Auswahl der wissenschaftlichen Literatur zum Thema „Cell-PCA“

This research intended to discover the significance of miR-138 on the espression profile, proliferation, and the associated regulatory mechanisms in prostate cancer (PCa). RT-qPCR was applied to compare the expression of miR-138 in the PCa cells with a non-cancer cell line, as well as PCa tissue samples with benign prostatic hyperplasia (BPH) samples. The expression of miR-138 notably diminished in PCa tissues and cell lines. Afterward, formerly documented genes, along with bioinformatics analysis, suggested seven possible target genes of miR-138. Among them, CCND1 seemed to have higher expression in the PCa cell lines and tissues. Also, the negative correlation of miR-138 and CCND1 in PCa cell line and tissues was validated using Pearson correlation. CCDN1 was revealed to be the target gene of miR138 in the PC3 cell line based on the results of the luciferase reporter gene assay. Over-expression of miR138-5p suppressed the expression of CCDN1 in PCa cell lines as exhibited by RT-qPCR. Finally, the results of the MTT assay exhibited the inhibitory impact of miR-138 on the proliferative capacities in PCa cell lines. Our research introduces miR-138 as a negative regulator of CCDN1 in the progression of PCa with an inhibitory impact on the proliferation rate of prostate cancer (PCa) cell lines. This regulatory mechanism could be utilized for the design and target selection of remedial miRNA-based approaches.

Abstract Background Prostate cancer (PCa) is one of the most common malignancies in men. YTHDF1 may play an important role in promoting PCa progression, but there is no reports to date on YTHDF1 function in PCa. Objective This study explored whether YTHDF1 could regulate TRIM44 in PCa cells. Methods By querying the TCGA database, we evaluated YTHDF1 expression in PCa, the OS and DFS of YTHDF1, and the correlation between YTHDF1 and TRIM44 in PCa. We constructed vectors to interfere with YTHDF1 expression and overexpress TRIM44 to examine the role of YTHDF1 and TRIM44 in PCa cells. Differentially expressed mRNAs were identified by mRNA sequencing. The levels of YTHDF1, TRIM44, LGR4, SGTA, DDX20, and FZD8 were measured by qRT-PCR and WB was used to determine YTHDF1 and TRIM44 expression. A CCK-8 assay was used to assess cell proliferation. A Transwell chamber assay was used measure cell migration and invasion ability. Results YTHDF1 was highly expressed in both Pca tissues and cells. PCa patient prognosis with high YTHDF1 expression was relatively poor. Cell function experiments showed that inhibiting YTHDF1 expression decreased cell proliferation, migration, and invasion. RNA sequencing analysis revealed that YTHDF1 may promote PCa cell proliferation, migration, and invasion by modulating TRIM44 expression. Cell function experiments further verified that YTHDF1 promoted PCa cell proliferation, migration, and invasion by regulating TRIM44. Conclusions YTHDF1 enhances PCa cell proliferation, migration, and invasion by regulating TRIM44.

Objective: Prostate cancer (PCa) is one of the most prevalent types of cancer among men worldwide. The incidence of PCa is increasing in China. Therefore, there is an urgent need to identify novel diagnostic and prognostic markers for PCa to improve the treatment of the disease. Methods: The Cancer Genome Atlas (TCGA) and GEO database were used to analyze the expression of miR-192, and the relationship between miR-192 and the clinical features of patients with PCa. Cell cycle and cell proliferation assay were used to detect the functional roles of miR-192 in PCa. Bioinformatic analysis for miR-192–5p was performed using gene ontology and KEGG analysis. Results: By analyzing the dataset of TCGA, we found that miR-192 was overexpressed in PCa samples compared to normal tissues and was upregulated in high-grade PCa compared to low-grade PCa. We also observed that higher miR-192 expression was associated with a shorter biochemical recurrence-free survival time. Our results also demonstrated that miR-192 promoted PCa cell proliferation and cell cycle progression. Conclusion: These results suggest that miR-192 may be considered for use as a potential diagnostic and therapeutic target of PCa.

Prostate cancer (PCa), the most incident cancer in men, is tightly regulated by endocrine signals. A number of different PCa cell lines are commonly used for in vitro experiments, but these are of diverse origin, and have very different cell-proliferation rates and hormone-response capacities. By analyzing the gene-expression pattern of main hormone pathways, we systematically compared six PCa cell lines and parental primary cells. We compared these cell lines (i) with each other and (ii) with PCa tissue samples from 11 patients. We found major differences in the gene-expression levels of androgen, insulin, estrogen, and oxysterol signaling between PCa tissue and cell lines, and between different cell lines. Our systematic characterization gives researchers a solid basis to choose the appropriate PCa cell model for the hormone pathway of interest.

Pogostemon cablin (PCa), an herb used in traditional Chinese medicine, is routinely used in the amelioration of different types of gastrointestinal discomfort. However, the mechanisms underlying the cancer suppression activity of PCa in colorectal cancer (CRC) cells have yet to be clarified. The aim of this study was to investigate the anticancer effects of PCa, specifically the induction of apoptosis in CRC cells. The growth inhibition curve of CRC cells following exposure to PCa was detected by an MTT assay. Moreover, PCa combined with 5-FU revealed a synergic effect of decreased cell viability. PCa inhibited cell proliferation and induced cell cycle arrest at the G0/G1 phase and cell apoptosis through regulation of associated protein expression. An in vivo study showed that PCa suppressed the growth of CRC via induction of cell apoptosis with no significant change in body weight or organ histology. Our results demonstrated that PCa inhibits the growth of CRC cells and induces apoptosis in vitro and in vivo, which suggests the potential applicability of PCa as an anticancer agent.

Purpose: To investigate the effects of Krüppel-like factor 8 (KLF8) in prostate cancer (PCa) cell viability and glycolysis, and explore its role as a regulatory factor.Methods: Immunoblot assays were conducted to assess the expression of KLF8 and proteins in AKT/mTOR pathway in PCa cell lines PC-3 and DU145. Cell Counting Kit-8 assays were performed to assess the effect of KLF8 on PCa cell viability. The glycolysis capacity of PCa cells was determined by measuring the levels of glucose intake, lactic acid production, and cellular ATP levels.Results: Depletion of KLF8 decreased the survival of PCa cells in vitro (p < 0.05). KLF8 depletion also inhibited aerobic glucose metabolism in PCa cells (p < 0.05). Further studies confirmed that KLF8 contributed to the growth and glycolysis of PCa cells via the regulation of AKT/mTOR pathway.Conclusion: KLF8 regulates glycolysis in PCa cells by regulating AKT/mTOR signaling pathway and is thus a promising therapeutic target for PCa treatment. Keywords: Krüppel-like factor 8 (KLF8), Prostate cancer (PCa), Aerobic glucose, AKT/mTOR signaling pathway, Therapeutic target

Prostate cancer (PCa) cells rely on the androgen receptor (AR) signaling axis to reprogram metabolism to sustain aberrant proliferation. Whether additional transcription factors participate to this reprogramming remains mostly unknown. To identify such factors, DNA motif analyses were performed in the promoter and regulatory regions of genes sensitive to androgens in PCa cells. These analyses identified two transcription factors, KLF5 and NFYA, as possibly associated with PCa cell metabolism. In clinical datasets, KLF5 and NFYA expression levels were associated with disease aggressiveness, being significantly decreased and increased, respectively, during PCa progression. Their expression was next investigated by qPCR and Western blot in human PCa cell models, revealing a positive regulation of KLF5 by androgens and a correlation between NFYA and AR protein expression status. siRNA-mediated knockdown of KLF5 increased human PCa cell proliferation rate in AR-positive cell models, suggesting a tumor suppressor function. Live-cell metabolic assays showed that knockdown of KLF5 promoted mitochondrial respiration, a key metabolic pathway associated with PCa progression. The opposite was observed for knockdown of NFYA regarding proliferation and respiration. RNA-seq analyses following the knockdown of either KLF5 and NFYA confirmed that both factors regulated distinct metabolic gene signatures, as well as other gene signatures, explaining their differential impact on PCa cell proliferation and metabolism. Overall, our findings identify KLF5 and NFYA as novel regulators of PCa cell metabolism.

Background: Prostate cancer (PCa) is a malignancy with high incidence and the principal cause of cancer deaths in men. GATA binding protein 5 (GATA5) belongs to the GATA gene family. GATA5 has a close association with carcinogenesis, but the role of GATA5 in PCa remains poorly understood. The aim of our present study was to probe into the effect of GATA5 on PCa progression and to elucidate the involved mechanism. Methods: The expression of GATA5 was detected in both PCa samples and PCa cell lines. GATA5 overexpression, PLAGL2 knockdown, and overexpression cell models were generated, then Western blotting experiments were utilized to validate the efficiency of transfection. The effects of GATA5 on PCa cell proliferation, metastasis, apoptosis, cell cycle progression, and EMT were detected in vitro or in vivo. Furthermore, the mechanism by which GATA5 inhibits prostate cancer progression through regulating PLAGL2 via the FAK/PI3K/AKT pathway was also explored. Results: GATA5 expression was downregulated in PCa samples and cell lines. GATA5 overexpression inhibited PCa cell proliferation and metastasis but increased the rate of apoptosis. In addition, we confirmed that GATA5 inhibited prostate cancer progression, including EMT, by regulating PLAGL2 via the FAK/PI3K/AKT pathway. Conclusion: We demonstrated that GATA5, as a tumor suppressor in PCa, inhibits PCa progression by regulating PLAGL2. These results showed that the GATA5/PLAGL2/FAK/PI3K/AKT pathway may become a new therapeutic direction for the treatment of PCa.

Abstract Prostate cancer (PCa) is curable if it is diagnosed and treated in localized and regional stage. However, PCa outcome is poor once it has distant metastasis. Approximately 70% to 100% of PCa deaths have bone metastasis, which may be associated with a specific bone microenvironment. In this study, we investigated the effect and molecular mechanism of osteoblast cells on stimulation of PCa cell migration and invasion and examined the effectiveness of 17α-estradiol on blocking osteoblast-induced PCa cell migration and invasion using in vitro cell analysis. PCa cells (PC3, LNCaP and DU145), osteoblast hFOB, kidney CV-1, breast tumor MCF-7 and liver cancer Huh-7 cells (ATCC) were cultured in RMPI-1640 or DMEM media supplemented with or without fetal bovine serum (FBS) at 37 oC in a 5% CO2-humidified incubator. hFOB condition media (HCM) without FBS were collected at different times of hFOB cell culture. Transwell and wound-healing experiments were used to determine PCa cell migration and invasion. Cell migration and invasion in PC3, DU-145 and LNCaP PCa cells were markedly promoted by co-culturing hFOB osteoblast cells or HCM, but not by cells or condition media originated from kidney (CV-1), liver (Huh-7) and breast (MCF-7). Compared to other non-osteoblast cell conditioned media, HCM had much higher levels of several cytokines and chemokines including tumor growth factor (TGF) β1. Both HCM and TGF-β1 produced a dose- and time-dependent induction of PCa cell migration and invasion as well as SMAD2 phosphorylation without altering cell proliferation. These HCM and TGF-β1 effects were inhibited by a specific TGFβ receptor inhibitor, LY2157299, as well as by 17α-estradiol in a dose-dependent manner. Most intriguing, 17α-estradiol significantly inhibited the HCM and TGF-β1-induced PCa cell migration and invasion at very low nanomolar concentrations, presumably mediated through estrogen receptor β. These findings suggest that TGF-β1 is a major factor in mediating hFOB cell stimulation of PCa cell migration and invasion, and 17α-estradiol is a potential agent to block PCa cell bone metastasis, probably through inhibition of TGF-β1/SMAD2 signal pathway.

Worldwide, 3.575 million people die each year from water-related diseases. The water and sanitation crisis claims more lives than any warfare and is predicted to be one of the biggest global challenges of this century. The rapid, accurate detection of viral pathogens from environmental samples is an ongoing and pertinent challenge in biological engineering. Currently employed methods are lacking in either efficiency or specificity. Here we explore a novel method for virus detection and concurrently use this method to learn more about the very early stages of the virus infection process. The method combines Fourier transform infrared (FTIR) spectroscopy, a method of visualizing molecules based on changes in vibration of particles, and mammalian cells as the biosensor. This method is used to detect and investigate viruses from the family picornaviridae, chosen due to their public health burden and their widespread presence in environmental samples, especially water sources. This family includes the Polioviruses, echoviruses and Coxsackieviruses, among others, many of which are human pathogens.The research outlined in this dissertation is aimed at developing and implementing a new cell-based biosensor that combines the advantages of FTIR spectroscopy with the ability of buffalo green monkey kidney (BGMK) cells to sense diverse stimuli, including infective enteroviruses. The goal of developing this biosensor is outlined in the first paper. The second paper focuses on the application of advanced statistical methods to analyze the spectra to discriminate different viral infections in BGMK cells. Finally, we designed a non-reactive metal biochamber to use with attenuated total reflectance-FTIR. This allowed near-continuous acquisition of real-time spectral data for the study of biochemical changes in mammalian cells caused by poliovirus (PV1) infection. This system is capable of tracking changes in cell biochemistry in minute intervals for many hours at a time.This work demonstrates the feasibility of FTIR spectroscopy in combination with the broad sensitivity of mammalian cells for potential use in the detection of infective viruses from environmental samples. We envision this method being extended to high throughput, automated systems to screen for viruses or other toxins in drinking water systems and medical applications.

L’identité et le devenir de chaque cellule dépend du contenu en protéines et, en particulier, des réseaux d'interactions protéine-protéine (IPP, également appelés interactomes). Les protéines ont la propriété générale de s'engager dans des assemblages macromoléculaires très variés, chacun ayant des fonctions bien distinctes. Par conséquent, identifier les IPP et les lier à des complexes particuliers est un enjeu crucial mais difficile en biologie. Cette problématique a été au cœur de mon travail de doctorat. Une première partie de mon travail est dédiée à l'amélioration d'une méthode existante pour capturer de nouvelles IPP dans le contexte de fonctions biologiques définies. Ce travail a été réalisé avec ERK1, un régulateur clé en aval de plusieurs voies de signalisation impliquées dans de nombreux cancers. Les nouveaux outils ont été testés dans le contexte de fonctions de ERK1 sensibles à deux molécules inhibitrices dans les cellules humaines HEK293T. Une interaction a été confirmée aux niveaux fonctionnel et moléculaire, ainsi qu’en utilisant une stratégie d'imagerie originale pour accéder à la dynamique des IPP dans les cellules vivantes. La deuxième partie de mon travail de doctorat est dédiée à l'établissement d'une méthodologie pionnière pour capturer les IPP endogènes établies par un complexe protéique dimérique spécifique dans les cellules humaines vivantes. Cette méthodologie couple la Complémentation de Fluorescence Bimoléculaire (BiFC) et les technologies démarquage par la biotine de proximité. Plus précisément, elle repose sur l’utilisation d’un petit anticorps (appelé aussi « nanobody ») dirigé contre le complexe BiFC et fusionné à la ligase biotine TurboID. Ces outils ont été établis avec les complexes TAZ/14-3-3e et TAZ/TEAD2, qui traduisent respectivement l'activité de la voie de signalisation Hippo dans le cytoplasme et le noyau. Notre approche a permis de capturer les interactomes spécifiques de ces deux complexes protéiques et d'identifier un nouveau régulateur clé du complexe TAZ/14-3-3e pour contrôler ses fonctions de prolifération cellulaire. Dans son ensemble, mon travail de doctorat a introduit deux méthodologies complémentaires pour déchiffrer les réseaux d'IPP au niveau de fonctions biologiques spécifiques ou pour un complexe protéique spécifique en contexte cellulaire vivant. Ces approches offrent une nouvelle dimension pour comprendre les fonctions des protéines et les interactomes sous-jacents dans des contextes cellulaires normaux ou pathologiques
Cell fate and fitness depend on the protein content, and in particular on the interaction networks (also called interactomes) connecting the different proteins. Proteins have the general property to engage in diverse and occasionally overlapping macromolecular assemblies, each serving distinct purposes. Therefore, identifying protein-protein interactions (PPIs) and linking them to complexes is a crucial yet challenging issue in biology. This issue was at the core of my PhD work. The first part of my work was dedicated to the improvement of an existing method for capturing novel PPIs in the context of defined biological functions. This work was established with ERK1, which is a key downstream regulator of several signaling pathways involved in many different cancers. The new tools were tested in the context of two different inhibitory molecules to capture drug-sensitive interactions of ERK1 in human HEK293T cells. One such interaction was confirmed at the functional and molecular levels, by using an original imaging strategy to access the PPI dynamics in live cells. The second part of my PhD work was dedicated to the establishment of a pioneer methodology to capture endogenous PPIs established by a specific dimeric protein complex in human live cells. This methodology couples Bimolecular Fluorescence Complementation (BiFC) and proximity biotin labelling technologies. More specifically, it is based on a GFP-nanobody directed toward the BiFC complex and fused to the TurboID biotin ligase. Tools were established to map TAZ/14-3-3 and TAZ/TEAD complexes interactome, which translate the activity of the Hippo signaling pathway in the cytoplasm and nucleus, respectively. Our approach allowed capturing specific interactomes of the two dimeric protein complexes and identifying a novel key regulator of TAZ/14-3-3 complexes in a cancer cell context. Collectively, my PhD work introduced two complementary methodologies for deciphering PPI networks in the context of specific biological functions or in the context of a specific protein complex in human live cells. These approaches provide a novel dimension for understanding protein functions and the underlying interactomes in normal or pathological cell contexts

Les virus manipulent la machinerie centrale de l’hôte à leur avantage. Afin de créer un environnement favorable pour leur survie et propagation ils bloquent les réponses antivirales de la cellule hôte. Le virus de la rougeole (MV) possède deux protéines non-structurales MV-V et MV-C, dont on a observé qu’elles jouent un rôle dans ce contournement la réponse interféron de l’hôte et la régulation des voies de signalisation de mort cellulaire. Plusieurs mécanismes moléculaires expliquant la régulation de l’immunité et la mort cellulaire par MV-V ont été proposés, alors que ceux de la protéine MV-C restent encore peu étudiés. On pense que certains facteurs cellulaires contrôlés par MV-C lors de la réplication virale font partie des voies de signalisation d’immunité innée et de mort cellulaire. Dans le but d’identifier ces facteurs ciblés par le virus, nous avons purifié les protéines cellulaires qui interagissent directement ou indirectement avec MV-C. Nous avons utilisé des virus recombinants exprimant certaines protéines virales tagguées et une méthode de purification par affinité couplée à l’identification par spectrométrie de masse. Nous avons pu en extraire une liste de protéines interagissant spécifiquement avec MV-C dans différentes lignées cellulaires. Nous avons ensuite sélectionné certaines protéines appartenant aux voies de signalisation d’immunité innée et de mort cellulaire. Pour tester leur interaction directe avec MV-C, des méthodes de complémentation protéique (PCA) et de transfert d'énergie de résonance de bioluminescence (BRET) ont été utilisées. Ainsi, nous avons mis en évidence l’interaction directe de la protéine C avec les protéines iASPP, p65 et p53 qui contrôlent à la fois les voies de mort cellulaire et d’immunité innée
Viruses manipulate central machineries of host cells to their advantage. They prevent host cell antiviral responses in order to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two non-structural proteins MV-V and MV-C, proposed to counteract the host interferon response and to regulate cell death pathways in various functional assays. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death responses have been proposed, whereas MV-C host protein partners are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. In order to determine which host factors are targeted by MV-C, we captured both direct and indirect host protein partners of MV-C protein. For this we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. A list of host proteins specifically interacting with MV-C protein in different cell lines was identified. Then we have selected proteins that belong to immunity and cell death biological pathways. Direct protein partners of MV-C were determined by applying protein complementation assay (PCA) and the bioluminescence resonance energy transfer (BRET) approach. As a result, we found that MV-C protein specifically interacts p65/iASPP/p53 protein complex that controls both death and innate immunity pathways

This research assessed landuse changes and trends in vegetation cover in the Sandveld, using remote sensing images. Landsat TM satellite images of 1990, 2004 and 2007 were classified using the maximum likelihood classifier into seven landuse classes, namely water, agriculture, fire patches, natural vegetation, wetlands, disturbed veld, and open sands. Change detection using remote sensing algorithms and landscape metrics was performed on these multi-temporal landuse maps using the Land Change Modeller and Patch Analyst respectively. Markov stochastic modelling techniques were used to predict future scenarios in landuse change based on the classified images and their transitional probabilities. MODIS NDVI multi-temporal datasets with a 16day temporal resolution were used to assess seasonal and annual trends in vegetation cover using time series analysis (PCA and time profiling).Results indicated that natural vegetation decreased from 46% to 31% of the total landscape between 1990 and 2007 and these biodiversity losses were attributed to an increasing agriculture footprint. Predicted future scenario based on transitional probabilities revealed a continual loss in natural habitat and increase in the agricultural footprint. Time series analysis results (principal components and temporal profiles) suggested that the landscape has a high degree of overall dynamic change with pronounced inter and intra-annual changes and there was an overall increase in greenness associated with increase in agricultural activity. The study concluded that without future conservation interventions natural habitats would continue to disappear, a condition that will impact heavily on biodiversity and significant waterdependent ecosystems such as wetlands. This has significant implications for the long-term provision of water from ground water reserves and for the overall sustainability of current agricultural practices.

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

To be able to make informed descicions regarding the research of new drug molecules (ligands), it is crucial to have access to information regarding the chemical interaction between the drug and its biological target (protein). Computer-based methods have a given role in drug research today and, by using methods such as molecular docking, it is possible to investigate the way in which ligands and proteins interact. Despite the acceleration in computer power experienced in the last decades many problems persist in modelling these complicated interactions. The main objective of this thesis was to investigate and improve molecular modelling methods aimed to estimate protein-ligand binding. In order to do so, we have utilised chemometric tools, e.g. design of experiments (DoE) and principal component analysis (PCA), in the field of molecular modelling. More specifically, molecular docking was investigated as a tool for reproduction of ligand poses in protein 3D structures and for virtual screening. Adjustable parameters in two docking software were varied using DoE and parameter settings were identified which lead to improved results. In an additional study, we explored the nature of ligand-binding cavities in proteins since they are important factors in protein-ligand interactions, especially in the prediction of the function of newly found proteins. We developed a strategy, comprising a new set of descriptors and PCA, to map proteins based on their cavity physicochemical properties. Finally, we applied our developed strategies to design a set of glycopeptides which were used to study autoimmune arthritis. A combination of docking and statistical molecular design, synthesis and biological evaluation led to new binders for two different class II MHC proteins and recognition by a panel of T-cell hybridomas. New and interesting SAR conclusions could be drawn and the results will serve as a basis for selection of peptides to include in in vivo studies.

Il tumore prostatico si sviluppa come una iper-proliferazione dipendente dagli androgeni delle cellule epiteliali ghiandolari e viene inizialmente affrontato con una terapia anti-androgenica. Tuttavia, dopo una regressione iniziale, il tumore si evolve in una forma più aggressiva indipendente dagli androgeni per cui ad oggi non è stata ancora trovata una cura (Grossmann et al., 2001). Nel nostro laboratorio è stata precedentemente descritta l’attivazione della tirosin chinasi Src in un gruppo di tumori prostatici avanzati, correlata alla fosforilazione in tirosina della RNA-binding protein Sam68 (Pronetto et al., 2004) appartenente alla famiglia STAR (Signal transduction and RNA metabolism), coinvolta nello splicing e nel processamento dei pre-mRNA (Lukong and Richard, 2003). Da qui abbiamo analizzato l’espressione e la funzione di Sam68 in cellule tumorali. Abbiamo osservato che, nei pazienti affetti da PCa, Sam68 è up-regolata sia a livello di mRNA che a livello di proteina. La down-regolazione di Sam68 tramite RNAi o interferire con la sua funzione in vivo con una proteina chimerica (GFP-Sam68GSG ) determinano un rallentamento della proliferazione di cellule tumorali prostatiche e le rendono più suscettibili all’apoptosi indotta da agenti chemoterapici. Questi dati mostrano quindi che l’espressione di Sam68 favorisce la proliferazione delle cellule tumorali di prostata e la sopravvivenza ad agenti chemoterapici (Busà et al., 2007). Ci siamo poi concentrati sullo studio del ruolo di Sam68 in questi eventi a livello molecolare. Abbiamo osservato che nelle cellule PC3, una linea di tumore prostatico non responsiva agli androgeni, in seguito a trattamento con l’agente chemoterapico mitoxantrone Sam68 rilocalizza all’interno di granuli nucleari. Abbiamo caratterizzato questi granuli nucleari ed abbiamo visto che in essi Sam68 colocalizza con diverse RNA-binding protein, sia appartenenti alla famiglia SR (SC35 e ASF/SF2) sia coinvolte nella risposta cellulare allo stress(hnRNP A1 e TIA1) (Guil et al., 2006). Sam68 si accumula anche in granuli citoplasmatici in cui co-localizza sia con hnRNP A1 che con TIA1, confermando si tratti dei cosiddetti stress granules (SGs). Questi dati suggeriscono che Sam68 faccia parte di una risposta cellulare allo stress “RNA-mediata”. Inoltre, poiché questa proteina è in grado di legare gli mRNA e di mediare lo splicing alternativo di pre-mRNA, abbiamo cercato di identificare i target modulati dal trattamento con il chemoterapico. In particolare ci siamo concentrati sullo splicing alternativo di un target già noto di Sam68, CD44 (Matter et al., 2002). Siamo andati ad analizzare lo splicing alternativo del pre-mRNA di CD44 in seguito a una dose-risposta con il mitoxantrone ed abbiamo riscontrato delle variazioni di splicing di alcuni esoni variabili, in particolare per v5 e v6, che sono noti essere regolati da Sam68 (Matter et al., 2002; Cheng and Sharp, 2006). Per valutare se le differenze osservate sono dovute alla rilocalizzazione di Sam68 effettueremo trattamenti con il chemoterapico su cellule silenziate per Sam68. Abbiamo individuato le vie biochimiche e di trasduzione del segnale che si accendono in risposta al trattamento con il mitoxantrone, la via del DNA damage di ATM e la via delle MAPkinasi indotta da stress di JNK1/2 e p38. Attraverso l’uso di inibitori specifici, per ATM, p38 e JNK, abbiamo osservato che queste vie non sono necessarie per la rilocalizzazione di Sam68. E ‘ dunque possibile che cambiamenti di conformazione della cromatina stimolino l’accumulo si Sam68 ed altri splicing factors nei granuli nucleari. Infine, alcune evidenze emerse nel corso dei nostri studi suggeriscono un nuovo ruolo di Sam68 nel metabolismo degli rRNA. In un esperimento di co-immunoprecipitazione per Sam68, tra le proteine lin grado di interagire con Sam68 abbiamo identificato Nucleolina, una proteina nucleolare coinvolta nel metabolismo del rRNA (Rickards et al., 2007). Abbiamo confermato quest’interazione e mappato la regione di legame nel dominio carbossi-terminale di Sam68. Inoltre, in un esperimento di co-immunoprecipitazione per Sam68 ed RNA, abbiamo identificato il 18S rRNA tra gli RNA legati da questa proteina. Abbiamo inoltre osservato, attraverso esperimenti di FISH confermati poi da real time PCR, che la down-regolazione di Sam68 determina un aumento significativo dei livelli del pre-rRNA in confronto alle cellule di controllo. Infine esperimenti di ChIP hanno dimostrato che Sam68 è in grado di legare il rDNA a cavallo della regione codificante per il 18S rRNA. Questi risultati suggeriscono un nuovo ruolo di Sam68 nel metabolismo del pre-rRNA. References: Busà R, Paronetto MP, Farini D, Pierantozzi E, Botti F, Angelini DF, Attisani F, Vespasiani G, Sette C., Oncogene 2007 26(30):4372-82. Cheng C, Sharp PA. (2006). Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol. 26(1):362-70. Grossmann ME, Tindall DJ (2001). Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst. 93:1687-97; Guil S, Long JC, Cáceres JF. (2006). hnRNP A1 relocalization to the stress granules reflects a role in the stress response. Mol Cell Biol. 26(15):5744-58. Lukong KE, Richard S (2003). Sam68, the KH domain-containing superSTAR. Bioch. Biophys. Acta 1653: 73-86. Matter N, Herrlich P, Konig H (2002). Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420:691-695. Paronetto MP, Farini D, Sammarco I, Maturo G, Vespasiani G, Geremia R et al (2004). Expression of a truncated form of the c-Kit tyrosine kinase receptor and activation of Src kinase in human prostatic cancer. Am. J. Path. 164:1243-1251; Rickards B, Flint SJ, Cole MD, LeRoy G. (2007). Nucleolin is required for RNA polymerase I transcription in vivo. Mol Cell Biol. 27(3):937-48.
Prostate carcinoma (PCa) is one of the main causes of death in the western male population. Although initially controlled by anti-androgenic therapies, PCa often evolves to become androgen-insensitive and highly metastatic. A predominant role in the development of androgen-refractoriness is played by the upregulation of signal transduction pathways that allow prostate cancer cells to autonomously produce their own requirements of growth factors and nutrients (Grossmann et al., 2001). The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and in our laboratory we have observed that its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68 (Paronetto et al., 2004), belonging to the STAR family (Signal transduction and RNA metabolism) and involved in RNA metabolism. In the first part of this PhD Thesis, we have investigated the expression and function of Sam68 in human prostate cancer cells. We observed that Sam68 is up-regulated both at protein and mRNA levels in patients affected by PCa. Moreover, it was observed that down-regulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression, reduced the proliferation rate and sensitized cells to apoptosis induced by DNA-damaging agents. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Finally, stable cell lines expressing a truncated GFP-Sam68GSG protein, that interacts with endogenous Sam68 affecting its activity, displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis, resembling down-regulation of Sam68 by RNAi. Together, these results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents (Busà et al., 2007). Stemming from this evidence, we then aimed to investigate the role played by Sam68 in the response to genotoxic drugs such as mitoxantrone (MTX), a topoisomerase II inhibitor.We observed that MTX caused a subcellular re-localization of Sam68 from nucleoplasm to nuclear granules. Co-staining experiments indicated that Sam68-positive nuclear granules are sites of accumulation of several RNA-binding proteins involved in alternative splicing, such as SR proteins like SC35 and ASF/SF2, and TIA-1 and hnRNP A1, involved in cellular stress responses to various stimuli (Guil et al., 2006). Sam68 also accumulated in cytoplasmic granules that were also co-stained with hnRNP A1 and TIA-1, suggesting that these structures are the well described cytoplasmic stress granules (SGs). These data strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell. Thus, we have begun to investigate whether changes in subcellular localization of Sam68 induced by genotoxic drugs affect alternative splicing of Sam68 target mRNAs, such as CD44 (Matter et al., 2002). Preliminary experiments have shown that MTX treatment in PC3 cells induces changes in alternative splicing of CD44 pre-mRNA. In particular, inclusion of variable exons v5 and v6, known to be regulated by Sam68 (Matter et al., 2002; Cheng and Sharp, 2006), was stimulated. We are current extending these studies to determine whether downregulation of Sam68 by RNAi affects these modifications of CD44 alternative splicing caused by MTX Since Sam68 is known to link signal transduction pathways to RNA metabolism (Lukong and Richard, 2003), we asked whether changes in Sam68 subcellular localization induced by MTX are determined by activation of specific signal transduction pathways. Our data show that although MTX triggers activation of DNA damage pathway, through ATM kinase, and stress-induced MAPKs p38 and JNK1/2 pathways, specific inhibition of these pathways did not affect the subcellular relocalization of Sam68. Thus, it is possible that direct changes in the chromatin structure or function trigger the observed accumulation of Sam68 and splicing factors in nuclear granules. Finally, a set of observations performed during our studies implicate Sam68 in nucleolar functions. In a co-immunoprecipitation experiment aimed at the identification of Sam68-interacting proteins in LNCaP cells we found Nucleolin, a nucleolar protein involved in rRNA metabolism (Rickards et al., 2007). This interaction has been confirmed and mapped to the carboxyterminal region of Sam68 by in vitro studies. Moreover, a RNA-protein co-immunoprecipitation experiment revealed that Sam68 binds 18S rRNA These observations lead us to investigate whether Sam68 plays a role in rRNA metabolism. First, we observed by FISH analysis, and then confermed by real time PCR, that downregulation of Sam68 caused a significant increase in the levels of pre-rRNA compared with control siRNA treated cells. Moreover, ChIP assays aimed at determining the site of the association of Sam68 with rDNA in PC3 cells revealed that Sam68 binds the 18S rRNA coding region. Thus, the results presented herein strongly suggest a novel role of Sam68 in the regulation of pre-rRNA maturation. Our current studies are aimed at investigating this hypothesis further. References: Busà R, Paronetto MP, Farini D, Pierantozzi E, Botti F, Angelini DF, Attisani F, Vespasiani G, Sette C., Oncogene 2007 26(30):4372-82. Cheng C, Sharp PA. (2006). Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol. 26(1):362-70. Grossmann ME, Tindall DJ (2001). Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst. 93:1687-97; Guil S, Long JC, Cáceres JF. (2006). hnRNP A1 relocalization to the stress granules reflects a role in the stress response. Mol Cell Biol. 26(15):5744-58. Lukong KE, Richard S (2003). Sam68, the KH domain-containing superSTAR. Bioch. Biophys. Acta 1653: 73-86. Matter N, Herrlich P, Konig H (2002). Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420:691-695. Paronetto MP, Farini D, Sammarco I, Maturo G, Vespasiani G, Geremia R et al (2004). Expression of a truncated form of the c-Kit tyrosine kinase receptor and activation of Src kinase in human prostatic cancer. Am. J. Path. 164:1243-1251; Rickards B, Flint SJ, Cole MD, LeRoy G. (2007). Nucleolin is required for RNA polymerase I transcription in vivo. Mol Cell Biol. 27(3):937-48.

In this section, we reviewed immunotherapy for metastatic castration-resistant PCa (mCRPC), which is a promising area of research. Sipuleucel-T, a vaccine, showed some survival benefit but limited PSA decline. Other vaccines and immune checkpoint inhibitors are being tested with mixed results. CAR-T cell therapy, where T cells are engineered to target cancer cells, is also under investigation. More studies are needed to determine the best immunotherapies for PCa.

AbstractEarly and accurate detection of prostate cancer (PCa) together with effective treatment can save lives. Despite the recent advances in its treatment, the mortality trends due to PCa, in the USA, continue to be alarming with annual increase of 3%. In this brief communication, we propose a novel approach to treat PCa, its metastasis and recurrence. This is guided by a large quantity of preliminary data driven by targeting an endogenous genetic product, VPAC, expressed in high density on the cell surface at the onset of genetic mutation that ignites the cancerous transformation.A large body of preclinical and clinical data generated in our laboratory used a positron-emitting radionuclide Copper-64 (Cu-64), conjugated to a small biomolecule, TP 3805 that has a high in vivo stability and high affinity (Kd 3.1 × 10−9 M) for VPAC. The data depict high uptake of Cu-64-TP3805 in primary and metastatic lesions and malignant lymph nodes with minimal uptake in normal tissues except the liver, prompted by the hepatobiliary excretion.Reflecting on the present and planning for the future, our quest is to systematically investigate the use of the beta-emitting sister radionuclide Cu-67 to target VPAC receptors for theranostic applications of primary and metastatic PCa.

Prostate cancer (PCa) is one of the leading causes of cancer-related deaths in American men. PCa patients often die of the aggressive forms of the malignancy at advanced stages such as distant metastasis. There are urgent need to understand the molecular mechanisms driving PCa progression and subsequently develop efficient therapeutics to improve men’s health in the US as well as the world. Tumor microenvironment (TME) has been realized to play a critical role in PCa progression and metastasis, and targeting key factors of the TME has become the logical strategy for efficiently controlling PCa malignancy. Stromal cells in prostate tumors secrete growth factors, cytokines, and extracellular matrix (ECM) proteins that provide the TME to fuel cell proliferation, invasion, and metastasis. This review will focus on several key factors influencing TME, which include cancer-associated fibroblasts (CAFs), ECM remodeling, androgen receptor (AR) signaling, inflammation, and hypoxia. We will explain and analyze the biological roles of these factors and their important contributions to PCa malignancy, targeted therapeutics, and drug resistance. Furthermore, we will discuss the contributions of the dysregulation of epigenetic regulators to the complexity of the TME in PCa.

Worldwide, prostate cancer (PCa) is the leading cause of morbidity and cancer-related mortality in men. The pathogenesis of PCa is complex and involves abnormal genetic changes, abrogation of cell growth with heterogeneous progression and predictive subgroups. In the last two decades there have been the exploration and development of molecular and genetic biomarkers for PCa due to limitations of traditional serum biomarkers such as prostate specific antigen (PSA) in screening and diagnosis. These biomarkers could possibly differentiate between PCa and benign prostatic hyperplasia (BPH) patients, and healthy controls as well as assist with prognosis, risk stratification and clinical decision-making. Such molecular biomarkers include serum (PHI and 4K score), urine (PCA3 and SelectMDx), and tumor tissue (Oncoytype DX, Decipher and Prolarix). microRNAs (miRNAs) deregulation where there is increased or decreased expression levels, constitute prospective non-invasive molecular biomarkers for the diagnosis and prognosis of PCa. There are also other emerging molecular biomarkers such as exosomal miRNAs and proteins that are in various stages of development and clinical research. This review is intended to provide a wide-ranging appraisal of the literature on current and emerging PCa biomarkers with robust evidence to afford their application in clinical research and by extension routine clinical practice.

Single-cell sequencing is a technique based on sequencing genomic, transcriptomic and epigenomic analysis at the level of individual cells. Compared with traditional sequencing techniques, it can study intercellular heterogeneity and provide in-depth information on cell distribution, cell types and cell function of a specific population. This paper used blood tissue samples to analyze single-cell sequencing. Cells were downscaled by principal component analysis (PCA) and used Leiden clustering to discover cell heterogeneity in the same cluster and annotated after screening marker genes. Finally, 13 marker genes MTRNR2L8, NKG7, FLNA, IL7R, CCR7, CD8B, MKI67, LGALS3, TYROBP, C0251_TotalSeqC, CD79A, HLA-C, CCL4, and MCM5 in the experimental samples were found by cellular enrichment, which are strongly associated with white stuffy syndrome, skin damage, giant cell virus infection and other diseases are closely linked. In the medical field, the experimental results can be used to study the molecular characteristics and functional differences of different cell types under physiological and pathological conditions, thus providing insight into the pathogenesis and treatment of diseases such as leukoaraiosis syndrome, skin damage, and cytomegalovirus infection.

Estrogens are steroid hormones that act through their receptors (ER) α and β. They are involved mainly in female physiology but, in males, influence the homeostasis of some tissues like the prostate. In this organ, estrogens promote or limit cell proliferation depending on the activated receptor, with implications for the pathophysiology of benign prostatic hyperplasia (BPH) and prostate cancer (PCa). ERα promotes proliferation while ER β is a protective factor against proliferative diseases. However, in the advanced stages of PCa, ERβ has a permissive role in prostate cells, increasing patient mortality. These effects are mediated by activating androgen-independent signaling pathways that promote proliferation. Another essential aspect of ER actions is the regulation of its expression. Steroid hormones participate in this process, but some non-steroid factors, like environment and epigenetic marks, influence the prostate’s physiopathology. Knowledge of these and other aspects of estrogenic action in the prostate will contribute to developing strategies for treating and preventing BPH and PCa. For this reason, this chapter will review the main aspects of estrogens’ permissive role in prostate diseases.

Abstract Spectral changes during malting included the development and disappearance of troughs. However, a strong peak at 2100 mm (starch or high molecular weight dextrin relating positively to extract) and a trough at 1980 mm (protein) persist through to kilning. The reconstruction at kilning had additional troughs at 2350 mm (cell walls) and its PCA-predicted values correlated highest (= 0.995) with extract values.

Interferon gamma (IFN), a lymphokine acting as biological response modifier, can induce morphological and phenotypic differentiation of some leukemic cell lines, specially along the monocytic pathway.Furthermore, myeloid leukemic cells and normal monocytes have been demonstrated to possess procoagulant activity (PCA).The aim of this study was to investigate the modulation of PCA induced by treatment with IFN on human promyelocytic cells from HL 60 cell line.Cells were cultured in suspension for 5days in RPMI 1640 medium supplemented with 10% fetal calf serum in the absence or in the presence of different concentrations of IFN (100-10.000 u/ml) at37°C and 5% CO2 Differentiation was assessed by morphological and cytochemical methods (MGG, ANAE, CAE, MPO) on cytospin preparations and surface marker analysis with monoclonal antibodies (0KMI, Mo2, MY9, MY7, MY4).PCA was measured as capacity to shortening ricalcification time of normal plasma and of factor VIII, VII and X deficient plasmas by the cells and the conditioned media.Untreated HL 60 cells exhibit high tissue factor-like PCA, related to the cellnumber. IFN treatment (1000 u/ml) induced at the same time, monocytic differentiation and significant increase in PCAboth in the cells and in the conditioned media.The PCA was not further affected by higher concentrations of IFN, unabletodetermine cell maturation.In conclusions the modulation by IFN seems to be dependent onmonocytic differentiation of HL 60 cells.

Cells of the monocyte/macrophage series develop thromboplastin procoagulant activity (PCA) when activated. This activity may be enhanced by T lymphocytes, but there have been few studies of the PCA of T lymphocytes themselves. In the present study, we have measured the PCA of four T lymphoma cell lines (JURKAT, CEM, HSB2 and MQLT4) as well as normal peripheral blood T lymphocytes, before and after stimulation with phyto-haemag-glutinin (PHA), using clotting and amidolytic methods.Of the four cell lines only one, JURKAT, gave enhanced PCA. after stimulation with PHA., with recalcification times in plastic tubes being shortened from 200 secs, to 90 secs, at a cell concentration of 4 × 106 ml. This activity was shown to be thromboplastin-like by its dependence on Factor VII in plasma and in an amidolytic assay with purified Factors VII and × . JURKAT was also the only one of the four cell lines to secrete interleukin-2. All four cell lines promoted the generation of large amounts of thrombin in platelet-free plasma in glass tubes; the peak thrombin concentration of 25 iu/ml was equivalent to that generated by a procoagulant phospholipid reagent. This activity was dependent on the presence of plasma Factor VIII, and was shown to be due to phospholipids in the cell membranes by its inhibition with phospholipase A2 and C and its activity in a purified system with Factors VIII, IXa and X. Normal T lynphocytes generated similar thromboplastin PCA to JURKAT after PHA stimulation, but their intrinsic PCA in the thrombin generation test was only 15% of that of the lymphoma cells.These results show that some T lymphocytes can develop PCA in both intrinsic and extrinsic systems and this should be taken into account in studies of the PCA of mixed leucocyte populations. The difference in intrinsic PCA between normal and lymphoma cells could be an interesting marker for malignant transformation.

Involvement of platelets and coagulation systems in the hematogenous metastasis of tumor cells has been suggested from in vivo and in vitro studies, however, there is still controversy about the exact role of hemostasis in metastasis. To date, at least three types of platelet aggregating mechanisms and three types of tumor cell procoagulants have been reported in different tumor cells.We investigated platelet aggregating activity (PAA), procoagulant activity (PCA) and the relationship between these two activities, using eight human neuroblastoma cell lines, three human leukemia cell lines and human mature lymphocytes. PCA in tumor cells was measured by the single stage recalcification time and the assay with chromogenic substrate S2222. PAA was determined turbidometrically with an aggregometer by adding cell suspensions of tumor cells to platelet rich plasma (PRP). The effects of protease inhibitors, enzymes and thrombin inhibitors on PAA and PCA were also studied.Neuroblastoma cell suspensions showed high PCAs which were reduced in Factor VII deficient human plasma, indicating a tissue factor-like activity. NCG line possessing the highest PCA also showed a high PAA, which was inhibited by pretreatment of cell suspensions with phospholipase A2 and abolished in the presence of heparin, hirudin or MD805 in the assay system. Human leukemia cell lines and mature lymphocytes had weak to moderate PCAs without showing PAA, but became active to express PAA after being removed of cell surface sialic acid by neuraminidase. These results suggest that in neuroblastoma, PCA closely linked with PAA may play a role in the hematogenous metastasis. In hemopoietic cells, PAA expressed when cell surface sialic acid is removed does not correlate with PCA, and sialic acid in these cells possibly prevents direct interaction with platelets in the hemostatic homeostasis.

Adhesion of leukocytes (WBC's) to vascular endothelial cells (EC's) is a component of inflammation, thrombosis and atherosclerosis. The purpose of this study was to assess the effect of WBC adhesion on the EC contribution to these pathologic events. Human WBC's were isolated and co-incubated with cultured human umbilical cord EC's. Supernatants and cell lysates (4 wells × 2) were obtained at 0.5,1,2, and 4 hours of incubation. EC's and WBC's (5 × 105) were-incubated alone or in combination. Supernatants and cell lysates were assayed for leukotriene B4 (LTB4), the thromboxane metabolite thromboxane B2 (TXB2) and the prostacyclin metabolite 6-keto prostaglandin FT alpha (6 keto) by RIA. Cell lysates were analyzed for cell associated procoagulant activity (PCA) by an APTT procedure, for plasminogen activator inhibitor (PAI) by an amidolytic assay and for IL-1 by a T-cell co-stimulator assay. Cellular and supernatant LTB4 was unmeasurable for both WBC's and WBC/EC cultures. WBC TXB2 showed a time dependent elevation which was unaffected by EC's. IL-1 activity was measurable at 2 hours and reach 14 U/ml in WBC's and 6 U/ml in EC/WBC cultures. Co-incubation of WBC's with EC's induced a 200% increase in both supernatant and cell associated 6 keto concentrations compared to EC's incubated alone. EC's and WBC's produced no PCA when incubated alone. PCA activity of the EC/WBC co-cultures was measurable at 2 hours and was 300-1500 U/ml after 4 hours. Coincubated EC's had a 50% decrease in cell PAI, suggesting an increased release of inhibitor from the cells. The prostacyclin and PAI release -along with the delayed expression of PCA activity are responses similar to those expected after EC exposure to cytokines. A source of these cytokines appears to be the WBC's which secreted measurable amounts of IL-1. WBC released IL-1 was sufficient to induce biochemical changes in EC's which can stimulate coagulation (PCA synthesis), inhibit fibrinolysis (PAI release), and enhance inflammation (prostacyclin synthesis). These results suggest that the release of WBC IL-1 can be sufficient to produce pro-thrombotic and inflammatory changes in EC's which are similar to those observed with the addition of exogenous IL-1 to EC cultures.

Mononuclear phagocytes, an integral part of the lymphoreticular infiltrate of human and experimental tumors, might contribute to fibrin deposition within malignant tissues through the production of procoagulant activity (PCA). We have studied the PCA of tumor-associated macrophages (TAM) in two poorly immunogenic, metastatic murine sarcomas (mFS6 and MN/MCA1); peritoneal macrophages (PM)from tumor-bearing and control animals were also studied, as reference cell populations. TAM were prepared from disaggregated tumor tissue and PM from ‘lavage’ fluid by adherence to plastic.PCA was evaluated by a one-stage clotting assay immediately after isolation (basal PCA) and following in vitro stimulatio.Basal PCA was very low(<1 u/104 M) inall cellpreparations. Exposure of PM from both normal and tumor-bearing animals to endotoxin (S. enteritidis LPS, 1 ug/ml f..), phorbol myristate acetate (PMA,0. ug/ml) or the chemotactic peptide FMLP(10-7 M) resulted in a 10-,7- and 3-fold increase of PCA respectively. In contrast TAM from mFS6 and MN/MCA1 consistently failed to generate PCAin response to different concentrationsof the same stimuli. Treatment of TAM with aspirin (10-3M) did not affect the cell unresponsiveness. Fluorescence microscopy showed that almost all PM were stain ed with fluorescein isothiocyanate (FITC)-LPS, while 10% of the TAM were stained. Moreover binding studies demonstrated that TAM had a loyer number of specific binding sites for phorbol esters than PM. These data suggest that the defective responsiveness of TAM to endotoxin, PMA and, possibly, to FMLP is due to the lack or very low expression of binding sites for these agents on the cell surface. The tumor environment may orient the functional status of in situ macrophages in a sense less favourable to the host.