Thesis (Ph.D.)--New York University, Graduate School of Arts and Science, 2007. / Typescript. Includes bibliographical references (leaves 182-227). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with licensed institutions.

Cell-extracellular matrix and cell-cell adhesion are essential for biological processes such as cell motility, signaling, proliferation, cytoskeletal organization and gene expression. For this reason, extensive effort has been devoted in the past few decades to measure cell adhesion as well as identify key molecules involved. This thesis focuses on two outstanding problems in this area, namely, how to quantitatively characterize the adhesion between neural cells and the substrate and how to model the turnover of adhesions in the intriguing phenomenon of stretch-induced cell realignment. First of all, using a combined atomic force (AFM) and total internal reflection fluorescence microscope (TIRFM) system a novel method was developed to systematically and quantitatively examine the adhesion between neurite branches and the extracellular matrix. Specifically, a tipless AFM cantilever was used to penetrate between a well-developed neurite and the functionalized substrate and then gradually peel the neurite from the surface. At the same time, a laser TIRFM was utilized to monitor the activities of different adhesion molecules during the detaching process. This approach provides a solution to the long-standing problem of how to quantitatively measure neuron-extracellular matrix interactions while, simultaneously, identify the roles of various adhesion proteins in the process. Besides heathy neurons, testes have also been conducted on cells affected by the Alzheimer's disease (AD) where the influence of such disease on the mechanical response of neural cells was demonstrated. Secondly, to better understand the observed peeling response of the neurite, as well as extract key information from it, finite element (FEM) simulation was carried out using ABAQUS. It was shown that a good fit between the simulation results and experimental data can be achieved by representing the adhesion between two surfaces with simple cohesive interactions. In particular, it was found that the apparent adhesion energy density, a quantity of central interest in cell adhesion studies, is in the range of 0.2-0.8 mj/m^2. Last but not the least, a mechanochemical modeling framework was developed to investigate the mechanism of cell reorientation induced by cyclic stretching on the substrate. It was shown that the final alignment of cells reflects the competition between stress fiber assembly or disassembly, focal adhesion growth or disruption, substrate stiffening and whole-cell rotation. Predictions from the model are consistent with a variety of experimental observations, suggesting that the main physics of this intriguing phenomenon may have been well captured. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Inactivation of the tumor suppressor protein p53 is a very important and common step in the process of carcinogenesis. The overall purpose of this project was to gain a better understanding of the mechanisms involved in the regulation of p53 function. To gain insight into these mechanisms, we chemically mutagenized A1-5 cells expressing high levels of temperature sensitive p53 val135 (tsp53) and selected for clones that were capable of growth at the permissive temperature for p53 activation. The clones generated, called ALTR (for A&barbelow;1-5 L&barbelow;ow T&barbelow;emperature R&barbelow;esistant), could grow at the permissive temperature. Using the ALTR cell system and the parent A1-5 cells, we determined that nuclear translocation of p53 could result in a change in the conformation from mutant to wild-type but that these may be two separable events. We also investigated, in depth, the mechanism by which p53 was inactivated in one ALTR cell line, ALTR 9. We identified calpain mediated degradation of p53 as a partial mechanism of p53 inactivation in these cells. Our results suggest that degradation of p53 by calpain can lead to the functional inactivation of p53 and that this degradation can be regulated by genomic stress. To gain insight into the significance of cytoplasmically sequestered p53 protein in tumors, we chose a neuroblastoma derived cell line, SK-N-SH, that expresses a wild-type but cytoplasmically sequestered p53 protein. We report here, that down regulation of p53 by HPV-16 E6 resulted in the morphological conversion of SK-N-SH cells to substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, a lack of responsiveness to RA induced neuronal differentiation, and loss of anchorage independent growth. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype. Both the ALTR cell system and the SK-N-SH cells provided us with insight into the mechanisms involved in p53 inactivation resulting in tumor formation.

Cells of the cardiovascular system express multiple connexins (Cx) with Cx40 and Cx43 being commonly coexpressed in many tissues. The expression levels of connexins are dynamic and can vary in response to a growth stimulus. It is not clear why cells express multiple connexins, or what advantage such dynamic regulation of expression patterns have on cell function. These issues are further complicated by the ability of some connexins to interact to form heterogeneous gap junction channels, with little being known regarding functional properties of such channels. The purpose of these experiments was threefold: (1) To determine whether Cx40 and Cx43 are capable of interacting to form heteromeric/heterotypic gap junction channels; (2) To characterize the functional properties of Cx40/Cx43 heteromeric/heterotypic channels; and (3) To determine the effect that changing Cx40:Cx43 expression ratio has on functional properties of heteromeric/heterotypic channels. Cell lines were developed that express only Cx43 (Rin43), Cx40 (Rin40), and Cx40 and Cx43 in varying Cx40:Cx43 expression ratios (6B5n, A7r5, A7r540C1, and A7r540C3). The Cx40:Cx43 expression ratios in the 6B5N, A7r5, A7r540C1, and A7r540C3 cells are approximately 1:1, 3:1, 5:1, and 10:1, respectively. Functional properties of the gap junction channels formed between these cells were determined using both electrophysiological and dye coupling techniques. Pairing of Rin43 and Rin40 cells demonstrated that Cx40 and Cx43 are capable of forming homomeric/heterotypic gap junctions with unique voltage-dependent gating and single channel behaviors. Rin43/A7r5 cell pairs displayed voltage-dependent gating and single channel conductance profiles that could only be explained by the presence of heteromeric/heterotypic gap junction channels between these cells. Pairing Rin43 cells with coexpressing cells of high Cx40:Cx43 expression ratio resulted in channel activities that were not predicted by the gating and conductance patterns of Cx40/Cx43 heterotypic channels. However, the dye coupling characteristics of these same cells in coculture demonstrated that the permeability of the channels formed between these cell types reflected that of Cx40 channels. In summary, Cx40 and Cx43 are capable of forming heteromeric/heterotypic gap junction channels. Increasing the Cx40:Cx43 ratio in coexpressing cells results in channels with unique gating and conductance properties, however dye permeability of these cells is predicted by their relative Cx40 content. Therefore, varying Cx40:Cx43 expression ratio provides cells with a mechanism to finely control the types of molecules shared between cells.

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Light, phase, and electron microscopic studies on the cytology of normal and dimethyl sulfoxide (DMSO)-treated amnii from days 101/2 through 191/2 of gestation were observed. This study emphasized the ontogeny of the structural moiety of normal Dimethyl sulfoxide (DMSO) induced various effects in amniotic tissue: cellular distortion, swelling of epithelial cells, endoplasmic reticuli and fibroblasts. Swelling and disruption of the mitochondria were also evident. Other effects such as t

In prostate cancer cells, TGFβ inhibits proliferation in earlier stages of the disease; however the cancer cells become refractory to growth inhibitory effects in advanced stages where TGFβ promotes cancer progression and metastasis. Inhibitor of Differentiation (Id) family of closely related proteins (Id 1- 1d4) are dominant negative regulators and bHLH transcription factors and in general promote proliferation, and inhibit differentiation. In the present study, we have investigated the role of Idi and Id3 proteins in the growth inhibitory effects of TGFβ on prostate cancer cells. The effect of TGF β on proliferation and Idl and Id3 expression were investigated in PZ-HPV7, DU145, and PC3 cells. Idi silencing through siRNA was also used in DU145 and PC3 cells to examine its role in anti-proliferative and migratory effects of TGFβ . TGFβ increased expression of Idi and Id3 in all cell lines followed by a later down regulation of Idi in PZ-HPV7 expression and DU145 cells but not in PC3 cells. Id3 expression remained elevated in all three cell lines. This loss of Idi protein correlated with an increase of CDKNI p21. Id1 knockdown in both DU145 and PC3 cells resulted in decreased proliferation. However, while TGFβ caused a further decrease in proliferation of DU145, but had no further effects in PC3 cells. Knockdown of Id1 or Id3 inhibited TGFβ 1 induced migration in PC3 cells. These findings suggest an essential role of Id1 and Id3 in TGFβ 1 effects on proliferation and migration in prostate cancer cells.

We previously identified a novel structural variant of the alpha6 integrin called alpha6p. This variant was produced on the cell surface and was missing the β-barrel extracellular domain. Using several different concentrations of amiloride, aminobenzamidine and PAI-1 and the urokinase-type plasminogen Activator (uPA) function blocking antibody (3689) we showed that uPA, acting as a protease, is responsible for production of α6p. We also showed that addition of uPA in the culture media of cells that do not produce α6p, resulted in a dose dependent α6p production. In contrast, the addition of uPA did not result in the cleavage of other integrins. Using α2-antiplasmin and plasmin depleted media, we observed that uPA cleaves the alpha6 integrin directly. Further, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced the production of alpha6p, and this induction was abolished by PAI-1 but not α2-antiplasmin. Using site directed mutagenesis we have identified the site of cleavage to be at arginines 594 and 595. We have also shown that while a fraction of α6 integrin is normally associated with CD151, the α6p form is not. In order to determine whether α6 integrin clipping occurs in tissue, we have found that α6p is present in human prostate cancer tissue, in normal mouse epidermis, in mouse papillomas and squamous cell carcinomas induced by DMBA, TPA and MNNG treatments and in mouse melanomas induced by activated ras. Interestingly, subcutaneous injection into athymic nude mice of a malignant mouse keratinocyte derived cell line (6M90) that is α6p negative, results in the development of tumors that contain α6p integrin. Furthermore, we have shown that PC3N cells transfected with an uncleavable mutant of the α6 integrin grew smaller tumors when injected subcutaneously in SCID mice compared to wildtype α6 transfected cells. In addition, the tumors from the uncleavable mutant alpha6 transfected PC3N cells had higher levels of activated caspase 3 indicating higher levels of apoptosis. This finding suggests that the α6 integrin clipping is important for integrin signaling for survival. Collectively, all these data suggest that the cell surface clipping of the α6 integrin is a novel mechanism for altering integrin-laminin interactions during skin tissue remodeling and carcinogenesis.

The (140 kD) α6 integrin is an essential gene product in epithelial cell maintenance and remodeling of the stratified epithelium. The prostate gland is an example of a glandular epithelium. In prostate cancer, alterations of integrins are observed. Specifically, a shift from α6β4 to persistent expression of α6β1 integrin occurs. Accompanying the loss of polarized α6β4 is loss of its extracellular ligand, laminin-5. Using immunofluorescence staining human prostate, breast and colon tissues, were examined for β4 integrin and laminin-5 expression. Loss of β4 and laminin-5 was apparent beginning in PIN lesions and was absent in prostate carcinoma, differing from retained expression in breast and colon carcinoma. These data suggested progressive loss of β4 integrin and laminin-5 occurs and that this combined defect is unique to prostate cancer progression. A novel 70 kD (non-reduced) variant of the α6 integrin, called α6p for the latin word parvus (small), was identified on the cell surface of normal epithelial and carcinoma cell lines. The α6p variant paired with either β1 or β4 subunits and retained sequences corresponding to the extracellular 'stalk region' and the cytoplasmic tail of the α6 integrin. The β-propeller domain postulated to mediate ligand binding, was missing from this variant. Protein levels of α6p increased three fold during calcium-induced terminal differentiation in a normal mouse keratinocyte model system. Production of the α6p variant was dependent upon an intact actin cytoskeleton. Cell surface α6p was less responsive to changes in the actin cytoskeleton, relative to that observed for α6 and β1 integrins, suggesting α6p did not participate in the focal contact. Additionally, inhibition of serine/threonine phosphatases decreased α6 integrin protein levels, but not α6p integrin, again suggesting the variant functioned as an inactive subunit for signaling. Finally, α6, but not α6p integrin co-immunoprecipitated with hemidesmosome components: laminin-5 and CD151. Preliminary data demonstrated adhesion to synthetic peptide integrin antagonists resulted in a 65 kD form of the alpha6p variant with no alteration of α6 integrin. Together the presented data were consistent with differential regulation of alpha6 and α6p integrins and suggested the α6p variant functioned as an inactive receptor.