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Little is known about how the size of a tissue is established during development and maintained subsequently. Proliferation-inhibiting signals secreted by cells within a tissue that act specifically on cells within that tissue can provide negative feedback on cell number, thus regulating tissue size. A better understanding of tissue-specific inhibitors of proliferation could be useful for designing therapies for cancer and other diseases. However, few signals of this sort have been identified, and little is known about how these signals function. Two examples of such signals are the proteins AprA and CfaD, which are secreted by the social amoeba Dictyostelium discoideum and inhibit cell proliferation in a concentration-dependent manner. Cells lacking either AprA or CfaD proliferate rapidly, and adding recombinant AprA or CfaD to cells reduces proliferation. However, little is known about the signal transduction pathways downstream of AprA and CfaD. I identified three proteins that are required for the normal function of AprA and CfaD: the kinase QkgA, the putative transcription factor BzpN, and the putative kinase PakD. Cells lacking any one of these proteins proliferate rapidly, and adding AprA or CfaD to cells lacking these proteins does not cause reduced proliferation, indicating that these proteins are involved in AprA/CfaD signal transduction. I also found that, in addition to its proliferation-inhibiting activity, AprA also functions as an autocrine chemorepellant. Colonies of cells lacking AprA expand less rapidly than wild-type colonies, despite the fact that individual cells lacking AprA show a random motility like that of wild-type cells. Further, two independent assays demonstrate that cells show a biased movement away from a source of AprA. The chemorepellant activity of AprA requires CfaD, QkgA, and PakD, but not BzpN, indicating that AprA affects proliferation and chemorepulsion through distinct but overlapping pathways. These results suggest that AprA functions as a readout of local cell density, to which cells respond by slowing proliferation and chemotaxing to regions of lower cell density, where nutrients are more likely to be present. The study of human AprA, CfaD, QkgA, BzpN, and PakD orthologs may serve to guide therapeutic approaches that modulate chemorepulsive or antiproliferative processes.

Angiogenesis is not only essential for tumor growth but is also implicated in invasion of the cancer cells into the circulation, and growth of dormant micro-metastases into frank metastatic lesions. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis as well as in solid tumors. It also has a role for VEGF in hematopoietic neoplasms; although has not been fully elucidated. This study will examine the VEGF secretary activity of malignant cells in the patients with hematologic malignancies. Supernatants of cell culture after 72 hours & 7th day were analyzed for VEGF value by ELISA method. The purposes of this study are to assay the VEGF activity and its correlation with disease prognosis in various hematological disorders; and to detect the VEGF autocrine activity of tumor cells in sequential culture without any stimulation in vitro. Results: our research samples were 75 specimens. The VEGF value was low in 13 cases of benign diseases, no obvious auto-secretary activity of those cells. There was no significant correlation between VEGF value and disease status in acute lymphoblastic leukemia; however, the cases were too small to had exact predict value (only 7 cases). In 17 Cases of malignant lymphoma, low D0 VEGF value (<300 pg) had good prognosis; those cases relapsed after treatment had high auto-secretary activity (high VEGF value of 7th culture day) and had bad disease prognosis, but there was no statistic significance. In 14 Cases of acute myelogenous leukemia, high D0 VEGF value (>150 pg) and high VEGF value of 7th culture day both presented bad disease outcome. In 16 cases of chronic myeloid leukemia (CML), low plasma VEGF level (D0<300pg) was all in chronic phase; all cases in accelerated phase (3 cases) and acute blastic crisis (5 cases) presented with high plasma VEGF level (>300pg). Patients with high plasma VEGF level had 70 % (7of 10) in worse disease status (P=0.010). Patients in chronic phase of CML had low VEGF auto-secretary activity and most of the blastic crisis patients were with high VEGF auto-secretary activity and also had bad prognosis. (P=0.248) Conclusion: although our study is a primary result, study cases are varied, but it still provide important information that VEGF has an important role in hematological malignancies. We will process further research of single and specific disease in the future to analyze the exact correlation of VEGF and hematological diseases.

(Uncorrected OCR) Abstract of thesis entitled NOVEL ASPECTS OF AUTOCRINEIP ARACRINE REGULATION OF GROWTH HORMONE SECRETION AND SYNTHESIS IN GRASS CARP PITUITARY CELLS Submitted by ZHOUHONG for the degree of Doctor of Philosophy at The University of Hong Kong in March 2003 In this study, autocrine/paracrine regulation of growth hormone (GH) synthesis and secretion by local interactions of gonadotrophs and somatotrophs was examined in vitro in pituitary cells prepared from Chinese grass carp (Ctenopharyngodon idellus). Treatment with exogenous OH and gonadotropin (OTH) resulted in a dose-dependent increase in basal GH release, GH production, and GH mRNA levels. However, the opposite effects were observed by removing endogenous OR and OTH using immunoneutralization. Furthermore, GR and OTH immunoneutralizations at the pituitary level were effective in blocking the stimulatory influence on GH mRNA expression induced by GH-releasing factors in fish, including GnRH, dopamine, and PACAP38�Apparently" GH-induced GH gene expression was mediated by increasing the T1/2 ofGH mRNA in the cytoplasm and enhancing the production of GH primary transcripts in the nucleus. Since GH-induced OR mRNA gene expression could be blocked by inhibiting JAK2, P42144MAPK, P38MAPK, and PI3K, it is likely that the JAK/MAPK and JAK/PI3K pathways are involved in the GH receptor signaling. Similarly, exogenous GTH increased the production ofGH primary transcripts. However, it did not improve OR mRNA stability but rather enhanced the turnover of GH transcripts. GTR also increased cAMP production in carp pituitary cells. GTH-induced GH mRNA expression Was mimicked by activating cAMP synthesis and blocked by inhibiting adenylate cyclase (AC) and PKA.. GTH-induced OR mRNA expression was also sensitive to inhibition of JAKz, P42/44MAPK, P3SM.AP1C and PI3K. Similar inhibitions, except for PI3K, were all effective in blocking OR mRNA expression induced by activation of cAMP synthesis. These results indicate that GTH may induce GR gene expression through the AC/ cAMP/PKA pathway secondary coupled to JAK.2 andlor MAPK. Apparently, a cAMP-independent PI3K component is also involved in the post-receptor signaling. Using a colunm perifusion approach, the dynamic interactions between somaotrophs and gonadotrophs were examined. In this case, exogenous OTR induced a rapid rise in basal GH secretion, whereas exogenous GR was found to inhibit basal GTR release. In parallel studies, GTHinduced OR mRNA expression was abolished by OR immunoneutralization. Similarly, GTR immunoneutralization blocked GR-induced OR mRNA expression in carp pituitary cells. These results, as a whole, indicate that endogenously secreted OH and GTR, besides their functions as endocrine hormones, serve as novel autocrine/paracrine factors at the pituitary level to modulate GH secretion, OH production, OH gene expression, and somatotroph sensitivity to stimulation by hypothalamic regulators. These stimulatory influences of GH and GTR on OR gene expression axe exerted at the level of GR rnRNA stability and OH gene transcription, presumably via a direct coupling to the JAK/MAPK and JAKiPI3K cascades or an indirect coupling via the AC/cAMP/PKA pathway. Apparently, a local il1trapituitary feedback loop is present. In this case, GTH released from gonadotrophs stimulates GH secretion in neighboring somatotrophs. GR release from somatotrophs is essential to maintain basal GH synthesis and secretion and also exerts a negative feedback on basal GTB release. This intrapituitary feedback loop formed by local interactions between gonadotrophs and somatotrophs may represent a novel mechanism to control OR gene expression in lower vertebrates. / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

Les lymphocytes B régulent la réponse immunitaire par la sécrétion de facteurs proinflammatoires ou immunosuppresseurs. Dans la Leucémie Lymphoïde Chronique (LLC),une sous-population de lymphocytes B CD5⁺ présente des propriétés immunosuppressives qui l’apparente aux lymphocytes B régulateurs notamment par la production d’IL-10. La survie des cellules leucémiques est, elle, associée à la réponse antigénique et à la production de cytokines dont l’IL-6. L’objectif de ce travail a été de caractériser dans la pathologie les populations produisant les cytokines pro-survie ou immunorégulatrices et d’analyser la relevance fonctionnelle de leur sécrétion. Nous avons identifié des sous-populations de cellules B leucémiques exprimant trois facteurs immunorégulateurs l’IL-10, le TGFβ1 et pour la première fois le facteur de transcription FOXP3, La proportion augmentée de cellules exprimant l’IL10 est associée à une diminution des cellules exprimant l’IL6. De manière importante, ce travail a identifié une boucle autocrine de stimulation de l’activité métabolique des cellules par l’IL10. La cytokine en se fixant à son récepteur permet l’activation des facteurs STAT3 et induit l’expression à la fois de protéines anti- apoptotiques de la famille Bcl2 mais surtout sa propre expression. Un blocage de cette boucle au niveau du récepteur à l’IL10 suspend l’avantage de survie des cellules tumorales. L’IL-6 ne déclenche pas ces mécanismes de maintien des cellules de LLC. Ce travail montre qu’en plus de son rôle sur les cellules du microenvironnement tumoral, l’IL-10 participe au maintien autocrine de la sous-population immunorégulatrice dans la LLC. / B cells produce pro-inflammatory or immunosuppressive factors to modulate the immuneresponse. In Chronic lymphocytic leukemia (CLL), a subset of the tumor lymphocytes produces IL10 and share immunoregulatory functions with regulatory B cells. CLL cell ssurvival is driven by antigenic response and pro-survival cytokines such as IL6. This project aimed at deciphering the cytokines profile of CLL subsets and analyzing their functional relevance. We identified immunoregulatory subsets producing IL-10, TGFβ1 and for the firsttime FOXP3. In patients, the increased proportion of cells expressing IL10 was correlated with decrease in IL6⁺ cells. Importantly we described an autocrine survival loop driven by IL10 in these cells. IL10 triggering led to STAT3 activation, induction of active pro-survival factors altogether with IL10 self-induction. Interrupting this loop with a blocking ab against IL10R prevented survival of the cells. IL6 did not manage such mechanisms. In conclusion,this work demonstrates that IL10 is an important mediator in CLL; the cytokine alters immune recognition of the tumor cells and sustains leukemic cells survival via the autocrine loop.

Le facteur de motilité autocrine (AMF) est une cytokine sécrétée par les cellules tumorales qui a été détectée dans le sérum et l'urine de patients cancéreux. Cette enzyme stimule la motilité des cellules cancéreuses in vitro et provoque des métastases in vivo. Elle peut être utilisée comme un biomarqueur métastasique.Dans cette étude, un biocapteur électrochimique sensible et spécifique a été conçu pour la détection et la quantification d'une enzyme modèle de l’AMF humain : la PGI de mammifère. Le biocapteur a été construit par liaison de 6-phosphate-D-fructose (F6P) sur une surface d'or d’électrode fonctionnalisée covalemment par des groupements oxyamine.La reconnaissance entre l’enzyme et le biorécepteur a été quantifiée par spectroscopie d'impédance électrochimique et voltammétrie dans une gamme de 10 fM à 100 nM. La limite de détection mesurée est de 6,6 fM. La sélectivité a été prouvée, ainsi que la reproductibilité. Notre biocapteur est une preuve de concept très prometteuse d'un futur dispositif analytique miniaturisé conçu pour la détection rapide, facile et précis de l'AMF. Il pourrait en outre contribuer à valider l'AMF en tant que nouveau biomarqueur du cancer métastatique.Afin d’étudier les interactions mises en jeu dans la reconnaissance entre l’enzyme et le biorécepteur, des études de mécanique moléculaire polarisable via le champ de forces SIBFA ont été réalisées. SIBFA est un champ de forces de seconde génération basé sur les résultats des décompositions ab-initio de l’énergie d’interaction et inclut donc la polarisation mais aussi l’énergie de transfert de charge.Pour cette étude nous avons mis en place deux modèles d’AMF pour SIBFA, une forme entière et une forme réduite, et nous avons construit un mime du biocapteur pour SIBFA. Pour cela, il a fallu concevoir et calibrer chaque fragment nécessaire à l’élaboration du mime. Ensuite différentes minimisations d’énergie ont été réalisées, en prenant en compte ou non la solvatation, puis des études sur les interactions mises en jeu ont été effectuées. / Autocrine motility factor (AMF) is a cytokine secreted by tumor cells that could be detected in the serum and the urine of cancer patients. This enzyme stimulates tumor cells motility in vitro and causes metastasis in vivo. It can be used as a biomarker of metastasis.In this study, a sensitive and specific electrochemical biosensor was designed for the detection and quantitation of a model of the human enzyme AMF: the mammalian PGI. The biosensor was constructed by covalently binding D-fructose 6-phosphate (F6P) on the oxyamine functionalized surface of a gold electrode.Recognition between the enzyme and the bioreceptor was quantified by electrochemical impedance spectroscopy and voltammetry in the range of 10 fM to 100 nM. The measured detection limit was 6.6 fM. Selectivity and reproducibility were also proven. Our biosensor is a promising proof of concept for the design of a future miniaturized analytical device for fast, easy and accurate detection of AMF. It could also help validate the AMF as a new biomarker of metastatic cancer.To study the interactions involved in the recognition process between the enzyme and the bioreceptor, we performed polarizable molecular mechanic studies using the force field SIBFA. SIBFA is a second-generation force field based on the results of ab- initio decomposition energy of interaction and therefore includes not only the polarization but also the charge transfer energy.For this study we have developed two models of AMF for SIBFA, an entire form and a reduced form, and we built a mime of the biosensor for SIBFA. For this, it was necessary to design and calibrate each fragment essential for the development of the mime. Then, different energy minimizations were carried out, some of which taking into account solvation parameters. Studies of interactions between the mime and the AMF model are being carried out.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

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