<p>Three-dimensional (3D) tumor models aim to reduce the need for animal models for drug and gene delivery studies. However, many models are not conducive to environmental manipulation and may not be easily adapted for <italic>in situ</italic> microscopic analysis of transport phenomena. One goal of this study was to develop a 3D tumor model that can mimic 3D cell-cell interactions to mimic native tumor tissues. </p><p>To this end, a novel 3D microfluidics-based tumor model was created which allowed the overnight culture of a high density of tumor cells and could be used for small molecule penetration studies. This microfluidic device facilitated the loading of B16.F10 tumor cells in a densely-packed three-dimensional arrangement in a micro-channel which was accessible for nutrient supply via channels on either side through which culture media was continuously infused. Cell volume fraction in the micro-channel was determined via nuclear staining and counting of cells immediately after loading and after a 12-hr culture period. The average volume fraction of cells in this model was 0.32 immediately after loading and 0.26 after 12-hr culture. The values are comparable to cell volume fractions of the <italic>in vivo</italic> B16.F10 tumor previously measured in our lab. The reduction in cell volume fraction after overnight culture was due to the change in cell morphology to become more elongated after time in culture. Cell-cell adhesions appeared to have formed during culture, resulting in more uniform packing. </p><p>Sodium fluorescein dye was used as a drug analog and the extent of penetration of this fluorescent molecule through the cell compartment was assessed through microscopy. The dye was introduced on one side of the cell micro-channel and fluorescence images were captured for generation of concentration profiles in the cell compartment. Results showed that dye penetration through the cell chamber was greatly limited by the presence of the 3D cell culture and a linear concentration profile was achieved across the cell compartment. Also, the concentration of sodium fluorescein in the cell compartment of the 12-hr microfluidic cell culture was appreciably lower than the concentration in the cell compartment when the dye was introduced immediately after loading cells. These results suggest that the proposed tumor model shows significant resistance to dye penetration and could prove to be extremely useful for mimicking tumor tissue resistance to drug penetration via diffusion.</p><p>There are many barriers to gene delivery to tumors which highlight the importance of selecting an effective gene carrier system. Some pathogenic bacteria have been investigated as gene delivery vectors because of their innate ability to selectively proliferate in tumor environments. However, pathogenicity concerns arise when trying to achieve therapeutic levels of gene expression. It has been shown that non-pathogenic bacteria such as <italic>E. coli</italic> can be engineered to invade mammalian cells and participate as gene delivery vehicles. Hence, the second part of this research project involved the use of the newly developed microfluidic 3D tumor model previously described to visualize the transport behavior of invasive (inv+) and non-invasive (inv-) <italic>E. coli</italic>. The inv+ bacteria harbored a plasmid containing the inv gene encoding the protein invasin that binds to &beta₁ integrin receptors on the surface of mammalian cells resulting in the phagocytosis of invasin-expressing bacteria by normally non-phaogcytotic cells. Two tumor cells lines were used: B16.F10 and EMT6, which have been shown to differ in expression of &beta₁ integrins. The bacteria were also engineered to express mCherry for fluorescent detection.</p><p>A suspension of tumor cells and bacteria was loaded into the microfluidic device and cultured for 12 hrs before imaging bacteria distribution throughout the cell culture. Proliferation of inv+ bacteria was generally uniform throughout the cell compartment in the B16.F10 model and bacterial cells were primarily concentrated outside of cells. Bacteria that were internalized did not appear to migrate far from the plasma membrane of the tumor cell. The non-invasive bacteria proliferated to a much greater extent than the invasive form and this proliferation was also generally uniform throughout the cell compartment. Proliferation of both invasive and non-invasive bacteria in the EMT6 model was less uniform than in the B16.F10 model. Overall bacterial concentration appeared to be lower in the EMT6 model. Viability staining after bacterial infection showed that tumor cells in the 3D model were able to maintain viability despite bacterial cell proliferation. </p><p>An additional assay was conducted in culture plate wells to determine the effect of chemical factors secreted by tumor cells on bacterial cell proliferation. The results of this assay revealed that tumor cells may be secreting anti-microbial factors that inhibit the proliferation of bacteria and that the binding of invasin-expressing <italic>E. coli</italic> to tumor cells may further promote the release of these factors.</p><p>The results of this study suggest that tumor cell type plays a major role in the distribution and proliferation of bacteria in a 3D environment. The ability to visualize bacterial spread throughout a 3D tumor model will prove to be useful for observing the effect of various genetic modifications on the transport and gene delivery efficiency of <italic>E. coli</italic>.</p> / Dissertation

In recent decade, lung cancers had the highest incidence and mortality rate among all cancers in Taiwan. Among lung cancers, adenocarcinoma was the most frequent type. The chemotherapy was still the main choice in treating lung cancer by the mechanism of destroying DNA, but the response rate kept low. The function of DNA repair makes cancer cells resistant to chemotherapy. Therefore, this study focused on the effect of cancer cell growth by silencing Mre11. The first part of this study was to make a tissue microarray consisting of adenocarcinoma from 57 patients. Immunohistochemistry staining for Mre11 was done. The correlation of Mre11 expression and clinical variables with survival was analyzed. The second part was tried to knockdown Mre11 in A549 cell by shRNA. Another A549 cell line containing empty vector was selected as control group. These cell lines were then ready for XTT method, soft agar colony formation assay, flow cytometry and nude mice assay. In the clinical data, the absence of lymph node and distant site metastasis were good prognosis factor for longer survival. Although the high expression on Mre11 had longer survival, this variable was not a true independent factor. On XTT method and soft agar colony formation assay, the A549 cells with Mre11 knockdown had a slower proliferation and fewer colony numbers, respectively. The cell cycle demonstrated an elevated G0/G1 and S phase and depressed G2/M phase in A549 cells with Mre11 knockdown. The tumor arising from A549 cells with Mre11 knockdown in the nude mice also had a smaller size. Based on the above study, inhibition of Mre11 may result in a reduction of tumor growth and provide another choice to treat lung cancer.

Deregulation of autophagy plays an important role in the pathogenesis of diseases such as cancer, neuronal degenerative or cardiovascular disease. Autophagy is a process to engulf the cytoplasmic contents into autophagosome and deliver them for lysosomal degradation. Its major function is to clear unfolded protein or damage organelles for maintaining proper metabolic homeostasis and normal cell physiological activities. Autophagy and multivesicular bodies, MVBs, cooperate to regulate the turnover of intracellular macromolecule, defective organelles and signaling receptor. Endosomal sorting complex required for transport, ESCRT, is important for the formation of MVBs, which regulates membrane receptor recycling, protein sorting and vesicular trafficking. Tumor Susceptibility Gene 101(TSG101) is a member of ESCRT-I that plays an important role on MVBs formation and maintaining ESCRT function. Previous report indicated that autophagosome accumulation upon deprivation of TSG101, implying possible role of TSG101 during autophagic process. In this study, we observed the increase of TSG101 and autophagic marker proteins, such as LC3-II and ATG upon nutrient starvation. Furthermore, knockdown TSG101 in cervical carcinoma HeLa cell resulted in the elevation of LC3-II, ATG3 and ubiquitinated protein aggregates marker protein p62, which is congruous to other reports. However, in neuroblastoma SH-SY5Y cell, transfection of siRNA led to the decrease of LC-II and ubiquitinated protein level. These results indicated that TSG101 might be critical for autophagy and the maintenance of steady-state level of cellular ubiquitinated proteins. Ectopic upregulatory expression of HA-TSG101 led to the increase of LC3-II in both cell type. The elevation of ATG3 level is also observed in HeLa cell. Therefore, we speculated that TSG101 might be important for the formation of autophagosome, but our data did not exclude the possible role of TSG101 in regulation of the fusion of autophagosome and lysosome, because the increase of ATG3 indicated ectopic HA-TSG101 might facilitate the execution of autophagic flow. In addition, we have established GFP-LC3 expression cell lines. Our imaging data showed the colocalization of TSG101 and GFP-LC3 in both cytoplasm and nucleus that might be an interesting research topic for investigation the role of TSG101 in autophagic pathway.

The Reelin gene located on the human chromosome region 7q22, encodes an extracellular matrix glycoprotein, a ligand for ApoER2 and low-density lipoprotein receptors (LDL) Receptor, is required for mediating the correct positioning of neurons during embryonic brain development1. In the current study, first we applied RT-PCR and immunohistochemistry analysis (IHC) analysis on tissue microarrays (TMA) to verify the Reelin expression patterns in a variety of adult tissues, suggesting additional roles for Reelin in stabling the cyto-architecture and controlling the remodeling of many organs during development. Second, we report the Reelin expression status in tumorigenesis. We discover that the loss of Reelin expression is associated with multiple types of cancers, including more than 80% of both breast and colorectal cancers. Interestingly, our study also found suspension small cell lung cancer (SCLC) cell lines that grow as large aggregates retained high Reelin expression, whereas attached non small cell lung cancer cultures do not. That may imply the Reelin expression may be also associated with cell culture morphology and growth characteristics in the in vitro culture system for lung cancers. Our results here also demonstrated that epigenetic silencing of Reelin expression by DNA hypermethylation in tumors directly correlates with loss of Reelin expression in many cancers. Reelinmethylation was reversed and expression restored by treating tumor cell lines with the demethylating agent 5-aza-2-deoxycytidine. In conclusion, from the molecular basis of Reelingene inactivation in human cancer here, we propose that the Reelinvariation in more than 80% of breast and colorectal cancers makes it a significant novel tumor marker.

Single minded 2 (Sim2) is a member of the basic helix-loop-helix Per-ARNT-Sim (Period-Arylhydrocarbon Nuclear Translocator-Single minded) family. Human SIM2 is involved in the etiology of the Down’s phenotype. In addition to the physical and mental deficiencies associated with DS, it has become apparent that women with DS are 10-25 times less likely to develop breast cancer in comparison to age-matched normal populations. Such significant effects on breast cancer susceptibility are thought to result from gene dosage effects of one or more tumor suppressor genes on chromosome 21. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Similar to full-length Sim2, Sim2s interacts with ARNT and to a lesser extent, ARNT2. The effects of Sim2s on transcriptional regulation through hypoxia-, dioxin- and central midline response elements are different than that of full length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s binds to and activates expression from a central midline response element-controlled reporter through an ARNT transactivation domain-dependent mechanism. Forced expression of SIM2s in MDA-MB-435 breast cancer cells significantly inhibited proliferation, reduced anchorage-independent growth, and decreased invasive potential. SIM2s directly decreased expression of matrix metalloprotease-3, a known mediator of breast cancer metastasis. In addition, loss of Sim2 in the mouse mammary gland increased ductal branching, accelerated lobuloalveolar-like precocious hyperplasia, and decreased cell apoptosis, suggesting that SIM2s is a mammary tumor suppressor. Sim2-/- mammary glands lose E-cadherin expression, suggesting that Sim2s plays a role in regulating E-cadherin/beta-catenin signaling. Loss of Sim2 in the mammary glands also resulted in dramatically increased MMP3 expression. The mechanism of SIM2smediated repression of MMP3 was found to be due to its ability to inhibit AP-1 binding to the MMP3 promoter. These results suggest that SIM2s contributes to the breast cancer protective effects observed in DS individuals.

Bladder cancer is a common malignancy affecting the genitourinary system. Although a large number of studies have been carried out on these areas for a long time, little is know about the molecular events which may involve in tumorigenesis. Until now, no profound immunohistological or molecular markers have been identified to define clinically relevant subsets of bladder cancer. The purpose of this thesis is to identify a novel bladder cancer carcinogenesis related genes. Chapter 1 attempts to illustrate the background, molecular markers, chromosomal abnormalities and genetic instability related to bladder cancer. In Chapter 2, various bioinformatics methodologies were used to annotate and identify candidate genes. Twenty-one genes were identified 1.5-fold up- or down-regulated in mRNA expression from RT4, TSGH8301 and J82, three different stages of bladder cancer cell lines by microarray chips (Dr. Liu, personal communications). Another eight candidate tumor suppressor genes were preliminarily identified from suppression subtractive hybridization (SSH) cDNA library of RT4 cell line based on an isoflavones-treated minus non-treated and further subjected to quantitative RT-PCR analyses to confirm the mRNA expression level in different stages of bladder cancer cell lines. Chapter 3 studies on the ANXA10 gene with special emphasis on its cloning, protein expression, subcellular localization and the preparation of polyclonal antibody. The result suggests that ANXA10 is a cytoplasmic protein in N18 cells. Chapter 4 analyzes the CDK2AP1 gene in mRNA and protein level at different bladder cancer cell lines and various specimens. In our preliminary observations, there are lost of CDK2AP1 expressions at invasive TCCs specimens when compared to noninvasive TCCs specimens. The mechanism of the tumor-associated loss of the CDK2AP1 expression is currently not clear. In Chapter 5, bladder cancer cell lines TSGH8301, UB37, TCCSUP and J82 in SCID mice xenograft model were established for further in vivo studies.

ABSTRACT Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta¡Vcatenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta¡Vcatenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.

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