Tn and NF-κB but not with expression of sTn. CONCLUSIONS: Our results indicate that a reciprocal relationship between Tn and sTn expression may serve as a reliable indicator for OSCC prognostic evaluation. In addition, expression of Tn rather than sTn may play an important role in deeply invasive OSCC via regulation of NF-κB signalling.

Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis.

Long-term cytokine-mediated inflammation is a risk factor for obesity and type 2 diabetes mellitus (T2DM). Our previous studies reveal significant associations between promoter single nucleotide polymorphisms (SNPs) of interleukin (IL)-4 and T2DM, as well as between SNPs in genes encoding IL-4/IL-4 receptor and high density lipoproteins. Our animal study reveals that IL-4 regulates glucose/lipid metabolism by promoting glucose tolerance and inhibiting lipid deposits. The above results strongly suggest the involvement of IL-4 in energy homeostasis. In the present study, we focus on examining the regulatory mechanism of IL-4 to lipid metabolism. Our results show that IL-4 inhibits adipogenesis by downregulating the expression of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein-α. Additionally, IL-4 promotes lipolysis by enhancing the activity and translocation of hormone sensitive lipase (HSL) in mature adipocytes, which suggests that IL-4 plays a pro-lipolytic role in lipid metabolism by boosting HSL activity. Our results demonstrate that IL-4 harbors pro-lipolysis capacity by inhibiting adipocyte differentiation and lipid accumulation as well as by promoting lipolysis in mature adipocytes to decrease lipid deposits. The above findings uncover the novel roles of IL-4 in lipid metabolism and provide new insights into the interactions among cytokine/immune responses, insulin sensitivity, and metabolism.

Nuclear monomeric integrin αv in cancer cells is a coactivator regulated by thyroid hormone.

Thyroid hormone induces tumor cell and blood vessel cell proliferation via a cell surface receptor on heterodimeric integrin αvß3. We investigated the role of thyroid hormone-induced internalization of nuclear integrin αv monomer. Physiological concentration of thyroxine (free T4, 10(-10) M), but not 3,5,3'-triiodo-l-thyronine (T3), induced cellular internalization and nuclear translocation of integrin αv monomer in human non-small-cell lung cancer (H522) and ovarian carcinoma (OVCAR-3) cells. T4 did not complex with integrin αv monomer during its internalization. The αv monomer was phosphorylated by activated ERK1/2 when it heterodimerized with integrin ß3 in vitro. Nuclear αv complexed with transcriptional coactivator proteins, p300 and STAT1, and with corepressor proteins, NCoR and SMRT. Nuclear αv monomer in T4-exposed cells, but not integrin ß3, bound to promoters of specific genes that have important roles in cancer cells, including estrogen receptor-α, cyclooxygenase-2, hypoxia-inducible factor-1α, and thyroid hormone receptor ß1 in chromatin immunoprecipitation assay. In summary, monomeric αv is a novel coactivator regulated from the cell surface by thyroid hormone for the expression of genes involved in tumorigenesis and angiogenesis. This study also offers a mechanism for modulation of gene expression by thyroid hormone that is adjunctive to the nuclear hormone receptor (TR)-T3 pathway.

Mechanisms of ceramide-induced COX-2-dependent apoptosis in human ovarian cancer OVCAR-3 cells partially overlapped with resveratrol.

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular-signal-regulated kinases (ERK)1/2- and p38 kinase-dependent apoptosis in human ovarian cancer OVCAR-3 cells, concomitant with an increase in the expression of COX-2 and p53 phosphorylation. Blockade of cyclooxygenase-2 (COX-2) activity by siRNA or NS398 correspondingly inhibited ceramide-induced p53 Ser-15 phosphorylation and apoptosis; thus COX-2 appears at the apex of the p38 kinase-mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide-treated cells. Ceramide-treated cells underwent a dose-dependent reduction in trans-membrane potential. Although both ceramide and resveratrol induced the expressions of caspase-3 and -7, the effect of inducible COX-2 was different in caspase-7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis-requiring, ERK1/2-dependent signal transduction pathway and induction of COX-expression as an essential molecular antecedent for subsequent p53-dependent apoptosis. In addition, expressions of caspase-3 and -7 are observed. However, a p38 kinase-dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide-induced apoptosis.

A novel synthetic bipartite carrier protein for developing glycotope-based vaccines.

Development of successful vaccines against glycotopes remains a major challenge. In the current studies, we have successfully developed a novel carrier protein for glycotopes based on the concept of antigen clustering and specific stimulation of T helper cells to mount strong antibody response to glycotopes. The bipartite carrier protein consists of a tandem repeat of a cysteine-rich peptide for docking of clustered glycotopes to effectively activate B cells and an Fc domain for antigen delivery to antigen presenting cells (APCs). To demonstrate its utility, we conjugated the tumor-specific monosaccharide antigen Tn to this novel carrier protein and successfully developed a Tn vaccine against cancer in animal models. The Tn vaccine effectively elicited high-titer IgG1 antibodies against Tn in immunized mice, and effectively suppressed the development of prostate cancer in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. Our results suggest that this novel bipartite carrier protein could be effectively used for developing anti-glycotope vaccines such as the anticancer Tn vaccine.

Phosphorylation-dependent SUMOylation of the transcription factor NF-E2.

Nuclear factor erythroid-derived 2 (NF-E2), a heterodimer composed of p45 and p18, is a transcriptional activator in hematopoietic progenitors. The transcriptional activity of NF-E2 is not only upregulated by SUMOylation but also stimulated by the cAMP-dependent protein kinase A (PKA). However, the relationship between SUMOylation and phosphorylation in the activation of NF-E2 is unclear. In the present studies, we have demonstrated that PKA enhances NF-E2 SUMOylation in an in vitro system using purified proteins, suggesting a possible mechanism for PKA-dependent activation of the NF-E2 transcription factor through SUMOylation.

Evodiamine stabilizes topoisomerase I-DNA cleavable complex to inhibit topoisomerase I activity.

Evodiamine (EVO), an alkaloidal compound isolated from Evodia rutaecarpa (Juss.), has been reported to affect many physiological functions. Topoisomerase inhibitors have been developed in a variety of clinical applications. In the present study, we report the topoisomerase I (TopI) inhibitory activity of EVO, which may have properties that lead to improved therapeutic benefits. EVO is able to inhibit supercoiled plasmid DNA relaxation catalyzed by TopI. Upon treatment 0-10 microM EVO TopI was depleted in MCF-7 breast cancer cells in a concentration-dependent and time-dependent manner in 0-120 min. A K-SDS precipitation assay was performed to measure the extent of Top I-trapped chromosomal DNA. The ability of EVO to cause the formation of a TopI-DNA complex increased in a concentration-dependent manner, in that the DNA trapped increased by 24.2% in cells treated with 30 microM. The results suggest that EVO inhibits TopI by stabilizing the enzyme and DNA covalent complex.

Subcellular transport of EKLF and switch-on of murine adult beta maj globin gene transcription.

Erythroid Krüppel-like factor (EKLF) is an essential transcription factor for mammalian beta-like globin gene switching, and it specifically activates transcription of the adult beta globin gene through binding of its zinc fingers to the promoter. It has been a puzzle that in the mouse, despite its expression throughout the erythroid development, EKLF activates the adult beta(maj) globin promoter only in erythroid cells beyond the stage of embryonic day 10.5 (E10.5) but not before. We show here that expression of the mouse beta(maj) globin gene in the aorta-gonad-mesonephros region of E10.5 embryos and in the E14.5 fetal liver is accompanied by predominantly nuclear localization of EKLF. In contrast, EKLF is mainly cytoplasmic in the erythroid cells of E9.5 blood islands in which beta(maj) is silenced. Remarkably, in a cultured mouse adult erythroleukemic (MEL) cell line, the activation of the beta(maj) globin gene by dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) induction is also paralleled by a shift of the subcellular location of EKLF from the cytoplasm to the nucleus. Blockage of the nuclear import of EKLF in DMSO-induced MEL cells with a nuclear export inhibitor repressed the transcription of the beta(maj) globin gene. Transient transfection experiments further indicated that the full-sequence context of EKLF was required for the regulation of its subcellular locations in MEL cells during DMSO induction. Finally, in both the E14.5 fetal liver cells and induced MEL cells, the beta-like globin locus is colocalized the PML oncogene domain nuclear body, and concentrated with EKLF, RNA polymerase II, and the splicing factor SC35. These data together provide the first evidence that developmental stage- and differentiation state-specific regulation of the nuclear transport of EKLF might be one of the steps necessary for the switch-on of the mammalian adult beta globin gene transcription.

Characterization of the B cell epitopes associated with a truncated form of Pseudomonas exotoxin (PE38) used to make immunotoxins for the treatment of cancer patients.

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.