Gestational choriocarcinoma is a malignant tumor derived from placental trophoblast and the most aggressive member of gestational trophoblastic disease (GTD). Apoptosis-stimulating protein of p53-2 (ASPP2) is a member of ASPP family that transactivates p53 and thereby functions as a tumor suppressor. In this study, the expression profile of ASPP2 in choriocarcinoma was examined in comparison with normal placentas and hydatidiform moles, the latter being a type of GTD that carries malignant potential. Downregulation of ASPP2 messenger RNA and protein was demonstrated in choriocarcinoma by quantitative PCR and immunohistochemistry. ASPP2-transfected choriocarcinoma cells (JEG-3 and JAR) showed an increase in apoptosis and a decrease in cell migration as detected by TdT-mediated dUTP nick end labeling and wound healing assays, respectively, illustrating the complex action of ASPP2 on cell functions other than programmed cell death. Activated Src is known to be important in tumor progression. Transfection of ASPP2 but not ASPP1, another tumor-suppressive ASPP, was found to be related to subsequent decreased Src-pY416 phosphorylation, suggesting an inactivating effect of ASPP2 on Src. Moreover, this ASPP2-mediated inactivation of Src could be abolished by RNA interference with C-terminal Src kinase (Csk), a kinase that can inhibit Src activation. Our findings suggested that the ability of ASPP2 to attenuate Src activation was specific to ASPP2 in a Csk-dependent manner. Taken together, we demonstrated a loss of tumor-suppressive ASPP2 in choriocarcinoma with effects on cell migration and apoptosis. We also unveiled a possible mechanistic link between ASPP2 and Csk/Src signaling pathway, implicating the multiple cellular functions of ASPP2.

Gestational trophoblastic disease encompasses a spectrum of trophoblastic lesions including true neoplasms such as choriocarcinomas and the potentially malignant hydatidiform moles, which may develop persistent disease requiring chemotherapy. ASPP1, a member of apoptosis-stimulating proteins of p53 (ASPPs), is a proapoptotic protein that can stimulate apoptosis through its interaction with p53. We evaluated the promoter methylation and expression profiles of ASPP1 in different trophoblastic tissues and its in vitro functional effect on two choriocarcinoma cell lines, namely JEG-3 and JAR. Significant downregulation of ASPP1 mRNA and protein levels was demonstrated in hydatidiform moles and choriocarcinomas, when compared with normal placentas by quantitative-PCR and immunohistochemistry. The ASPP1 mRNA level was significantly correlated with its hypermethylation status, evaluated with methylation-specific PCR, in placenta and gestational trophoblastic disease samples (P ¼ 0.024). Moreover, lower ASPP1 immunoreactivity was shown in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those that regressed (P ¼ 0.045). A significant correlation was also found between expression of ASPP1 and proliferative indices (assessed by Ki67 and MCM7), apoptotic activity (M30 CytoDeath antibody), p53 and caspase-8 immunoreactivities. An in vitro study showed that ectopic expression of ASPP1 could trigger apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression. The latter suggests a hitherto unreported novel link between ASPP1 and the extrinsic pathway of apoptosis. Our findings suggest that downregulation of ASPP1 by hypermethylation may be involved in the pathogenesis and progress of gestational trophoblastic disease, probably through its effect on apoptosis.

Rheumatoid arthritis (RA) is a common disease characterized by chronic inflammation and irreversible destruction of articular cartilage and bone. In this report, we examined the anti-inflammatory and antiarthritic effects of a novel leflunomide analogue, UTL-5b (also known as GBL-5b), for potential RA treatment. Using a carrageenan-induced edema study in rats, UTL-5b exhibited a better anti-inflammatory effect as compared with leflunomide and its metabolite. The chronic efficacy of UTL-5b was examined using type II collagen-induced arthritis (CIA) mouse model. UTL-5b exerted an anti-arthritic effect in a dosedependant manner with mice given 30 mg/kg exhibiting amelioration of disease early in the trial, but losing statistical significance over time. In contrast, mice treated with 60 mg/kg showed reduced clinical disease parameters early in the trial and these effects were sustained over the ten week trial period. Mechanistic studies indicate that UTL-5b is an inhibitor of TNF- production in vivo. Oral administration of UTL-5b prior to i.p. injection with lethal dose of lipopolysaccharide (LPS)/D-galactosamine markedly reduced the levels of serum TNF- and increased survival rates of animals from septic shock-induced death. Acute toxicity study using mice receiving increasing doses of UTL-5b showed that no animals were killed by UTL5b at 2,000 mg/kg (LD 50 >2,000 mg/kg). Our studies show that UTL-5b represents a novel anti-inflammatory and anti-arthritic agent with potential therapeutic application for RA treatment.

The US Center for Disease Control and Prevention (CDC, 2009) reports that each year 76 million cases of foodborne illness occur with over 300,000 people hospitalized and 5000 deaths. This study, phase I of a continuing study, identifies key areas for food safety improvement. This study is designed as an exploratory evaluation of independently owned and operated delicatessen operations, using Escherichia coli and Staphylococcus aureus as indicators to assess food handling and the public’s risk for pathogenic contamination from commonly served ready-to-eat/take-away foods. The analysis consisted of a comprehensive strategy of laboratory testing of samples for pathogenic contamination, informal field observation of food handling procedures, and the examination of the most recent health inspection reports for each of the 18 operations visited. The deli turkey, cream cheese and lettuce were tested using bacteria indicator plates. The results showed widespread levels of contamination. Of the 54 samples tested for Escherichia coli, 26 showed positive results which are ~45% for E. coli contamination. Of the 54 samples tested for Staphy-lococcus aureus, 31 showed positive results which are ~57% for S. aureus contamination. One issue discovered while conducting the study was the lack of consistent uniform international standards of contamination tolerance levels. The informal field observations and health report analyses revealed widespread temperature violations and numerous instances of poor food handling. The study offers independent practitioners a strategy designed to improve their health inspections scores, food handling, and mitigation of operator liability. Independent operators traditionally do not enjoy the resources of centralized supervision and expert on-staff training; yet in aggregate they account for significant volume in both dollars and the quantity of product served. This study adds needed scrutiny to this important food niche.

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Gestational choriocarcinoma is a malignant tumour derived from placental trophoblast and is the most aggressive member of gestational trophoblastic disease (GTD). Apoptotic stimulating protein of p53-2 (ASPP2) is a tumor suppressor member of the ASPP family which transactivates p53-dependent apoptotic pathway. In this study, we examined the expression profile of ASPP2 in choriocarcinoma in comparison with normal placentas and hydatidiform moles which is a type of GTD that carries malignant potential. Results from quantitative PCR and immunohistochemical studies showed that downregulation of ASPP2 was detected in choriocarcinoma at both RNA and protein levels. ASPP2-transfected choriocarcinoma cells (namely JEG-3 and JAR) showed an increase in apoptosis and a decrease in cell migration as detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and wound healing assays respectively, illustrating the complex action of ASPP2 on cell functions other than program cell death. Transfection of ASPP2 but not ASPP1, another tumor suppressive member of the ASPP family, decreased Src-pY416 phosphorylation, suggesting an inactivating effect of ASPP2 on Src. Moreover, this ASPP2-induced inactivation of Src could be abolished by RNA interference using Csk small interfering RNA (siRNA). All these findings suggested that the ability of ASPP2 to attenuate Src activation was specific to ASPP2 in a Csk dependent manner. Taken together, we demonstrated a loss of tumor suppressive ASPP2 in choriocarcinoma with effects on cell migration as well as apoptosis. We also unveiled a possible mechanistic link between ASPP2 and Csk/Src signaling pathway, implicating the multiple cellular functions of ASPP2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2107. doi:10.1158/1538-7445.AM2011-2107