<p>Mast cells are essential effector cells in the immune system as they release several inflammatory mediators. An accumulation of mast cells has been described in inflammatory conditions such as asthma and allergic rhinitis. Increased mast cell number, in the skin and other organs, is also a characteristic in mastocytosis, a disease without an effective treatment. One explanation for the increase in mast cell number is migration of mast cells in the tissue. In our studies we utilised mast cell lines, including HMC-1; cell lines transfected with the <i>c-kit</i> gene; and <i>in vitro</i> developed mast cells.</p><p>Our aim was to characterise, two variants of the HMC-1 cell line; the signalling pathways essential for mast cell migration towards TGF-β and SCF; and the mechanism regulating mast cell accumulation in mastocytosis.</p><p>Our results help to explain inconsistent findings regarding mast cell biology when HMC-1 cells have been used as a model system. The two variants, which we name HMC-1⁵⁶⁰ and HMC-1^{560, 816}, are used in different laboratories around the world. HMC-1⁵⁶⁰ and HMC-1^{560, 816} exhibited different characteristics regarding their karyotype, phenotype as well as their set of activating point mutations in the Kit receptor. Furthermore, divergent signalling pathways are of importance for mast cell migration towards TGF-β and SCF. The classical MAP kinase-signalling cascade was found to be of major relevance for TGF-β-induced migration. In contrast, this pathway had a modest impact on SCF-induced migration, which instead was highly dependent on p38 MAP kinase signalling. Finally, one mechanism for mast cell accumulation in mastocytosis appeared to be an activating point mutation in the gene for the Kit receptor. This mutation appeared to prone transfected cells and mast cell progenitors to a higher rate of migration towards SCF if compared with cells expressing wt Kit receptor.</p><p>In conclusion, our results show the importance of two different MAP kinase signalling pathways and mutations in the Kit receptor for mast cell migration induced by various types of stimuli. This knowledge helps us to understand the mechanism </p>

Mast cells are essential effector cells in the immune system as they release several inflammatory mediators. An accumulation of mast cells has been described in inflammatory conditions such as asthma and allergic rhinitis. Increased mast cell number, in the skin and other organs, is also a characteristic in mastocytosis, a disease without an effective treatment. One explanation for the increase in mast cell number is migration of mast cells in the tissue. In our studies we utilised mast cell lines, including HMC-1; cell lines transfected with the c-kit gene; and in vitro developed mast cells. Our aim was to characterise, two variants of the HMC-1 cell line; the signalling pathways essential for mast cell migration towards TGF-β and SCF; and the mechanism regulating mast cell accumulation in mastocytosis. Our results help to explain inconsistent findings regarding mast cell biology when HMC-1 cells have been used as a model system. The two variants, which we name HMC-1560 and HMC-1560, 816, are used in different laboratories around the world. HMC-1560 and HMC-1560, 816 exhibited different characteristics regarding their karyotype, phenotype as well as their set of activating point mutations in the Kit receptor. Furthermore, divergent signalling pathways are of importance for mast cell migration towards TGF-β and SCF. The classical MAP kinase-signalling cascade was found to be of major relevance for TGF-β-induced migration. In contrast, this pathway had a modest impact on SCF-induced migration, which instead was highly dependent on p38 MAP kinase signalling. Finally, one mechanism for mast cell accumulation in mastocytosis appeared to be an activating point mutation in the gene for the Kit receptor. This mutation appeared to prone transfected cells and mast cell progenitors to a higher rate of migration towards SCF if compared with cells expressing wt Kit receptor. In conclusion, our results show the importance of two different MAP kinase signalling pathways and mutations in the Kit receptor for mast cell migration induced by various types of stimuli. This knowledge helps us to understand the mechanism

P68 RNA helicase is a RNA helicase and an ATPase belonging to the DEAD-box family. It is important for the growth of normal cells, and is implicated in diverse functions ranging from pre-mRNA splicing, transcriptional activation to cell proliferation, and early organ development. The protein is documented to be phosphorylated at several amino-acid residues. It was previously demonstrated in several cancer cell-lines that p68 gets phosphorylated at threonine residues during treatments with TNF-α and TRAIL. In this study, the role of threonine phosphorylation of p68 under the treatment of anti-cancer drug, oxaliplatin in the colon cancer cells is characterized. Oxaliplatin treatment activates p38 MAP-kinase, which subsequently phosphorylates p68 at T564 and/or T446. P68 phosphorylation, at least partially, influences the role of the drug on apoptosis induction. This study shows an important mechanism of action of the anti-cancer drug which could be used for improving cancer treatment. This study also shows that p68 is an important transcriptional regulator regulating transcription of the cytoskeletal gene TPPP/p25. Previous analyses revealed that p68 RNA helicase could regulate expression of genes responsible for controlling stability and dynamics of different cytoskeletons. P68 is found to regulate TPPP/p25 gene transcription by associating with the TPPP/p25 gene promoter. Expression of TPPP/p25 plays an important role in cellular differentiation while the involvement of p68 in the regulation of TPPP/p25 expression is an important event for neurite outgrowth. Loss of TPPP expression contributes to the development and progression of gliomas. Thus, our studies further enhance our understanding of the multiple cellular functions of p68 and its regulation of the cellular processes.

Transforming growth factor-β (TGF-β) is the proto-type member of a super family of secreted proteins comprised of several structurally related, but functionally divergent proteins like the BMP, activin, inhibin, mullerian inhibitory substance etc. TGF-β was originally identified as a secreted factor, which in the presence of EGF was capable of transforming normal rat kidney fibroblasts. Studies over the years have shown that this protein is multifunctional that influences several processes including development, immune function, epithelial cell growth and motility, wound healing etc. TGF-β plays important role in the normal physiology as well as in pathological conditions in mammals. There are three mammalian isoforms that are involved in several developmental processes as has been shown by the knockout mice models. An important role for TGF-β has been implicated in several disease processes like fibrotic disorders (of liver, lung, kidney), inflammatory disorders (rheumatoid arthritis), autoimmune disorders (systemic lupus erythematosus) and cancer. TGF-β has a dual role in carcinogenesis. Initially it acts as a tumor suppressor and causes growth arrest of epithelial cells and cells in the early stages of cancer. But in an established tumor, TGF-β exerts an effect which is favorable for the survival, progression and metastasis of the tumor by promoting epithelial-mesenchymal transition (EMT), angiogenesis and escape from immune surveillance. Studies using mouse models have shown that an intact TGF-β signaling is essential for the metastasis of breast cancer. These observations indicate that the normal epithelial cells show differential response to TGF-β as compared to the tumor they give rise to. Supporting this, it has been shown that prostate tumor cells show invasive behavior in response to TGF-β and not non-tumorigenic cells. Most actions of TGF-β are brought about by regulation of gene expression and differential gene expression mediated by TGF-β has been reported in tumor cells and normal cells. For example, in response to TGF-β, tumorcells show increase in the production of proteases like uPA, MMPs etc and down regulation of the inhibitors of proteases TIMP isoforms, whereas this is not observed in the normal cells. However, there is no clear understanding of the mechanism (s) responsible for differential responses of various cell types to TGF-β. Since a role for TGF-β has been established in several pathological conditions particularly cancer and fibortic disorders, this pathway are a very attractive target for therapeutic intervention. Hence, if the TGF-β pathway has to be targeted for therapy of any disease, it becomes essential to identify the targets of TGF-β in different cell-types and their mechanism of regulation, particularly in un-transformed and transformed cells. Over the past few years, there have been several independent transcriptome analyses of cells in response to TGF-β treatment in various cell types such as HaCaT, fibroblasts, corneal epithelial cells etc. From a comparison of these studies, it is noted that TGF-β regulates genes in a cell type specific manner. Considering the dual role of TGF-β on normal and transformed cells, identification of genes and/or biochemical pathways regulated by TGF-β in these cells may allow identification of therapeutic targets for diseases involving TGF-β signaling pathway. With this background, the following objectives were set for the current investigation: 1. Identification of targets of TGF-β in normal and tumor cells and also the genes differentially regulated by TGF-β 2. Understand the mechanism of regulation of a few selected genes 3. Characterize novel targets of TGF-β with respect to their regulation by TGF-β and also their function Towards the aim of identification of targets of TGF-β in different cell-lines, expression profiling of genes in response to TGF-β was performed in a lung adenocarcinoma cell line (A549) and a matched immortalized lung epithelial cell line (HPL1D). Our data showed similar regulation of 267 genes in HPL1D and A549 cells by TGF-β. This suggests that the genes commonly regulated in both HPL1D and A549 are not tumor specific. Some of these genes were also reported to be regulated by TGF-β in other studies using micro array in various cell types. While 1757 genes are exclusively regulated by TGF-β in A549, only 733 genes are exclusively regulated in HPL1D cells. The reasons for this differential response are not known. However, some of the genes exclusively regulated in A549 such as Integrin αV, thrombospondin 1 have been shown to aid tumor survival, maintenance and metastasis. In contrast, in HPL1D, TGF-β regulates tumor suppressor genes like WT1, ECM proteins like collagen which are responsible for arrest of cell growth and apoptosis. This differential gene regulation in normal and tumor cells may explain the dual role of TGF-β in carcinogenesis. The differences in the effects of TGF-β on these two cell-lines could be due to the phenotypic properties of these cells, HPL1D being a non-transformed cell-line and A549 being a transformed cell-line. It is also possible that the differences are due to cell-type specific effects. In order to address this question, expression profiling in response to TGF-β was carried out using another cell-line namely HaCaT, which is an immortalized skin keratinocyte cell-line. When the expression profiles of the three celllines namely HPL1D, HaCaT and A549 in response to TGF-β treatment were compared, it was found that the genes regulated by TGF-β can be divided into seven categories based on the cell-line in which they are regulated. In this comparison, it was seen that there were several genes which were regulated by TGF-β in A549 and HaCaT despite the fact that these two cell-lines have little in common. The reason for these two celllines to show similarities in their gene expression profile in response to TGF-β is unclear. When the genes regulated by TGF-β in the three cell-lines were categorized based on their annotated functions using the DAVID tool, it was found that signaling pathways like MAP kinas, focal adhesion, Wnt signaling are regulated by TGF-β in all the celllines. On the other hand, Integrin αV was found to be regulated in A549 and HaCaT cells and very marginal regulation was seen in HPL1D cells. This could be one of the reasons for the similarities between A549 and HaCaT. There are studies which show the role of Integrin αV in some of the TGF-β mediated actions although the mechanism by which Integrin signaling modulates gene expression is not well understood. Our data shows that indeed thrombospondin 1 which is regulated by TGF-β in A549 and HaCaT is regulated through the integrin signaling pathways as blocking this pathway partially blocks the induction of this gene by TGF-β. TGF-β actions on cells are to a large extent are carried out by the phosphorylation of SMAD 2/3 by activated TGF-β type I receptor upon TGF-β signaling. Several genes that are transcriptionally regulated by TGF-β contain a SMAD complex binding element (SBE). However, over the last few years, evidences have accumulated which suggest that some actions of TGF-β could be independent of SMADs, mediated by the other signaling pathways like the MAP kinas, PKC and others. In order to understand the mechanism of regulation of a few selected genes by TGF−β, inhibitors for the three MAP kinas pathways (p38, ERK and JNK) were used prior to treatment with TGF-β. The expression of these genes was assessed by qRT-PCR analyses. These studies showed that most of the genes regulated by TGF-β require one or more of the MAP kinas pathways. In HaCaT and A549, the number of genes dependent on the MAP kinas pathways is more compared to HPL1D. Based on our data, we propose that activated MAP kinas pathway could be one of the essential determining factors for the various differential actions of TGF-β in tumor cells. However, the reason for the behaviour of HaCaT cells, which are untransformed cells in a manner similar to the A549 cells, is still unclear. One of the reasons for the similarity could be the activation of the integrin signaling pathway as described before. The expression profiling data identified several novel targets of TGF-β. One such target is S100A2, a calcium binding protein containing an EF hand motif that has been implicated in cancer. A progressive reduction in the expression of this gene has been reported with increasing grade of the tumor. Our studies show that this gene is regulated by TGF-β in HaCaT and HPl1D, but not in A549 cells. The induction of S100A2 by TGF-β in HaCaT cells is likely to be transcriptional as it is sensitive to actinomycin treatment. We further investigated role of other signaling pathways in the regulation of S100A2 by TGF-β and found that the regulation of this gene by TGF-β depends on the ERK and also the integrin signaling pathways. In order to characterize this gene with respect to its functions, A549 cells were chosen as they have very low endogenous expression of S100A2. Hence, in order to explore if there is any role for the loss of S100A2 expression in the progression of A549 cells, we cloned the DNA of S100A2 in a mammalian expression vector, transected A549 cells with this and isolated clones stably expressing this gene. We performed assays to assess cell proliferation, cell migration and potential to form colonies in soft agar. The data suggests phenotypic differences in the colonies that formed in soft agar and no major differences in other assays. Overall, our data has identified several novel targets regulated by TGF-β other than S100A2 like IGFBP7, FGFR1, and SPUVE etc. Further, regulation of several genes was found to be in a cell type specific manner involving MAP kinase and integrin signaling pathways. This study also identified major differences in the genes regulated by TGF-β in transformed and non-transformed lung epithelial cells.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xii, 216 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

To identify a model legume experimental system for studying low temperature signaling and cold acclimation, cold-induced expression and regulation of homologues of alfalfa (Medicago sativa) cold acclimation-specific genes cas15 and cas30 were examined in M. arborea (relatively frost tolerant) and M. truncatula (relatively frost sensitive). Both cas15 and cas30 genes are present in the genomes of both species but whereas both genes are cold-induced in M. arborea, only cas15 is induced in M. truncatula. Cold-induced expression of these genes is inhibited by calcium chelators and channel blockers and by the membrane fluidizer benzyl alcohol. Treatment of leaves with dimethylsulfoxide, a membrane rigidifier, induced both genes at 25°C. A cold-activated MAP kinase activity was expressed in both species. These results suggest that M. truncatula, an annual, self-pollinated species may be successfully used as model experimental systems in studies of cold signaling and role of cas genes in cold acclimation in legumes.

L'objectif de travail a été d'évaluer l'effet de deux types de polluants, le tributylétain (TBT) et les hydrocarbures aromatiques polycycliques (HAPs), sur l'activation de la voie des MAP kinases et sur l'induction de l'apoptose chez deux invertébrés marins, la moule Mytilus galloprovincialis et l'éponge Suberites domuncula. Il a été montré, chez ces deux espèces exposées aux deux composés, une activation systématique de p38 en réponse à toutes les conditions expérimentales testées. JNK est également activée suite à leur exposition au TBT. En revanche, une exposition aux HAPs, dans les conditions expérimentales choisies, induit l'activation de JNK, chez la moule et de ERK, chez l'éponge. En outre, une induction de l'expression de Bcl-xS a été observée chez la moule, protéine impliquée dans la voie intrinsèque de l'apoptose. Chez l'éponge, l'induction de l'apoptose est dépendante de l'activation de la caspase 3 alors que chez la moule, comme chez d'autres bivalves, le processus apoptotique n'est dépendant de la caspase 3 que pour certaines concentrations de polluant. Par ailleurs, l'analyse des échantillons de moules prélevées in situ dans dix neuf stations de la côte adriatique (Croatie), polluées à des degrés divers notamment par le TBT et les HAPs, durant l'hiver et l'été, a montré une activation des trois MAPKs p38, JNK et ERK. Le niveau d'activation est corrélé au degré de pollution et à la température. Pour conclure, ce travail permet de noter l'intérêt de la p38 comme biomarqueur d'exposition et celui de l'apoptose comme marqueur d'effet.

<p>Hematopoiesis is the continual process of blood cell generation that primarily occurs in the bone marrow of adult animals. Hematologic neoplasms can also occur in the bone marrow and often result from dysregulation of signal transduction pathways. One example is the activation of the Ras oncogene, which has been linked to a variety of different cancers, including hematologic neoplasms. Ras is located proximal to the cell membrane and can activate many downstream effector pathways, thus it is difficult to determine which downstream pathway is mediating oncogenic Ras function. My thesis work focused on the effect of inappropriate activation of MEK/ERK, a downstream Ras effector pathway, in the hematopoietic system.</p><p>Using a retroviral transduction system, we expressed a constitutively active form of MEK1 in hematopoietic stem cells (HSCs). Mice transplanted with HSCs expressing active MEK developed a lethal myelodysplastic syndrome/myeloproliferative disease (MDS/MPN) characterized by the expansion of granulocytes/macrophages (GM) at the expense of lymphoid cell development. Transplantation of active MEK-induced MDS/MPNs into naïve mice did not result in further disease, suggesting that the MDS/MPN is not a frank leukemia.</p><p>Bcl-2 is an anti-apoptotic molecule that has been shown to play a role in leukemia development and maintenance. Coupling expression of active MEK and Bcl-2 resulted in MDS/MPNs that were phenotypically identical and had very similar disease onset compared to active MEK-induced MDS/MPNs. However, transplantation of Bcl-2/active MEK-induced MDS/MPNs did not result in a myeloid disease; rather, it resulted in the development of T-acute lymphoblastic leukemia (T-ALL) that was marked by activated Notch signaling. </p><p>These results led us to conclude that activation of MEK/ERK was sufficient to cause a pre-leukemic myeloid disease; however, additional oncogenic factors, such as Bcl-2 and Notch, were necessary for frank leukemia development. Moreover, additional oncogenic factors can alter the disease phenotype and disease course. Future analysis of the interplay between oncogenic factors will help shed light on disease development and aid in the development of more effective cancer treatments.</p> / Dissertation

The coxsackie-adenovirus receptor (CAR) is a viral receptor for Group B coxsackieviruses (CVB). Physiologically, CAR is a cellular adhesion protein. I report that upregulation of cardiac CAR in the young adult mouse (CAR+/MtTA+ ) caused a cardiomyopathy that was characterized by inflammation and hypertrophy. In the hearts of CAR+/MtTA+ mice c-Jun N terminal kinase (JNK) was specifically activated. JNK activation is known to promote hypertrophy of cardiomyocytes, and disrupt proteins at the intercalated disc. CVB3-infected CAR+/MtTA+ mice did not exhibit increased cardiac viral load or myocarditis severity, but did demonstrate a greater cardiac interferon-γ (IFN-γ) response when compared to littermate controls. CAR-induced expression of this antiviral cytokine may have prevented the increase in myocarditis susceptibility. Further investigation into the activation of protein kinase signaling, and antiviral signaling will provide better understanding of how CAR participates in the pathogenesis of both viral and non-viral heart diseases.

The coxsackie-adenovirus receptor (CAR) is a viral receptor for Group B coxsackieviruses (CVB). Physiologically, CAR is a cellular adhesion protein. I report that upregulation of cardiac CAR in the young adult mouse (CAR+/MtTA+ ) caused a cardiomyopathy that was characterized by inflammation and hypertrophy. In the hearts of CAR+/MtTA+ mice c-Jun N terminal kinase (JNK) was specifically activated. JNK activation is known to promote hypertrophy of cardiomyocytes, and disrupt proteins at the intercalated disc. CVB3-infected CAR+/MtTA+ mice did not exhibit increased cardiac viral load or myocarditis severity, but did demonstrate a greater cardiac interferon-γ (IFN-γ) response when compared to littermate controls. CAR-induced expression of this antiviral cytokine may have prevented the increase in myocarditis susceptibility. Further investigation into the activation of protein kinase signaling, and antiviral signaling will provide better understanding of how CAR participates in the pathogenesis of both viral and non-viral heart diseases.