Pascale Adami

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

Pokeweed antiviral protein (PAP), a ribosome‐inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin‐releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH‐PAP shows an IC50 of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID50 of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.

Identification of a biological inactive complex form of pokeweed antiviral protein

Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80°C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.

High expression of QSOX1 reduces tumorogenesis, and is associated with a better outcome for breast cancer patients

IntroductionThe gene quiescin/sulfhydryl oxidase 1, QSOX1, encodes an enzyme directed to the secretory pathway and excreted into the extracellular space. QSOX1 participates in the folding and stability of proteins and thus could regulate the biological activity of its substrates in the secretory pathway and/or outside the cell. The involvement of QSOX1 in oncogenesis has been studied primarily in terms of its differential expression in systemic studies. QSOX1 is overexpressed in prostate cancers and in pancreatic adenocarcinoma. In contrast, QSOX1 gene expression is repressed in endothelial tumors. In the present study, we investigated the role of QSOX1 in breast cancer.MethodsWe analyzed QSOX1 mRNA expression in a cohort of 217 invasive ductal carcinomas of the breast. Moreover, we investigated QSOX1s potential role in regulating tumor growth and metastasis using cellular models in which we overexpressed or extinguished QSOX1 and xenograft experiments.ResultsWe showed that the QSOX1 expression level is inversely correlated to the aggressiveness of breast tumors. Our results show that QSOX1 leads to a decrease in cell proliferation, clonogenic capacities and promotes adhesion to the extracellular matrix. QSOX1 also reduces the invasive potential of cells by reducing cell migration and decreases the activity of the matrix metalloproteinase, MMP-2, involved in these mechanisms. Moreover, in vivo experiments show that QSOX1 drastically reduces the tumor development.ConclusionsTogether, these results suggest that QSOX1 could be posited as a new biomarker of good prognosis in breast cancer and demonstrate that QSOX1 inhibits human breast cancer tumorogenesis.

The autophagy GABARAPL1 gene is epigenetically regulated in breast cancer models

BackgroundThe GABARAP family members (GABARAP, GABARAPL1/GEC1 and GABARAPL2 /GATE-16) are involved in the intracellular transport of receptors and the autophagy pathway. We previously reported that GABARAPL1 expression was frequently downregulated in cancer cells while a high GABARAPL1 expression is a good prognosis marker for patients with lymph node-positive breast cancer.MethodsIn this study, we asked using qRT-PCR, western blotting and epigenetic quantification whether the expression of the GABARAP family was regulated in breast cancer by epigenetic modifications.ResultsOur data demonstrated that a specific decrease of GABARAPL1 expression in breast cancers was associated with both DNA methylation and histone deacetylation and that CREB-1 recruitment on GABARAPL1 promoter was required for GABARAPL1 expression.ConclusionsOur work strongly suggests that epigenetic inhibitors and CREB-1 modulators may be used in the future to regulate autophagy in breast cancer cells.

Improved TRAP-silver staining versus conventional radioactive TRAP assays: quantification of telomerase activity during immortalization and in pathological human endometrium.

OBJECTIVES To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification. DESIGN AND METHODS TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls. RESULTS TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions. CONCLUSIONS TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.

Development of a double sandwich ELISA able to discriminate between free PAP (pokeweed antiviral protein) and complexed PAP in leaf extracts

Abstract Pokeweed antiviral protein (PAP) is a soluble leaf protein produced by the plant Phytolacca americana . It is able to specifically remove an adenine residue from the 28 S ribosomal RNA of eukaryotic ribosomes, thus blocking protein synthesis. Although PAP seems to be synthesized constitutively at very high concentrations during plant development, the mechanism of its synthesis remains unclear since the protein is toxic to pokeweed ribosomes. Recently, our group identified an inactive complexed form of PAP (PAPi) which could play a significant role in the plant protection mechanism during PAP synthesis. In order to estimate the concentration of PAP in plant extracts, a double sandwich ELISA was developed. This test enabled the detection and the quantification of PAP in a crude leaf extract at concentrations ranging from 2–1000 ng ml −1 . It was specifically developed to discriminate between free PAP and PAPi. This report shows that the use of a monoclonal antibody specific to the C-terminal extremity of mature PAP, combined with an anti-PAP polyclonal antibody did not detect PAPi, unless it was denaturated prior to ELISA. Thus, the specificity of this test was restricted to free PAP and the test proved to be a reliable and unique tool to estimate the integrity of PAPi.

Olfactory epithelium destruction by ZnSO4 modified sulfhydryl oxidase expression in mice.

Experimental destruction of olfactory neurons stimulates proliferation and differentiation of local neural precursors and is used as a model to study in vivo mechanisms for degeneration and regeneration of the nervous system. Quiescin-sulfhydryl oxidases (QSOX) have a potential role in the control of the cell cycle or growth regulation and have recently been described in the central nervous system. In mice, we show an expression of QSOX in olfactory mucosa. Northern- and western-blot analysis show that the destruction of olfactory epithelium is associated with a reversible reduction in QSOX expression. Interestingly, QSOX is not localized in olfactory neurons (ON) but in cells of the lamina propria, suggesting that olfactory epithelium destruction may act as a signal of down-regulation of QSOX expression.