A High-throughput Screening of a Chemical Compound Library in Ovarian Cancer Stem Cells.

BACKGROUND: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of "cancer stem cells" could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. METHOD: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. RESULTS AND CONCLUSION: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.

Cytokine profile, HLA restriction and TCR sequence analysis of human CD4+ T clones specific for an immunodominant epitope of Mycobacterium tuberculosis 16-kDa protein.

The identification of immunodominant and universal mycobacterial peptides could be applied to vaccine design and have an employment as diagnostic reagents. In this paper we have investigated the fine specificity, clonal composition and HLA class II restriction of CD4+ T cell clones specific for an immunodominant epitope spanning amino acids 91-110 of the 16-kDa protein of Mycobacterium tuberculosis. Twenty-one of the tested 28 clones had a Th1 profile, while seven clones had a Th0 profile. None of the clones had a Th2 profile. While the TCR AV gene usage of the clones was heterogeneous, a dominant TCR BV2 gene family was used by 18 of the 28 clones. The CDR3 regions of BV2+ T cell clones showed variation in lengths, but a putative common motif R-L/V-G/S-Y/W-E/D was detected in 13 of the 18 clones. Moreover, the last two to three residues of the putative CDR3 loops, encoded by conserved BJ sequences, could also play a role in peptide recognition. Antibody blockade and fine restriction analysis using HLA-DR homozygous antigen-presenting cells established that 16 of 18 BV2+ peptide-specific clones were DR restricted and two clones were DR-DQ and DR-DP restricted. Additionally, five of the 18 TCRBV2+ clones recognized peptide 91-110 in association with both parental and diverse HLA-DR molecules, indicating their promiscuous recognition pattern. The ability of peptide 91-110 to bind a wide range of HLA-DR molecules, and to stimulate a Th1-type interferon (IFN)-gamma response more readily, encourage the use of this peptide as a subunit vaccine component.

C-terminal peptides of interleukin-6 modulate the expression of junB protooncogene and the production of fibrinogen by HepG2 cells.

Interleukin-6 (IL-6) is a 185 amino acid residue helical cytokine with various biological activities (e. g. B cell development, acute phase reaction). We have investigated the role of the 168-185 C-terminal region of IL-6 in the induction of fibrinogen synthesis and expression of junB mRNA using synthetic peptides corresponding to this region. Circular dichroism spectroscopy data suggest that even truncated peptides have a strong tendency to adopt an ordered conformation. Peptides were tested alone or in combination with recombinant hIL-6 on an IL-6 responsive human hepatoma HepG2 cell line. The expression of the protooncogene junB monitored by competitive RT-PCR represents an early, while the fibrinogen production detected by sandwich ELISA a late, marker of IL-6 initiated events. We found that peptides--depending on their structure--modulate spontaneous as well as IL-6 induced fibrinogen production and/or mRNA expression of junB by exhibiting inhibition (in the presence of IL-6) or stimulation (in the absence of IL-6).

Synthesis, solution conformation and interleukin-6-related activities of interleukin-6 peptides.

Interleukin-6 (IL-6) is a member of the cytokine superfamily characterised by a wide variety of biological activities on various cell types. IL-6 exerts pleiotropic activities on hematopoiesis in the immune response and it is the main regulator of acute-phase protein synthesis in liver cells. According to structure-function studies, residues of helix A located at the N-terminal part and/or helix D of the C-terminal part of the protein are involved in the induction of acute-phase responses. Two groups of synthetic peptides corresponding to the 18-46 N-terminal and the 168-185 C-terminal regions of the IL-6 were prepared by solid-phase synthesis to identify structural requirements for induction of fibrinogen or complement factor B synthesis. These peptides were characterised by amino acid analysis, analytical reversed-phase high-performance liquid chromatography, fast atom bombardment mass spectrometry, and circular dichroism (CD) spectroscopy. CD results showed that under appropriate conditions both 18-46 and 168-185 related peptides are able to adopt markedly ordered conformation. We demonstrated that even octapeptides from the N-terminal part and truncated derivatives of the C-terminal region preserved some tendency to display the CD curve of periodic conformation. The ability of the peptides to induce de novo synthesis of acute-phase proteins was evaluated by measuring fibrinogen and complement factor B levels in the supernatants of human HepG2 cells. These results showed that residues 21-34 are critical for eliciting fibrinogen synthesis in the presence or absence of IL-6. In contrast, the full-length 168-185 peptide is required for the induction of complement factor B response.

The effect of WSEWS pentapeptide and WSEWS-specific monoclonal antibodies on constitutive and IL-6 induced acute-phase protein production by a human hepatoma cell line, HEPG-2.

Interleukin-6 receptor (IL-6R) is a member of the cytokine receptor superfamily characterised by the obligatory presence of WSXWS (Trp-Ser-X-Trp-Ser) sequence motif near the transmembrane domain. To more clearly understand the role of this motif, we treated the HepG2 hepatoma cell line with synthetic WSEWS peptide (E is glutamic acid) and checked the spontaneous and IL-6-induced production of acute-phase protein fibrinogen and C1-inhibitor (C1-INH). The peptide revealed a definitely stimulatory effect both on the constitutive synthesis of C1-INH and on the IL-6-induced fibrinogen synthesis of HepG2 cells. Monoclonal antibody specific for WSEWS pentapeptide was stimulatory for the spontaneous secretion of both fibrinogen and C1-INH. However, the IL-6-induced elevations of these acute-phase proteins were oppositely regulated, since the anti-WSEWS monoclonal antibody was inhibitory on the production of fibrinogen induced by IL-6 but strongly augmented the IL-6 induced production of C1-INH. Our study indicates that the WSEWS motif is critical in the effect of IL-6 on the acute-phase protein production influencing either the ligand binding by the WSEWS-containing receptor molecule or the signal transduction.

Characterisation of a recombinant Fv fragment of anti-MUC1 antibody HMFG1.

A recombinant Fv (variable fragment) has been produced for the murine monoclonal antibody HMFG1. This antibody was raised against human milk fat globules and reacts with an epitope (PDTR) in the protein core of MUC1 mucins, which are up-regulated in human breast and other carcinomas. Binding specificity of the Fv fragment has been demonstrated through immunoaffinity purification, and by radioimmunoassay. The affinity constants for this Fv fragment and for the proteolytically produced Fab (antigen binding fragment) of the related humanised antibody HuHMFG1 were determined by monitoring the fluorescence quenching of the antibody fragments whilst adding aliquots of MUC1 related antigenic peptides KAPDTRPAPG and VTSAPDTRPAPG. Using these techniques it has been demonstrated that the products of these different methods of antibody fragmentation are comparable, and suitable for solution structure analysis using nuclear magnetic resonance (NMR) spectroscopy.

Chromatographic characterization of HSV-1 gD 268-284 and IL-6 179-185 synthetic oligopeptides by reversed-phase high-performance liquid chromatography, automated Edman degradation and mass spectrometric analysis.

Two groups of synthetic oligopeptides (namino acid = 7 and 17) were prepared by solid-phase peptide synthesis using the Boc-polystyrene strategy. After deprotection, cleavage and gel permeation, the crude products were analysed by conventional RP-HPLC methods. Separation and isolation of major components were performed on a semi-preparative RP-HPLC column. In order to clarify the primary structure of these products, amino acid analysis, Edman degradation sequence determination and analytical RP-HPLC characterization were applied. The isolated fractions were further assessed by direct molar mass investigation utilizing the fast atom bombardment and 252Cf plasma desorption mass spectrometry. The results with an interleukin-6 oligopeptide corresponding to the 179LRALRQM185 sequence indicate that the single peak product obtained by RP-HPLC separation contains only one component, as verified by amino acid analysis and mass spectrometry. In contrast, the analysis of 268LAPEDPEDSALLEDPVG284-NH2 from HSV-1 gD protein suggests that this large peptide amide showing a single peak after repeated purification by RP-HPLC contains microheterogeneities as revealed by mass spectrometry and sequencing, but not by amino acid analysis.

Epitope mapping of the 273-284 region of HSV glycoprotein D by synthetic branched polypeptide carrier conjugates.

To investigate the antigenicity of a predicted epitope region of herpes simplex virus gD, peptides comprising the 273-284 sequence have been synthesized and conjugated to a branched polypeptide with polylysine backbone (poly[L-Lys-(DL-Alam)], AK). In order to analyze the effect of the carrier on the solution conformation of the potential peptide-epitopes, three peptides (273-284, 273-281 and 276-284) and their polypeptide conjugates were studied by CD spectroscopy in PBS or in TFE. In immunized BALB/c and CBA mice, the level of peptide-, conjugate- and carrier-specific antibody responses were measured. Conjugates with synthetic polypeptide carrier AK induced epitope-specific IgG responses, accompanied by the appearance of a low level of carrier-specific antibodies. The cross-reactivity pattern of induced antibodies revealed the presence of at least two functionally distinct, overlapping epitopes, the availability of which was influenced by flanking residues at the N-terminus. Preimmunization of BALB/c or CBA mice with the [276-284]-AK conjugate resulted in the production of HSV-specific antibodies and in prolonged survival of animals infected with a lethal dose of herpes simplex virus. The degree of protection was comparable to that of [1-23]-AK conjugate (30).