published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

The basis of this investigation is to characterize growth property differences in normal versus virally transformed cells. Using a temperature-sensitive mutant of Rous sarcoma virus, the cells' transformation state is regulated by the growth temperature; at 33°C the cells are transformed, while at 39°C the cells have normal characteristics. The morphology of NRK cells is elongated and fibroblastic; when transformed the cells are rounded. Normal cells grow to a monolayer and stop, while transformed cells grow to saturation densities greater than just a monolayer amount. Transformed cells can form foci when grown in mixture with normal cells. Normal cells must be in contact with the culture vessel in order to grow, but transformed cells lack anchorage dependence for growth.

In response to DNA damage, the ATM and ATR kinases proliferate a signal that is transduced, either directly or via Chk2 and Chk1, to effector proteins, forming the DNA damage response (DDR). The effector proteins delay cell cycle progression, through checkpoints, and activate specific DNA repair mechanisms essential for preserving genome integrity and preventing cancer formation. Bloom's syndrome (BS) patients, which lack the BLM protein show genome instability and have a predisposition to cancer. BLM is phosphorylated by the DDR kinases ATM, ATR and Chk1. These phosphorylation events are essential for BLM to maintain replication fork integrity, preserve the S phase checkpoint and activate BLM to interact with other DDR proteins. In this study I have shown that BLM, isolated from mitotic cells, is phosphorylated on amino acid residue serine 26 (S26). BS cells lacking native BLM, but expressing a variant of BLM protein that cannot be phosphorylated at S26, fail to fully activate the G2/M checkpoint following UV irradiation or treatment with inhibitors of DNA topoisomerase H. Consequently, these cells are more sensitive to killing by these agents than are BS cells expressing wildtype BLM. The Chk1 and Aurora B kinases are able to phosphorylate BLM on S26 in vitro. Moreover, loss of Aurora B kinase activity leads to reduction of S26 phosphorylation in mitotic cells. Cells treated with inhibitors of Aurora B fail to fully active the G2/M checkpoint after UV DNA damage. Taken together, these data suggest, that Aurora B kinase phosphorylates BLM on S26 and that this is required to fully activate the G2/M checkpoint.

Understanding of cell death mechanisms is important to identifying therapeutic approaches to treat excess cell growth, as seen in tumors, or to inhibit excess cell death, as seen in neurodegenerative disease and ischemia. In the first part of this work, we aim to extend the understanding of a non-apoptotic cell death phenotype, ferroptosis, through use of a genome-wide siRNA screen. We identified knockdown of CARS, or cysteinyl-tRNA synthetase, as an inhibitor of erastin-induced ferroptosis. Loss of CARS led to upregulation of the transsulfuration pathway, where methionine is used as the source of sulfur for cysteine synthesis, as a suppressive mechanism. Upregulation of the transsulfuration pathway may serve as a biomarker to identify tumor types that may be insensitive to ferroptosis-inducing therapeutics. On the other hand, induction of the transsulfuration pathway may be beneficial in disease contexts that involve excess cell death. In the second part of this work, we elucidate the mechanism of action of a small molecule Mdm2 inhibitor, or MEL. Mdm2 is a negative inhibitor of p53; therefor, an inhibitor of Mdm2 may be useful in treating tumors driven by Mdm2 overexpression. We found MEL to inhibit the E3-ligase activity of Mdm2/MdmX heterocomplex, proving to be a useful tool to probe the importance of the heterocomplex in normal physiology and disease development. We also explored the structural scaffold of MEL compounds, an indole, and identified a novel ferroptosis inducer, increasing the chemical toolbox available to study ferroptosis.

In this thesis, we develop and apply top-down, quantitative approaches to gain novel insights into various complex biological systems. Beginning at the multicellular level, we study human gut microbiome dynamics from an ecological perspective. We develop computational frameworks to enable a global understanding of the spatiotemporal variability of gut bacterial abundances. We demonstrate the utility of our frameworks to elucidate the ecological processes governing abundance changes of gut microbiota. We then shift our focus to the intracellular level by investigating the metabolic limitations of cancer cell growth. We use coarse-grained mathematical modeling to identify a major growth limitation of cancer cells associated with electron acceptor deficiency, which we then experimentally validate. Collectively, these set of approaches help to decipher the organizing principles of complex biological systems at both the individual and multicellular levels.

Hepatoma-derived growth factor (HDGF) is commonly over-expressed in human cancer cells. It was able to stimulate cell growth. The expression level of HDGF was reported to correlate with poor prognosis of cancer therapy. It was found that HDGF is over-expressed in the fractionated gamma radiation conditioned HepG2 cells, which have higher growth rate, lower radiosensitivity and higher drug sensitivity. The aim of the present study was to investigate the role of HDGF in mediating these changes in human cancer cells and the underlying mechanisms. The results indicate that transfection of HDGF cDNA carrying vector stimulated the growth of cancer cells while knock-down of HDGF by transfection of HDGF antisense oligos not only suppressed the growth but also triggered apoptosis in human cancer cells. It suggests that HDGF stimulates cancer cell growth and acts as a survival factor for human cancer cells. Mechanistic study showed that knock-down of HDGF may trigger apoptosis through the regulation of the apoptotic pathways. The apoptosis induced by HDGF knock-down was mediated by the BAD regulated intrinsic apoptotic pathway and the Fas regulated extrinsic apoptotic pathway. The HDGF knock-down induced apoptosis was also mediated by the changes in the activity of the cell survival pathways, including the Ras/Raf/MEK/ERK, PI3K/Akt, NFkappaB and Jak/STAT pathways. In addition to the growth promoting function, HDGF was found to regulate the radiosensitivity and chemosensitivity of cancer cells. Overexpression of HDGF reduced the radiosensitivity and the level of apoptosis induced by gamma radiation. On the contrast, overexpression of HDGF increased the chemosensitivity and the level of apoptosis induced by anti-cancer drugs, including Taxol, doxorubicin (Dox) and tamoxifen. The results indicated that HDGF may stimulate the growth, reduce the radiation sensitivity and increase the drug sensitivity of cancer cells. HDGF may also be responsible for the changes in cancer cell properties after fractionated gamma radiation treatment. The present findings suggest that HDGF may be a potential target for cancer therapy. / Tsang, Tsun Yee. / Adviser: Tim Tak Kwok. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3497. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Although there is strong evidence that thyroid tumors occur more frequently in females than in males, few studies have investigated the role sex hormones play in thyroid carcinogenesis, especially the role of estrogen (E2). This laboratory has previously shown that estrogen receptors (ERs) exist in thyroid papillary carcinoma cells. Continuing along this line of research, we studied the role of E2 and its receptors on the regulation of human thyroid cancer. / In conclusion, we have demonstrated (1) a novel mechanism by which E2 contributes to the proliferation and growth of thyroid cancer cells, (2) that E2 influences the expression of ERalpha and ERbeta differently, causing an imbalance between them, which may change the biological behavior of thyroid cancer cells, giving them the ability to proliferate and resist apoptosis by influencing the level of ERK1/2 activity and subsequently the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax, and (3) that the subcellular localization of ERalpha and ERbeta may be a factor that contributes to the differing pathogeneses of papillary and anaplastic thyroid cancers. / To further clarify the mechanism by which E2 promotes cellular proliferation in thyroid cancer cells, we studied the localization of ERalpha and ERbeta in both KAT5 and anaplastic carcinoma cells (FRO) by immunofluorescence staining and by immunoblotting of the proteins in subcellular fractions. Cell proliferation and apoptosis were examined together with the expression of selected apoptotic proteins such as Bax, AIF and cytochrome c. We showed that the subcellular localization of ERalpha and ERbeta differed in papillary and anaplastic thyroid cancer. E2 administration led to an increase in the level of ERalpha in the nuclei of papillary cancer cells while the levels of ERbeta remained unchanged. However, the level of mitochondrial ERbeta surpassed that of ERalpha in anaplastic cancer cells. We also showed that E2 affected caspase-dependent and/or independent apoptosis via ERs in thyroid cancers. / We first studied the molecular pathways by which E2 promotes cellular proliferation in thyroid cancer cells using a human thyroid cancer cell line (KAT5) treated with E2, a selective E2 alpha receptor (ERalpha) agonist (PPT), a selective E2 beta receptor (ERbeta) agonist (DPN), an ERalpha antagonist (MPP), an E2 antagonist (ICI182780) and siRNA, which blocks ERalpha and ERbeta, by MTT assay, DNA fragmentation ELISA, BrdU cell proliferation assay and Western blot. We found that E2 and PPT gradually promoted cell proliferation by increasing the expression of ERalpha and by up-regulating the expression of Bcl-2 and pERK1/2. In contrast, we found that DPN had a negative effect on cell growth by enhancing the expression of ERbeta and Bax and by down-regulating pERK1/2 expression. At the same time, blocking ERalpha significantly reduced the E2-mediated Bcl-2 and pERK1/2 expression. On the other hand, blocking ERbeta markedly enhanced their expression. These results suggest that E2 regulates cellular growth of KAT5 cells by an ER-ERK1/2-MAPK pathway and also that E2 affects mitochondrial homeostasis. / Zeng, Qiang. / "September 2007." / Adviser: George Gong Chen. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4616. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 136-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Chronic Lymphocytic Leukemia (CLL) is defined by the accumulation of clonally expanded CD5+ and CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. CLL represents one third of all leukemia cases with an average age of 72 years at diagnosis making it the most common adult leukemia. The Eµ-Tcl1 mouse serves as an excellent model to study the development of CLL as they progress to a CLL like disease by 9-14 months of age, due to overexpression of an oncogene, T cell Leukemia 1(Tcl1), specifically in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to the development of a CLL like disease within 3-8 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, spleen ultrasonography and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 protein (Par-4), a known pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers and has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. We found that CLL cells have constitutively active B-cell receptor signaling (BCR) and that inhibition of BCR signaling with FDA approved drugs causes a decrease in Par-4 protein, mRNA levels, and an increase in apoptosis. In particular, activities of Src family kinases, spleen tyrosine kinase and Bruton’s tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling in both Eµ-Tcl1 CLL cells and primary human CLL samples. Consistent with this, lenti-viral shRNA mediated knockdown of Lyn kinase leads to a decrease in Par-4 expression in MEC-1 cells, a human CLL derived cell line. Igα (CD79a) silencing in primary human CLL cells also results in down regulation of Par-4 expression. Additionally, we knocked down expression of Par-4 in MEC-1 cells which resulted in a decrease in cell growth that could be attributed to an increase in p21 expression and a reduction in the G1/S cell cycle transition. We have also observed this phenomenon by crossing mice deficient in Par-4 with the Eµ-Tcl1 mouse where lack of Par-4 delays CLL growth in the mouse significantly (time to euthanization due to poor body condition - Eµ-Tcl1: 8.9mo vs Par4-/-EµTcl1: 11.97 mo, p = 0.0472) and splenic B-CLL cells from these mice also have increased expression of p21. Since mice in this cohort are whole body knockout for Par-4, the difference in survival times between the Par-4 +ve and Par-4 –ve EµTcl1 mice could be due to the influence of Par-4 on CLL cells as well as the effect of Par-4 secreted by the CLL cells on the microenvironment. There could be other potential roles for Par-4 in the context of CLL which are under further investigation. We have also investigated the site of CLL growth in mouse models to determine that the spleen is the primary organ to accumulate the CLL tumor burden. We have found that splenectomy significantly delays the development of CLL in the primary Eμ-Tcl1 mouse model and prevents growth and development in the adoptive transfer model. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for Par-4 compared to C57BL/6 wild type mice. Par-4 appears to regulate a specific microenvironment required for CLL growth. Current studies are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.

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